Roles of Endothelial Cells During Infection

Abstract

Endothelial cells (ECs) integrate immune and vascular functions to promote host defense against pathogens. While previously studied as forming passive flow conduits, ECs are now recognized as active contributors to maladaptive inflammation. During acute infection, ECs may promote tissue pathologies including hypoxia, acidosis, electrolyte disturbances, and capillary barrier breakdown. These pathologies significantly contribute to organ dysfunction and septic shock. In chronic vascular infection, ECs may promote loss of vascular quiescence, contributing to vascular malformations and vaso-occlusive lesions. In this review, we discuss the interplay between the immune and vascular roles of blood ECs, an emerging area of interest with therapeutic implications. While previously overlooked, therapies targeting dysregulation of EC function(s) may show clinical benefit towards improving outcomes related to both acute and chronic forms of infection.

INTRODUCTION

Blood endothelial cells (ECs) form a single cell monolayer that lines vascular conduits, providing an interface between the blood and the interstitium. At this interface, ECs perform functions related to host immunity and tissue homeostasis. There are an estimated 1-6x10¹³ ECs in humans with a cumulative surface area of ~1000m².¹ Due to their broad distributions and surface areas, ECs are positioned for immune surveillance, systemically sensing pathogen-derived signals. Concurrently, due to their juxtaposition to smooth muscle cells and/or underlying parenchymal cells, ECs may regulate hemodynamic and vascular functions related to tissue homeostasis.

Infectious disease involves xenogen colonization within a host compartment, and this process encompasses diverse pathogens and cell types. Acute infection manifests as a continuum where mild infection induces systemic inflammatory response syndrome (SIRS) that if untreated may progress to impaired organ function or sepsis, and culminating in profound circulatory and metabolic abnormalities or septic shock.² Sepsis/septic shock, collectively termed ‘sepsis,’ accounts for ~11 million deaths each year, alarmingly representing ~20% of deaths worldwide.³ In comparison to acute infection leading to sepsis, effects of chronic EC infection is a relatively understudied process. Numerous studies indicate broad pathologic effects of chronic EC infection including increased angiogenesis and loss of EC quiescence, and these chronic changes may lead to adverse vasculopathic effects.

Full understanding of the intersection between the immune and vascular functions of ECs has implications for treating sequelae of acute and chronic infection. The first section of this review discusses immune and vascular functions of ECs during acute infection (Figure 1). The second section of this review describes how chronic infection may alter EC quiescence to contribute to vascular pathologies.

Figure 1. EC Functions During Acute and Chronic Infection.

During acute infection, EC-mediated perturbations in vascular functions may modulate immunity at sites of infection. Chronic infection of ECs may promote processes leading to pathologic vascular remodeling.

I. EC Contributions to Acute Infection.

A. Immune Functions of ECs.

Many excellent articles have provided in-depth reviews of the immune roles of blood ECs during acute infection, variably focusing on pathogen-sensing,⁴ T cell priming,⁵ and immune cell recruitment functions.^6,7 We briefly summarize these immune features below. Acronyms in the text are fully spelled out in Table 1. Luminal surfaces of ECs face the blood environment, enabling ECs to sample pathogen-associated patterns and microbial antigens in blood to initiate immune responses. To enable innate immunity, ECs express surface and endosomal pattern recognition receptors including Toll-like receptors (TLR1/2/3/4/5/6/9/10),⁸ NOD-like receptors (NLRP1, AIM2, NLRP3),^9-12 and nucleotide-binding sensors, RIG-I and cGAS.¹³ Along with these sensors, ECs express intracellular machinery for propagating inflammation including all 4 stress kinase pathways (ERK1/2, JNK, P38, ERK5),⁴ canonical and non-canonical NF-κB,¹⁰ inflammasomes^9,11-13 (ASC, caspase-1), and type I interferon signaling.¹³

Table 1. Abbreviations.

AIM2	Absent in Melanoma 2
AP-1	Activator Protein-1
ASC	Apoptosis-associated Speck-Like Protein Containing a Caspase Recruitment Domain
cGAS	Cyclic GMP-AMP Synthase
CMV	Cytomegalovirus
DIC	Disseminated Intravascular Coagulation
EC	Endothelial Cell
ELF	E74-Like Factor
ERK1/2	Extracellular Signal-Regulated Kinase
ET-1	Endothelin-1
ETS	Erythroblast Transformation-Specific
FOXP3	Forkhead Box Protein 3
GPR	G Protein Coupled Receptor
HCV	Hepatitis C Virus
HIF-1a	Hypoxia Inducible Factor-1 Alpha
HSV	Herpes Simplex Virus
IBD	Inflammatory Bowel Disease
ICAM-1	Intercellular Adhesion Molecule-1
ICOS	Inducible T Cell Costimulator
IFN-g	Interferon-gamma
IL	Interleukin
INOS	Inducible Nitric Oxide Synthase
IRR	Insulin Receptor-Related Receptor
JAM	Junctional Adhesion Molecule
JNK	Jun N-Terminal Kinase
LOF	Loss-Of-Function
LPS	Lipopolysacchardie
MAdCAM-1	Mucosal Vascular Addressin Cell Adhesion Molecule-1
MHC I/II	Major Histocompatibility Complex I/II
NF-kB	Nuclear Factor-Kappa B
NLRP1	Nod-Like Family Receptor Pyrin Domain Containing 1
NLRP3	Nod-Like Family Receptor Pyrin Domain Containing 3
NO	Nitric Oxide
PAI-1	Plasminogen Activator Inhibitor-1
PAMP	Pathogen-Associated Microbial Pattern
PD-L1/2	Programmed Cell Death Protein Ligand 1/2
RIG-I	RetinoicAcid-Inducible Gene I
ROCK	Rho coiled/coiled kinase
RORgt	RAR-Related Orphan Receptor Gamma
SGK1	Serum and Glucocorticoid-Regulated Kinase 1
SIRS	Systemic Inflammatory Response Syndrome
SP1	Specificity Protein 1
STAT1	Signal Transducer and Activator of Transcription 1
TJ	Tight Junction
TLR	Toll-Like Receptor
TNF-a	Tumor Necrosis Factor Alpha
Treg	Regulatory T cell
TRPV	Transient Receptor Potential Cation Channel Subfamily V
VCAM-1	Vascular Cell Adhesion Molecule-1

Humans ECs function as semi-professional antigen presenting cells that can directly prime CD4+ and CD8+ effector memory T cells.^5,14 Following STAT1 agonism, canonically by IFN-γ, ECs upregulate both MHC I/II along with costimulatory molecules including ICOS, PD-L1/2 and CD58.¹⁴ The ability of human ECs to prime lymphoid cells is often overlooked as mouse ECs in contrast lack sufficient costimulatory molecules to directly mediate priming.¹⁴ In certain cases, ECs themselves may become substrates for infection by EC-tropic viruses including hantavirus,¹⁵ ebolavirus,¹⁶ and dengue¹⁷ viruses causing hemorrhagic fever syndromes. In such cases, direct antigen presentation by ECs may play a pivotal role in the host adaptive response. Pathogen-associated molecular patterns (PAMPs) as well as bacterial and viral antigens typically skew CD4+ T cells towards type 1 responses characterized by elaboration of IFN-γ,⁵ but ECs can also induce type 2 responses marked by IL-4 as well.¹⁸

Following initiation of innate and/or adaptive immune responses, ECs rapidly degranulate, releasing inflammatory factors stored within Weibel-Palade bodies including P-selectin, IL-8, and endothelin-1.¹⁴ Additionally, ECs express various effector molecules including cytokines (IL-1β, IL-6, and IL-15), type I interferons (IFN-α/β), and chemokines [IL-8 (CXCL8), RANTES, MIP1a, IP10] as well as a diverse battery of cognate receptors for such mediators, allowing both autocrine and paracrine modulation of downstream responses.

An additional, well-established role of ECs is their ability to regulate immune cell recruitment. In addition to functioning as semi-professional antigen presenting cells as above, blood ECs may bind and regulate circulating levels of IgG via the neonatal FC receptor (FcRn), and blood ECs may present tissue homing molecules including adhesion molecules (VCAM-1, ICAM-1, MAdCAM-1) and selectins (E-selectin) to recruit immune cells to sites of infection.^6,7,14 Inhibitors blocking FcRn as well as cognate interactions with ICAM-1 and MAdCAM-1^{19, 20} have been trialed clinically for inflammatory conditions involving EC-mediated inflammation (Table 2) and are particularly relevant for recruitment of immune cells to mucosal sites where there is ongoing exposure to microbes. While not yet fully defined, via mechanisms above ECs may promote retention of tissue-resident immune cells, and this remains a burgeoning area of interest.

Table 2.

EC-Targeted Therapies

Drug	Targeted Molecule	Targeted Pathway
Drotrecogin alfa	Activated Protein C	EC-Mediated Coagulopathy
Eculizumab	Complement C5	EC Activation
Nipocalimab	Neonatal Fc Receptor	EC Activation
Vedolizumab	α4β7 Integrin	EC-Mediated Immune Cell Recruitment

B. EC-Mediated Autoregulation of Blood Flow During Infection and Sepsis.

Perfusion pressure is the pressure gradient driving blood flow through an organ, and proper blood flow supports adequate tissue oxygenation and metabolism during infection. ECs regulate perfusion pressure during sepsis by releasing vasodilatory mediators, many of which are induced by infection-associated stimuli including lipopolysaccharide (LPS) and pro-inflammatory cytokines like TNF-α, IL-1β, IL-6, and IL-8.⁵ During sepsis, nitric oxide (NO), a vasodilatory gas derived from ECs and macrophages, decreases systemic vascular resistance (SVR) by acting on contiguous smooth muscle cells (Figure 2). Concurrently, EC-derived endothelin-1 (ET-1) and prostanoids (prostaglandins, thromboxanes, leukotrienes) promote microvascular dysfunction and increased recruitment of immune cells, furthering release of vasodilatory mediators (Figure 2). The combined macro- and micro-vascular effects of EC-derived factors overall decrease perfusion pressure, promoting tissue hypoxia and metabolic acidosis during sepsis.

Figure 2. Immune Mediators Promote Loss of Systemic Vascular Resistance.

During acute infection, local immune mediators promote release of EC-derived factors that act on smooth muscle cells to reduce systemic vascular resistance (SVR) during sepsis.

Tissue hypoxia, a hallmark of sepsis and septic shock, has been shown to enhance antimicrobial immunity. CD4+ T cells infiltrate into hypoxic tissue, show a gene signature compatible with cellular adaptation to hypoxia including upregulation of HIF1α and demonstrate increased effector responses. Hypoxia may potentiate immunity to enhance intestinal homeostasis, allowing clearance of bacterial,^21,22 viral,²³and fungal^24,25 pathogens; and these responses are attributable to effects of hypoxia on immune cells including macrophages,²⁶ T cells,^27,28 and B cells.^29,30 At the same time, these metabolic and hypoxia-associated pathways need to be properly modulated. For example, Hedl et al found that with respect to the glycolytic and HIF1α pathways activated upon microbial ligand stimulation of macrophages, variants in the immune-mediated disease risk gene IRF5 show gain-of-function, thereby potentiating glycolysis to dysregulate immune signaling.²⁶

During progression from SIRS to sepsis, EC-mediated hypoxia stimulates metabolic changes in tissue-infiltrating immune cells. Metabolic changes in glycolysis,^26,27 oxidative phosphorylation,^31-33 fatty acid biosynthesis,^31-33 and amino acid metabolism³⁴ have been observed in various immune cells exposed to hypoxic microenvironments. These metabolic changes may subsequently lead to pathologic effector states including prolonged neutrophil survival,³⁵ M1 polarization of macrophages,^26,36 Th17 polarization of CD4+ T cells,^37,38 and initiation of T cell programs causing functional exhaustion.³⁹ These processes have been attributed to metabolic switching induced by hypoxia,⁴⁰ and may collectively contribute to uncontrolled infection,⁴¹ abscess formation,⁴² and worsened tissue injury.⁴³

During infection, hypoxic niches induce programs in blood ECs that promote new blood vessel growth or angiogenesis. Hypoxia specifies programs including increased EC proliferation and expression of spatial positioning and adhesion molecules, and these programs collectively promote immune cell recruitment to allow extracellular matrix remodeling for vessel outgrowth. Dysregulation of such signals during prolonged ischemia or during ischemia reperfusion injury (IRI), may lead to a wide range of maladaptive phenotypes in ECs including ENOS uncoupling and oxidative injury, unconstrained membrane attack complex (MAC) formation,⁵² and immune signal dysregulation, leading to cell death.⁴⁵ Anti-MAC therapies have been trialed in infectious settings involving Shiga-toxin E. coli infection (O7:H157) and COVID-19 (Table 2).

In addition to hypoxia, extracellular ATP released by bacteria potently drives both angiogenesis and immune processes in ECs, and bacteria have evolved metabolic adaptations to subvert immune signaling driven by extracellular ATP. Bacteria may promote degradation of extracellular ATP via bacteria intrinsic enzymes, induction of host ectonucleotidases, and downregulation of purinergic receptors on host ECs. These responses dampen host sensing by NOD-like receptors (NLRs) and impair mucosal IgA responses of T follicular helper cells⁴⁴ and have the overall effect of impairing host immunity.

C. EC-Mediated Acidosis During Infection and Sepsis.

During progression from SIRS to sepsis, metabolic supply:demand mismatch generates metabolites including ketone bodies, succinate, and lactate. These species contribute to tissue acidosis, particularly in skeletal muscle. In acidotic tissues, acid pH is sensed by proton-binding (GPR4, GPR65, GPR68, GPR132) and ketone-binding (GPR81, GPR91, GPR99) G protein-coupled receptors, acid-sensing ion channels, transient receptor potential cation channel subfamily V members (TRPVs), and the recently discovered, alkali-sensing insulin receptor-related receptor, IRR.⁴⁶ These sensors, widely expressed on immune cells,⁴⁷ activate programs supporting pathogen clearance.

Acidosis biases hematopoiesis towards the myeloid lineage and promotes survival of bone marrow emigrants.⁴⁷ Acidosis additionally enhances endo/phagocytic capacity of myeloid cells, dually augmenting pathogen internalization⁴⁸ and antigen presentation⁴⁹ while suppressing excess T cell effector function.⁵⁰ Finally, acidosis may vigorously stimulate the complement pathway⁵¹ whose terminal activation forms anaphylatoxins and membrane attack complexes⁵² to facilitate pathogen clearance. Loss of pH sensing in genetic models has been linked to worsened mucosal infections and/or chronic inflammation of the colon⁵³ and skin.⁵⁴

D. EC-Mediated Coagulopathy During Infection and Sepsis.

Disseminated intravascular coagulation (DIC) is a life-threatening condition occurring during severe sepsis/septic shock and is characterized by unconstrained activity of coagulation factors, leading to bystander cleavage and activation of complement proteins and subsequent depletion of coagulation proteins. This promotes cytokine storm and a pro-thrombotic state. ECs become substrates for microvascular thrombosis during DIC by releasing pro-thrombotic molecules like tissue factor and factor VII and anti-fibrinolytic molecules like plasminogen activator-inhibitor (PAI-1) while downregulating anti-thrombotic molecules like thrombomodulin.⁵⁵ Affirming the relevance of EC-mediated coagulopathy, activated protein C, an inhibitor of the coagulation cascade, is clinically used to obviate thrombotic sequelae of severe sepsis (Table 2).

E. EC-Mediated Regulation of Interstitial Sodium Balance During Infection and Sepsis.

ECs form a barrier that dynamically regulates electrolytes within interstitial fluid. ECs express sodium transporters including Na+/H+ exchangers, ENaC channels, and Na+/K+/Cl− cotransporters, and expression of many of these transporters are altered during infection.⁵⁶ Accordingly, sodium imbalances are among the most common electrolyte disturbances seen among septic patients. Among tissues, the interstitium of the skin has been posited as a physiologic reservoir for sodium. Quantification of Na+ concentrations in intravascular vs cutaneous interstitial compartments using ²³Na-magnetic resonance imaging spectroscopy showed that plasma sodium concentrations remained stable while sodium content within the interstitium highly fluctuated without accompanying changes in fluid content.⁵⁷ This indicated that cells confined to interstitial space including tissue-infiltrating immune cells are subjected to dynamic sodium concentrations exceeding that of intravascular space.

High sodium microenvironments may modulate immune responses. Increased sodium concentrations preferentially induce Th17 polarization of CD4+ T cells in a manner requiring the salt-sensitive kinase, SGK1.^58,59 This can, in turn, lead to inflammation at tissue sites such as the intestine⁵⁸. Regulatory T cells (Tregs) exposed to high sodium microenvironments show a FoxP3-RORγt+ phenotype, lose their regulatory properties, and acquire the ability to elaborate IL-17.⁶⁰ Interestingly, high sodium exposures have also been shown to generate a population of FoxP3+RORγt+ T cells dually elaborating IL-17 and retaining regulatory properties.⁶¹ Such T cells express CCR6⁶¹, enabling homing to skin, a postulated sodium reservoir.^57,62 Human macrophages exposed to high sodium microenvironments similarly show effector functions including increased inducible nitric oxide synthase (INOS)⁶³, upregulated stress kinase pathways, and M1 polarization characterized by IL-12^HiIL-4^Lo expression.⁶⁴

F. EC-Mediated Barrier Disruption During Infection and Sepsis.

In homeostasis, capillary ECs form inter-EC tight junctions (TJs) that establish a permselective barrier between blood and tissues. TJs are formed via proteins including claudins, occludins, and junctional adhesion molecules (JAMs). Unlike capillaries, venular ECs form fewer TJs and may become activated by infection to more readily allow cells, solutes and fluid into the infected tissue space.⁶⁵

Due to its massive cumulative surface area, breakdown of the capillary EC barrier is pathologic and frequently clinically significant. Leakage of fluid into tissue causes tissue edema, impairing diffusion of oxygen, glucose, and other nutrients. Of note, vascular barrier disruption at mucosal sites such as the intestine⁶⁶ and skin⁶⁷ may lead to significant and clinically relevant inflammation.

ECs act both as targets and mediators of the complex cytokine milieu associated with disrupted EC barriers. During progression from SIRS to sepsis, the capillary tight junctional barrier breaks down, causing indiscriminate recruitment of immune cells and EC intrinsic programs subsequently dysregulate immune cell function. Numerous studies analyzing the cytokine milieu of sepsis have largely observed great patient and disease heterogeneity, and underlying mechanisms remain incompletely elucidated. What is clear, is that numerous pro- and anti-inflammatory cytokines wax and wane in a given individual over time with an unpredictable course.

In response to sepsis, microbial ligands and damage-associated molecules activate pathways in ECs that converge on RhoGTPases and Rho coiled/coiled kinase (ROCK), resulting in actin stress fibers (Figure 3).^68,69 Stress fibers comprised of actin increase cell tension, disrupting ECs conjoined by TJs to promote decreased barrier function. Adherens junctions such as VE-cadherin are phosphorylated, internalized, and/or shed,^69,70 further contributing to junction instability. Concurrently, inflammatory gene programs are initiated by NF-κB and AP-1, resulting in increased adhesion molecule presentation and worsening capillary leak. Compounding the above, during sepsis increased cell death causes EC denudation from the basement membrane, leaving large gaps for cells, solutes and fluid to leak into tissue.

Figure 3. EC Barrier Disruption During Sepsis.

During acute infection, immune mediators promote myriad signaling cascades promoting EC barrier disruption. EC barrier disruption, in addition to compromising tissue function, potentiates immune cell recruitment at sites of infection.

II. Chronic Infection and Vasculopathy.

A. Subacute/Chronic Infection and Angiogenesis.

Angiogenesis is an EC-mediated process forming new microvessels. While an indispensable component of normal physiology, angiogenesis may show pathological effects during subacute and chronic infection. Both viral and bacterial infections increase angiogenesis in vivo, and dysregulated angiogenesis may form vascular malformations during chronic papovirus family (SV40, human papillomavirus, polyoma), herpesvirus family (Epstein-Barr virus, human herpesvirus 8), and Bartonella infections.⁷¹ Infection-related vascular malformations contribute to significant pain and cause significant bleeding in isolated cases.

Infection-related factors including cytokines, chemokines, and hypoxia promote EC proliferation, perturbing vascular quiescence. Cellular quiescence is governed by transcription factors like ETS. These molecules have garnered significant interest as therapeutic targets, particularly in inflammatory conditions like solid organ malignancies where pathologic angiogenesis may occur.

ETS transcription factors are a large and conserved group comprised of ~28 family members implicated in both EC and immune functions.⁷² ETS transcription factors are categorized into 3 classes and 6 subfamilies including the ELF subfamily. Among the ELF subfamily, ELF4 has garnered increased attention based on its roles in EC and immune quiescence. Loss-of-function (LOF) of ELF4 has been reported to lead to loss of EC quiescence, causing enhanced EC proliferation.⁷² Furthermore, LOF of ELF4 promotes loss of immune cell quiescence, causing pathologically enhanced immune responses to microbial ligands. Indeed, human deficiency in ELF4 results in an autoinflammatory disease affecting the gastrointestinal tract, skin, and joints.^73,74 Aside from ELF4, many ETS transcription factors including ETS1, SP1, PU.1, and ELF1, are heavily studied for their roles in modulating immune functions and EC activation. Ongoing studies may further clarify the relationship of these factors in ECs during infection.

B. Subacute/Chronic EC Infection and Atherosclerosis.

Atherosclerosis is a condition where chronic inflammatory processes promote flow-limiting stenoses and ischemic complications. Hallmarks of atherosclerosis include EC activation and chronic vascular inflammation. While dysregulated lipid, glucose, and sodium metabolism are widely studied as major contributors, multiple studies have suggested that infection-related inflammation may be a significant contributor.

Using nucleic acid or antigen detection methods,⁷⁵ numerous studies have detected viral (CMV, HCV, HIV, HSV, EBV, enteroviruses, and parvoviruses) and bacterial (Chlamydia pneumonia, Mycoplasma pneumonia, Helicobacter pylori, periodontal anaerobes) pathogens. In human atherosclerotic plaques. Follow-up studies have shown that antecedent infection may worsen EC activation, inflammation, and plaque burden in murine models of atherosclerosis.⁷⁵ Moreover, atherosclerosis can serve as a risk factor for inflammation at mucosal sites where there is ongoing microbial exposure, particularly in older adults, as is the case with IBD.⁷⁶

Murine ‘B-1’ B cells and the more recently identified CD20+CD27+CD43+CD70− B cells in humans are positively selected during development and secrete natural IgM antibodies (Abs).⁷⁷ Natural Abs show broad polyreactivity against both modified and apoptotic host cells as well as pathogens. Natural Abs are believed to be evolutionarily conserved as housekeeping effector molecules. An anti-phosphorylcholine-binding natural antibody, termed T15, was isolated and showed cross-reactivity to S. pneumoniae and oxidized low density lipoprotein (ox-LDL), an inflammatory neo-epitope in ECs in atherosclerotic lesions. This led to the hypothesis that molecular mimicry due to infection may contribute to propagation of inflammation during atherosclerosis.

While many bacterial pathogens have been detected in a sizable proportion (25-50%) of atherosclerotic lesions, culture isolation has been reported only for Chlamydia pneumoniae, Enterobacter hormaechei, and herpesvirus.⁷⁵ Antibiotics including macrolides and quinolones have not demonstrated a clear benefit in secondary prevention, i.e., halting progression of established atherosclerotic disease. However, inflammation is attendant to all subacute/chronic infections, and inflammatory states in particular collagen vascular disease are well-known to accelerate atherosclerotic lesions. Precisely how chronic or persistent infection or associated inflammation may contribute to atherosclerosis remains an ongoing area of study.

CONCLUSIONS & FUTURE DIRECTIONS

ECs pivotally modulate immune and vascular functions during infection. During acute infection, EC interplay among these dual functions contribute to host pathologies frequently seen during progression of SIRS to sepsis including hypoxia, acidosis, electrolyte alterations, and capillary barrier disruption. During chronic infection, ECs may contribute to clinically significant vascular pathologies including vascular malformations and vaso-occlusive lesions. Current sepsis treatments have focused on antimicrobial measures including antibiotics, and there is a paucity of therapies focused on reducing maladaptive inflammation to preserve tissue homeostasis. We propose that blocking EC-mediated immune cell recruitment, coagulopathy, and barrier dysfunction may offer avenues for blocking tissue-level sequelae of infectious disease. Further interrogation to clarify such roles of ECs in infection may lead to targeted therapies for improving patient outcomes.