Targeting of G-protein coupled receptors in sepsis

Abstract

The Third International Consensus Definitions (Sepsis-3) define sepsis as life-threatening multi-organ dysfunction caused by a dysregulated host response to infection. Sepsis can progress to septic shock—an even more lethal condition associated with profound circulatory, cellular and metabolic abnormalities. Septic shock remains a leading cause of death in intensive care units and carries a mortality of almost 25%. Despite significant advances in our understanding of the pathobiology of sepsis, therapeutic interventions have not translated into tangible differences in the overall outcome for patients. Clinical trials of antagonists of various pro-inflammatory mediators in sepsis have been largely unsuccessful in the past. Given the diverse physiologic roles played by G-protein coupled receptors (GPCR), modulation of GPCR signaling for the treatment of sepsis has also been explored. Traditional pharmacologic approaches have mainly focused on ligands targeting the extracellular domains of GPCR. However, novel techniques aimed at modulating GPCR intracellularly through aptamers, pepducins and intrabodies have opened a fresh avenue of therapeutic possibilities. In this review, we summarize the diverse roles played by various subfamilies of GPCR in the pathogenesis of sepsis and identify potential targets for pharmacotherapy through these novel approaches.

1. Introduction

Historically, the term “sepsis” was first used by Hippocrates (470–360 BC) to describe a disease-producing process by which flesh rotted and wounds festered. The modern concept of sepsis is an abnormal host response to infection that results in multi-organ dysfunction. This condition is associated with a high rate of mortality and morbidity with an estimated 5.3 million deaths per year being attributable to sepsis (Adhikari, Fowler, Bhagwanjee, & Rubenfeld, 2010). In the United States, sepsis was estimated to account for more than $20 billion (5.2%) of the total hospital costs in the year 2011 (Angus, et al., 2001). Sepsis is reported to have a prevalence of 12% in hospital-derived cohorts in the United States and may be even higher in lower-to-middle income countries (Mayr, Yende, & Angus, 2014). Sepsis was made a global health priority by the World Health Organization in 2017 and a resolution was adopted by both the World Health Organization and the World Health Assembly to improve the diagnosis, treatment and prevention of sepsis (Reinhart, et al., 2017).

According to the Third International Consensus Definitions (Sepsis-3) (Singer, et al., 2016), sepsis is defined as life-threatening end-organ dysfunction caused by a dysregulated host response to infection. Organ dysfunction in patients with sepsis is assessed using the Sequential Organ Failure Assessment (SOFA) score (J.-L. Vincent, et al., 1996). Presence of an infection coupled with an increase in SOFA score by 2 points is sufficient to diagnose sepsis. In a small subset of patients, sepsis results in profound cellular and circulatory abnormalities severe enough to substantially increase mortality; these patients are deemed to have septic shock and have a reported in-hospital mortality of 40–60% (Cecconi, Evans, Levy, & Rhodes, 2018). Despite significant advances in our understanding of the pathobiology of sepsis, therapeutic interventions have not translated into tangible differences in overall outcomes for patients. The mortality from sepsis remains at 25%, while that for patients in septic shock is even higher ranging from 30% to 50% in most series (Fleischmann, et al., 2016). This may well be a conservative estimate as evidence suggests that mortality from sepsis and septic shock is even higher in the developing world where close to 87% of the world’s population lives (Reinhart, et al., 2017). Scarcity of data from the developing world makes it difficult to make accurate epidemiological estimations regarding the incidence and mortality of sepsis and septic shock. However, it is estimated that mortality from sepsis is likely around 50–60% and that for septic shock is close to 80% in the developing world (Jawad, Lukšić, & Rafnsson, 2012). In this review, we summarize our current understanding of the pathology of sepsis followed by a review of prior unsuccessful therapeutic strategies in sepsis. We then turn our attention to G-protein coupled receptors (GPCRs) and discuss those that could be potentially targeted in sepsis. Finally, we shed light on novel methods of intracellularly targeting those receptors in sepsis.

2. Pathophysiology of sepsis

Sepsis is a dysregulated host response to infection and is initiated by hyper-activation of the innate immune system. Cells of the innate immune system (macrophages, monocytes, granulocytes, natural killer cells and dendritic cells [DCs]) continuously sample the local tissue environment at various microbial portals of entry. Multiple microbial products and their derivatives (pathogen-associated molecular patterns [PAMPs]) as well as endogenous danger signals (damage-associated molecular patterns [DAMPs]) can bind to cell surface receptors (pattern recognition receptors) on innate immune cells and trigger increased expression of pro-inflammatory cytokines. Simultaneous binding of multiple PAMPs and DAMPs to Toll-like receptors, mannose-binding lectin (MBL), scavenger receptors, nucleotide-binding oligomerization domain (NOD)-like receptors, retinoic acid-inducible gene (RIG)-like receptors, C-type lectin receptors and other receptors leads to complementary activation and amplification of multiple signaling pathways (Gentile & Moldawer, 2013). Intracellular phosphorylation of Janus kinases, signal transduction and activators of transcription, mitogen-activated protein kinases (MAPKs) and other intermediates leads to recruitment of interferon regulatory factor and nuclear factor-κB (NFκB) (Kumar, 2018). Interferon regulatory factor increases the expression of interferon (IFN), while NFκB and activator protein 1 (AP-1) increase the transcription of tumor necrosis factor (TNF), interleukin (IL)-1, IL-12 and IL-18 (among other cytokines) as well as endothelial adhesion molecules (Wiersinga, 2011). Irrespective of the initial repertoire of PAMPs and DAMPs implicated, redundant and complementary signaling pathways are activated in sepsis that converge on the enhanced expression of a common pool of pro-inflammatory cytokines. These cytokines have further diverse downstream effects including activation of complement and coagulation pathways, endothelial barrier dysfunction, alteration of cellular metabolism, and suppression of the adaptive immune system (T. van der Poll & Opal, 2008). Mitochondrial dysfunction has also been identified as a core pathophysiologic feature of sepsis-induced organ dysfunction in more recent studies (Joseph, et al., 2017). Chemokines (including IL-6, IL-8, IFNγ, CXC-chemokine ligand 10 [CXCL10], CC-chemokine ligand 2 [CCL2] and CC-chemokine ligand 3 [CCL3]) induce the chemotaxis and recruitment of phagocytes (Schulte, Bernhagen, & Bucala, 2013). A shift in the endothelial expression of various procoagulant proteins (von Willebrand factor, thrombomodulin, tissue factor and activated protein C [APC]) results in the transformation of a healthy (anticoagulant) endothelium to a prothrombotic endothelium in sepsis (Ince, et al., 2016). Moreover, internalization of the vascular endothelial (VE)-cadherin, as a consequence of pro-inflammatory protease activity leads to a leaky endothelium with increased vascular permeability.

2.1. Complement activation

PAMPs and DAMPs can lead to activation of the complement cascade. The complement cascade is an integral part of the innate immune response and acts as a bridge between innate and acquired immunity. This system consists of a series of proteins that mediate responses to inflammatory triggers through a co-ordinated and sequential enzyme cascade, eventually leading to clearance of foreign cells through pathogen recognition, opsonization and lysis. The complement system also possesses anti-inflammatory functions in that it binds to immune complexes and apoptotic cells, and assists in their removal from the circulation. This important system is involved in the eradication of invading microbes, but, also contributes to the inflammatory response during sepsis.

The complement cascade in humans can be activated through three distinct pathways (as illustrated in Figure 1): (a) the classical pathway; (b) the alternate pathway; and (c) the lectin pathway (Lupu, Keshari, Lambris, & Coggeshall, 2014). C1q in the classical pathway acts as a pattern recognition receptor and can bind to PAMPs or DAMPs, thereby resulting in activation of the classical pathway. PAMPs or DAMPs can also activate the lectin pathway by binding to MBL or ficolins, which in turn can activate MBL-associated serine proteases and lead to the formation of C3 convertase. Cleavage of C3 by C3 convertase leads to the formation of C3a (an anaphylatoxin) and C3b (an opsonin). C3b then participates in the formation of C5 convertase, which cleaves C5 into C5a (another anaphylatoxin) and C5b. C5a is one of the most potent inflammatory peptides produced in the complement pathway and results in chemotaxis of phagocytic cells. C5a also amplifies the production of pro-inflammatory cytokines by innate immune cells and triggers the oxidative burst within neutrophils. Production of free radicals by neutrophils leads to widespread tissue injury and contributes to the multi-organ dysfunction typical of sepsis. Moreover, C5a can cause endothelial activation, vasodilation and activation of the coagulation cascade that may initiate disseminated intravascular coagulation (DIC). DIC further compromises tissue perfusion through the formation of microthrombi and aggravates multi-organ dysfunction. Complement proteins C3 and C5 can also be cleaved by thrombin and plasmin (produced in the coagulation cascade) to activate the complement cascade (so-called “extrinsic protease pathway”), and in turn, complement proteins C3a and C5a can bind to their receptors on platelets to regulate platelet activation and thrombus formation. This cross-talk between the coagulation and complement cascades represents an important link between coagulation and inflammation in the setting of sepsis, and both processes can perpetuate each other leading to worsening multi-organ dysfunction (Foley & Conway, 2016; Marcel Levi & van der Poll, 2017).

Figure 1: Complement cascade.

Complement cascade can be activated by three pathways viz. classical, alternate and lectin pathways. Classical pathway can be activated by a number of factors including antigen-antibody complexes and binding of PAMPs to C1q (a PRR). Likewise, the lectin pathway is activated when DAMPs bind to MBL or ficolins to activate MASPs. Activation of both the lectin and the classical pathways results in the cleavage of complement proteins C2 and C4 to form classical pathway C3 convertase (C4b2a), which is composed of C2a and C4b. C1 inhibitor inhibits the activation of the classical pathway by inhibiting cleavage of C2 and C4 by C1s. The alternate pathway of complement activation involves the spontaneous hydrolysis (‘tick over’) of C3 to form a structurally altered form of C3 [C3(H₂O)], which can bind to factor B and enable its cleavage by factor D. This results in the formation of the alternate pathway C3 convertase (C3bBb) that is composed of C3b and Bb. Both C3 convertases can cleave C3 to form C3a and C3b, which in turn can participate in the formation of classical pathway C5 convertase (C4b2a3b) and alternate pathway C5 convertase (C3bBb3b). Both C5 convertases act on complement protein C5 to form C5a and C5b. C5b can combine with complement proteins C6, C7, C8 and C9 to form an amphiphilic membrane attack complex that can create physical pores in cell membranes and lead to cell lysis. Complement proteins C3a and C5a can both act as anaphylatoxins by binding to their respective receptors (C3aR1 and C5aR1) to enhance chemotaxis, degranulation and vascular permeability. Similarly, C3b can act as an opsonin by binding to complement receptors CR1, CR2 and CRIg. Ab = Antibody; Ag = antigen; C3aR1 = complement protein 3a receptor 1; C5aR1 = complement protein 5a receptor 1; CR = complement receptor; CRIg = complement receptor of the immunoglobulin family; DAMP = damage-associated molecular pattern; MAC = membrane attack complex; MASP = mannose-binding lectin–associated serine protease; MBL = mannose-binding lectin; PAMP = pathogen-associated molecular patterns; PRR = pattern recognition receptor.

2.2. Endothelial dysfunction

The normal endothelium under homeostatic conditions has an anticoagulant property to allow smooth blood flow and permit the exchange of various solutes between the blood, endothelium and interstitium (Hack & Zeerleder, 2001). Integrity of the endothelial barrier is essential to maintain a state of homeostasis as the underlying subendothelial collagen is highly pro-thrombotic. This barrier is maintained consistently by a number of features including the glycocalyx, actin cytoskeleton and intercellular tight junctions maintained by adhesion molecules (VE cadherins) (Aird, 2001). Nitric oxide is synthesized in endothelial cells by the activity of nitric oxide synthase and plays an important role in maintaining the physiologic barrier function of the endothelium (J. L. Vincent, Zhang, Szabo, & Preiser, 2000). Endothelial cells also regulate the composition of the interstitium—the milieu for different types of cells across various tissues—by maintaining a dynamic and interactive interface between the blood and the interstitium (W. L. Lee & Slutsky, 2010).

In patients with sepsis, release of pro-inflammatory mediators increases the expression of selectins and integrins on endothelial cells, which promotes leukocyte adhesion and migration (Ince, et al., 2016). Injury to the glycocalyx, actin cytoskeleton and intercellular tight junctions results in increased vascular permeability and a leaky endothelium (Ait-Oufella, Maury, Lehoux, Guidet, & Offenstadt, 2010). This allows the interaction of tissue factor with circulating factor VII, thereby initiating the tissue factor pathway of the coagulation cascade. Likewise, subendothelial collagen fibers bind to von Willebrand factor, which in turn binds platelets (through the glycoprotein Ib/IX receptor) and leads to thrombosis (M. Levi, ten Cate, & van der Poll, 2002). Additionally, endothelial dysfunction leads to systemic vasodilatation and loss of intravascular proteins and plasma fluids into the extravascular space with disruption of capillary flow and tissue hypoperfusion (Fernandez-Sarmiento, Salazar-Pelaez, & Carcillo, 2020). Nitric oxide production markedly increases in sepsis as a result of increased expression of inducible nitric oxide synthase and contributes to endothelial dysfunction (J. M. Wong & Billiar, 1995). Widespread activation of the coagulation cascade leads to the formation of platelet microthrombi in the circulation (DIC), which further compromises tissue perfusion and manifests as septic shock.

2.3. Coagulation cascade

As mentioned before, increased vascular permeability and exposure of subendothelial collagen can lead to binding of von Willebrand factor, which in turn can bind platelets. Additionally, many pro-inflammatory cytokines and chemical mediators released in sepsis can lead to activation of platelets and the coagulation cascade. Release of tissue factor from the endothelium in response to sepsis can activate the tissue factor pathway and result in formation of fibrin. Glycoprotein IIb/IIIa receptors on platelets can bind fibrin and cross-link multiple platelets together. Formation of fibrin strands and their cross-linking along with aggregation of platelets leads to the formation of a stable thrombus. Moreover, formation of thrombin through the tissue factor pathway leads to activation of the contact pathway, which further amplifies the coagulation process. There is significant “cross-talk” between the coagulation, kinin and complement cascades—activation of the contact pathway produces factor XIIa, which can form kallikrein. Kallikrein not only produces bradykinin but can also amplify the coagulation cascade by producing more factor XIIa. Likewise, kallikrein can cleave plasminogen to form plasmin, which can activate the complement cascade. Many coagulation factors (specifically, factors XIa, Xa and IXa) can also cleave complement proteins C3 and C5, thereby activating the complement cascade (Amara, et al., 2010). Conversely, complement activation can lead to cleavage of thrombinogen to form thrombin, further amplifying the coagulation cascade. Thrombin itself can amplify the overall inflammatory response by multiple mechanisms, such as induction of tissue ischemia (through thrombosis), production of down-stream mediators (for instance, APC) and signaling through protease-activated receptors (PARs) which leads to release of inflammatory mediators and, recruitment and chemotaxis of inflammatory cells. Platelets themselves are a source of P-selectin, which serves as an attachment for neutrophils. Neutrophils in turn can form neutrophil extracellular traps (NETs) through the process of NETosis, which provides a scaffold for further clot formation (Delabranche, et al., 2017). Inflammation is closely linked to coagulation and both processes can amplify each other in the setting of tissue injury. When such tissue injury is localized, these processes can act synergistically to wall off the site of injury and protect the rest of the host from infection. However, in the setting of sepsis, widespread activation of coagulation and inflammatory processes lead to DIC and multi-organ failure.

2.4. Phases of sepsis

Sepsis is a dysregulated host response to infection that results in multi-organ dysfunction. Traditionally, this dysregulation has been perceived as a hyper-activation of the innate immune system with a cascade of pro-inflammatory cytokines and mediators that result in uncontrolled inflammation. However, a whole body of evidence suggests that immunosuppression occurs both in the early and delayed phases of sepsis (Gentile, et al., 2012). Studies of sepsis survivors have shown that patients who survive the initial “hyper-inflammatory” phase of sepsis go on to develop a prolonged state of “immune paralysis” and chronic inflammation (termed persistent inflammation/immunosuppression and catabolism syndrome). This delayed phase of sepsis is associated with profound changes in functioning of the immune system (Rubartelli & Lotze, 2007; Walton, et al., 2014) including a predominance of immature neutrophils, recruitment of myeloid-derived suppressor cells, peripheral lymphopenia, increased proportion of T_reg cells (CD4⁺/CD25⁺/FOXP3⁺ phenotype), impaired antimicrobial activity of innate immune cells, preferential differentiation to the macrophage M2 phenotype, elevated levels of anti-inflammatory cytokines (chiefly IL-10 and transforming growth factor-β) and reduced expression of MHC (major histocompatibility complex)-II molecules on DCs (Boomer, et al., 2011; Taneja, Sharma, Hallett, Findlay, & Morris, 2008). Experimental studies have also demonstrated increased expression of programmed death ligand 1 (PD-L1) on antigen presenting cells and stromal cells, which can interact with the programmed death protein 1 (PD1) receptor on T cells, thereby leading to broad T cell anergy (Drewry, et al., 2014). Similarly, studies from patients with sepsis identified profound apoptosis of DCs, T cells and B cells (Hotchkiss, et al., 1999). In fact, the degree of apoptotic loss of lymphocytes has been shown to be correlated with the severity of sepsis (Drewry, et al., 2014). Pharmacological approaches that block the interaction of PD-L1 with PD1 and reduce lymphocytic apoptosis have been shown to be beneficial in experimental models of sepsis (Patil, Guo, Luan, & Sherwood, 2017). Immune checkpoint inhibitors that block PD-L1 have shown promising results in cancer immunotherapy trials and hold great promise for use in the treatment of sepsis (van Ton, Kox, Abdo, & Pickkers, 2018).

2.5. Subtypes of sepsis

Sepsis is known to be an extremely heterogeneous condition with variations in the type and severity of host response depending on the repertoire of PAMPs and DAMPs implicated in its pathogenesis. This poses significant challenges in designing randomized trials and assessing response to various therapeutic modalities. Consequently, the importance of delineating accurate nosology for designing personalized therapies tailored to the individual patient has been recognized for long. In 2017, the MARS (Molecular Diagnosis and Risk Stratification of Sepsis) consortium published a study describing four molecular endotypes of sepsis (termed MARS1, MARS2, MARS3 and MARS4) based on array-based transcriptomics analysis (Scicluna, et al., 2017). Using a 140-gene expression signature, patients were reliably stratified into one of the four molecular endotypes. When these endotypes of sepsis were combined with clinical data (APACHE [Acute Physiology and Chronic Health Evaluation] scores), they provided robust predictions of 28-day mortality risk. Similar to the MARS consortium study, Sweeney and colleagues identified three distinct clusters of sepsis across multiple datasets using unsupervised machine learning algorithms of transcriptomics data (Sweeney, et al., 2018); the authors termed these clusters as the “Inflammopathic”, “Adaptive” and “Coagulopathic” subtypes of sepsis. The “Inflammopathic” subtype was associated with activation of the innate immune system and higher mortality rate, while the “Adaptive” subtype was associated with activation of the adaptive immune system and a lower mortality rate. The different molecular subtypes of sepsis described in these studies are useful in providing a unifying framework for understanding the molecular heterogeneity of sepsis and applying precision medicine approaches to sepsis.

With the discovery of various molecular endotypes of sepsis, efforts were made to describe clinical phenotypes of sepsis that could be identified using routine clinical parameters (such as vital signs and laboratory investigations). Four different clinical phenotypes of sepsis were recently described in the literature (Seymour, et al., 2019). These phenotypes of sepsis (named α, β, γ and δ) were derived from multiple large datasets by Seymour and colleagues using unsupervised machine learning techniques—most notably, clustering. These phenotypes were associated with mortality and were unique in their defining characteristics (29 clinical variables, such as vital signs and laboratory parameters) when compared to the commonly used severity scales for sepsis (SOFA and APACHE scores). In-hospital mortality for α, β, γ and δ phenotypes were 2%, 5%, 15% and 32% respectively.

The recognition of molecular endotypes and clinical phenotypes of sepsis highlighted the importance of considering sepsis as a heterogeneous syndrome (constellation of signs and symptoms) rather than a single disease entity. Inaccurate and vague nosology for a heterogeneous clinical syndrome results in dumping of multiple different pathologic entities into a single basket group. This one-size-fits-all approach partly accounts for the myriad number of negative clinical trials in sepsis as discussed in the next section.

3. Prior therapeutic strategies in sepsis

Since 1982, more than 80 phase II and phase III clinical trials involving patients with sepsis have been conducted. Despite this, the only interventions consistently shown to have any tangible impact on the survival from sepsis and septic shock have been early administration of appropriate antimicrobials, source control and hemodynamic stabilization. The current treatment of sepsis is centered around limiting the development of end-organ dysfunction by providing rapid source control and hemodynamic stabilization, and when needed, organ support to ensure the recovery of end-organ function. Based on differing results from numerous trials evaluating the use of corticosteroids in sepsis, the Surviving Sepsis Campaign guidelines recommend administration of glucocorticoid therapy only for those patients with septic shock who remain hemodynamically unstable despite adequate fluid resuscitation and vasopressor therapy (Rhodes, et al., 2017). The negative clinical trials in sepsis also warrant attention in that they improved our understanding of its pathophysiology and shed light on the challenges of conducting clinical trials in sepsis. Prior therapeutic strategies in sepsis initially focused primarily on thwarting the vicious circle of inflammation and controlling the cytokine storm that typifies sepsis. However, over the past decade, a paradigm shift occurred in sepsis research as immune paralysis was identified as a central theme leading to mortality in a vast majority of septic patients (Leentjens, Kox, van der Hoeven, Netea, & Pickkers, 2013). This shifted the emphasis from designing pharmacotherapies to block the initial hyper-inflammatory phase to therapies aimed at stimulating the adaptive immune response. Many such immune stimulatory therapies have been explored in randomized controlled trials with mixed results. More recently, randomized trials evaluating the role of immune checkpoint inhibitors for the treatment of sepsis have been started (NCT02960854 and JapicCTI-173600). Given the encouraging results of immune checkpoint inhibitors in oncological trials, legitimate optimism exists in the critical care community regarding the use of these therapies in patients with sepsis. Table 1 provides a summary of various interventions that have been tested hitherto in clinical trials for sepsis.

Table 1 —

Prior major clinical trials assessing various interventions in sepsis

Intervention	First author and year	PubMed ID	Primary outcome(s)	Study name
Activated protein C	Abraham E et al. (2005)	16192478	All-cause 30-day mortality	ADDRESS trial
Anti-endotoxin antibody	Angus DC et al. (2000)	10755499	All-cause 14-day mortality	E5 study
Anti-thrombin III	Warren BL et al. (2001)	11597289	All-cause 28-day mortality	KyberSept trial
Anti-TNF antibody	Abraham E et al. (1998)	9734938	All-cause 28-day mortality	NORASEPT II trial
Bactericidal/permeability-increasing protein	Levin M et al. (2000)	11041396	Mortality, amputations and change in pediatric overall performance category at 60 days	rBPI21 study
Deltibant	Fein AM et al. (1997)	9020273	Risk-adjusted 28-day survival	CP-0127 SIRS study
Esmolol	Morelli A et al. (2013)	24108526	Sustained reduction in heart rate over a 96-hour period
G-CSF	Cheng AC et al. (2007)	17599307	All-cause 28-day mortality
High-dose corticosteroids	Hinshaw L et al. (1987)	2888017	All-cause 14-day mortality	VA Systemic Sepsis Cooperative Study
Ibuprofen	Bernard GR et al. (1997)	9070471	All-cause 30-day mortality	Ibuprofen in Sepsis Study
IFNγ	Dries DJ et al. (1994)	7944932	Major infection or death at 60 days
IL-1 receptor antagonist	Opal SM et al. (1997)	9233735	All-cause 28-day mortality	IL-1 receptor antagonist sepsis group
Immuno-nutrition	Heyland D et al. (2013)	23594003	All-cause 28-day mortality	REDOXS study
Inhaled nitric oxide	Trzeciak S et al. (2014)	25080051	Change in microcirculatory flow index
Intravenous immunoglobulin	Tugrul S et al. (2002)	12225613	All-cause 28-day mortality
Ketoconazole	ARDS Network (2000)	10789668	Ventilator-free days and ventilator-free survival at 28 days
Levocarnitine	Jones AE et al. (2018)	30646314	All-cause 28-day mortality	RACE trial
LPS analog	Opal SM et al. (2013)	23512062	All-cause 28-day mortality	ACCESS trial
N-acetylcysteine	Spies CD et al. (1994)	7956276	Overall survival rate
PAF acetyl-hydrolase	Opal SM et al. (2004)	14758145	All-cause 28-day mortality
PAF antagonist	Dhainaut JF et al. (1998)	9875905	All-cause 28-day mortality	BN 52021 Sepsis Study
Pentoxifylline	Staubach KH et al. (1998)	9438767	All-cause 28-day mortality
PLA2 inhibitor	Zeiher BG et al. (2005)	16096451	All-cause 28-day mortality	EZZI study
Polymyxin B hemoperfusion	Dellinger RP et al. (2018)	30304428	All-cause 28-day mortality	EUPHRATES trial
Prostaglandin E2	Vincent JL et al. (2001)	11685297	All-cause 28-day mortality	TLC C-53 study
Selenium	Valenta J et al. (2011)	21347869	All-cause 28-day mortality
Selepressin	Laterre PF et al. (2019)	31577035	Vasopressor- and ventilator-free days within 30 days
Thrombomodulin	Vincent JL et al. (2019)	31104069	All-cause 28-day mortality	SCARLET trial
Tissue factor pathway inhibitor	Abraham E et al. (2003)	12851279	All-cause 28-day mortality	OPTIMIST trial
TNF receptor	Abraham E et al. (1997)	9153367	All-cause 28-day mortality	Ro 45-2081 Study
Vitamin C	Fowler III AA et al. (2019)	31573637	Change in SOFA score and biomarkers of inflammation & vascular injury	CITRIS-ALI trial

ARDS = acute respiratory distress syndrome; G-CSF = Granulocyte-colony stimulating factor; IFN = interferon; IL = interleukin; LPS = lipopolysaccharide; PAF = platelet activating factor; PLA₂ = phospholipase A₂; SOFA = sequential organ failure assessment; TNF = tumor necrosis factor.

4. G-protein coupled receptors

GPCRs are the largest family of transmembrane receptors in humans and are involved in signal transduction of numerous chemical and physical stimuli including olfaction, vision, gustation, neurotransmission, hormonal control, metabolic regulation and modulation of local mediators. There are close to ~800 GPCRs encoded by the human genome with the vast majority of them being involved in olfaction. Among the ~369 GPCRs not involved in basic sensations (i.e. gustation, vision and olfaction), another ~150 are orphan receptors in that their ligands and regulatory functions remain uncertain (Fredriksson, Lagerstrom, Lundin, & Schioth, 2003; Glukhova, Draper-Joyce, Sunahara, Christopoulos, & Wootten, 2018). GPCRs have regulatory functions in all organ-systems of the body and dysregulation of GPCR signaling has been implicated in the pathogenesis of numerous diseases (Heng, Aubel, & Fussenegger, 2013). Phylogenetically, GPCRs have been classified into five distinct subfamilies: (i) Glutamate; (ii) Rhodopsin; (iii) Adhesion; (iv) Frizzled/Taste2; and (v) Secretin. For the purposes of this review, we will be mainly focusing on GPCRs that are implicated in the pathogenesis of sepsis and have not been previously evaluated as pharmacologic targets in clinical trials for sepsis.

The general structure of all GPCRs consists of highly conserved hepta-helical transmembrane α-helix folds, an extracellular N-terminus and an intracellular C-terminal cytoplasmic tail. These cell-surface receptors transduce extracellular signals into intracellular biochemical effector pathways via G-protein (guanine nucleotide binding protein) activation. G-proteins associated with the receptor typically exist in the form of a heterotrimer composed of a Gα subunit interacting with a Gβγ complex. The first step in signal transduction is the binding of a ligand to the GPCR, which results in conformational changes in the receptor, typically involving disruption of strong ionic interactions between various transmembrane helices (Rosenbaum, et al., 2007). These changes expose an otherwise hidden cytoplasmic surface of the receptor that facilitates guanine nucleotide exchange on the Gα protein (release of guanosine diphosphate [GDP] and binding of guanosine triphosphate [GTP]) to which the receptor is coupled. Binding of GTP results in activation of the heterotrimeric G-protein complex and allows the dissociation of both Gα subunit and Gβγ complex from the receptor (Rosenbaum, Rasmussen, & Kobilka, 2009). Depending on the types of G-protein, the dissociated Gα subunit and Gβγ complex can then modulate a variety of downstream signaling pathways through either stimulation or inhibition of enzymes and/or ion channels. Gα proteins have the capability to both bind and hydrolyze GTP through their intrinsic GTPase activity. Binding of GTP activates the G-protein—a process that may be facilitated by guanine nucleotide exchange factors, while hydrolysis of GTP to GDP inactivates the G-protein—which may be regulated by GTPase-activating proteins. However, hydrolysis of GTP occurs at a relatively slow rate, which allows the activated G-protein (GTP bound) to have a much longer half-life in the cell than the activated receptor itself. This allows amplification of the transduced signal such that the initial ligand-receptor interaction may last for a short of period (e.g. few milliseconds), while the activated G-protein may remain active in the cell for a much longer period of time (e.g. many seconds) (Homan & Tesmer, 2014). Following hydrolysis of GTP to GDP, Gα proteins can associate again with Gβγ complexes to form heterotrimers and re-couple with GPCRs. Despite this, the response mediated by GPCRs to their ligands tends to attenuate over time, even in the continued presence of the ligand—a phenomenon referred to as desensitization. The chief reason for this is that activation of the GPCR not only activates G-proteins, but it also activates a family of protein kinases called GPCR kinases (GRKs). The intracellular C-terminus of the GPCR contains many serine and threonine residues whose hydroxyl (–OH) groups can be phosphorylated by GRKs (Gurevich & Gurevich, 2019). This phosphorylation leads to diminished receptor–G-protein coupling and increases the affinity of the receptor to bind a protein called β-arrestin. Binding of β-arrestin to the receptor further diminishes the ability of the receptor to bind ligands and promotes endocytosis of the receptor through recruitment of clathrin and adaptor protein-2 that leads to the formation of clathrin-coated pits. Dissociation of ligand from the internalized receptor reduces its affinity for β-arrestin and allows dephosphorylation of the receptor through the action of protein phosphatases (Bahouth & Nooh, 2017). Recycling of the internalized receptor to the cell membrane allows it to bind ligands again and elicit cellular responses (resensitization). However, repeated or continued exposure to ligands favors the lysosomal degradation of internalized receptors, thereby leading to down-regulation of receptor density and persistent desensitization. It should be noted here that despite similarities among different GPCRs, individual GPCRs have unique combinations of signal transduction activities involving multiple G-protein-dependent and G-protein-independent signaling pathways along with complex regulatory processes.

A variety of G-protein heterotrimers can be coupled to a diverse array of GPCRs to elicit divergent responses in various cells. There are at least 18 different Gα proteins to which GPCRs can be coupled. In turn, these Gα proteins form heterotrimeric complexes with Gβ and Gγ subunits, both of which have at least 5 and 11 different types respectively (Kroeze, Sheffler, & Roth, 2003). Gα proteins have been broadly classified into four families: Gα_i/o, Gα_s, Gα_q/11 and Gα12/13. Gα_s proteins chiefly stimulate the activity of adenylyl cyclase, while Gα_i/o proteins principally inhibit adenylyl cyclase. Adenylyl cyclase is an enzyme responsible for the formation of cyclic adenosine monophosphate (cAMP) from ATP (adenosine triphosphate). cAMP, in turn, can modulate a plethora of functions within different cells through the activation of cAMP-dependent protein kinases (Lorenz, Bertinetti, & Herberg, 2015). On the other hand, Gα_q/11 proteins mainly act by activating phospholipase C (PLC), whereas Gα_12/13 proteins chiefly stimulate the activity of the Rho family of GTPases through RhoGEFs (Suzuki, Hajicek, & Kozasa, 2009). PLC is responsible for breaking down PIP₂ (phosphatidylinositol-4,5-biphosphate), a component of membrane phospholipids, into diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP₃). DAG, which is confined to the cell membrane, can activate protein kinase C (a phospholipid- and calcium-sensitive protein kinase), while IP₃ diffuses through the cytoplasm and binds to ligand-gated calcium channels, thereby triggering the release of calcium ions into the cytosol. Calcium ions can stimulate the activity of calcium-dependent protein kinases through multiple mechanisms, such as by binding to calmodulin (Mizuno & Itoh, 2009). Numerous experimental studies have demonstrated that each GPCR can be activated by a variety of ligands and a single GCPR can couple with multiple G-proteins; such properties of a particular receptor may be partly modified through alternative splicing and/or post-translational modifications (Markovic, 2013). Moreover, biased ligands may preferentially activate a particular signaling pathway by activating certain G-proteins coupled with the receptor (Rankovic, Brust, & Bohn, 2016). Additionally, a single Gα protein can modulate the activity of multiple effectors in response to varying concentrations of a single ligand (for instance, Gα_s can stimulate the activity of PLC in addition to stimulating the activity of adenylyl cyclase) (Hermans, 2003). Besides the canonical pathways of GPCR signaling mentioned here, GPCRs may also interact with a wide variety of GPCR-interacting proteins, such as PDZ-scaffold proteins, receptor-activating modifying proteins and allosteric mediators (Romero, von Zastrow, & Friedman, 2011). Likewise, although many GPCRs exist and function as monomers, GPCRs can form homo- or heterodimers in association with other GPCRs (Bulenger, Marullo, & Bouvier, 2005). All these factors can fine-tune GPCR signaling and influence receptor interactions with their major orthosteric ligands.

With this background of GPCRs, we now serially discuss some of the GPCRs that have not been fully explored in clinical trials for sepsis but hold promise as potential therapeutic targets.

4.1. Adrenergic receptors

Adrenergic receptors or adrenoceptors are GPCRs that mediate the effects of catecholamines including adrenalin (epinephrine) and noradrenalin (norepinephrine). Adrenoceptors are expressed on nearly all tissues of the body and mediate a diverse variety of physiologic functions including blood pressure regulation, myocardial contractility, energy mobilization, metabolic processes and bronchial responsiveness (Ahles & Engelhardt, 2014). Adrenoceptors are of two broad classes viz. α- and β-adrenoceptors, each of which has further subtypes (α_1A, α_1B, α_1D, α_2A, α_2B, α_2C and β₁, β₂, β₃). α₁-adrenoceptors couple principally to G_q/11 proteins and activate PLC, which in turn leads to an increase in the cytosolic concentration of calcium ions and activation of calcium-dependent protein kinases. α₁-adrenoceptors are expressed on a wide variety of tissues and cells in the body, such as iris, ciliary body, blood vessels (tunica media), smooth muscles (primarily, internal anal sphincter and urethral sphincter), and cardiac myocytes. Stimulation of α₁-adrenoceptors on various cells leads to a diverse array of physiologic responses including mydriasis, systemic vasoconstriction, and tightening of anal and urinary sphincters (Michel & Vrydag, 2006). α₂-adrenoceptors couple primarily to G_i proteins and their stimulation leads to inhibition of adenylyl cyclase and a reduction in the intracellular concentration of cAMP. α₂-adrenoceptors are primarily expressed on presynaptic autonomic neurons and their stimulation leads to a net inhibition of sympathetic outflow. Clonidine, a prototype α₂ agonist, has sympatholytic effects in the body including hypotension, bradycardia and bronchoconstriction (Jamadarkhana & Gopal, 2010). α_2B receptors are mainly involved in neurotransmission at the level of the spinal cord, while α_2C receptors modulate the release of catecholamines from adrenal medullary chromaffin cells (Gyires, Zadori, Torok, & Matyus, 2009).

β-adrenoceptors are of three types (β₁, β₂ and β₃) and are widely distributed throughout various tissues and cells of the human body. All β-adrenoceptors couple chiefly to G_s proteins and their stimulation leads to an increase in the intracellular concentration of cAMP through activation of adenylyl cyclase (Baker, 2010). β₁-adrenoceptors are densely expressed on cardiac myocytes and their stimulation leads to positive inotropic (increased contractility), chronotropic (increased heart rate), dromotropic (increased conduction) and lusitropic (rapid relaxation) effects. β₁-adrenoceptor stimulation has also been implicated in stimulating the release of renin from the juxtaglomerular apparatus. β₂-adrenoceptors are expressed on hepatocytes, adipocytes, blood vessels, detrusor muscle, intestinal smooth muscle and bronchial smooth muscle. Stimulation of β₂-adrenoceptors leads to vasodilation in skeletal muscle beds, increased gluconeogenesis, enhanced lipolysis, bronchodilation, urinary retention and constipation. Both β₁- and β₂-adrenoceptors are expressed on the ciliary epithelium and their stimulation enhances the production of aqueous humor. β₃-adrenoceptor expression has been demonstrated in adipose tissue, intestinal smooth muscles, detrusor muscle, prostate gland, and coronary endothelial cells (Ursino, Vasina, Raschi, Crema, & De Ponti, 2009). Stimulation of β₃-adrenoceptors leads to lipolysis, constipation, urinary retention, and coronary vasodilation.

A number of pharmacologically active drugs targeting α- and β-adrenoceptors are currently in use for the treatment of various disorders. Phenylephrine, a selective α₁-agonist, has potent vasoconstrictor effects and is used as a vasopressor of choice in patients with hypertrophic obstructive cardiomyopathy (Gajewski & Hillel, 2012). Selective α1-antagonists (prazosin, terazosin, tamsulosin) are used in the treatment of patients with bladder outlet obstruction secondary to prostatic enlargement. Clonidine, an α₂-agonist, is used for treatment of patients with opioid withdrawal and is also used as an antihypertensive agent. Yohimibine, an α₂-agonist, is used for the treatment of erectile dysfunction. Another α₂ agonist, mirtazapine, is used as an antidepressant (Dekeyne & Millan, 2009). Selective β₂-agonists (albuterol, terbutaline, salmeterol) are potent bronchodilators and are the cornerstone of management of asthma and emphysema (Broadley, 2006). Mirabegron, a selective β₃-agonist, is approved for the treatment of detrusor overactivity (Cernecka, Sand, & Michel, 2014). Likewise, dobutamine, a selective β₁-agonist, is a potent inotropic agent and is useful in the management of cardiogenic shock. Conversely, selective β₁-blockers (such as metoprolol and bisoprolol) have anti-arrhythmic, antianginal and anti-hypertensive effects and form the backbone of pharmacotherapy for coronary artery disease and congestive heart failure. Labetalol, a non-selective β-blocker, is used in the management of pre-eclampsia and eclampsia. Moreover, propranolol, another non-selective β-blocker, is utilized for the treatment of a variety of diseases including essential tremor, thyrotoxicosis, portal hypertension, performance anxiety disorder, and migraine headaches (D. W. Wang, et al., 2010). Carvedilol, a non-selective α- and β-antagonist, is commonly used in the management of patients with congestive heart failure. Additionally, ophthalmic preparations of certain non-selective β-blockers, such as timolol, are efficacious in the management of patients with glaucoma (Winn, Culhane, Gilmartin, & Strang, 2002). Given the diverse physiologic processes mediated by adrenoceptors, it is not surprising that pharmacologic agents targeting these receptors have found wide applications in numerous diseases.

Adrenergic receptors have been shown to modulate inflammatory and immunological processes, which makes them potential targets for pharmacotherapy in sepsis (Hasko & Szabo, 1998; Hasko, Szabo, Nemeth, & vizi, 1997). The sympathetic nervous system plays an important role in controlling the function of the immune system and modulating inflammation (Hasko, 2001). Neurotransmitters of the sympathetic nervous system—norepinephrine and epinephrine—are released in the vicinity of immune cells in response to various stressful stimuli and fine-tune the immune response by binding to adrenoceptors on immune cells (Sperlágh, Dóda, Baranyi, & Haskó, 2000). Presynaptic adrenoceptors are implicated in inhibiting the release of neurotransmitters and serve as a feedback loop (Vizi, Orso, Osipenko, Hasko, & Elenkov, 1995). Presynaptic α₂-adrenoceptors have a much greater affinity for their ligands than post-synpatic α₂-adrenoceptors. Consequently, ligand binding to α₂-adrenoceptors occurs predominantly on the presynaptic side and the effects of such ligands are principally determined by their interactions with presynaptic receptors. α₂-adrenoceptor stimulation in vivo can increase the production of pro-inflammatory cytokines (TNFα and IL-12) (Elenkov, Hasko, Kovacs, & Vizi, 1995). Conversely, blockade of α₂-adrenoceptors can suppress the production of TNFα, MIP-1α and IL-12, while increasing the production of anti-inflammatory cytokines, such as IL-10 (Hasko, Elenkov, Kvetan, & Vizi, 1995). As mentioned previously, these effects are likely to be mediated through stimulation of presynaptic α₂-adrenoceptors. In experimental models, inhibition of presynaptic α₂-adrenoceptors was shown to increase the release of norepinephrine in lymphoid organs (including the thymus and spleen) (Hasko, Elenkov, & Vizi, 1995). The high local concentration of norepinephrine can stimulate β-adrenoceptors on immune cells and alter the cytokine expression profile of these immune cells. These observations further confirm the role of presynaptic α₂-adrenoceptors as regulators of neuro-immune interactions.

β-adrenoceptors are present on immune cells and can modulate a variety of their effector functions including leukocyte trafficking, lymphocyte proliferation, phagocytosis of pathogens and release of cytokines (Hasko, Shanley, et al., 1998; Hasko, Szabo, Nemeth, & Deitch, 2002; Pastores, Hasko, Vizi, & Kvetan, 1996). Experimental studies in mice showed that stimulation of β-adrenoceptors inhibited the LPS-induced release of IL-12 (Hasko, Szabo, Nemeth, Salzman, & Vizi, 1998a). In another study, exogenous and endogenous catecholamines were found to inhibit the release of MIP-1α induced by LPS infusion in mice. This effect was reversed by propranolol, but not phentolamine, suggesting that the effect was mediated through activation of β-adrenoceptors. Isoproterenol, a non-selective β-agonist, was found to inhibit the production of TNFα and nitric oxide, while stimulating the production of IL-10, in the RAW 264.7 macrophage cell line (Hasko, Nemeth, Szabo, et al., 1998). In vivo, isoproterenol was also found to protect against the development of endotoxin-induced vasoplegia in rats through inhibition of LPS-induced TNFα and nitric oxide release, while stimulating LPS-induced release of IL-10 (Szabo, et al., 1997). Stimulation of β-adrenoceptors leads to an increase in the intracellular concentration of cAMP in immune cells. Consequently, increase in intracellular cAMP concentration by phosphodiesterase inhibitors and dopamine receptor agonists has been demonstrated to have a similar impact on cytokine secretion from immune cells (Hasko, Szabo, Merkel, et al., 1996; Hasko, Szabo, Nemeth, Salzman, & Vizi, 1998b). Stimulation of β-adrenoceptors also has an impact on the adaptive immune response by inhibiting the release of IFNγ and stimulating the release of IL-10; this inhibits the formation of T_H1 cells and promotes differentiation into T_H2 cells (Panina-Bordignon, et al., 1997). The experimental studies discussed so far suggest a potentially beneficial role of β-adrenoceptor agonists and α₂-adrenoceptor antagonists in sepsis.

More recent experimental evidence has shown discordant effects of α- and β-adrenoceptors activation and blockade in different cells and tissues. In a CLP model of polymicrobial sepsis in rats, dexmedetomidine (an α₂-adrenoceptor agonist) was shown to reduce mortality and alleviate renal dysfunction by decreasing the expression of IL-1β, IL-6, TNFα, NFκB and TLR4; the protective effects of dexmedetomidine were blocked by atipamezole (an α₂-adrenoceptor antagonist) suggesting the involvement of α₂-adrenoceptors (Qiu, et al., 2018). On the other hand, other experimental studies suggested that the protective effect of dexmedetomidine may be related to its anti-inflammatory effects independent of the α₂-adrenoceptor as these effects were not antagonized by yohimbine (J. Zhang, Wang, Wang, Zhou, & Li, 2015). Conversely, Hsing and colleagues demonstrated that dexmedetomidine attenuated sepsis-induced acute kidney injury by reducing expression of histone deacetylase-2 and histone deacetylase-5, while increasing expression of acetyl-histone H3 (Hsing, et al., 2012). Interestingly, these effects of dexmedetomidine were antagonized by yohimbine, which suggests the possible involvement of α₂-adrenoceptors in these effects. Further studies utilizing the CLP model of sepsis in rats showed that α_2A-adrenoceptor blockade reduces levels of HMGB1 (high-mobility group box protein 1) and attenuates sepsis-induced acute lung injury by reducing levels of TNFα and IL-6 (Ji, et al., 2012). Likewise, erythropoietin was shown to reverse sepsis-induced vasoplegia by preserving α_1D-adrenoceptor expression in murine aorta, possibly through inhibition of GRK2 (Kandasamy, et al., 2016). With respect to β-adrenoceptors, the effects of agonists and antagonists are divergent in different tissues. Both epinephrine infusion and propranolol were found to have deleterious effects in mice with polymicrobial sepsis induced by CLP (Oberbeck, et al., 2004). On the other hand, another study showed that propranolol treatment alone was found to have lung protective effects and favorable metabolic effects in rats with sepsis induced by CLP (J. Wilson, et al., 2013). Further experimental studies have shown that blockade of myocardial β₁-adrenoceptors improves cardiac and vascular function in sepsis (Kimmoun, et al., 2015). On the other hand, stimulation of β₁-adrenoceptors by dobutamine was found to improve survival in septic rats by improving hepatic microcirculation; this effect was completely abolished by esmolol (Fink, et al., 2013). Isoproterenol was also shown to have anti-inflammatory effects through down-regulation of HMGB1 (high-mobility group box protein 1) in mice with sepsis induced by CLP (Ha, et al., 2011). In other experimental studies, terbutaline (a selective β₂-adrenoceptor agonist) was shown to improve sepsis-induced organ dysfunction, circulatory failure and diaphragmatic dysfunction (Ito, Fujimura, Omote, & Namiki, 2006; Tsao, et al., 2010; Uzuki, Yamakage, Fujimura, & Namiki, 2007). Overall, most studies suggest a protective effect of β₁-adrenoceptor antagonism and β₂-adrenoceptor stimulation in sepsis (de Montmollin, Aboab, Mansart, & Annane, 2009). The discrepancies between the results of individual studies may be related to inter-specie differences, variations in experimental protocols, different dosages of drugs used, and phenotypic heterogeneity in sepsis induced by CLP.

Despite the theoretical promise of targeting adrenoceptors in sepsis, clinical use of drugs targeting adrenoceptors is limited to vasopressor therapy. Norepinephrine is recommended as the first-line vasopressor for patients with septic shock (Rhodes, et al., 2017). Norepinephrine is used preferentially over other vasopressors, such as dopamine and phenylephrine, as the latter are associated with more adverse effects (De Backer, Aldecoa, Njimi, & Vincent, 2012; Vail, et al., 2017). Norepinephrine is also preferred over epinephrine as experimental evidence suggests that epinephrine may increase myocardial oxygen consumption with potentially deleterious consequences (Ducrocq, et al., 2012). From a theoretical perspective, this observation is of interest as epinephrine has greater affinity for β₂-adrenoceptors than norepinephrine, and experimental evidence suggests a beneficial role of β₂-adrenoceptor stimulation in sepsis (de Montmollin, et al., 2009). Conversely, the high affinity of epinephrine for α₂-adrenoceptors may also counterbalance its beneficial effects in sepsis. Having said this, it seems unlikely that traditional agonists and antagonists of adrenoceptors will be of much clinical benefit in patients with sepsis and septic shock. Evidence accumulated from recent studies shows that adrenoceptors are down-regulated in patients with septic shock due to activity of GRKs and up-regulation of phosphodiesterases and phospholipases (Sakai, et al., 2017; Thangamalai, et al., 2014). Novel methods of targeting adrenoceptors intracellularly through pepducins and aptamers may circumvent these problems and hold theoretical promise for use in sepsis.

4.2. Adenosine receptors

Adenosine is an endogenous purine nucleoside that is elaborated in response to tissue injury and inflammation (Hasko & Cronstein, 2004). Adenosine is constitutively present in the extracellular space at low concentrations, but, its concentration increases markedly in response to tissue injury. Newby classified adenosine as a ‘retaliatory metabolite’ and postulated that adenosine, which is released in response to a wide variety of stressful stimuli, mediates an auto-regulatory loop that serves to limit end-organ injury (Newby, 1984). Adenosine is believed to exert its protective effects through multiple mechanisms including reduction in the energy demand of tissues (for instance, negative inotropic effects in cardiac muscle), promotion of a more favorable tissue environment (for instance, coronary vasodilation leading to improved nutrient and oxygen delivery) and modulation of the immune response (Antonioli, Blandizzi, Pacher, & Hasko, 2013; Hasko, Deitch, Szabo, Nemeth, & Vizi, 2002).

Extracellular concentration of adenosine is tightly regulated in tissues through modulation of its production, release and metabolism as well as regulation of intracellular purinergic metabolic pathways. During tissue hypoxia, ATP is degraded to AMP (adenosine monophosphate) and the dephosphorylation of AMP to adenosine by the enzyme 5’-nucleotidase is up-regulated while the re-phosphorylation of adenosine by adenosine kinase is inhibited (M. D. Nguyen, Ross, Ryals, Lee, & Venton, 2015). As the intracellular concentration of adenosine increases, adenosine is exported to the extracellular space by the function of highly specialized equilibrative nucleoside transporters (Csoka, et al., 2015). Another important source of extracellular adenosine is through the action of ectonucleotidases (CD39 and CD73) on extracellular ATP, ADP (adenosine diphosphate) and AMP that is released from cells during tissue hypoxia and inflammation (Antonioli, Pacher, Vizi, & Hasko, 2013). Adenosine is chiefly catabolized to inosine by the enzyme adenosine deaminase, which itself has immunomodulatory and neuroprotective effects (Hasko, Kuhel, Nemeth, et al., 2000; Hasko, Sitkovsky, & Szabo, 2004; Liaudet, et al., 2002; Liaudet, et al., 2001; Marton, et al., 2001; Soriano, et al., 2001). In experimental models, the principal sources of extracellular adenosine have been determined to be neutrophils, endothelial cells and platelets (Eltzschig, et al., 2004).

Adenosine can bind to one of four different GPCRs designated A₁, A_2A, A_2B and A₃. These receptors are encoded by the genes ADORA1 (1q32.1), ADORA2A (22q11.23), ADORA2B (17p12-p11.2) and ADORA3 (1p13.2) respectively in humans. A₁ and A₃ receptors are principally coupled to G_i proteins that lead to inhibition of adenylyl cyclase with a concomitant decrease in intracellular cAMP concentration. On the other hand, A_2A receptor is chiefly coupled to G_s proteins that lead to stimulation of adenylyl cyclase with a concomitant increase in the intracellular concentration of cAMP. A_2B receptor can transduce signals through both G_q and G_s proteins that lead to activation of both adenylyl cyclase and PLC. This is demonstrated in Figure 2. In mast cells, increase in intracellular cAMP concentration can up-regulate the accumulation of NFATc1, which in turn can increase the transcription of IL-4 in response to a rise in cytosolic calcium concentration (mediated by PLCβ). Mast cells have a high density of A_2B receptors and activation of these receptors can lead to mast cell degranulation and bronchospasm. A₁ receptors are concentrated in the central nervous system and are implicated in inhibition of neurotransmitter release (Hasko, Pacher, Vizi, & Illes, 2005). A₃ receptors are present in the heart and, together with A₁ receptors, are involved in ischemic preconditioning of myocardium. A_2A receptors are expressed on the surface of platelets, neutrophils and endothelial cells; they are implicated in the pathogenesis of inflammation and sepsis (Csoka, et al., 2014; Hasko, et al., 2011; Hasko & Pacher, 2008).

Figure 2: Intracellular signal transduction pathways of adenosine receptors.

Adenosine is produced in the cell through degradation of ATP. During hypoxic states, ATP is dephosphorylated to AMP, which in turn is dephosphorylated to adenosine by the enzyme 5’-nucleotidase. Conversely, adenosine can be phosphorylated to AMP by the enzyme adenosine kinase, which can be further phosphorylated to ATP. Both adenosine and ATP can move transcellularly along their concentration gradients through equilibrative nucleoside transporters. Extracellularly, adenosine can be produced by the action of ectonucleotidases (CD39 and CD73) on extracellular ATP and AMP. Adenosine can act through four different G-protein coupled receptors (GPCRs) that couple to various Gα proteins. Adenosine A₁ and A₃ receptors inhibit the activity of adenylyl cyclase and decrease intracellular concentration of cAMP by coupling to Gα_s proteins. In contrast, adenosine A_2A receptors couple to Gα_s and increase the intracellular concentration of cAMP by stimulating adenylyl cyclase. Adenosine A_2B receptors couple to both Gα_s and Gα_q proteins, and stimulate the activity of adenylyl cyclase and phospholipase C respectively. Activation of phospholipase C results in cleavage of PIP₂ to produce IP₃ and DAG. IP₃ can stimulate release of calcium ions from the sarcoplasmic reticulum into the cytosol. Additionally, DAG and IP₃ can both lead to activation of protein kinase C. Likewise, intracellular cAMP concentration can stimulate the activity of protein kinase A. Both protein kinase A and C can phosphorylate multiple down-stream targets and regulate the expression of numerous genes involved in diverse physiologic processes. A = Adenosine; ADP = adenosine diphosphate; AMP = adenosine monophosphate; ATP = adenosine triphosphate; β_arr = β-arrestin; cAMP = cyclic adenosine monophosphate; DAG = diacylglycerol; IP₃ = inositol-1,4,5-triphosphate; PIP₂ = phosphatidylinositol-4,5-biphosphate; PKA = protein kinase A; PKC = protein kinase C; PLC = phospholipase C.

Adenosine A_2A and A_2B receptors are involved in modulation of the inflammatory response (Hasko & Cronstein, 2013; Hasko, Linden, Cronstein, & Pacher, 2008). Endothelial cells are known to be a source of adenosine due to their ability to dephosphorylate AMP and ADP to adenosine (Eltzschig, et al., 2003). In particular, during inflammation, transmigrating neutrophils release AMP which is dephosphorylated by endothelial cells to adenosine (Lennon, Taylor, Stahl, & Colgan, 1998). In experimental studies, adenosine has been shown to inhibit expression of adhesion molecules (vascular cell adhesion molecule-1 and E-selectin) and down-regulate secretion of IL-6 and IL-8 by human umbilical vein endothelial cells, probably through stimulation of A_2A receptors (Bouma, van den Wildenberg, & Buurman, 1996; Ohta & Sitkovsky, 2001). In another study, adenosine stimulated IL-8 production from human microvascular cells—but not from human umbilical vein endothelial cells—through stimulation of A_2B receptors (Feoktistov, et al., 2002). This suggests that both A_2A and A_2B receptors are involved in the modulation of the inflammatory response in sepsis, although the net effect of adenosine in a given tissue is likely related to its inflammatory milieu.

Adenosine receptors A_2A and A_2B are also expressed on innate immune cells and are important for regulating the immune response in sepsis (Hasko, Csoka, Nemeth, Vizi, & Pacher, 2009; Hasko, Pacher, Deitch, & Vizi, 2007). A_2A receptors are present on macrophages and their stimulation can modulate the release of pro-inflammatory cytokines (Hasko & Pacher, 2012; Pinhal-Enfield, et al., 2003). Studies on knock-out mice have shown that A_2A receptor activation on macrophages leads to inhibition of TNFα release from macrophages (Hasko, Kuhel, Chen, et al., 2000; Kreckler, Wan, Ge, & Auchampach, 2006). Moreover, in Adora2a^−/− mice, use of selective A_2B receptor antagonists suggests an additional role of the A_2B receptor in inhibiting the release of TNFα (Ryzhov, et al., 2008). However, A_2B receptors are only operational when their effect is not masked by A_2A receptors as genetic deletion of Adora2b does not influence the secretion of TNFα in the presence of functional A_2A receptors. A_2A receptors also play a key role in augmenting the release of IL-10 from macrophages as has been demonstrated in studies utilizing the RAW264.7 macrophage cell line (Hasko, Szabo, Nemeth, et al., 1996; Németh, et al., 2005). Likewise, studies in knockout mice demonstrated a crucial role of A_2A receptors in stimulating the release of IL-10 from Escherichia coli-exposed macrophages (Csoka, et al., 2007). Additionally, A_2B receptors also have been implicated in amplifying the release of IL-10 from LPS-stimulated macrophages through post-transcriptional mechanisms (Hasko, Kuhel, Nemeth, et al., 2000; Koscsó, et al., 2013; Koscsó, et al., 2012). A_2B receptors have also been demonstrated to stimulate the release of IL-6 from macrophages in Adora2b^+/+ but not in Adora2b^−/− mice. Moreover, activation of A_2B receptors on macrophages results in the differentiation of macrophages to the anti-inflammatory M2 phenotype, while A_2A receptors also play a complementary role in this process (Csoka, et al., 2014; Csoka, et al., 2012; Ferrante, et al., 2013; Grinberg, Hasko, Wu, & Leibovich, 2009; Koscsó, et al., 2013).

Adenosine is also an important regulator of neutrophil function as it modulates phagocytosis and the generation of reactive oxygen species in activated neutrophils (Cronstein, Rosenstein, Kramer, Weissmann, & Hirschhorn, 1985). Through A_2A receptors, adenosine decreases the production of TNFα, MIP-1α (CCL3), MIP-1β (CCL4), MIP-2α (CXCL2) and MIP-3α (CCL20) (McColl, et al., 2006; Szabó, et al., 1998). Moreover, adenosine has also been shown to modulate the adhesiveness of neutrophils to vascular endothelium by altering the expression of adhesion molecules; specifically, A₁ and A₂ receptors enhance and reduce (respectively) the adhesiveness of neutrophils to endothelial cells (Cronstein, et al., 1992).

Adenosine receptors are expressed on the surface of DC and their activation can regulate the immune response in sepsis. Adenosine A_2A receptors are present on myeloid and plasmacytoid DCs, and their stimulation can lead to a shift from a pro-inflammatory to an anti-inflammatory cytokine profile (i.e. increased IL-10 production and reduced secretion of IL-6, IL-12 and IFNγ) (Schnurr, et al., 2004). This change in cytokine profile leads to a preferential differentiation of naïve CD4⁺ T cells to T_H2 cells (humoral immune response) as opposed to T_H1 cells (cell-mediated immune response) (Panther, et al., 2003). Moreover, in animal studies, administration of DCs treated ex vivo with an A_2A receptor agonist protected mice from ischemia-reperfusion injury through suppression of IFNγ production and regulation of DC co-stimulatory molecule expression (L. Li, et al., 2012). Conversely, activation of A_2B receptors on hematopoietic progenitor cells in mice leads to DC differentiation towards a distinct phenotype characterized by the expression of both monocyte and DC surface markers (Novitskiy, et al., 2008). In addition, A_2B receptor stimulation on DCs augmented IL-6 secretion, which resulted in increased T_H17 polarization of naïve T cells (Wei, et al., 2013). Additionally, adenosine A₁ receptors may also play a role in DC maturation as activation of A₁ receptor inhibits vesicular MHC class I cross-presentation by resting DCs (L. Chen, Fredholm, & Jondal, 2008). Likewise, stimulation of adenosine A₃ receptors has been demonstrated to have anti-inflammatory effects through inhibition of IL-6 and TNFα release (Vincenzi, et al., 2013). In another study, agonists of A₃ receptors were found to be protective in endotoxemic mice by decreasing levels of IL-12 and IFNγ (Hasko, Nemeth, Vizi, Salzman, & Szabo, 1998). These studies suggest that adenosine plays a complex role in the differentiation and functioning of DCs and, depending on the state of the DC and the type of receptor activated, adenosine may induce differential responses in effector cells.

Adenosine can indirectly affect lymphocyte function through modulation of DC maturation as discussed previously. However, adenosine can also act directly on lymphocytes by binding to adenosine A_2A receptors on the surface of lymphocytes. Activation of A_2A receptors on the surface of naïve CD4⁺ T cells leads to inhibition of IL-2 secretion, which suppresses proliferation of T lymphocytes (Naganuma, et al., 2006). Moreover, A_2A receptor activation can also lead to up-regulation of negative co-stimulatory molecules (viz. PD-1 [programmed death protein-1] and CTLA-4 [cytotoxic T lymphocyte antigen 4]), down-regulation of CD40L and suppression of IFNγ and IL-4 release; all these actions culminate in overall suppression of the adaptive immune system (Csoka, et al., 2008). At the same time, A2A receptor activation on T cells suppresses both Th1 and Th2 differentiation and activation-induced cell death (Himer, et al., 2010). A_2A receptors are also expressed on natural killer (NK) cells and regulatory T (T_reg) lymphocytes. Stimulation of A_2A receptors inhibits the cytolytic activity of IL-2 activated NK cells (Raskovalova, et al., 2005). Moreover, stimulation of A_2A receptors on T_reg cells leads to enhanced immunosuppressive effects through the amplification of FOXP3 expression, which drives the co-expression of CD39 and CD73—both of which are involved in the generation of adenosine from dephosphorylation of exogenous ADP and AMP (Deaglio, et al., 2007). Lastly, invariant natural killer T cells are also receptive to the effects of adenosine in that stimulation of A_2A receptors on invariant natural killer T cells inhibits the release of pro-inflammatory cytokines, principally IFNγ (Lappas, Day, Marshall, Engelhard, & Linden, 2006).

Experimental studies exploring the role of adenosine receptors in the CLP model of sepsis have shown somewhat discordant results as compared to other experimental models. In one study, the combination of an adenosine A_2A receptor agonist and P2X7 antagonist was hepatoprotective during the acute phase of sepsis (Savio, et al., 2017). Likewise, A_2A and A_2B receptors were shown to attenuate ischemia-reperfusion injury in septic rat hearts (Busse, et al., 2016). On the other hand, A_2A receptor antagonism was observed to afford protection against sepsis-induced lymphopenia (Riff, et al., 2017). Moreover, A_2A receptor blockade and A_2B receptor stimulation increased survival in polymicrobial sepsis induced by CLP (Cohen & Fishman, 2019; Csoka, et al., 2010). In another study, A_2B receptor blockade was shown to enhance macrophage-mediated bacterial phagocytosis and improve survival in polymicrobial sepsis induced by CLP (Belikoff, et al., 2011). Moreover, the A₁ receptor antagonist L-97–1 was shown to protect against renal dysfunction and improve survival from sepsis (C. N. Wilson, Vance, Lechner, Matuschak, & Lechner, 2014). Experimental studies have also demonstrated that A₃ receptor stimulation can decrease renal and hepatic injury in mice with sepsis induced by CLP, thereby leading to a reduction in mortality (H. T. Lee, et al., 2006).

Adenosine receptors are widely expressed on numerous cell types and have pleiotropic effects on the human body. A₁ receptor stimulation can cause both cardiovascular and pulmonary adverse effects, while A₃ receptor stimulation appears to be safe (Conti, Monopoli, Gamba, Borea, & Ongini, 1993; Fishman, Bar-Yehuda, Liang, & Jacobson, 2012). These considerations and the protective role of A_2A receptor blockade and A₃ receptor stimulation in animal models of sepsis indicate that selective A_2A receptor antagonists (pbf-509 and v81444) and selective A₃ receptor agonists (piclidenoson [cf101] and namodenoson [cf102]) hold great promise for use in sepsis (Antonioli, et al., 2014; Cohen & Fishman, 2019; Koscsó, Csóka, Pacher, & Haskó, 2011; Németh, et al., 2005) (see Table 2).

Table 2 —

Chemical compounds targeting adenosine receptors

Drug	Pharmacological effects	Clinical effects	References	Stage of development
AMP579	Agonist of A2B receptor	Cardio-protection	PMID: 19730798	Preclinical
Capadenoson (BAY 68-4986)	Partial agonist of A1 receptor	Anti-anginal	PMID: 22370739 NCT00568945	Phase 1 trials
Derenofylline (SLV-320)	Antagonist of A1 receptor	Diuresis	PMID: 21641345 NCT00744341	Phase 2 trials
FK-352	Antagonist of A1 receptor	Improves intra-dialytic hypotension	PMID: 16395260	Phase 2 trials
GS-9667 (CVT-3619)	Partial agonist of A1 receptor	Anti-insulin sensitizer	PMID: hyperlipidemic, 23427000 PMID: 18494808	Phase 1 trials
MRS-5698	A3 receptor agonist	Analgesia	PMID: 26111639	Preclinical
Namodenoson (CF-102)	A3 receptor agonist	Antineoplastic	PMID: 23299770	Phase 2 trials
Neladenoson	Partial agonist of A1 receptor	Cardio-protection	PMID: 27624622	Phase 1 trials
PBF-509	Antagonist of A2A receptor	Antineoplastic	NCT02403193	Phase 2 trials
PBF-680	Antagonist of A1 receptor	Bronchodilation	NCT03774290	Phase 2 trials
PBF-999	Antagonist of A2A receptor	Antineoplastic	NCT03786484	Phase 1 trials
Piclidenoson (CF-101)	A3 receptor agonist	Immuno-modulatory	NCT03168256	Phase 3 trials
Preladenant (SCH-420814 or MK-3814)	Antagonist of A2A receptor	Neuroprotection	NCT01227265	Phase 3 trials
Rolofylline (KW-3902)	Antagonist receptor of A1	Diuresis	PMID: 20925544	Phase 3 trials
Selodenoson (RG-14202)	Partial agonist of A1 receptor	Coronary vasodilator	PMID: 8496817	Preclinical
T-62	Partial agonist of A1 receptor	Analgesia	NCT00809679	Phase 2 trials
Tecadenoson (CVT-510)	Partial agonist of A1 receptor	AV node blockade	PMID: 15956124	Phase 3 trials
Tonapofylline (BG-9928)	Partial agonist of A1 receptor	Diuresis	PMID: 20926754	Phase 1 trials
Trabodenoson (INO-8875)	Partial agonist of A1 receptor	Reduces intra-ocular pressure	PMID: 27046445	Phase 2 trials
V-81444	Antagonist of A2A receptor	ADHD Parkinson’s disease	NCT02253745 NCT02764892
VCP-746	Partial agonist of A1 and A2B receptors	Cardio-protection	PMID: 27377874	Preclinical
WRC-0571	Inverse agonist of A1 receptor	Tachycardia	PMID: 8632314	Preclinical

ADHD = Attention deficit-hyperactivity disorder; NCT = National ClinicalTrials.gov number; PMID = PubMed identifier.

4.3. Complement peptide receptors

Complement receptors are expressed on multiple blood cells (including erythrocytes, platelets, neutrophils, monocytes, macrophages, eosinophils, mast cells and lymphocytes) and can be broadly classified into two categories: (a) receptors that bind fluid-phase cleavage products of complement proteins (e.g. receptor for C5a); and (b) receptors that bind to complement products deposited on the surface of other cells (e.g. CR1), essentially forming a bridge that links the target cell to the receptor (Karsten & Köhl, 2012). Of the first category, the most well-characterized receptor is the receptor for C5a (C5aR1 or CD88). C5aR1 is a GPCR that is expressed on neutrophils, monocytes and macrophages. Activation of the C5aR1 on neutrophils and macrophages promotes chemotaxis. Some experimental studies suggest that C5aR1 may interact cooperatively with Fcγ receptors on macrophages to enhance phagocytosis and microbial killing (Atkinson, 2006). Another receptor for C5a is C5L2—a G-protein independent receptor that may serve as a decoy receptor for C5a with regulatory functions (R. Li, Coulthard, Wu, Taylor, & Woodruff, 2013). The receptor for C3a (C3aR1) is expressed on B cells, mast cells, adipocytes and endothelial cells. C3aR1 has been implicated in activation of the adaptive immune response and vascular changes characteristics of acute inflammation (Mathern, K. Horwitz, & Heeger, 2018). Moreover, evidence from experiments in mice suggests that both C3aR1 and C5aR1 play vital roles in the maturation and differentiation of T_reg lymphocytes (Kwan, van der Touw, Paz-Artal, Li, & Heeger, 2013; Strainic, Shevach, An, Lin, & Medof, 2013). The second category of complement receptors includes receptors for cleavage products of C3 and C4 (CR1, CR2, CR3, CR4 and CRIg) and C1qR. C1qR is a carbohydrate-rich protein expressed on the surface of lymphocytes and phagocytes. Activation of C1qR on these cells modulates phagocytosis, cytotoxicity and release of pro-inflammatory cytokines (Ross & Medof, 1985). C1qR can be activated by a number of ligands including C1q, MBL, surfactant protein A and conglutinin. CR1 (receptor for C3b/C4b) is expressed on erythrocytes, neutrophils, monocytes, lymphocytes and follicular DCs. CR1 has been shown to be involved in clearance of immune complexes, ingestion of C3b/C4b-bearing particles and modulation of lymphocytic function (J. G. Wilson, Andriopoulos, & Fearon, 1987). CR2 (receptor for C3d and C3dg) is present on the surface of B lymphocytes and follicular DCs. Association of CR2 with CD19 in B cells plays an important role in the activation of B cells in response to complement activation (Matsumoto, et al., 1991). CR2 also plays a role in targeting immune complexes to lymphocyte-rich areas in the spleen and lymph nodes. Both CR3 and CR4 are members of the integrin family and can bind to iC3b (implicated in the alternate complement pathway); CR3 can also bind to C3b and C3dg. CR3 is implicated in neutrophil adhesion, while both CR3 and CR4 are involved in phagocytosis of microbes (Myones, Dalzell, Hogg, & Ross, 1988). CRIg can bind to C3b and iC3b, and is expressed on the surface of macrophages, especially Kupffer cells. This receptor can block the activity of C3 and C5 convertases, thereby inhibiting the complement cascade (Wiesmann, et al., 2006).

Modulation of the complement cascade in sepsis can be a double-edged sword with over-activation leading to microbial eradication at the expense of worsening inflammation and multi-organ dysfunction, while inhibition may limit host tissue damaged at the cost of unchecked proliferation of microbial pathogens. This is substantiated by evidence from experiments where inhibition of C5a signaling improved survival (Ward, 2008), while C3 deficiency was associated with worsening mortality from sepsis (Fischer, et al., 1997). These seemingly paradoxical effects may be explained by the fact that different levels of complement activity are needed during the progression of sepsis: complement activation in the early phases is necessary to curtail the spread of microbes and limit microbial invasion; however, in later stages, complement over-activity in concert with the cytokine storm may lead to host tissue damage and multi-organ dysfunction. Given the pivotal role of the complement cascade in diverse physiologic activities, a number of therapeutic targets have been explored in clinical trials for various diseases (including sepsis, paroxysmal nocturnal hemoglobinuria, thrombotic microangiopathy, C3 glomerulopathy, neuromyelitis optica, antineutrophil cytoplasmic antibody-associated vasculitis, macular degeneration and others) (Morgan & Harris, 2015). Most notably, infusion of C1 esterase inhibitor was shown to improve survival in patients with sepsis who had the lowest C1-esterase inhibitor activity levels (Igonin, et al., 2012). Further studies continue to explore the potential utility of C1 esterase inhibitor in the treatment of patients with sepsis and septic shock (Bobkov, Tikhonov, Shuster, Poteryaev, & Bade, 2017). With respect to complement receptors, a number of agonists and antagonists are currently being explored in clinical trials. Avacopan (CCX168), an oral C5aR1 antagonist, is currently being tested in phase II and III trials as a treatment for antineutrophil cytoplasmic antibody-associated vasculitis, hidradenitis suppurativa, IgA nephropathy and C3 glomerulopathy (NCT03301467, NCT03852472, NCT02994927 and NCT02384317). Two other antagonists of the C5aR1 receptor have also been described in the literature viz. NDT9513727 (a non-peptide inverse agonist) and PMX53 (a peptide antagonist) (H. Liu, et al., 2018). Another potent, selective and non-competitive antagonist of C5aR1, DF2593A, has been studied in mice for its anti-inflammatory and anti-nociceptive effects (Moriconi, et al., 2014). These agents hold promise for use in patients with sepsis as C5a blockade in rats and mice in the cecal ligation and puncture (CLP) model of sepsis was highly effective in diminishing the severity of sepsis (Rittirsch, et al., 2008). CCX140, an inhibitor of CCR2, is currently being explored in randomized trials for diabetic nephropathy and focal segmental glomerulosclerosis (NCT03703908 and NCT01440257). Table 3 lists the pharmacologic agents currently being developed against various complement proteins.

Table 3 —

Pharmacologic agents targeting the complement cascade

Drug	Pharmacological effects	Clinical indication	References	Stage of development
ALXN-5500	Antibody against C5	Immunological disorders	Alexion Pharmaceuticals, Inc. website	Phase 1 trials
AMY-101 (Cp40)	Inhibits C3 cleavage	Gingivitis C3 glomerulopathy	NCT03694444 PMID: 25982307	Phase 2 trials
AMY-201	Factor H analog	PNH	PMID: 23616575	Phase 1 trials
APL-1	Inhibits C3 cleavage	Periodontitis	PMID: 27021500	Phase 2 trials
APL-2	Inhibits C3 cleavage	Age-related macular degeneration PNH	NCT02503332	Phase 2 trials
ARC-1905	Anti-C5 aptamer	Age-related macular degeneration	NCT00950638	Phase 1 trials
Avacopan (CCX-168)	C5aR1 antagonist	ANCA vasculitis C3 glomerulopathy Hidradenitis suppurativa	NCT02994927 NCT03301467 NCT03852472	Phase 3 trials
Bikaciomab	Antibody against factor Bb	Age-related macular degeneration	Patent US8981060	Phase 1 trials
C6-antisense oligonucleotide	Decreased expression of C6	Traumatic brain injury	PMID: 24489093	Preclinical
C6-specific antibody	Inhibits C6	Traumatic brain injury	PMID: 26493766	Preclinical
Cemdisiran (ALN-CC5)	siRNA directed against C5	IgA nephropathy Thrombotic microangiopathy	NCT03841448 NCT02352493	Phase 2 trials
CLG561	Antibody against properdin	Age-related macular degeneration	NCT02515942	Phase 2 trials
Compstatin	Inhibits C3 cleavage	Age-related macular degeneration	PMID: 25678219	Phase 2 trials
Coversin (rVA576)	Inhibits C5 cleavage and inactivates LTB4	PNH HUS Atopic keratoconjunctivitis	NCT03829449 NCT04037891	Phase 2 and 3 trials
Danicopan (ACH-4471)	Factor D inhibitor	C3 glomerulopathy	NCT03459443	Phase 2 trials
DF2593A	C5aR1 antagonist	Chronic pain syndromes	PMID: 25385614	Phase 1 trials
DF2593A	C5aR1 antagonist	Chronic pain syndromes	PMID: 25385614	Phase 1 trials
DF2593A	C5aR1 antagonist	Chronic pain syndromes	PMID: 25385614	Phase 1 trials
Eculizumab	Antibody against C5	PNH Atypical HUS MG NMO	NCT00867932 NCT02205541 NCT02301624 NCT01892345	Approved (Soliris®)
IFX-1	Antibody against C5a	Sepsis Hidradenitis suppurativa ANCA vasculitis	NCT02246595 NCT03001622 NCT03712345	Phase 2 trials
Lampalizumab (FCFD4514S)	Antibody against factor D	Age-related macular degeneration	NCT02288559	Phase 2 trials
LFG-316	Antibody against C5	PNH Age-related macular degeneration Thrombotic microangiopathy	NCT02534909 NCT01527500 NCT02763644	Phase 2 trials
LNP023	Factor B inhibitor	C3 glomerulopathy PNH	NCT03439839 NCT03896152	Phase 2 trials
Mirococept (APT-070)	CR1 analog (inhibitor)	Protection against ischemia-reperfusion injury	PMID: 28587616	Phase 1 trials
Narsoplimab (OMS-721)	Antibody against MASP-2	Thrombotic microangiopathy IgA nephropathy	NCT03205995 NCT03608033	Phase 3 trials
NDT-9513727	C5aR1 inverse agonsit	Inflammatory diseases	PMID: 18753409	Preclinical
NM-9401	Antibody against properdin	PNH	Patent US8664362B2	Phase 1 trial
NOX-D19	Anti-C5a aptamer	Asthma	PMID: 23530212	Preclinical
NOX-D20	Anti-C5a aptamer	Sepsis	PMID: 23887360	Preclinical
Olendalizumab (ALXN1007)	Antibody against C5a	Antiphospholipid antibody syndrome	NCT02128269	Phase 2 trials
PMX-53	C5aR1 antagonist	Inflammatory diseases	PMID: 21441599	Preclinical
Ravulizumab (ALXN-1210)	Antibody against C5	PNH, MG, atypical HUS	NCT03406507 NCT03920293	Phase 3 trials
Recombinant human C1 inhibitor	C1 esterase inhibitor	Hereditary angioedema	NCT01108848	Approved (Berinert®, Cinryze® and Ruconset®)
SOBI-002	Inhibits C5	Immunological disorders	NCT02083666	Phase 1 trials
Sutimlimab (BIVV009)	Antibody against C1s	Cold agglutinin disease	NCT03347396	Phase 3 trials
TNT-003	Antibody against C1s	Warm AIHA, cold agglutinin disease	PMID: 25904443	Preclinical
TNT-009	Antibody against C1s	Warm AIHA, cold agglutinin disease	PMID: 27716293	Phase 1 trials
TP10 (CDX-1135)	Soluble CR1	Glomerulonephritis	NCT01791686	Phase 1 trials
TT30 (ALXN-1102)	Fusion protein of factor H and CR2	PNH	NCT01335165	Phase 1 trials

AIHA = Autoimmune hemolytic anemia; ANCA = antineutrophil cytoplasmic antibody; C5aR1 = C5a complement receptor 1; CR1 = complement receptor 1; CR2 = complement receptor 2; HUS = hemolytic uremic syndrome; LTB₄ = leukotriene B₄; MASP = mannose-binding lectin–associated serine protease; MG = myasthenia gravis; NCT = ClinicalTrials.gov number; NMO = neuromyelitis optica; PMID = PubMed identifier; PNH = paroxysmal nocturnal hemoglobinuria; siRNA = small interfering ribonucleic acid.

4.4. Chemokine receptors

Chemokines are a family of cytokines that primarily promote leukocyte chemotaxis to the site of inflammation. The general structure of chemokines consists of a C-terminal α-helix, three β-pleated sheets and a short N-terminus that plays an important role in chemokine receptor activation. Variation in the configuration of cysteine residues closest to the N-terminal domain confers specificity to a particular chemokine and has been used to classify chemokines into four different classes: CC, CXC, CX3C and XC chemokines (Nomiyama, Osada, & Yoshie, 2013). CXC chemokines harbor a single amino acid between the two cysteine residues, while the two cysteine residues are directly juxtaposed in CC chemokines. Likewise, CX3C chemokines have three amino acids between the two cysteine residues, while XC chemokines lack the first and third cysteines of the motif. Moreover, a diverse array of genes encode for CC and CXC chemokines with considerable copy number variation and allelic isoforms, which creates significant genetic diversity and influences susceptibility to various pathologies (Guergnon & Combadiere, 2012).

Chemokines bind to both conventional chemokine receptors (cCKRs) and atypical chemokine receptors (ACKRs) to elicit a variety of responses including chemotaxis (directional movement towards a chemical gradient in a soluble medium), chemokinesis (random movement induced by a chemical gradient in a soluble medium), haptotaxis (directional movement up a gradient of cellular adhesion molecules) and haptokinesis (random movement while adhering to cellular adhesion substrates). The nomenclature for chemokine receptors is based on the predominant type of chemokine they bind followed by the letter ‘R’ and a number denoting the order of their discovery (Bachelerie, et al., 2014). Signal transduction through cCKRs typically involves Gα_i subunits and β-arrestins, although down-stream signaling pathways may also involve cross-talk and overlap with JAK-STAT pathways (Kufareva, Salanga, & Handel, 2015). Moreover, in the case of chemotaxis, Gβγ subunits also play an important role as compared to Gα_i subunits (Neptune & Bourne, 1997). Additionally, G_12/13 subunits may also play important roles in regulating actin-dependent functions including endocytosis and cytoarchitectura l remodeling (Kim, Kim, & Ko, 2010). Once chemokines tether to the extracellular loops and N-terminal domain of their cognate cCKR, the N-terminus of the cCKR interacts with its heptahelical bundle and induces conformational changes in the receptor that leads to its activation and intracellular signal transduction. ACKRs are structurally related to cCKRs but do not couple to the same signal transduction pathways as cCKRs. Although ACKRs can bind to chemokines with high affinity, it remains controversial whether chemokine–ACKR interaction actually leads to transduction of any intracellular signals at all (Nibbs & Graham, 2013). Having said that, all ACKRs do play an important role in regulating chemokine abundance, distribution and localization; this can indirectly influence interactions between chemokines and cCKRs, and regulate their physiologic and pathophysiologic responses (Nibbs & Graham, 2013).

Additionally, although cCKRs exist as homodimers, they can aggregate with ACKRs, other cCKRs and non-chemokine-binding GPCRs (e.g. opioid receptors) to form functionally distinct heterodimers (Hauser, et al., 2016). While leukocyte movement and migration were initially thought to be the dominant responses mediated by chemokines, pleiotropic effects of chemokines on a variety of cells (such as endothelial cells, epithelial cells, mesenchymal cells, neurons and astrocytes) have been demonstrated in numerous studies (López-Cotarelo, Gómez-Moreira, Criado-García, Sánchez, & Rodríguez-Fernández, 2017).

Chemokines mediate multiple homeostatic and inflammatory responses in sepsis and chemokine receptors can serve as potential therapeutic targets for pharmacotherapy. The homeostatic functions of chemokines include cell survival, proliferation, endocytosis, actin polymerization, cytoskeletal remodeling, integrin activation, cell-cell adhesion, chemotaxis, chemokinesis, chemorepulsion, haptotaxis, haptokinesis, haptorepulsion and trans-endothelial migration. On the other hand, the inflammatory functions of chemokines include NET formation, respiratory burst stimulation, phagocytosis, degranulation and exocytosis. Cells of the innate immune system (namely, neutrophils, monocytes, macrophages, DCs and NK cells) express cCKRs that regulate inflammatory responses. CXCR1 and CXCR2 receptors on neutrophils promote the formation of NETs (Hazeldine, et al., 2014). Moreover, CXCR1 and CXCR2 receptors on both monocytes and neutrophils amplify the respiratory burst (Walz, Meloni, Clark-Lewis, von Tscharner, & Baggiolini, 1991). Likewise, CCR4 expressed on the surface of macrophages up-regulates the respiratory burst in these cells (Ness, Ewing, Hogaboam, & Kunkel, 2006). Bactericidal protease release can be enhanced by a variety of chemokine receptors on neutrophils (CXCR1, CXCR2 and CCR5), monocytes (CCR1 and CCR5), macrophages (CCR4), NK cells (CCR5) and dendritic cells (CCR1, CCR2, CCR3 and CCR5) (Chabot, et al., 2006; Jin, Batra, Douda, Palaniyar, & Jeyaseelan, 2014; Matsukawa, et al., 2000; Sallusto & Lanzavecchia, 2000).

Additionally, eosinophils express the CCR2 and CCR3 receptors, which promote degranulation and the respiratory burst in these cells (Badewa, Hudson, & Heiman, 2002). Mast cells also express CCR1 and CCR2 receptors, which promote their activation and recruitment during inflammatory responses (Lippert, et al., 1998). Apart from innate immune cells, chemokines can also influence the adaptive immune response. CCL19 can induce proliferation of CD4⁺ T cells and also stimulates monocytes to elaborate IL-10, which inhibits T_H1 cells and has anti-inflammatory effects (Byrnes, et al., 1999; Ploix, Lo, & Carson, 2001). Likewise, leukocyte and lymphocyte differentiation, survival and cytokine expression profiles can be influenced by a variety of chemokines through CCR1, CCR2, CCR3, CCR5, CXCR2, CXCR3, CXCR6 and CX3CR1 receptors (Locati, et al., 2002).

Experimental studies suggest that chemokine receptors play important roles in sepsis and their pharmacologic stimulation or inhibition may be potentially useful for treatment. Castanheira and colleagues studied the role of ACKR2 in a CLP model of polymicrobial sepsis (Castanheira, et al., 2018). They demonstrated that ACKR2 deficient mice had more severe lung and kidney lesions as compared to wild-type mice, which suggested a protective role for ACKR2 in sepsis. In another experimental study, Castanheira et al. showed that CCR5^−/− knockout mice have increased severity of systemic inflammatory responses suggesting that CCR5 may also have a protective role in sepsis (Castanheira, et al., 2019). In a case-control study, Klaus et al. showed that plasma concentrations of CCL20 and CCR6 correlated with the severity of illness in patients with sepsis and septic shock (Klaus, et al., 2016). In another case-control study, Xiu and others showed that increased expression of CCR2 on monocytes and DCs was a reliable marker of the development of sepsis in patients with burns (Xiu, Stanojcic, Wang, Qi, & Jeschke, 2016). Likewise, CX3CR1 was shown to be protective in a murine model of septic acute kidney injury and humans with the pro-adhesive I249 CX3CR1 allele had a lower incidence of acute kidney injury in the context of sepsis (Chousterman, et al., 2016). Although multiple chemokine receptors are potential targets for pharmacotherapy in sepsis, no pharmacological agent is currently being tested for sepsis in human randomized trials. Three CCR1 antagonists (CP-481,715, BX-471 and MLN-3897) had been evaluated in randomized clinical trials for rheumatoid arthritis and multiple sclerosis with negative results (Clucas, Shah, Zhang, Chow, & Gladue, 2007). Reparixin—an allosteric inhibitor of CXCR1 and CXCR2—is currently being investigated in phase II trials as a treatment for metastatic triple-negative breast cancer (NCT02370238). Table 4 provides a list of various pharmacologic agents currently being developed that target chemokine receptors.

Table 4 —

Pharmacologic agents targeting chemokines and their receptors

Drug	Pharmacological effects	Clinical indication	References	Stage of development
ALX-0651	Anti-CXCR4 nanobody	Anti-inflammatory	NCT01374503	Phase 1 trials
AMD-11070 (AMD-070)	CXCR4 antagonist	HIV-1 infection	NCT00361101	Phase 1 trials
AMD-3465	CXCR4 antagonist	Antineoplastic HIV-1 infection Anti-inflammatory	PMID: 23484027 PMID: 16011832 PMID: 19540208	Preclinical
AZD-2423	CCR2 antagonist	Neuropathic pain COPD	NCT01200524 NCT01215279	Phase 2 trials
AZD-4818	CCR1 antagonist	COPD	PMID: 20466530	Phase 2 trials
BAY86-5047 (ZK-811752 or BX-471)	CCR1 antagonist	Endometriosis	PMID: 28168715	Phase 2 trials
BL-8040	CXCR4 antagonist	Stem cell mobilization	NCT03246529	Phase 3 trials
Cal-1 (LVsh5/C46)	Lentiviral vector delivering shRNA down-regulating CCR5	HIV-1 infection	NCT03593187	Phase 2 trials
CCL2-LPM	CCR2 antagonist	IgA nephropathy	NCT00856674	Phase 1 trials
CCX-140B	CCR2 antagonist	FSGS	NCT03703908	Phase 2 trials
CCX-354-C	CCR1 antagonist	Rheumatoid arthritis	NCT01027728	Phase 2 trials
CCX-507	CCR9 antagonist	IBD	ChemoCentryx, Inc. website	Phase 2 trials
CCX-587	CCR6 antagonist	Psoriasis	ChemoCentryx, Inc. website	Phase 2 trials
CCX-872	CCR2 antagonist	Antineoplastic	NCT02345408	Phase 2 trials
Cenicriviroc (tbr-652)	CCR2 antagonist	NAFLD	NCT02217475 NCT01338883	Phase 2 trials
CNTX-6970	CCR2 antagonist	Chronic pain disorders	NCT03787004	Phase 2 trials
CP-481,715	CCR1 antagonist	Rheumatoid arthritis	PMID: 17713973	Phase 2 trials
CTCE-9908	CXCR4 antagonist	Antineoplastic	PMID: 24472670	Phase 2 trials
Danirixin	CXCR2 antagonist	COPD	NCT02130193	Phase 2 trials
Elubrixin (SB-656933)	CXCR2 antagonist	IBD CF	NCT00748410 NCT00903201	Phase 2 trials
Emapticap pegol (NOX-E36)	Anti-CCL2 aptamer	Diabetic nephropathy	NCT01547897 PMID: 25901662	Phase 2 trials
INCB-3284	CCR2 antagonist	Rheumatoid arthritis	PMID: 24900329	Phase 2 trials
Ladarixin	CXCR1/2 antagonist	Diabetes mellitus	NCT02814838	Phase 2 trials
Leronlimab (PRO 140)	Anti-CCR5 antibody	HIV-1 infection Antineoplastic	NCT02483078 NCT03838367	Phase 3 trials
Maraviroc	CCR5 antagonist	HIV-1 infection HCV infection	NCT00844519 NCT02881762	Approved (Selzentry® and Celsentri®)
Mavorixafor (AMD070 or X4P-001)	CXCR4 antagonist	WHIM syndrome Antineoplastic	NCT03995108 NCT02823405	Phase 3 trials
MLN3897	CCR1 antagonist	Rheumatoid arthritis	PMID: 19950299	Phase 2 trials
Mogamulizumab	Anti-CCR4 antibody	Antineoplastic	NCT02705105	Phase 2 trials
MSX-122 (Q-122)	CXCR4 antagonist	Vasomotor symptoms	NCT03518138	Phase 2 trials
Nanobodies	Anti-CXCR2 nanobodies	Anti-inflammatory	PMID: 25468882	Preclinical
Navarixin (MK-7123 or SCH-527123)	CXCR2 antagonist	Asthma COPD	NCT01006161 NCT01068145	Phase 2 trials
PA-401	CXCR1/2 antagonist	COPD	NCT01627002	Phase 1 trials
Plerixa for (AMD3100)	CXCR4 antagonist	Stem cell mobilization HIV-1 infection Antineoplastic	PMID: 30776910 PMID: 9427609 PMID: 28817386	Approved (Mozobil®)
Plozalizumab (MLN-1202)	Anti-CCR2 antibody	Antineoplastic	NCT01015560	Phase 2 trials
Reparixin	CXCR1/2 antagonist	Diabetes mellitus Antineoplastic	NCT01220856 NCT02001974	Phase 2 trials
TG-0054	CXCR4 antagonist	Stem cell mobilization	NCT02104427	Phase 2 trials
TPI-ASM8	CCR3 antisense oligonucleotide	Asthma	NCT00550797	Phase 2 trials
Ulocuplumab (BMS-936564 or MDX-1338)	Anti-CXCR4 antibody	Antineoplastic	NCT03225716	Phase 2 trials
Vercirnon (Traficet-EN, CCX-282 or GSK1605786A)	CCR9 antagonist	Crohn’s disease	NCT01536418	Phase 3 trials
Vicriviroc (SCH-417690 or MK-7690)	CCR5 antagonist	HIV-1 infection Antineoplastic	NCT00686829 NCT03631407	Phase 3 trials

CCL = CC-chemokine ligand; CCR = CC-chemokine receptor; COPD = chronic obstructive pulmonary disease; CXCR = CXC-chemokine receptor; FSGS = focal segmental glomerulosclerosis; HCV = hepatitis C virus; HIV = human immunodeficiency virus; IBD = inflammatory bowel disease; NAFLD = non-alcoholic fatty liver disease; NCT = ClinicalTrials.gov number; PMID = PubMed identifier; shRNA = short hairpin ribonucleic acid; WHIM = warts, hypogammaglobulinemia, infections and myelokathexis.

4.5. Protease-activated receptors

PARs belong to a family of highly conserved GPCRs that are expressed on a variety of cells. A characteristic feature of PARs is that they are activated upon proteolysis by the action of extracellular proteases and the distinct structural conformation adopted upon receptor proteolysis dictates the direction and type of intracellular signal transduction (Nieman, 2016). The first member of the PAR to be discovered was the thrombin receptor (PAR-1) on platelets. This was followed by the identification of three other PARs viz. PAR-2, PAR-3 and PAR-4 (Rezaie, 2014). Each PAR shares significant structural and sequence homology with other PARs. In general, the structure of a PAR consists of an extracellular N-terminal domain linked to a heptahelical transmembrane structure, which is in turn linked by intra- and extracellular loops to a cytoplasmic C-terminal domain (tail) (Coughlin, 2005). Proteolysis of the N-terminal domain at defined protease-specific sites by various proteases results in an exposed amino acid sequence (the so-called ‘tethered ligand’) that can interact with the extracellular loops (in the main body of the receptor) and induce conformational changes and elicit intracellular signal transduction. Each protease has distinct requirements for activating a PAR including cleavage sites and co-factors (H. Lin, Liu, Smith, & Trejo, 2013). Moreover, individual PARs can be cleaved by multiple different proteases at various cleavage sites, which in turn allow the transduction of a multitude of signaling events and modulation of various physiologic processes. In fact, one of the notable features of PARs is their ability to stimulate opposing signaling pathways depending on the proteolytic stimulus (‘biased signaling’) (Zhao, Metcalf, & Bunnett, 2014). Another remarkable feature is the ability of PARs to physically interact with other PARs and result in their direct transactivation through the formation of heterodimers; this allows one type of PAR to influence signaling through other PARs and adds a whole new dimension to PAR signal transduction (Gieseler, Ungefroren, Settmacher, Hollenberg, & Kaufmann, 2013). For instance, direct transactivation can occur through the formation of heterodimers between PAR1 and PAR4. Thrombin bound to PAR1 within the heterodimer can ‘reach over’ and cleave PAR4 with subsequent calcium influx inside platelets (Leger, et al., 2006). Likewise, formation of a PAR1-PAR2 heterodimer on endothelial cells can switch the effect of thrombin from a pro-inflammatory mediator (promoting increased vascular permeability) to an anti-inflammatory factor (preserving the endothelial barrier).

In sepsis, significant cross-talk occurs between the processes of coagulation and inflammation as coagulation factors can promote inflammation and vice versa. Cleavage of PAR1 by thrombin and other proteases plays an important role in triggering DIC—a phenomenon that may be seen in 30%–60% of patients with sepsis (Tom van der Poll, 2019). Thrombin activates PAR1 by cleaving a peptide bond between Arg-41 and Ser-42 that liberates a ‘tethered ligand’ leading to activation of PAR1 and intracellular signal transduction through Gα_12/13, Gα_q and Gα_i subunits (Tiruppathi, et al., 2000). Phosphorylation of the C-terminal domain of PAR1 by G-protein coupled receptor kinase (GRK)-3 or GRK-5 leads to signal termination. Moreover, thrombin-mediated PAR1 activation is significantly prolonged in β-arrestin 1-deficient murine fibroblasts, which suggests a key role of β-arrestin 1 in PAR1 desensitization after activation of PAR1 by thrombin (Paing, Stutts, Kohout, Lefkowitz, & Trejo, 2002). Activation of PAR1 by thrombin on platelets leads to platelet aggregation, release of platelet granules, activation of adhesion proteins and morphological changes. In endothelial cells, activation of PAR1 by thrombin leads to exocytosis of Weibel-Palade bodies, expression of adhesion proteins, loss of barrier function and induction of angiogenesis. Moreover, neurons, immune cells, fibroblasts, smooth muscle cells and epithelial cells all undergo substantial changes in response to thrombin-mediated PAR1 activation (Petäjä, 2011). Apart from thrombin, many other proteases can also activate PAR1 including APC, endothelial protein C receptor and matrix metalloproteinases (MMPs) with multiple pleiotropic effects. It is also important to note that PAR1 activation can have dual effects depending on the cleavage site; activation of PAR1 by thrombin and MMP-1 elicits a pro-inflammatory response (increased vascular permeability), while cleavage of PAR1 by APC and endothelial protein C receptor leads to anti-inflammatory effects (endothelial barrier protection) (Roy, Ardeshirylajimi, Dinarvand, Yang, & Rezaie, 2016). MMP-1 has been found to be implicated in DIC and can disrupt the endothelial barrier through activation of PAR1; blockade of MMP1-PAR1 interaction can potentially attenuate these adverse consequences in sepsis (Tressel, et al., 2011). Development of drugs and agents that specifically target PARs has been challenging in that the receptor ligand is tethered to the receptor itself and cannot diffuse away. Nevertheless, cell-penetrating peptides (pepducins), small molecules and therapeutic proteases have been used experimentally to successfully target PARs (Flaumenhaft & De Ceunynck, 2017).

With respect to endothelium, regulation of vascular permeability and expression of tight junction linkers between endothelial cells is dependent on multiple signaling mechanisms and factors. One of these factors is the relative expression of two G-protein-linked GTPases—RhoA and Rac1 (Radeva & Waschke, 2018). RhoA is a GTPase that can induce actin filament breakdown and internalization of VE-cadherin, thereby leading to the breakdown of endothelial barrier. Rac1 has opposing effects in that it stabilizes the actin cytoskeleton and protects against endothelial cell apoptosis. The differential activity of RhoA and Rac1 can be regulated through the activation of PARs on the surface of endothelial cells (Klarenbach, Chipiuk, Nelson, Hollenberg, & Murray, 2003). In sepsis, thrombin generation leads to the activation of PAR1 on endothelial cells, which promotes RhoA signaling and increases vascular permeability through the breakdown of endothelial barrier function. Conversely, activation of PAR2 by a variety of proteases can have opposing effects through Rac1 signaling and protection of the endothelial barrier. Using a pepducin approach, Kaneider and colleagues showed that PAR1 switched from being a vascular disruptive receptor to a vascular protective receptor during progression of sepsis in mice (Kaneider, et al., 2007). This switch in the behavior of PAR1 required transactivation of PAR2 signaling pathways, which suggests that pharmacotherapies selectively activating PAR1-PAR2 complexes may be potentially efficacious in the treatment of sepsis.

4.6. Cannabinoid receptors

Cannabinoid (CB) receptors CB₁ and CB₂ were identified as members of the GPCR family more than two decades ago (Howlett & Abood, 2017). These receptors mediate the effects of Δ⁹-tetrahydrocannabinol, an exogenous ligand derived from the plant Cannabis sativa. Endogenous ligands (called endocannabinoids) can also stimulate these receptors and have been found to be involved in a wide variety of physiologic processes (Araque, Castillo, Manzoni, & Tonini, 2017).

The two principal endocannabinoids are anandamide and 2-arachidonoyl glycerol, both of which are derivatives of arachidonic acid. Anandamide can be catabolized by the enzyme fatty acid amide hydrolase to arachidonic acid, while 2-arachidonoyl glycerol can be degraded to arachidonic acid through the action of monoacylglycerol lipase (Biro, Toth, Hasko, Paus, & Pacher, 2009; Cao, et al., 2013; Di Marzo & Piscitelli, 2015).

CB₁ and CB₂ receptors are widely distributed throughout different cells and tissues of the body (Stella, 2010). CB₁ receptors are most abundant in the brain with high expression noted in the basal ganglia, hippocampus, cerebellum and cortex. Within the nervous system, CB₁ receptors are chiefly localized on the terminals of central and peripheral neurons. This distribution correlates with the function of these receptors in memory, cognition, analgesia and mood. Outside of the nervous system, CB₁ receptors have been detected in numerous tissues including heart, lung, liver, prostate, vas deferens, uterus, ovary, adrenal glands, bone marrow, thymus and tonsils. CB₂ receptors have been found to be expressed heavily on macrophages, neutrophils and lymphocytes in the spleen, thymus and tonsils. In contrast with CB₁ receptors, CB₂ receptor expression in the healthy central nervous system is minimal, although CB₂ receptors are up-regulated in various diseased states (Pal Pacher, Steffens, Haskó, Schindler, & Kunos, 2018).

A whole body of literature suggests that the endocannabinoid system plays important roles in inflammatory processes including sepsis (Csoka, et al., 2009; Mukhopadhyay, Horváth, et al., 2011; M Rajesh, et al., 2008). CB₁ receptors are expressed by neurons in the hypothalamus and these receptors are involved in the initiation of LPS-induced hypotension (Varga, Wagner, Bridgen, & Kunos, 1998). Experimental evidence suggests that LPS-induced hypotension involves a process in which an inflammatory signal is conveyed from the periphery to the brain via autonomic sensory nerves, which then precipitates vasoplegic shock through a central mechanism requiring activation of neurons in the preoptic/anterior hypothalamic area (Villanueva, et al., 2009). Given that rimonabant (a CB₁ inverse agonist) can attenuate the fall in arterial pressure evoked by LPS infusion in mice and also lower plasma concentrations of pro-inflammatory cytokines, this suggests a crucial role played by endocannabinoids in mediating LPS-induced hypotension (Godlewski, Malinowska, & Schlicker, 2004). Moreover, experimental evidence suggests that vasopressin release from the hypothalamus may be reduced in septic shock, possibly through the action of endothelin-1 on endothelin A receptors.

Interestingly, endocannabinoids are involved in mediating the inhibitory effects of endothelin on vasopressin release, primarily through stimulation ofCB1 receptors (M. C. Leite-Avalca, et al., 2016; Vercelli, Aisemberg, Billi, Wolfson, & Franchi, 2009). In another study, anandamide was found to be implicated in mediating LPS-induced nitric oxide production, which was antagonized by CB₁ and CB₂ receptor antagonists (Gardiner, March, Kemp, & Bennett, 2002). Other experiments have shown that endocannabinoids exert vascular tone regulatory effects through an increase in sympatho-adrenal activity (mediated centrally by CB₁ receptors), which could be abolished through antagonism of β-adrenoceptors (Gardiner, March, Kemp, & Bennett, 2005). In fact, hyporesponsiveness to norepinephrine observed in an experimental model of polymicrobial sepsis could be reversed through inhibition of CB₁ receptors by AM-251 (a selective CB₁ receptor antagonist). Apart from the effects on blood vessels, anandamide may also mediate LPS-induced hypothermia in rats which can be abolished by rimonabant (Steiner, et al., 2011). In another study, endocannabinoids were found to be implicated in LPS-induced septic ileus in mice, which was sensitive to blockade with selective CB₁ and CB₂ receptor antagonists (HU210 and JWH133 respectively) (Y. Y. Li, et al., 2010). In a mouse model of colitis, inhibition of monoacylglycerol lipase by JZL 184 (with consequent increase of 2-arachidonoyl glycerol levels) resulted in reduction of histologic evidence of colitis and decreased levels of pro-inflammatory cytokines (Alhouayek, Lambert, Delzenne, Cani, & Muccioli, 2011). This effect was abolished by co-administration of CB₁ and CB₂ receptor antagonists (SR141716A and AM630), which confirmed the involvement of cannabinoid receptors. In the CLP model of sepsis, rimonabant decreased markers of septic multi-organ dysfunction (M. Leite-Avalca, et al., 2019). Likewise, antagonism of the CB₁ receptor was found to protect against hepatic ischemia-reperfusion injury in experimental endotoxemia (Caraceni, et al., 2009), mainly through prevention of endotoxin-related hypotension and inhibition of neutrophil recruitment (in turn driven by a reduction in levels of CXCL1 and MIP-2α) (Smith, Denhardt, & Terminelli, 2001). In another study, stimulation of CB₁ receptors by CB receptor agonists decreased levels of TNFα and IL-12, and increased level of IL-10 in an experimental model of endotoxemia in mice (P Pacher & Hasko, 2008; Pertwee, 2012; Smith, Terminelli, & Denhardt, 2000; Tschöp, et al., 2009). Moreover, stimulation of CB₂ receptors was also found to protect against ischemia-reperfusion injury in an in vivo mouse model by decreasing inflammatory cell infiltration and reducing levels of TNFα, MIP-1α (CCL3) and MIP-2α (CXCL2) (Batkai, et al., 2007; Mukhopadhyay, Rajesh, et al., 2011; Mohanraj Rajesh, Pan, et al., 2007). Additionally, CB₂ receptor activation was shown to attenuate TNFα-induced human endothelial cell activation and transmigration of monocytes in a mouse model of LPS-induced hypotension (Mohanraj Rajesh, Mukhopadhyay, et al., 2007; M Rajesh, et al., 2008).

Despite the theoretical promise of targeting cannabinoid receptors, pharmacologic targeting of cannabinoid receptors for their anti-inflammatory effects has not been materialized hitherto. The main reason for this stagnation is that systemic antagonism of CB₁ and CB₂ receptors is fraught with neuropsychiatric adverse effects. Rimonabant was initially introduced in the European market as an anorectic drug; however, the drug was withdrawn later after serious psychiatric adverse effects were reported. This discouraged pharmaceutical companies from developing further drugs targeted at the cannabinoid receptors. However, there are multiple different pharmacological strategies that could still be theoretically used to design drugs targeting these receptors. Such strategies could include the use of drugs that do not cross the blood-brain barrier, use of partial CB₁ receptor agonists, use of selective CB₂ receptor agonists, combined use of a selective CB₁ receptor antagonist with a selective CB₂ receptor agonist, modulation of local endocannabinoid metabolism, and/or targeting CB₁ or CB₂ receptor signaling in conjunction with drugs targeting other intracellular signaling pathways (Klein, 2005).

4.7. Endothelin receptors

Endothelin (ET)-1 is a hypoxia inducible peptide that plays important roles in circulatory regulation. Endothelin receptors (ET_A, ET_B1 and ET_B2) are GPCRs that mediate vasoconstriction (ET_A and ET_B2 receptors) and vasodilation (ET_B1 receptors) in certain microcirculatory beds (Inscho, Imig, Cook, & Pollock, 2005). In experimental models of endotoxemia, ET-1 has been shown to be implicated in alteration of respiratory mechanics, pulmonary hypertension, myocardial depression and systemic hypotension through differentially expressed ET_A and ET_B receptors in various tissues (Forni, et al., 2005). Likewise, in patients with sepsis, plasma ET-1 concentration was correlated with myocardial depression and overall severity of sepsis (Pittet, et al., 1991). Rutai and colleagues recently showed that in a CLP model of experimental sepsis, a combination of IRL-1620 (ET_B1 receptor agonist) and ETR-p1/fl peptide (ET_A receptor antagonist) improved sepsis-induced hypotension, normalized microcirculatory perfusion and restored mitochondrial oxidative phosphorylation (Rutai, et al., 2019). This suggests that selective ET_A receptor antagonists and selective ET_B1 receptor agonists may be potentially useful in the treatment of sepsis. Given that ET receptor antagonists (such as ambrisentan, macitentan and bosentan) are already approved for use in pulmonary hypertension, these agents should be explored in randomized trials for patients with sepsis (Kowalczyk, Kleniewska, Kolodziejczyk, Skibska, & Goraca, 2015).

4.8. Lysophospholipid receptors

Lysophospholipid receptors are another group of GPCRs that could be potentially targeted in patients with sepsis. As the name suggests, endogenous ligands for these receptors are lysophospholipids viz. lysophosphatidic acid (LPA), sphingosine-1-phosphate (S1P), lysophosphatidylinositol (LPI) and lysophosphatidylserine (LysoPS). The receptors for these lysophospholipids are LPA_1–6, S1P_1–5, LPI₁ and LysoPS_1–3 respectively. Lysophospholipids have been implicated in a diverse array of physiological and pathophysiological processes including fibrosis, pain, inflammation, fertility, osteogenesis, immune modulation and angiogenesis (Kihara, Mizuno, & Chun, 2015). Specifically, S1P receptors are involved in lymphocyte development, maturation and recirculation (Tsai & Han, 2016). S1P₂ receptors have also been shown to modulate vascular development and remodeling as well as alter cell motility (Rosen, Stevens, Hanson, Roberts, & Oldstone, 2013). S1P₂ receptors couple to Gα_12/13 proteins and activate Rho signaling while inhibiting Rac signaling. This enhances actin filament breakdown and promotes increased vascular permeability by breaking down VE-cadherin between endothelial cells (as discussed previously in section 4.5). In the experimental CLP model of sepsis, elevated S1P₂ receptor levels in peripheral blood mononuclear cells were positively correlated with the severity of sepsis; S1P₂ receptor signaling was found to suppress macrophage phagocytosis (Hou, et al., 2015). Moreover, in another experimental study using the CLP model of sepsis, ileal expression of sphingosine kinase 1 (enzyme responsible for synthesis of S1P by phosphorylation of sphingosine) was up-regulated six-folds in septic mice; pharmacological inhibition of sphingosine kinase 1 alleviated symptoms of sepsis (Ugwu & Ho, 2019). Likewise, S1P₁ receptor agonists were found to be beneficial in improving renal microcirculation in mice with sepsis induced by CLP (Z. Wang, Sims, Patil, Gokden, & Mayeux, 2015). In another experimental study, S1P was found to be implicated in thymic involution and lymphocyte apoptosis in mice with sepsis secondary to CLP (Kuchler, et al., 2017). Additionally, the S1P analogue, FTY720, was found to reduce vascular permeability and plasma extravasation in mice with CLP-induced sepsis (Lundblad, Axelberg, & Grande, 2013). Fingolimod, a non-selective S1P receptor agonist and selective down-regulator of S1P₁ receptors, is already approved for use in patients with remitting-relapsing multiple sclerosis (Chaudhry, Cohen, & Conway, 2017). Other selective agonists and antagonists of S1P receptors may be potentially useful in the treatment of sepsis.

Apart from S1P receptors, LPI₁ receptor (GPR55) is another GPCR that may be a potential target for pharmacotherapy in sepsis. This receptor was first described as a novel endocannabinoid receptor because of its sequence homology to cannabinoid receptors CB₁ and CB₂ (Yang, Zhou, & Lehmann, 2016). Later, LPI was found to be the endogenous ligand binding to this receptor, which led to its re-classification as a receptor for LPI (i.e. LPI₁ receptor). GPR55 is expressed on a variety of tissues including endothelium, adrenal glands, intestines, spleen and leukocytes (Henstridge, et al., 2011). GPR55 mediated signaling is found to be implicated in energy metabolism as well as innate immunity through stimulatory effects on neutrophils, monocytes, NK cells and mast cells (Chiurchiu, Lanuti, De Bardi, Battistini, & Maccarrone, 2015; Simcocks, et al., 2014). In an experimental model of colitis, GPR55 knockout mice exhibited less severe inflammation (Schicho & Storr, 2012). Moreover, increased level of GPR55 expression was noted in the gastrointestinal tracts of septic mice (X. H. Lin, et al., 2011). Experiments in mice with sepsis induced by endotoxemia, GPR55 inhibition (using antagonists CID16020046 or O-1918) resulted in decreased levels of pro-inflammatory cytokines (TNFα and IL-6) and reduced leukocyte adherence in submucosal venules (Zhou, Yang, & Lehmann, 2018). These experimental studies suggest that selective targeting of the GPR55 may be of value in sepsis, although further research is needed to understand the pleiotropic effects mediated by this receptor and to design selective agonists and antagonists for this receptor.

4.9. Melatonin receptors

Melatonin (5-methoxy-N-acetyltryptamine) is the primary chronobiotic hormone produced by the pineal gland and plays a role in diverse physiologic processes including regulation of sleep and circadian rhythms, pubertal development, seasonal adaptation, learning, memory, glucose metabolism, hemostasis, antioxidant defenses and modulation of the innate immune system (Carrillo-Vico, Lardone, Alvarez-Sanchez, Rodriguez-Rodriguez, & Guerrero, 2013). Melatonin exerts its physiological effects through two different GPCRs viz. MT₁ and MT₂ receptors. Both MT₁ and MT₂ receptors couple to G_i and G_q/11 proteins, and inhibit adenylyl cyclase, stimulate phosphorylation of MAPK and extracellular signal-regulated kinase, and increase potassium conductance through inwardly rectifying potassium channels (Emet, et al., 2016). Like other GPCRs, MT₁ and MT₂ receptors can form homo-dimers or hetero-oligmers, which modifies the physiologic and pharmacological properties of these receptors. MT₁ and MT₂ receptors are expressed on a variety of tissues including the brain (principally hypothalamus), retina, heart, blood vessels, testes, ovary, skin, liver, kidney, adrenal cortex, immune cells, pancreas and spleen (Slominski, Reiter, Schlabritz-Loutsevitch, Ostrom, & Slominski, 2012). Melatonin has been shown to be elaborated by human lymphocytes and induces the secretion of IL-2 (Carrillo-Vico, et al., 2004). Moreover, daily rhythms of melatonin and IL-2 are transiently lost in inflammatory diseases with the recovery of IL-2 rhythm following restoration of daily melatonin rhythm (Pontes, Cardoso, Carneiro-Sampaio, & Markus, 2007). These observations suggest the existence of a pineal gland–immune system axis that modulates the immune response.

Sepsis has been shown to disrupt circadian rhythms resulting in abnormalities in melatonin secretion (Bellet, et al., 2013). Chronodisruption, in turn, has been associated with alterations of the immune system that could potentially worsen outcome from sepsis (Acuna-Castroviejo, et al., 2017). Experimental evidence suggests that mice may be at an elevated risk of sepsis at night as compared to during daytime because of variations in melatonin levels and its effects on the immune system (K. D. Nguyen, et al., 2013). In the LPS model of experimentally induced sepsis, melatonin inhibited the inflammatory response induced by LPS infusion in mice in a dose-dependent manner (Escames, Lopez, Ortiz, Ros, & Acuna-Castroviejo, 2006). Moreover, melatonin was shown to alleviate sepsis-induced liver damage in mice through inhibition of the NFκB pathway (Garcia, et al., 2015). In the CLP model of experimental sepsis, melatonin was also shown to have anti-oxidant effects and direct effects on the mitochondria that boosts the production of ATP and impedes the activation of the NLRP3 (Nucleotide-binding oligomerization domain-like receptor family, pyrin domains-containing protein 3) inflammasome (Escames, et al., 2006). Likewise, melatonin was also shown to enhance the antibacterial activity of neutrophils in the CLP model of experimentally induced sepsis (Xu, et al., 2019). Moreover, melatonin has also been shown to have stimulatory effects on nearly all innate immune cells including monocytes, NK cells and macrophages (Calvo, Gonzalez-Yanes, & Maldonado, 2013). These results suggest that melatonin signaling may be a potential therapeutic target in sepsis and pharmacotherapies that increase the local concentrations of melatonin may be beneficial for patients with sepsis. At present, melatonin receptor agonists (ramelteon, agomelatine and tasimelteon) are already approved for the treatment of sleep and mood disorders. A phase II clinical trial (Eudract # 2008–006782-83) is currently evaluating the anti-inflammatory effects of an injectable formulation of melatonin (PCT/ES2015070236) for patients with sepsis. Therapies directed at melatonin signaling may be potentially useful in the management of patients with sepsis.

4.10. Resolvin receptors

Resolution of acute inflammation was traditionally thought to be a passive process with dilution of pro-inflammatory mediators and local chemo-attractants. Evidence published over the past two decades has shown that inflammation is a tightly regulated process and, its initiation and termination is governed by fine-tuned chemical mediators including lipoxins and specialized pro-resolving mediators (SPMs) (Serhan, 2014). SPMs are lipid derivatives derived from polyunsaturated fatty acids which play important roles in resolving tissue inflammation (termed catabasis). Catabasis consists of a number of discrete steps including removal of cellular debris and dead microbes by phagocytes (termed efferocytosis), restoration of vascular integrity and regeneration of tissues. SPMs are divided into four classes viz. D-series resolvins (R_VD), E-series resolvins (R_VE), protectins and maresins (Basil & Levy, 2016). R_VDs are derivatives of docosahexaenoic acid, while R_VEs are derivatives of eicosapentaenoic acid.

R_VDs act through GPCRs and actively promote resolution of inflammation through enhanced efferocytosis and restoration of tissue integrity. R_VD₁ acts through the formyl peptide receptor 2 (ALX/FPR2) and GPR32 receptor—also known as R_VD₁ receptor. FPR2 receptor is expressed on a variety of cells including monocytes, neutrophils, epithelial cells, hepatocytes and astrocytes (Schmid, Gemperle, Rimann, & Hersberger, 2016). Pro-resolving effects mediated through the FPR2 receptor involve suppression of Ca²⁺-calmodulin-dependent protein kinase and subsequent inhibition of p38 MAPK phosphorylation. R_VD₁ receptor is expressed on macrophages and is activated by a number of D-series resolvins viz. R_VD₁, R_VD₃ and R_VD₅. Activation of the R_VD₁ receptor on macrophages results in enhanced efferocytosis and differentiation of macrophages into M2 phenotype (Schmid, et al., 2016). Moreover, activation of R_VD₁ receptor on T cells results in decreased differentiation into T_H1 and T_H17 phenotypes (Chiurchiu, et al., 2016). R_VD₂ acts through the GPR18 receptor—now termed the DRV2 receptor. Interaction of RvD₂ with DRV2 receptor results in inhibition of neutrophil chemotaxis, decreased monocyte adhesion to adipocytes, and induces efferocytosis of apoptotic neutrophils (Spite, et al., 2009). In an experimental model of sepsis induced by CLP, RvD₂ significantly improved survival through activation of DRV2 receptors and enhanced phagocytosis-mediated bacterial clearance (Chiang, de la Rosa, Libreros, & Serhan, 2017). In patients with sepsis, resolvins were also found to be predictive of the development of acute respiratory distress syndrome and overall survival (Dalli, et al., 2017).

R_VE₁ acts as a full agonist of the chemokine-like receptor 1 for which reason this receptor is often referred to as the ERV1 receptor. R_VE₂ also acts as a partial agonist of the same receptor. Interaction of R_VE₁ with ERV1 receptor on neutrophils leads to neutrophil apoptosis and efferocytosis (El Kebir, Gjorstrup, & Filep, 2012). Macrophages derived from mice deficient in the ERV1 receptor have an enhanced ability to produce pro-inflammatory cytokines, which is consistent with a pro-resolving effect of R_VE₁ (López-Vicario, et al., 2017). In human monocyte-derived macrophages, these pro-resolving effects of R_VE₁ were abolished by an antibody directed against ERV1 (Ohira, et al., 2010). It is worth noting here that binding of different ligands to the same resolvin receptors can elicit pro-inflammatory responses. For instance, binding of chemerin to ChemR23 and binding of serum amyloid A to FPR2 can have pro-inflammatory effects. Protectins and maresins are two other classes of SPMs that are both derived from docosahexaenoic acid. Protectins are docosatrienes derived from docosahexaenoic acid, while maresins (macrophage mediators in resolving inflammation) are produced in macrophages through 14-lipoxygenation of docosahexaenoic acid (Serhan, et al., 2009). Maresin 1 has been shown to limit neutrophil infiltration in murine peritonitis models and enhance efferocytosis by human macrophages by binding to ALX/FPR2 (Serhan, et al., 2012). Moreover, maresin 1 was also shown to attenuate sepsis induced in mice by CLP and potentially alleviated mitochondrial dysfunction in patients with sepsis (Gu, et al., 2018). Protectin D1 (also referred to as neuroprotectin D1) has been shown to play important roles in efferocytosis, wound healing, neovascularization, neuro-protection, and corneal protection from thermal injury (Serhan, Dalli, Colas, Winkler, & Chiang, 2015). Neuroprotectin D1 exerts its actions by binding to the GPCR GPR37. Given that pro-resolving effects of SPMs are mediated through GPCRs, these receptors represent potential targets for pharmacotherapy in sepsis.

4.11. Ghrelin receptor

Obestatin and ghrelin are two peptides encoded by the GHRL gene and produced by proteolytic cleavage of pre-proghrelin. These peptide hormones bind to the ghrelin receptor (also known as GPR39 or growth hormone secretagogue receptor [GHS-R]) and exert antagonistic activities with regards to satiety and hunger (obestatin signals satiety, while ghrelin stimulates hunger). GHS-R is expressed in a variety of organs including the gastrointestinal tract, pancreas, liver, adipose tissue, thyroid, kidney, lung and heart. Ghrelin and obestatin regulate a wide variety of physiologic responses including glucose metabolism, energy expenditure, food intake, growth hormone secretion, fat deposition, muscle build-up and bone formation (Pradhan, Samson, & Sun, 2013). Moreover, ghrelin has also been shown to modulate the cardiac sympathetic nerve activity and may improve prognosis after a myocardial infarction by reducing the incidence of arrhythmias (Mao, et al., 2012).

GHS-R is expressed on innate immune cells including macrophages and couples to G_q/11 proteins. Experimental studies have shown that GHS-R may be involved in the pathogenesis of many diseases including metabolic syndrome, depression and colitis (Petersen, et al., 2011; Sunuwar, Medini, Cohen, Sekler, & Hershfinkel, 2016). In an experimental model of sepsis induced by LPS, GHS-R expression was up-regulated in peritoneal macrophages and stimulation of GHS-R enhanced LPS-induced IL-10 secretion (Muneoka, et al., 2018). In this study, oral administration of a GHS-R agonist afforded improved survival in mice with LPS-induced sepsis through its anti-inflammatory effects. Likewise, ghrelin was shown to attenuate the release of IL-6 in response to LPS stimulation in the murine dopaminergic SN4741 cell-line (Beynon, et al., 2013). In the CLP model of experimentally induced sepsis, ghrelin had protective effects mediated in part by the restoration of CD4⁺ T cell proliferation (M. Zhou, et al., 2018). Experimental studies on knockout mice have shown that GHS-R deletion affects macrophage polarization and alters the ratio between M1 and M2 phenotypes (L. Lin, et al., 2016). Ghrelin has also been shown to inhibit the NFκB pathway of inflammation in rats with sepsis-induced acute lung injury (Wu, et al., 2007). Studies in patients with sepsis have revealed that elevated ghrelin levels portend a favorable prognosis in sepsis (Koch, et al., 2010). Moreover, expression of GHS-R in rat aorta, heart and small intestine was markedly up-regulated in sepsis along with increased vascular sensitivity to ghrelin (Wu, Zhou, Cui, Simms, & Wang, 2004). Results of these studies suggest that GHS-R may play diverse roles in the inflammatory response in various tissues and may be a potential target for pharmacotherapy in sepsis.

4.12. Hydroxycarboxylic acid and free fatty acid receptors

Short-chain fatty acids (SCFAs) are produced by colonic bacteria as byproducts of fermentation of non-digestible and partially digested polysaccharides. SCFAs are a subset of fatty acids containing six or less carbon molecules, such as acetate, butyrate and propionate. The quantity and repertoire of SCFAs produced in the colon are in part determined by composition of the gut microbiome and reflect the importance of the gut microbiome in physiologic and pathophysiologic processes (Morrison & Preston, 2016). SCFAs are taken up by colonic enterocytes and either metabolized locally or transported across the gut epithelium into the portal circulation. SCFAs have been implicated in a variety of physiologic and pathophysiologic processes including metabolism, inflammation and immune responses to infection. SCFAs are believed to signal through two main mechanisms viz. inhibition of histone deacetylases and GPCRs. A number of GPCRs are involved in SCFA signaling including GPR41 (free fatty acid receptor 3 [FFAR3]), GPR43 (free fatty acid receptor 2 [FFAR2]) and GPR109A (hydroxycarboxylic acid receptor 2 [HCAR2]) (Tan, et al., 2014).

FFAR2 is the primary receptor for acetate in that acetate is the most selective ligand for this receptor, while the most potent ligand for this receptor is propionate. FFAR2 is expressed throughout the gastrointestinal tract on a variety of cells including enterocytes, innate immune cells, pancreatic islet cells and neuroendocrine cells (Tolhurst, et al., 2012). FFAR2 is expressed on neutrophils, monocytes, eosinophils, macrophages and B lymphocytes. Intracellular signal transduction through FFAR2 occurs primarily through G_q/11 proteins and phospholipase C stimulation, while G_i/o proteins also play a secondary role in inhibiting the activity of adenylyl cyclase (Nilsson, Kotarsky, Owman, & Olde, 2003). FFAR2 stimulation on neutrophils results in their recruitment and activation (Le Poul, et al., 2003). Experimental studies have shown that FFAR2 knock-out mice suffer from non-resolving or prolonged inflammation in models of colitis, asthma and arthritis (Maslowski, et al., 2009). A phase I trial of GLPG0974 (FFAR2 antagonist) in patients with ulcerative colitis found GLPG0974 to be safe and well-tolerated (Namour, et al., 2016), although results from subsequent phase II trials did not demonstrate clinical benefits (NCT01829321). A possible explanation for this apparently negative result could be a compensatory increase in FFAR3 expression as seen in FFAR2 knock-out mice (Bjursell, et al., 2011). FFAR3 is expressed widely on immune cells including T cells, B cells, monocytes and macrophages (Brown, et al., 2003). Although FFAR3 is highly related to FFAR2 (52% similarity) and is activated by similar ligands, it has differing specificity for SCFA of different carbon lengths; for instance, pentanoate is the most potent ligand for this receptor.

FFAR3 chiefly transduces signals through G_i/o proteins and inhibits adenylyl cyclase to modulate cytoplasmic cAMP concentration in innate immune cells (Xiong, et al., 2004). Activation of FFAR3 in conjunction with FFAR2 mediates the anti-inflammatory effects of SFCAs by reducing IL-6 and IL-8 production (M. Li, van Esch, Henricks, Folkerts, & Garssen, 2018). HCAR2 is the target of niacin (nicotinic acid) and is sometimes referred to as the nicotinic acid receptor, although niacin is not believed to be the natural ligand for this receptor; butyrate is instead the natural ligand for this receptor (Thangaraju, et al., 2009). Expression of HCAR2 has been demonstrated in splenic macrophages, neutrophils, Langerhans cells, adipocytes, retinal pigment epithelial cells, keratinocytes and intestinal epithelial cells. Stimulation of HCAR2 in the colon by butyrate (produced by gut microbes) suppresses intestinal inflammation by inducing differentiation of T_reg cells, and inhibiting colonic macrophages and DCs (Singh, et al., 2014). Intracellular signaling through HCAR2 is mediated through G_i/o proteins, which inhibit adenylyl cyclase and decrease the intracellular concentration of cAMP. Additionally, HCAR2 can also stimulate phospholipase A₂, activate the MAPK cascade and inhibit the Akt/mTOR signaling pathway (Z. Li, et al., 2017; Richman, et al., 2007). Activation of HCAR2 by β-hydroxybutyrate on monocytes and macrophages affords neuroprotection in a stroke model in mice (Rahman, et al., 2014). Moreover, HCAR2 stimulation suppressed IL-23 production by colonic DCs and inhibited colonic inflammation in a mouse model of colitis (Bhatt, et al., 2018). In experimental models of sepsis induced by CLP, HCAR2 knockout mice were noted to have distinct gut microbiota composition, altered intestinal permeability and increased mortality (G. Chen, et al., 2018). Interestingly, blood–mucosal barrier was reconstituted in HCAR2 knockout mice after these mice received gut microbiota from wild-type mice. These findings suggest that HCAR2 regulates the gut microbiota and plays a crucial role in maintaining the integrity of intestinal epithelial barrier. All these studies indicate that receptors for SCFAs may be attractive targets for potential pharmacotherapy in sepsis.

4.13. Urotensin II receptor

Urotensin II is an 11-amino acid peptide that is known to be the most potent vasoconstrictor. Urotensin was named so as it was originally isolated from the urophysis (endocrine organ) of teleost fish (Ames, et al., 1999). Urotensin II receptor (UTR) is a GPCR that transduces intracellular signals primarily by coupling to G_q/11 proteins and leading to activation of phospholipase C with an increase in the cytoplasmic concentration of calcium ions, although inhibition of adenylyl cyclase and decrease in intracellular cAMP concentration may also be implicated (Ziltener, Mueller, Haenig, Scherz, & Nayler, 2002). Expression of UTR is nearly ubiquitous and has been detected in cardiac myocytes, fibroblasts, endothelial cells, skeletal muscle cells, neurons and innate immune cells. Numerous experimental studies suggested the role of urotensin II and UTR in the pathogenesis of a variety of cardiovascular disorders including hypertension, heart failure, atherosclerosis, pre-eclampsia, diabetes mellitus and cerebrovascular disease (Maryanoff & Kinney, 2009). UTR is also believed to be implicated in inflammation, principally leukocyte recruitment and migration (Castel, et al., 2017). UTR is expressed on the surface of B lymphocytes, NK cells, monocytes and macrophages. Urotensin II acts as a chemoattractant for human monocytes (Maguire, Kuc, Wiley, Kleinz, & Davenport, 2004) and induces increased vascular permeability in rats and mice (Vergura, et al., 2004). Urotensin II induces the secretion of pro-inflammatory cytokines (such as IL-6), while pro-inflammatory cytokines (TNFα and IFNγ) up-regulate the expression of UTR (Segain, et al., 2007). Moreover, urotensin II also increases the synthesis of pro-thrombotic and inflammatory markers (intercellular adhesion molecule-1, tissue factor and plasminogen activator inhibitor-1) in human coronary endothelial cells (Cirillo, et al., 2008). In an experimental model of sepsis induced by CLP, urotensin II aggravated sepsis-induced lung injury in diabetic mice through UTR, which was antagonized by palosuran (UTR antagonist) (Ugan, Cadirci, Halici, Toktay, & Cinar, 2018). Our knowledge of urotensin II, UTR and their role in the pathophysiology of sepsis is still evolving. As our understanding of the urotensinergic system improves, it may become a potentially feasible target for pharmacotherapy in sepsis (Svistunov, et al., 2018). Having said that, the UTR antagonist palosuran has been tested in phase I human trials and found to be well-tolerated at a dose of 500 mg twice daily in healthy volunteers (Sidharta, van Giersbergen, & Dingemanse, 2018).

5. Intracellular targeting of GPCRs

Of the ~800 GPCRs identified within the human proteome, about ~369 are implicated in the pathophysiology of various diseases and represent potential targets for pharmacotherapy. The current market share of drugs targeting GPCRs is estimated to be approximately 40%, even though the overall number of GPCRs targeted by current drugs is ~30 (Wise, Jupe, & Rees, 2004). This suggests that the true potential of targeting GPCRs has not been fully realized to this date.

The general structure and features of signal transduction through GPCRs have been discussed previously in section 4. As mentioned before, GPCRs can couple to a limited number of G proteins and transduce signals through these proteins. Despite marked structural diversity at the intracellular regions, GPCRs couple to only about 18 different subtypes of G proteins (belonging to the four major families i.e. G_s, G_i, G_q and G_12/13). These G proteins can be activated by a wide variety of different cationic α-helical structures, although the selectivity of GPCR coupling to particular G proteins seems to be encoded by a combination of two functional domains at the intracellular regions (Hedin, Duerson, & Clapham, 1993). The first activation domain can activate multiple G protein subtypes, while the second selectivity domain restricts coupling to a single signaling pathway. Slight changes in the structural conformation at these functional domains can affect signal transduction through the GPCR by altering G protein selectivity. This helps to explain how GPCRs translate a diverse array of extracellular inputs into a limited number of intracellular biochemical signals (S. K.-F. Wong, 2003).

Studies employing multiscale computational approaches have revealed that GPCRs can exist in numerous inactive and active conformations, and the balance between these conformations is altered upon binding of orthosteric ligands by changing the thermodynamic stability of the molecular system (Niesen, Bhattacharya, & Vaidehi, 2011). Consequently, traditional pharmacologic approaches to GPCR targeting have focused on designing orthosteric agonists and antagonists that alter the conformational state of the receptor. This has proven challenging in view of the fact that many GPCRs have a high degree of sequence and structural homology, particularly within the same receptor. Moreover, orthosteric modulators that tonically inhibit or stimulate signaling are more likely to have side effects. Allosteric modulators that bind to less conserved regions of GPCRs may be more selective in their action and potentially have fewer adverse effects by modulating a narrow range of physiologic responses (X. Liu, et al., 2017).

Fortunately, recent studies have reported the crystal structures of a few GPCRs complexed with allosteric modulators bound to the cytoplasmic side of the receptor (Oswald, et al., 2016; Zheng, et al., 2016). These studies have improved our understanding of the structure and mechanistic basis for intracellular signal transduction through GPCRs, thereby providing a potential framework for targeting GPCRs intracellularly (see Figure 3). In the following lines, we briefly discuss some of the strategies that may be potentially useful for targeting GPCRs intracellular.

Figure 3: Intracellular targeting of G-protein coupled receptors.

A schematic diagram depicting the conceptual framework of intracellular targeting of G-protein coupled receptors through the use of pepducins, aptamers and intrabodies. Pepducins are cell-penetrating lipidated peptides that can “flip-flop” through the cell membrane and affect G protein-coupled receptor signaling by interacting with the receptor–G protein intracellular interface. Aptamers are small molecules of nucleic acid that can bind to the intracellular face of G protein-coupled receptors and affect intracellular signal transduction. Intrabodies are proteins composed of the variable fragment of single heavy-chain antibodies. Plasmids encoding intrabodies are delivered to target cells through lentiviral vectors and cytoplasmic expression of these intrabodies is achieved following integration of the plasmid into the host genome as part of a lysogenic cycle. Intrabodies then bind to the cytoplasmic face of the GPCR and modulate intracellular signaling. β_arr = β-arrestin; GCPR = G protein-coupled receptor.

5.1. Pepducins

Pepducins are a group of cell-penetrating lipidated peptides that can be used to modulate the activity of GPCRs by acting at the intracellular receptor–effector interface (P. Zhang, Covic, & Kuliopulos, 2015). These molecules have a peptide backbone that is typically derived from a sequence of the cytoplasmic part of the target GPCR and a lipid moiety is conjugated with the peptide to make the molecule permeable. Because of this lipid moiety, pepducins can penetrate through the intact plasma membrane and anchor in the cytosolic interface, thereby modulating the interaction of the target receptor with its effector G proteins (as illustrated in Figure 4) (Carr & Benovic, 2016). Pepducins are different from conventional GPCR agonists and antagonists in that they are allosteric modulators and affect receptor–effector G protein interactions at the intracellular interface. For instance, ICL3–9, a pepducin based on the third intracellular loop of the β₂-adrenergic receptor, can stimulate interactions between the β₂-adrenergic receptor and Gα_s proteins. However, the receptor conformation induced by ICL3–9 is different from the activated conformational state induced by isoproterenol—the orthosteric agonist for β₂-adrenergic receptor (Carr, et al., 2014). Moreover, intracellular activation of G proteins by pepducins is typically not subject to desensitization by β-arrestin or GRKs. In fact, certain pepducins can directly stimulate or inhibit Gα proteins independent of GPCRs (Carr, et al., 2016). Pepducins can also act as biased agonists or antagonists of one particular class of Gα proteins. For example, the CXCR4 pepducin ATI-2431, derived from the first intracellular loop of CXCR4, selectively activates Gα_i signaling but not Gα_12/13 signaling (Quoyer, et al., 2013). Likewise, the PAR2 pepducin P2pal-18S, based on the third intracellular loop of PAR2, was strongly biased towards inhibiting PAR2-Gα_q and PAR2-Gα_i signaling, but had no effect on PAR2-ligand activated endocytosis (Sevigny, et al., 2011). Although the precise details of how pepducins affect GPCR–G protein interactions remain to be elucidated, a number of pepducins have been designed against a variety of GPCRs.

Figure 4: Modus operandi of cell-penetrating lipidated peptides (pepducins).

β_arr = β-arrestin. GPCR = G protein-coupled receptor.

F2Pal16 is a pepducin that acts as an agonist of FPR2. This pepducin is composed of a peptide that has a sequence identical to the third intracellular loop of FPR2 and has a palmitic acid (16-carbon) attached to the peptide (Forsman, et al., 2013). F2Pal16 can activate FPR2 in phagocytes and transfected HL-60 cells, similar to conventional FPR2 agonists. Another pepducin, F1Pal16, was composed of a peptide with sequence identical to the third intracellular loop of FPR1 and linked to palmitic acid. Surprisingly, this pepducin was found to have no effect on FPR1 signaling, but inhibited FPR2-mediated cellular responses (Winther, Gabl, Welin, Dahlgren, & Forsman, 2015). A shorter variant pepducin, F2Pal10, was shown to act as a partial agonist for the FPR2 receptor, but acted as a full agonist for cross-talk triggered FPR2 activation mediated by platelet activating factor and ATP (P2Y₂) receptors (Gabl, et al., 2014).

PZ-128 (P1pal-7) is a pepducin based on the third intracellular loop of PAR1 that can inhibit the interaction of PAR1 with its effector G proteins (Leger, et al., 2006). PZ-128 is highly efficacious in blocking PAR1-dependent platelet aggregation as it inhibits p38 MAPK activation and blocks Gα_12/13-Rho kinase activation. In experimental studies, PZ-128 had an onset of action within 15 minutes of intravenous administration and suppressed PAR1-mediated platelet aggregation in guinea pigs and baboons (P. Zhang, et al., 2012). PZ-128 was the first pepducin to be tested in a human clinical trial (NCT01806077) and it was found to have a rapid, specific and dose-dependent effect on PAR1-mediated platelet aggregation (Gurbel, et al., 2016). Moreover, PZ-128 was also shown to reduce atherosclerotic plaque burden in patients with coronary artery disease by inhibiting MMP1-PAR1 signaling (Rana, et al., 2018). Larger clinical trials assessing the safety and efficacy of PZ-128 in coronary artery disease are currently being planned. Given that both thrombin- and MMP1-mediated PAR1 activation is implicated in the pathogenesis of sepsis (Tressel, et al., 2011), PZ-128 holds promise for use in patients with sepsis. Thrombin-mediated activation of PAR4 is mechanistically different from that of PAR1 and PAR1-mediated platelet aggregation is generally transient, unless additional signals from PAR4 or P2Y₁₂ receptors strengthen it (Covic, Singh, Smith, & Kuliopulos, 2002). Consequently, a pepducin was designed based on the third intracellular loop of PAR4, namely P4pal-10. P4pal-10 was found to be a dual inhibitor of PAR1 and PAR4, and inhibited 85% of human platelet aggregation in response to thrombin (Covic, Misra, Badar, Singh, & Kuliopulos, 2002). Given that PAR1 and PAR4 form heterodimers in human platelets, pepducins with dual inhibitory effects on PAR1 and PAR4 may also be of therapeutic value for treatment of sepsis (Leger, et al., 2006).

PZ-235 (P2pal-18S) is a pepducin designed against the PAR2 and is based on the third intracellular loop of PAR2. PZ-235 acts as a full antagonist of PAR2 and was evaluated for its protective effects in a mouse model of nonalcoholic steatohepatitis (Shearer, et al., 2016). PZ-235 significantly suppressed hepatic fibrosis, inflammatory cytokine release, reactive oxygen species production, stellate cell proliferation, and nonalcoholic fatty liver disease activity scores by 50–100%. Given that PAR2 plays an important role in the pathogenesis of atopic dermatitis, PZ-235 was also evaluated in laboratory models of atopic dermatitis (Barr, et al., 2019). PZ-235 significantly suppressed total leukocyte and T-cell infiltration, epidermal thickness and total lesion severity scores in filaggrin-deficient mice exposed to dust mite allergens. Moreover, PZ-235 also inhibited PAR2-mediated expression of inflammatory factors by human mast cells and keratinocytes. Given the role played by PARs in the pathogenesis of sepsis, targeting of PARs by pepducins in patients with sepsis may be potentially beneficial.

Chemokine receptors have also been targeted successfully by pepducins in experimental studies. CXCR1 and CXCR2 receptors share an identical third intracellular loop, and the pepducin x1/2pal-i3, derived from the third intracellular loop, targets both of these receptors. In human neutrophils, x1/2pal-i3 completely inhibited IL-8–induced calcium influx and blocked neutrophil migration toward chemotactic gradients of IL-8 (Kaneider, Agarwal, Leger, & Kuliopulos, 2005). Another pepducin x1/2LCA-i1, derived from the first intracellular loop of CXCR1 and CXCR2, blocked chemotactic responses of human and mouse neutrophils by inhibiting CXCR1-and CXCR2-mediated signaling (Kaneider, et al., 2005). When tested in the CLP model of sepsis in mice, both x1/2pal-i3 and x1/2LCA-i1 pepducins afforded marked protection against death from sepsis (Kaneider, et al., 2005).These results suggest that targeting of chemokine receptors by pepducins may be a potential therapeutic strategy for patients with sepsis. Pepducins targeting CXCR4 have also been designed. ATI-2341 is a pepducin based on the first intracellular loop of CXCR4 and induced CXCR4-dependent signaling and chemotaxis in leukocytes (Tchernychev, et al., 2010). In mice and cynomolgus monkeys, AT-2341 dose-dependently increased the release of granulocyte-macrophage progenitor cells from the bone marrow. Conversely, the pepducin x4pal-i1, also based on the first intracellular loop of CXCR4, inhibited CXCR4 signaling and blocked CXCL12-mediated migration of lymphocytes (O’Callaghan, et al., 2012). These studies suggest that targeting of chemokine receptors through pepducins is feasible and may be potentially useful in the treatment of sepsis.

S1P receptors play important roles in the pathogenesis of sepsis and are potential therapeutic targets as discussed previously in section 4.8. KRX-725 is a pepducin that activates S1P₃ receptors and is based on the second intracellular loop of the S1P₃ receptor (Licht, Tsirulnikov, Reuveni, Yarnitzky, & Ben-Sasson, 2003). KRX-725 causes activation of S1P₃ receptors on mouse aortic rings, which induces Gi-dependent ERK activation and endothelium-dependent vasodilation mediated by nitric oxide. Severino and colleagues synthesized a pepducin (peptide sequence Myr-GRPYDAN-NH₂) that antagonized S1P₃ receptors (Severino, et al., 2013). Given the role played by S1P in sepsis, pepducins targeting S1P receptors may be potentially useful for patients with sepsis.

A number of peculiarities regarding pepducins should be noted here. Firstly, it has been observed that pepducins are not entirely specific for their “designated” target receptor (Winther, et al., 2017). As an example, two pepducins (P2Y₂PalIC2 and P2Y₂PalIC3) containing sequences from the second and third intracellular loops (respectively) of the ATP (P2Y₂) receptor were found to be agonists for FPR2 on neutrophils (Gabl, et al., 2016). Interestingly, this phenomenon involved cross-talk between ATP bound-P2Y₂ receptor and P2Y₂PalIC2 bound-FPR2 receptor. Likewise, a pepducin designed as an agonist for the CXCR4 receptor, ATI-2341, was found to have stimulatory effects on neutrophils through activation of FPR2 (Holdfeldt, Winther, Gabl, Dahlgren, & Forsman, 2016). Secondly, small substitutions in the amino acid sequence of certain pepducins leads to complete abrogation of their targeting activity (Gabl, et al., 2016). Additionally, FPR2 targeted pepducins have no effect on FPR1 despite significant similarity in the amino acid sequences of intracellular loops of FPR1 and FRP2 (He & Ye, 2017). These observations suggest that the intracellular targeting of GPCRs by pepducins may be related to their ability to target specific dimeric or oligomeric forms of the target GPCR. The precise details of how pepducins intracellularly interact with their cognate receptors have not been fully elucidated (Carr & Benovic, 2016). Despite this, pepducins hold great promise for targeting GPCRs as these cell-penetrating peptides can access receptor conformations that are not otherwise accessible by orthosteric targeting.

5.2. Small molecule allosteric modulators

The interaction of small molecule allosteric modulators with GPCRs is most well-described for chemokine receptors. The structure of CCR9 crystallized in complex with vercirnon has been described, which revealed that the binding site of vercirnon (CCX282) is on the cytoplasmic face of the receptor (Oswald, et al., 2016). Vercirnon has been shown to be efficacious for treatment of inflammatory bowel disease in phase II clinical trials, and is currently being tested in phase III clinical trials (Wendt & Keshav, 2015). Likewise, the crystalline structure of CCR2 complexed with the allosteric modulator CCR2-RA-[R] has also been described (Zheng, et al., 2016). CCR2-RA-[R] binds to a highly druggable pocket that is the most intracellular allosteric site observed in any class A GPCR. Apart from chemokine receptors, the crystal structure of the β₂-adrenergic receptor complexed with an allosteric inhibitor cmpd-15A has also been published (X. Liu, et al., 2017). These studies describing the crystalline structures of GPCRs complexed to allosteric modulators have provided important insights with implications for future drug discovery. One important observation is the fact that these allosteric antagonists appear to act through steric hindrance as they preclude the interaction of the GPCR with G-proteins and β-arrestin. Another important observation is that the intracellular ligand-binding pocket harbors a balanced combination of polar and hydrophobic moieties, which makes it a potentially druggable target. Lastly, and most importantly, these crystal structures provide high-resolution details of the intracellular ligand-binding pockets, which may serve as a platform for virtual ligand screening for identifying other allosteric modulators.

5.3. Intrabodies

Nanobodies is the name given to the variable fragment of single-chain antibodies. Human immunoglobulins are composed of heavy and light chains, and differ from single-chain antibodies that are typically produced by members of the camelid family. Pardon and colleagues generated a set of nanobodies against the β₂-adrenergic receptor by immunizing Ilamas with an agonist-bound purified β₂-adrenergic receptor (Pardon, et al., 2018). Staus et al. expressed a set of these nanobodies in the cytoplasm of HEK293 cells as intrabodies and assessed their effects on β₂-adrenergic receptor-mediated signaling (Staus, et al., 2014). Many of the intrabodies were found to inhibit cAMP accumulation, β-arrestin recruitment, GRK-mediated receptor phosphorylation, and/or receptor endocytosis with a preference for either active (agonist occupied) or inactive (antagonist occupied) conformation of β₂-adrenergic receptors. The ability of these intrabodies to fine-tune ligand-induced GPCR trafficking and signaling opens a unique avenue of opportunities to intracellularly target GPCRs in a precise manner. However, the main barrier to their use in clinical settings would be the delivery or expression of these intrabodies in live cells. A number of putative methods including self-internalizing peptides, cationic liposome encapsulation and nanoparticle-mediated delivery are currently being developed, but, these methods are not yet ready for prime time (Cardinale, Merlo, Giunchedi, & Biocca, 2014).

5.4. Aptamers

The word “aptamer” comes from the Latin word aptus meaning fit and the Greek word meros meaning part. Aptamers are actually single-stranded molecules of RNA or DNA that can bind to specific interfaces on proteins with high specificity and affinity that is determined by their secondary and tertiary structures (Nimjee, White, Becker, & Sullenger, 2017). These oligonucleotides are typically produced by the SELEX (systematic evolution of ligands by exponential enrichment) method. This iterative method entails incubating a target protein with a large library of nucleic acid molecules and separating nucleic acid molecules that bind to the target protein. The bound RNA molecules are then amplified by real time-polymerase chain reaction and resulting DNA templates are transcribed. The new pool of nucleic acids is again incubated with the target protein and the whole cycle is repeated 8–12 times until an RNA pool with a high affinity for the target protein isolated. Sullenger and colleagues demonstrated that these aptamers could be isolated to virtually any target and may serve as potential therapeutic agents (Sullenger, Gallardo, Ungers, & Gilboa, 1990). Since then, numerous aptamers have been designed against a variety of proteins and are in preclinical or clinical phases of development. Of note, an aptamer against vascular endothelial growth factor, pegaptanib, was approved by the FDA in 2000 for use in patients with wet age-related macular degeneration.

Kahsai and colleagues employed the SELEX (systematic evolution of ligands by exponential enrichment) method to identify RNA aptamers that bind to allosteric sites of the β₂-adrenergic receptor with nanomolar affinity (Kahsai, et al., 2016). They started with an RNA library containing 10¹⁵ unique sequences and used an iterative selection process employing next-generation sequencing and comparative bioinformatics to isolate candidate aptamers with desirable binding properties. These aptamers further underwent nine rounds of positive selection against unliganded and agonist-bound β₂-adrenergic receptors in order to isolate high-affinity aptamers binding at structurally relevant sites. At the end of the selection process, the pool of final aptamers was able to stabilize unliganded and ligand-specific conformations of the β₂-adrenergic receptor with nanomolar affinities. In particular, aptamers A1, A2 and A13 significantly inhibited agonist-induced cAMP accumulation. This study demonstrated that aptamers could be potentially developed as pharmacological agents for the modulation of GPCR-mediated signaling.

6. Conclusions and future directions

GPCRs play diverse physiologic roles in the body and are implicated in the pathogenesis of sepsis. Traditional pharmacologic approaches of targeting GPCRs employed orthosteric ligands, which has a number of shortcomings. Novel pharmacologic approaches can target GPCR signaling intracellularly through the use of aptamers, intrabodies and pepducins. This has opened a fresh avenue of pharmacological possibilities that were not previously feasible with traditional methods of drug discovery. On the other hand, numerous clinical trials in sepsis have failed to show a survival benefit for any particular drug or intervention. A number of reasons may partly account for the myriad number of failed trials in sepsis. Firstly, sepsis is a heterogeneous syndrome caused by a wide spectrum of diverse infectious entities. The traditional approach of enrolling patients in sepsis trials who meet the broadly defined criteria of the sepsis syndrome is likely contributing to failed trials. The description of various phenotypes and molecular endotypes of sepsis have provided new insights and potential opportunities for precision medicine in sepsis. Secondly, the natural course of sepsis is biphasic in that early sepsis is characterized by a hyperinflammatory response followed by a delayed state of immuno-paralysis. Interventions that are tested in clinical trials need to be tailored in a time-sensitive manner.

Certain interventions that may be beneficial in the hyperinflammatory phase of sepsis may be detrimental in the immuno-paralysis phase of sepsis and vice versa. Moreover, the hyper- or hypoactive immune response in sepsis is highly heterogeneous and accurate diagnostics that dynamically capture the state of the immune response in real-time are crucial for developing tailored, individualized therapies. Thirdly, not all preclinical models of sepsis accurately parallel the pathogenesis of sepsis in humans, and certain interventions that appear beneficial in preclinical studies have been found to have no real benefit in human trials. In this regards, the endotoxemia model of experimentally induced sepsis is particularly problematic and does not accurately recapitulate the true pathogenesis of polymicrobial sepsis. The CLP model of experimental sepsis is often considered the gold standard method for studying sepsis. To further address this issue, an expert consensus group has developed the MQTiPSS (Minimal Quality Thresholds in Preclinical Studies for Sepsis) guidelines for conducting pre-clinical studies in sepsis (Osuchowski, et al., 2018). This will help to improve the quality of preclinical studies in sepsis and may provide better experimental models for testing pharmacotherapy in sepsis. Fourthly, multiple redundant pathways are concurrently activated in sepsis and testing singular interventions in traditional randomized controlled trials may be a reason for their failure. Consequently, newer trials should test a “cocktail” or package of multiple pharmacological interventions concomitantly. Finally, traditional design of randomized trials may not be the best method to test interventions in sepsis. Newer adaptive trial designs that incorporate Bayesian probabilities to modify and evolve the conduct of the trial as data is generated, may be better suited to identify beneficial interventions in sepsis. In this regard, the present review identified a number of GPCRs that may be potentially useful targets for pharmacotherapy in sepsis (Table 5). Novel approaches that utilize pepducins, aptamers and intrabodies to target these GPCRs may provide an unprecedented opportunity for altering the trajectory of morbidity and mortality from sepsis.

Table 5 —

GPCRs that are potential targets for pharmacotherapy in sepsis

Endogenous target	Pharmacothe rapy	Anticipated effects
Adenosine receptors	A2A receptor blockade	Immune stimulation and mitigation of inflammation
	A3 receptor activation
Adrenoceptors	β-adrenoceptor stimulation	Modification of cytokine expression profile of immune cells
Complement receptors	C3aR1 modulation	Re-establishment of vascular endothelial barrier
	C5aR1 modulation
Chemokine receptors	ACKR2 activation	Blockade of cytokine storm during hyper-inflammatory phase of sepsis
	CCR2 blockade
	CCR5 activation
	CX3CR1 stimulation
Coagulation cascade	PAR1 modulation	Restoration of physiologic endothelial function
Cannabinoid receptors	CB1 and CB2 receptor modulation	Alleviation of sepsis-induced multi-organ dysfunction
Endothelin receptors	ETA receptor blockade and ETB1 receptor stimulation	Restoration of mitochondrial oxidative phosphorylation and alleviation of sepsis-induced hypoperfusion
LPI receptor	LPI1 receptor blockade	Reconstitution of the blood-gut barrier
Sphingosine-1-phosphate	S1P1 receptor stimulation	Re-establishment of vascular endothelial barrier
Melatonin receptors	MT1 and MT2 receptor stimulation	Normalization of pineal gland-immune axis
Resolvin receptors	FPR2 and DRV2 receptor stimulation	Enhancing bacterial clearance and promoting resolution of inflammation
Ghrelin receptor	GHS-R stimulation	Immune stimulation and mitigation of inflammation
Hydroxycarboxylic acid receptors	HCAR2 stimulation	Reconstitution of the blood-gut barrier and intestinal microbiome
Urotensin II receptors	Urotensin II receptor blockade	Alleviation of sepsis-induced multi-organ dysfunction

ACKR2 = Atypical chemokine receptor 2; C3aR1 = complement protein 3a receptor 1; C5aR1 = complement protein 5a receptor 1; CB = cannabinoid; CCR = CC-chemokine receptor; CX3CR = CX3C chemokine receptor; DRV2 = D-series resolvin 2 receptor; ET = endothelin; FPR = formylpeptide receptor; GHS-R = growth hormone secretagogue receptor; GPCR = G protein-coupled receptor; HCAR = hydroxycarboxylic acid receptor; LPI = lysophosphatidylinositol; MT = melatonin; PAR = protease-activated receptor; S1P = sphingosine-1-phosphate.

List of abbreviations

ACKR

Atypical chemokine receptor

ADP

Adenosine diphosphate

AMP

Adenosine monophosphate

APACHE

Acute Physiology and Chronic Health Evaluation

APC

Activated protein C

ATP

Adenosine triphosphate

C3aR1

Complement protein 3a receptor 1

C5aR1

Complement protein 5a receptor 1

cAMP

Cyclic adenosine monophosphate

CB

Cannabinoid

CCL2

CC-chemokine ligand 2

CCL3

CC-chemokine ligand 3

cCKR

Conventional chemokine receptor

CCR

CC-chemokine receptor

CLP

Cecal ligation and puncture

CR1

Complement receptor 1

CR2

Complement receptor 2

CXCR

CXC-chemokine receptor

DAG

Diacylglycerol

DAMP

Damage-associated molecular protein

DC

Dendritic cell

DIC

Disseminated intravascular coagulation

ET

Endothelin

FFAR

Free fatty acid receptor

FPR2

Formylpeptide receptor 2

GDP

Guanosine diphosphate

GHS-R

Growth hormone secretagogue receptor

G-protein

GTP-binding protein

GPCR

G-protein coupled receptor

GRK

GPCR kinase

GTP

Guanosine triphosphate

HCAR

Hydroxycarboxylic acid receptor

IFN

Interferon

IL

Interleukin

IP₃

Inositol-1,4,5-triphosphate

LPA

Lysophosphatidic

LPI

Lysophosphatidylinositol

LPS

Lipopolysaccharide

LysoPS

Lysophosphatidylserine

MAPK

Mitogen activated protein kinase

MBL

Mannose-binding lectin

MMP

Matrix metalloproteinase

NCT

National ClinicalTrials.gov number

NET

Neutrophil extracellular trap

NFκB

Nuclear factor kappa (κ)-B

NK

Natural killer

PAMP

Pathogen-associated molecular pattern

PAR

Protease-activated receptor

PLC

Phospholipase C

RVD

D-series resolvin

RVE

E-series resolvin

S1P

Sphingosine-1-phosphate

SCFA

Short-chain fatty acid

SOFA

Sequential Organ Failure Assessment

SPM

Specialized pro-resolving mediator

TNF

Tumor necrosis factor

VE

Vascular endothelial