Immunology of Sepsis

Introduction

The current definition of sepsis as “life-threatening organ dysfunction caused by a dysregulated host response to infection” recognizes that sepsis is fundamentally an immunologic disorder.¹ Humans have evolved highly conserved innate and adaptive immune responses to different types of pathogens that are essential to survival on our microbially-diverse planet. However, the magnitude and character of those responses are highly variable based on genetic variability, host-pathogen interactions, and the environment. The type of immune response to infection that ultimately leads to the optimal resolution of infection is unclear. This is complicated by the fact that there is a wide range of biologic responses to different types of infections at different stages of injury that contribute to clinical outcomes. One of the most important shifts in our understanding of sepsis in the past twenty years has been that it is a heterogenous immunologic process, but is defined as a single clinical entity. Inclusion of a broad cohort of patients meeting sepsis clinical criteria into an interventional trial may dilute the mean effect of a particular immune targeting therapy, thus concealing a potentially responsive subpopulation of patients.

In this article, we provide a review of how different aspects of the immune system respond to infection in the context of sepsis and how modulating that response might improve outcomes. We start by providing an overview of how different immune cell types respond to infection and how they trigger downstream systemic responses. We then discuss some of the landmark clinical trials that have tested agents to modulate the immune response to sepsis, and provide context for how some of these “failed” therapies might be reconsidered to treat subpopulations of patients identified by immunologic biomarkers that might receive benefit. An underlying premise throughout this article is that the development of new therapies for sepsis should be targeted at specific host immunologic derangements that can be robustly identified through tissue-specific and systemic biomarkers.

Sepsis Manifests in Three Overlapping Immune Phases

Sepsis can be conceptualized as immune dysfunction occurring in three overlapping phases (Figure 1). The first phase is pathogen recognition and response. Tissue-resident immune cells (e.g. epithelial cells, macrophages) at the sites of infection such as the lung mediate the initial inflammatory responses seen in sepsis. These innate immune cells sense the presence of microbial components (pathogen-associated molecular patterns or PAMPs) or byproducts of tissue injury (damage-associated molecular patterns or DAMPs) by pattern recognition receptors (PRRs). There are five general types of PRRs on the cell-surface or in the cytoplasm of innate immune cells: 1) Toll-like receptors (TLRs); 2) RIG-I-like receptors (RLRs); 3) NOD-like receptors (NLRs); 4) C-type lectin receptors (CLRs); and 5) Aim2-like receptors (ALRs).² Engagement of PRRs leads to activation of a cascade of downstream transcription factors that promote release of proinflammatory cytokines and chemokines that can trigger the second phase of sepsis – the immune activation phase. The immune activation phase, when activated locally, promotes pathogen killing and eradication. Sepsis manifests when the host response to infection exceeds organ containment and drives deleterious systemic responses. A third phase of sepsis called immune paralysis can simultaneously occur with immune activation. Immune paralysis is driven by innate immune cell exhaustion, epigenetic modifications, and lymphocyte apoptosis, and can manifest with unresolved infection and increased incidence of secondary infections. In the next section, we highlight the role of immune cell types in different tissues during these three phases of sepsis.

Figure 1. Sepsis-Related Immune Dysregulation.

Sepsis-related immune dysregulation occurs in three overlapping phases, including pathogen recognition/response, inflammation, and immune paralysis. Downstream mediators including “pro-inflammatory cytokines,” “anti-inflammatory cytokines,” chemokines, antimicrobial peptides, and other effector molecules are outlined in Table 1.

AEC: alveolar epithelial cell; ALR: Aim2-like receptor; AMP: antimicrobial peptides; CLR: C-type lectin receptor; MPO: myeloperoxidase; NLR: NOD-like receptor; RLR: RIG-I-like receptor; Sp-A: surfactant protein A; Sp-D: surfactant protein D; TAM: Tyro3, Axl, Mer receptors; TLR: toll like receptor; Treg: regulatory T cell

Epithelial Cells

The first line of host defense against microbial entry are the epithelial layers of the skin and linings of the respiratory, gastrointestinal, and genitourinary tracts. Epithelial cells are polarized, which allows them to maintain secure barriers and regulate transportation of solutes, water, and other products between different cellular and tissue compartments. Epithelial cells are also highly immunogenic and can trigger multiple inflammatory pathways in response to pathogen exposure.

Keratinocytes:

The skin is the primary site of infection in 6–10% of patients with sepsis.^3,4 Keratinocytes express multiple PRR including TLRs/RLRs/NLRs, which recognize components of some of the most important skin pathogens such as staphylococcus and streptococcus.⁵ Upon stimulation with various PAMPs, keratinocytes have been shown to secrete key immune mediators such as TNF-α, IL-1 family members, CXCL8 (IL-8), CCL2 (MCP-1), CCL20 (MIP-3α) (Table 1). Keratinocytes are also a key source of antimicrobial peptides (AMPs), most importantly β-defensins and cathelicidins.⁶ IL-17A and IL-22 have been shown to be key regulators of AMP secretion by keratinocytes, highlighting the importance of the IL-17 pathway in mucosal and skin defenses.⁷ Although keratinocytes are generally tolerogenic,⁸ they can express MHC class II molecules under certain conditions (e.g. IFN-γ or bacterial enterotoxin exposure).⁹

Table 1.

Immune Cell Subsets and Downstream Mediators Associated with Sepsis

Immunologic Domain	Cell Type	Relevant Subset Markers	Mediators/Relevant Biomarkers
Pathogen Recognition and Response	Epithelial (keratinocytes)	NA	Cytokines: TNF-a, IL-1, MIP-3α, IL-17A, IL-18, IL-22 Chemokines: IL-8, MCP-1 AMPs: β-defensins, cathelicidins
	Epithelial (bronchial)	NA	mucins, immunoglobulins (esp. IgA), lactoferrin, lysozymes AMPs: b-defensins, cathelicidins
	Epithelial (Type II pneumocyte)	NA	Cytokines: GM-CSF, IL-6 Chemokines: GRO-α, MIP-2α, MCP-1, CCL5, IL-8 Surfactant proteins, collectins, AMPs, tubular myelin, complements, lysozyme, complement proteins
	Epithelial (GU)	NA	Cytokines: IL-1b, IL-6, TNF-α Chemokines: IL-8 AMPs, NGAL, uromodulin
	Macrophages (barrier – lung, gut, skin)	PD-L1, CD163	Cytokines: IL-1RA, IL-1β PAMPs/DAMPs: LPS/endotoxin, HMGB1 Complement proteins
	Monocytes (infiltrating)	CD14	PAMPs/DAMPs: HMGB1, presepsin, sCD14 Cytokines: IL-1RA, IL-1β
Immune Activation	Neutrophils	CD10, PD-L1, TREM1	Chemokines: CXCL12, IL-8, G-CSF, GRO-α Effector mediators: NETs, proteases, calprotectin Apoptotic mediators: IL-10, PBEF, lipoxin receptors, annexin A1, caspases, Bcl-2 family members
	Monocytes (circulating)	CD11b, CD14, CD16, CCR2, CD40, CD48, HLA-DR, TREM1	Cytokines: IL-6, sTNFR1 Chemokines: MCP-1, MIP-1α, MIP-1β, CCL5
	Effector Macrophages	CD80, CD86	Cytokines: IFN-α, IFN-β, IFN-γ, IL-6, IL18, MIF, sTNFR1 Chemokines: MCP-1, MIP-1α, MIP-1β, CCL5
	T Cells	CD48, CCR4, CCR6	Cytokines: IFN-γ, IL-6, IL-17, MIF, sTNFR1 Chemokines: CCL5
Immune Paralysis	Monocyte/Macrophages CD4 and CD8 T Cells	HLA-DR, PD-L1, ICOS-L, CD200R TCR, Tregs, PD-1, TRAIL, LAG3, BTLA, CTLA4, ICOS, CD28, CRTH2	Cytokines: IL-10 Growth factors: IGF1, HGF, TGF-β Apoptosis: lymphopenia Ag specificity: TCR-β chain, proliferation Diminished cytokine production: TNF-α, IFN-γ, IL-6, IL-10

AMP: antimicrobial peptides; BTLA: B and T lymphocyte associated; CTLA: chronic T lymphocyte associated protein; DAMPs: damage-associated molecular patterns; G-CSF: granulocyte colony-stimulating factor; GM-CSF: granulocyte macrophage colony-stimulating factor; GU: genitourinary; HGF: hepatocyte growth factor; HMGB: high mobility group box; ICOS: inducible co-stimulator; IGF: insulin-like growth factor; IFN: interferon; IL: interleukin; LAG: lymphocyte activation gene; LPS: lipopolysaccharide; MCP: monocyte chemoattractant protein; MIF: melanocyte inhibiting factor; MIP: macrophage inflammatory protein; NETs: neutrophil extracellular traps; NGAL: neutrophil gelatinase-associated lipocalin; PAMPs: pathogen-associated molecular patterns; PBEF: pre-B cell colony enhancing factor; PD-1/PD-L1: programmed cell death protein/ligand; TCR: T cell receptor; TNF: tumor necrosis factor; TRAIL: TNF-related apoptosis inducing ligand; Treg: regulator T cell; TREM: triggering receptor expressed on myeloid cells

There are multiple ways that local skin infections can overwhelm epithelial cell driven defenses and progress to sepsis. First, there are certain microorganisms that are more virulent that are associated with sepsis. Some gram-positive cocci derive pathogenicity from virulence factors and can infect individuals despite normal host defense.¹⁰ Polymicrobial infections with pathogens such as bacteroides, clostridium, or peptostreptococcus are more common in older, morbid, and immunocompromised individuals.¹¹ Second, breakdown of the epithelial barrier (e.g. trauma, insect bites) is an important risk factor for development of sepsis.¹² Injection drug use is one of the strongest risk factors for developing necrotizing fasciitis caused by methicillin-resistant staph aureus.¹³ Third, abnormalities in non-barrier host defense (e.g. HIV infection) predispose individuals to develop systemic organ injury from skin infections.^13,14

Lung Epithelial Cells:

The lung is the most common site of infection that is associated with the development of sepsis, with pneumonia representing approximately 40–60% of patients with sepsis.^3,4 Defense of the respiratory tract starts with the mucus lining the nasal, pharyngeal, and bronchial epithelial cells. Mucins are the most important component of mucus, which helps trap particulates and microbes. This lining is maintained at a low pH and contains multiple antimicrobial constituents such as AMPs (β-defensin and cathelicidin), immunoglobulins (especially IgA), lactoferrin, and lysozymes.¹⁵ The immunologic properties of different subsets of human airway epithelial cells have been recently reviewed.¹⁶ Airway infections, including bacterial/viral bronchitis, bronchiectasis, and bronchiolitis often have systemic manifestations such as fever, but rarely lead to severe sepsis or organ failures unless they progress to pneumonia.

In contrast, infection of the gas-exchanging units of the lung (by definition, pneumonia) is one of the most important causes of severe sepsis. Type I epithelial cells have been shown to produce inflammatory cytokines in cell culture,¹⁷ but likely have limited immunologic functions in vivo. In cell-targeted genetic deletion experiments, NF-κb RelA knock-out of alveolar epithelial cells only impacted 2 of 84 cytokine transcripts measured in mice treated with LPS, strongly suggesting a very limited role for type I alveolar epithelial cells in innate immune responses.¹⁸ Type II alveolar epithelial cells, on the other hand, play a large role in immune defense mechanisms. Human type II alveolar epithelial cells constitutively express TLR4 as well as TLR2 under stimulatory conditions.¹⁹ They are also a key source of GM-CSF, which is a primary alveolar macrophage growth factor. Type II alveolar epithelial cells secrete multiple PRRs into the alveolar space, most importantly collectins (SP-A, SP-D), tubular myelin, and complement (Table 1). At homeostasis, SP-A and SP-D bind SIRP-α to tonically suppress alveolar inflammation.²⁰ However, when the carboxy-terminal lectin-like domains of SP-A and SP-D bind to PAMPs, they activate pathogen killing functions such as phagocytosis, ROS production, and cytokine secretion to activate professional phagocytes such as alveolar macrophages via CD91.^20–23 Complement is another essential component of the alveolar milieu. The complement system includes more than 30 plasma and membrane bound proteins in the circulating blood, however local complement activation is a key feature of lung host defense.²⁴ Complement, particularly C3a and C5a, is present in human BALF and is secreted by type II alveolar epithelial cells, macrophages, and mesothelial cells.^25–28 Complement peptides can recognize some PAMPs but are primarily activated by binding to the Fc region on IgM and IgG antibodies. Complement peptides help opsonize microbes and are some of the most potent chemoattractants for neutrophils, monocytes, and macrophages. Humans with complement deficiencies are highly susceptible to pneumonia (especially from encapsulated bacteria),²⁹ highlighting their role in pulmonary host defense.

We can speculate on factors for why local inflammatory response mechanisms to alveolar infections progress to sepsis and systemic organ injury, however a complete conceptual model is currently lacking. The alveolar barrier is necessarily thin to allow for gas exchange but is constantly exposed to inhaled pathogens. Compared with the skin and other mucosal surfaces, there is relatively little anatomic “reserve” in the alveolar environment before invading pathogens can access more immunologically protected areas. As a result, the lung is particularly vulnerable to exaggerated immune responses to more serious infections. Although these factors are clearly important, the relative contributions of pathogen and host response factors to the development of pulmonary-associated sepsis are still not well understood.

GU Epithelial Cells:

Genitourinary infections are the cause of ICU-associated sepsis in approximately 11% of cases.^3,4 Similar to skin and lung epithelial cells, GU epithelial cells constitutively secrete AMPs such as β-defensin into the urine to restrict bacterial growth.³⁰ Other constituents of the urine that are secreted in part by epithelial cells like lipocalin-2 (NGAL) have antimicrobial properties such as iron sequestration (Table 1). Uromodulin (Tamm-Horsfall protein) is the most abundant urinary protein and has been shown to have key antibacterial properties.^31,32 Bladder epithelial cells also contain multiple PRRs and have been shown to secrete proinflammatory cytokines and chemokines such as IL-1β, IL-6, IL-8, and TNF-α (Table 1). Two unique immunologic properties of bladder epithelial cells compared with other epithelial cells are their exocytic and “shedding” activities. Bladder epithelial cells are able to autonomously expel intracellular bacteria via exosome-encased bacteria.^33–35 Bladder epithelial cells also regularly shed infected cells into the urine, and are replaced with highly proliferative urothelial cells to limit infection and to activate immune responses.^36–38

The primary risks for developing urinary tract associated sepsis include abnormalities of the urinary anatomy (e.g. catheters) and obstructive diseases,³⁹ both of which directly impact the normal epithelial defense mechanisms and/or predispose to higher than normal microbiologic inoculum. Studies have demonstrated that prolonged acute infections of the lower urinary tract epithelial lining predispose future infections.⁴⁰ Despite the high incidence of urinary tract associated sepsis, the associated mortality remains below most other types of infections.^3,4

Neutrophils

Neutrophils are the most abundant immune cell in the bone marrow and are one of the first systemic responders to local infections. These short-lived cells can rapidly reach all perfused tissues and contain a plethora of antimicrobial properties to help clear pathogens. Peripheral blood neutrophils are recruited to infected tissues via chemokine gradients. Once neutrophils arrive at infected tissues, they undergo diapedesis out of capillaries through a sequence of margination, rolling, and transmigration that is mediated by multiple selectins and adhesion molecules.⁴¹ Neutrophils kill pathogens through phagocytosis, release of antimicrobial mediators (e.g. proteases), or generation of neutrophil extracellular traps (NETs). Recent studies have demonstrated there is significantly more neutrophil diversity than previously appreciated based on ontogeny, pace of granulopoiesis, and other environmental signals.⁴²

Peripheral Blood Neutrophils:

Neutrophils account for approximately 50% of nucleated blood cells at steady-state, a proportion that dramatically increases in response to infection. Emergency granulopoiesis is a key source of neutrophil diversity.⁴² Infection and sepsis-induced immature neutrophils recruited from the bone marrow have been shown to have less chemotactic, inflammatory cytokine production, and phagocytic capabilities compared with more mature neutrophils.^43,44 CXCL12/CXCR4 mediated neutrophil migration has robustly been shown to play a key role in animal models of sepsis, along with G-CSF, the main cytokine responsible for neutrophil maturation (Table 1). Interestingly, immature neutrophil subsets from patients with sepsis appear to inhibit T cell activation and proliferation compared with other subsets. In co-culture experiments, CD66b+ neutrophils from patients with sepsis inhibit CD4 T cell proliferation and are associated with a lower proportion of PD-1+ or CD69+ CD4 T cells.⁴⁵ In separate studies, neutrophil subsets that express PD-L1 have been shown to distinguish sepsis patients compared with controls.^44,46,47 Taken together, granulopoiesis plays a key role in sepsis pathophysiology.

Recent studies using single-cell transcriptional approaches have identified previously underappreciated diversity in neutrophil function.^42,45,48 For example, interferon-stimulated neutrophils have been shown to expand during bacterial or viral infections.^43,49–51 IL1R2⁺ neutrophils and epigenetic reprogramming have been associated with patients within more severe subphenotypes of sepsis.⁴⁵ In sepsis and ARDS, neutrophils have long been known to be in a state of delayed apoptosis, prolonging their lifespan and potentially prolonging inflammation.⁵² Lipoxin receptors, annexin A1, caspases, and Bcl-2 family members have all been implicated in impaired neutrophil apoptosis in sepsis (Table 1). Although surface markers to robustly and efficiently identify cells predisposed to impaired apoptosis in patients with sepsis have not been identified, measurable circulating cytokines such as Pre-B cell colony-enhancing factor and IL-10 have been shown to play a key role in delayed neutrophil apoptosis.

Tissue-Infiltrating Neutrophils:

The vast majority of neutrophils in the body are circulating and migrate to sites of infection. However, there are neutrophil subsets that are variably present in different organs at steady state and have recently been shown to help regulate basal physiology.^53,54 Interestingly, homeostatic tissue-infiltrating neutrophils retain their unique functional properties throughout their lifespan.⁵⁴ For example, lung and intestinal neutrophils appear to be less immunologic compared with peripheral blood neutrophils, but express significantly more angiogenic properties.⁵⁴ The implications for these recent findings of distinct functions for tissue-infiltrating neutrophils to sepsis are unclear. However, it is possible that some of these unique functions – for instance, angiogenesis in the intestine – may play a key role in recovery from sepsis-related ischemia and hypoxemia and merit future investigations.

NETs:

One of the biggest advances in our understanding of neutrophil function has been the discovery of NETs. NETs are web-like chromatin structures that form when the contents of neutrophils are released, and contain antimicrobial mediators including neutrophil elastase, myeloperoxidase, calprotectin, cathelicidins, defensins, and actin.⁵⁵ There is a host benefit from NET formation in some, but not all, types of local infections. For instance, NETs are essential to clearing large pathogens such as fungal hyphae and extracellular bacterial aggregates.⁵⁶ In contrast, small yeast or single bacteria do not elicit NET formation. The benefit of NETs to clearing local infections seems particularly relevant to fungal (aspergillus),⁵⁷ oral commensal bacteria,⁵⁸ and gram positive and negative bacterial pathogens,^59–62 although the degree to how much they protect from sepsis is debated.⁶³

There are multiple reasons why neutrophil activation during infection may contribute to sepsis. First, it is well-established that excessive activation and/or a lack of regulation of NETs can contribute to local tissue injury. However, NETs have also been shown to play a key role is distant/systemic organ injury that is mediated in part through thrombosis.^64,65 The tipping point of when NET formation transitions from protective to pathogenic is not clear. Although NETs appear to help trap circulating bacteria in the liver and aid clearance, NET formation can also contribute to significant hepatic injury.⁶⁶ A link between neutrophils, NETs, vascular dysfunction, and organ perfusion is likely a fundamental component of sepsis biology. Second, excessive migration of neutrophils to organs likely contributes to damage.⁶⁷ Neutrophilic chemotactic factors, particularly IL-8 and CXCL12, have been associated with excessive organ dysfunction in critically ill patients. Finally, although we have discussed neutrophil migration and activation as a primary trigger for sepsis, significant neutrophil deficits are likely a stronger risk factor for developing life-threatening systemic organ dysfunction from infection than activation. Neutrophil impairment via neutropenia, medications that inhibit neutrophil activity, age, and other comorbidities such as diabetes with known dramatic effects of neutrophils have robustly been some of the strongest epidemiologic risk factors for severe sepsis.^68,69

Monocytes and Macrophages

Monocytes and tissue-resident macrophages play a central role in all aspects of sepsis biology because of their ability to recognize pathogens, their antimicrobial properties, and their capacity to coordinate disparate features of the innate immune response.⁷⁰ Monocytes originate in the bone marrow, circulate in the blood, and are recruited to different tissues and organ systems. Tissue-resident macrophages can either arise locally from embryonic precursors or can develop from recruited blood monocytes. During infection, macrophages in barrier tissues recognize a wide variety of PAMPs and DAMPs and trigger early effector responses including inflammation and immune cell chemotaxis (Figure 1). These macrophages also play a key role in phagocytosing pathogens and clearing cells that have undergone apoptosis or necrosis. Blood monocyte are recruited to infected organs and can mature into different subsets of macrophages that play different roles in different phase of infection. Macrophage subsets with pro-resolving and regenerative properties can secrete mediators and growth factors that promote tissue repair after injury.

Peripheral Blood Monocytes:

Monocytes account for approximately 5% of nucleated blood cells at steady-state. CD14+CD16− “classical” monocytes represent approximately 85% of circulating monocytes and are primed for PAMP/DAMP recognition (classical monocytes highly express all 5 classes of PRRs), migration to tissues, phagocytosis, and inflammation.⁷¹ Nonclassical (CD14-CD16+) and intermediate (CD14+CD16+) monocytes constitute the remaining 15% circulating monocytes and play a role in vascular homeostasis and antigen presentation. Recent studies have demonstrated that classical monocytes progressively give rise to intermediate and nonclassical monocytes over the course of a few days.⁷² In patients with sepsis, the proportion of nonclassical and intermediate monocytes can increase to as much as 50% of all monocytes.⁷³ Although the lineage of different monocyte subsets likely accounts for relatively distinct cell functions, there is a wide spectrum of activation that can overlap depending on context.⁷⁴

The degree of HLA-DR expression on monocytes has repeatedly been associated with outcomes in patients with sepsis, and helps support the concept of “endotoxin tolerance” and sepsis-associated immunoparalysis. HLA-DR is responsible for antigen presentation, and its expression on classical monocytes is significantly depressed in patients with sepsis and predicts development of secondary infections and poor outcomes.^75–78 One of the most important studies in sepsis in the past 15 years analyzed splenic antigen presenting cells and tissue macrophages, and found that HLR-DR cell surface expression was significantly lower in cells collected from patients with sepsis compared with non-septic controls.⁷⁸ The binding of monocyte/macrophage PD-L1 to PD-1 on T cells decreases T cell proliferation, decreases cytokine production, increases regulatory T cell expansion, and impairs antigen responsiveness. Expression of the checkpoint ligand PD-L1 on antigen-presenting cells and tissues is significantly higher in patients with sepsis compared with controls,⁷⁸ and multiple subsequent clinical studies have observed similar findings.^79–81 Consistent with the PD-L1/PD-1 studies, blockade of other checkpoint proteins such as CTLA-4 in sepsis models has also been shown to decrease sepsis-induced T cell apoptosis and improve outcomes at certain doses.^82,83 Together, these findings strongly suggest that immunoparalysis – coordinated in large part through monocyte subsets – is a fundamental aspect of sepsis pathobiology that contributes to secondary infection and worse overall survival.

Tissue Resident Macrophages:

Tissue macrophages represent approximately 1–5% of tissue-resident cells and play a fundamental role in modifying organ function during stress such as severe infection.⁸⁴ They can be classified in multiple ways, however for the discussion of sepsis an important distinction can be made between macrophages located in barrier tissues (e.g. lung, intestine, skin) and macrophages located in more immunologically privileged organs (e.g. brain). Barrier macrophages in general contain more surface receptors associated with phagocytic activity are much more immunologically tolerant (e.g. less response to TLR ligands, higher expression of inhibitory mechanisms) than non-barrier macrophage subsets.⁸⁵ Almost all barrier macrophages secrete cytokines that promote epithelial cell growth at homeostasis as well as have the capacity to secrete proinflammatory chemokines and cytokines in response to PAMPs and DAMPs (Table 1). An area of investigation is the degree to which recruited monocyte, monocyte-derived macrophage, and resident macrophage subsets contribute to inflammation during stress and infection. For example, some,^86–88 but not all,^89,90 studies have shown that resident intestinal macrophages have limited proinflammatory capacity. The balance of studies of lung macrophages suggest recruited monocytes and monocyte-derived macrophages are more “proinflammatory” than resident populations.^91–96 Overall, barrier macrophages and recruited monocytes play a fundamental role to pathogen recognition and downstream immune activation in sepsis.

Monocyte-derived macrophages and resident macrophage also play a key role in the resolution of inflammation, which may also contribute to immune paralysis in sepsis. Monocytes and macrophage efferocytosis of apoptotic neutrophils activates numerous downstream transcription factors including LXRα, LXRβ, and PPARγ that attenuate expression of proinflammatory cytokines and chemokines.^97–99 In addition to downregulating proinflammatory mediators, efferocytosis promotes release of molecules that interact with the epithelium and promote tissue repair (Table 1). These programs are essential to resolution of injury and repair, but it is not clear if or when these mechanisms might become deleterious in sepsis. Innate immune training¹⁰⁰ – epigenetic regulation of gene expression – likely plays a key role in sepsis-associated immunoparalysis and risk for secondary infections. Landmark studies have demonstrated that LPS-induced monocyte/macrophage exhaustion is regulated in large part through histone modifications.^101,102 Many of these epigenetic signatures have been observed in patients with bacterial sepsis,¹⁰³ in some cases as early as 1-day post initial injury, and have been shown to persist as long as 6-months post-injury.^104,105 Further research in determining how monocyte and macrophage reprogramming via epigenetic changes within the context of sepsis is needed given the clear role immunoparalysis plays in sepsis outcomes.

Given their role in all three broad aspects of sepsis immune biology (host response, downstream activation, and immunoparalysis), modulation of monocyte and macrophage function is a promising target for sepsis therapies. As mentioned above, the balance of evidence suggests recruitment of blood monocytes to infected organs is often associated with worse sepsis-associated outcomes. Attenuating chemotaxis of blood monocytes to sites of injury may mitigate exuberant tissue injury, provided a local infection is contained through antibiotics and other host defense mechanisms. Dampening the early host monocyte response to infection is another possible approach to prevent the onset of severe sepsis. For example, blockade of the monocyte PRR CD14 leads to significantly reduced systemic inflammatory mediator levels in humans treated with LPS.¹⁰⁶ On the other end of the spectrum, there is intense interest in augmenting monocyte function during later phases of sepsis through approaches such as checkpoint pathway inhibition. This concept is based on the findings suggesting the majority of patients with sepsis die from unresolved infection.

T cells

T cells are an important part of the adaptive immune system and make up the majority of the lymphocyte population in blood. T cells can also be tissue resident and provide immune protection against invading pathogens. While T cells play a key role in the clearance of pathogens, persistent T cell dysfunction has been implicated in poor outcomes in sepsis. In particular, sepsis-associated immune paralysis and risk for secondary infection is thought to be related to impaired T cell immune function.

Decreased numbers of circulating lymphocytes, or lymphopenia, has been repeatedly associated with increased sepsis-associated mortality.^107,108 The primary mechanism of this lymphopenia is thought to be due to increased lymphocyte apoptosis. Both CD4 T cells and CD8 T cells decrease after the onset of sepsis. Particularly in elderly patients, this lymphopenia can be quite prolonged and is associated with mortality.¹⁰⁹ If cell numbers do return to baseline, there may be longer lasting functional impairments that can lead to viral reactivation or new infections.^110,111

It is not simply the number of T cells but their diversity and functionality that change over the course of sepsis. T cells recognize their cognate antigen through the T-cell receptor (TCR) and the broad and diverse assembly of TCR sequences leads to a broad repertoire of T cell responses. One study has shown that the T cell pool in sepsis has a decreased repertoire, particularly in the TCR-β chain that returns back to normal over time.¹¹² This low TCR diversity has been associated with the development of nosocomial infections.^112,113 Similarly, T cells appear to have an exhausted phenotype during sepsis that may impair their effector functions. A sentinel study showed that splenocytes secreted lower amounts of TNF-α, IFN-ɣ, IL-6, IL-10 in response to anti-CD3/CD28 mAb stimulation.⁷⁸ Human studies and murine models of sepsis have shown that CD4 T cells express higher levels of inhibitory receptors that dampen immune responses including PD-1, TRAIL, LAG3, and BTLA suggesting an exhausted phenotype (Table 1). T cells also have decreased capacity for proliferation.^114–116 There are also suggestions that suppressive T cell subsets in circulation, like Treg, are increased in frequency in sepsis, particularly in relationship to other more proinflammatory T cell subsets like Th1 and Th17 cells.^117–119 Together, these suggest impaired T cell function during sepsis with the potential for subsequent risk for nosocomial infection and poor outcomes. T cell exhaustion or improving lymphopenia in sepsis have been proposed as therapeutic targets in sepsis.

It is important to note that while T cells have been characterized in circulation frequently in patients with sepsis, tissue resident memory T cells (T_RM) occupy tissues without recirculation and play an important role in immune responses at these sites. How sepsis affects long-term changes in tissue resident lymphocytes is less well known. One recent study showed that sepsis survivors had a lower cumulative incidence of cancer compared to patients with hospitalizations for less severe infections.¹²⁰ This was attributed to an accumulation of a particular CXCR6 tissue-resident T cell population in murine models that were similarly detected in human sepsis survivors. Further studies are necessary to determine how sepsis effects T cell populations at tissue sites.

Approaches to Modulate the Immune Response in Sepsis

The rationale for targeting the immune system as a therapeutic strategy for sepsis is based on decades of molecular, immunologic, and clinical data summarized above. Unfortunately, almost all large Phase II and III studies of immunomodulating therapies in sepsis have failed to demonstrate efficacy despite promising preclinical and early clinical data (Table 2). One of the most important reasons for these prior failures has likely been the inclusion of patients meeting sepsis clinical criteria into interventional trials who may or may not manifest the specific biologic target under investigation.¹²¹ Our proposed model of sepsis immunology is inclusive of significant biologic variability, fluctuating phases of immunologic response, and variable clinical trajectories (Figure 1). The relative contribution of different immunologic derangements (e.g. “immune activation” vs. “immune paralysis”) to two patients with similar clinical signs and symptoms may be highly distinct.

Table 2.

Selected Clinical Studies of Immune-Modulating Therapies for Sepsis

Immunologic Domain	Target	Rationale	References
Pathogen Recognition and Response	Endotoxin	Endotoxin activates the host inflammatory response	126–132
	TLR4	TLR4 is one of the most proximal receptors on monocytes/macrophages to PAMPs/DAMPs	133,134
	CD14	CD14 is one of the most proximal receptors on monocytes/macrophages to PAMPs/DAMPs	135
Immune Activation	TNF-α	TNF-α and IL-1 are proinflammatory cytokines that have been shown to mediate sepsis pathophysiology	136–141
	IL-1		122,142,143
	Acute phase reactants (Statins)	Statins have been shown to have pleiotropic downregulatory effects on proinflammatory mediators such as NFκB	144,145
	Ibuprofen	Ibuprofen inhibits cyclooxygenase leading to lower levels of prostaglandins and thromboxanes	146,147
Immune Paralysis	G-CSF	Growth factor for neutrophils	148,149
	GM-CSF	Induces proliferation and differentiation of monocytes, macrophages, and DCs; upregulation of HLD-DR	150–153
	Thymosin-α	Data suggesting thymosin-α augments T cell proliferation and function	154
	IL-7	Growth factor for B and T cells	155
	PD-L1/PD-1	Checkpoint proteins that decrease T cell proliferation, cytokine production, antigen responsiveness, and promote cytotoxic T cell apoptosis and Treg expansion	156–158

It is essential for future interventional trials to propose enrollment criteria that are based on immunologic derangements that are shared between the majority of individuals enrolled into the study. Although these types of study designs may limit generalizability to all patients with “sepsis”, they will at least identify subgroups of patients that may be responsive to specific therapies. For example, the Phase III reIL1RA Sepsis Syndrome Study enrolled individuals meeting SIRS criteria with evidence of hypoperfusion into a placebo arm vs. two different dosing strategies of rhIL1RA.¹²² This study was amongst several to focus on the IL-1 target based on its robust association with the “immune activation” aspect of sepsis biology and poor clinical outcomes. The original trial did not detect a signal for efficacy of the primary outcome (28-day all-cause mortality) at either treatment dose. In a retrospective analysis of a subgroup of patients from this trial who had baseline plasma collected, individuals with a high baseline plasma IL-1RA levels had a statistically significant mortality benefit from IL-1RA antagonist.¹²³ In contrast, individuals with lower baseline plasma IL-1RA did not receive a benefit from IL-1RA antagonist treatment. The heterogeneity of treatment effect observed in this study, and in others with similar post-hoc analyses,¹²⁴ demonstrate the promise of “predictive enrichment” approaches to future sepsis trial study designs.¹²⁵

Summary

One of the most frustrating paradoxes in critical care medicine over the past few decades has been that despite our improved understanding of sepsis immunology, almost no effective new therapies have been developed. We now understand that this failure is due in large part to the inclusive clinical definition of sepsis that has often led to a misalignment between individuals who may or may not be manifesting a specific immunologic target, even while meeting criteria for “sepsis.” Recent successful treatments that have been developed for asthma offer an important lesson for future interventional trials in sepsis – patient selection for enrollment should be informed by endotypes that reflect underlying biology and predict treatment response. As the scalability of molecular platforms to quickly and accurately better define patient subgroups evolves, we hope the promise of precision medicine for sepsis will be fulfilled.

Key Points:

• Sepsis-related immune dysregulation occurs in three overlapping phases, including pathogen recognition/response, inflammation, and immune paralysis.

• Despite our improved understanding of sepsis immunology, almost no effective new therapies have been developed.

• Future interventional trials in sepsis should propose enrollment criteria that are based on immunologic derangements that are shared between the majority of individuals enrolled into the study.

Synopsis.

Sepsis-related immune dysregulation occurs in three overlapping phases, including pathogen recognition, inflammation, and immune paralysis. There are many immune cells that underlie these three phases, with the most well-studied being epithelial cells, neutrophils, monocytes, macrophages, and T cells. These cells regulate multiple downstream mediators such as cytokines and chemokines that can be measured and used as immunologic biomarkers. Past failures in sepsis immune modulating treatment trials have likely resulted from inclusion of patients meeting sepsis clinical criteria but do not sufficiently express the immunologic target under investigation. More precise approaches to trial designs may uncover effective immune modulating therapies.

Clinics Care Points.

• Sepsis is a coordinated cellular immune response with multiple phases including pathogen recognition, inflammation, and immune paralysis

• Targeting both sustained immune activation as well as the relative immunosuppression are important considerations in sepsis patients for future clinical trials

• There is a clear need to identify biomarkers of inflammation and identify subgroups of patients who will respond to specific immune therapies