Effect of ethanol exposure on innate immune response in sepsis

Abstract

Alcohol use disorder, reported by 1 in 8 critically ill patients, is a risk factor for death in sepsis patients. Sepsis, the leading cause of death, kills over 270,000 patients in the United States alone and remains without targeted therapy. Immune response in sepsis transitions from an early hyperinflammation to persistent inflammation and immunosuppression and multiple organ dysfunction during late sepsis. Innate immunity is the first line of defense against pathogen invasion. Ethanol exposure is known to impair innate and adaptive immune response and bacterial clearance in sepsis patients. Specifically, ethanol exposure is known to modulate every aspect of innate immune response with and without sepsis. Multiple molecular mechanisms are implicated in causing dysregulated immune response in ethanol exposure with sepsis, but targeted treatments have remained elusive. In this article, we outline the effects of ethanol exposure on various innate immune cell types in general and during sepsis.

Ethanol exposure dysregulates innate immune response in sepsis.

1. Introduction

Sepsis, defined as life-threatening organ dysfunction caused by a dysregulated host response to infection, is the leading cause of death in hospitalized patients, killing over 270,000 patients annually in the United States alone.^1,2 Currently, no sepsis-specific therapies are available, and the management heavily relies on supportive treatments. Decades of research has suggested a variety of molecular mechanisms; however, currently there are no targeted therapies to treat sepsis. Multiple organ dysfunctions positively correlate with sepsis mortality.³ It is well accepted that the comorbidities modulate the frequency and severity of multiple organ dysfunction in sepsis. Several modifiable and nonmodifiable comorbidities, such as age, obesity, diabetes mellitus, cirrhosis of the liver, underlying immune status, and alcohol use disorder (AUD), are known to affect sepsis mortality.^4,5

Specifically, AUD, a modifiable risk factor reported by 1 in 8 critically ill patients, is an independent risk factor for sepsis mortality.⁶ During the COVID-19 pandemic, the incidence of AUD increased even further.⁷ Multiple preclinical experimental models have shown that acute and chronic ethanol drinking impairs bacterial clearance. While several mechanisms and molecular targets are identified, therapies to reverse this phenomenon have remained elusive. AUD progressing to liver diseases and/or cirrhosis of liver further adds to the complexity of host immunity in sepsis. Evidence suggests that the comorbidities and complications are more prevalent compared with male counterparts in female patients with alcohol-associated chronic liver disease.⁸ Increased incidence or worse outcomes in younger females with AUD with sepsis is not evident from existing clinical studies.^6,9 However, studies focused on gender and age differences in ethanol with sepsis are in order.

An ideal inflammatory response to an infection would initiate quickly upon pathogen invasion, be of proportionate severity to the infectious insult, have a limited impact on organ function, and resolve in a timely manner back to homeostasis, in which it is poised to respond to the next pathogen invasion. During sepsis, however, the immune response is far from ideal and ios dysregulated with a transition from an early/hyperinflammation to late persistent inflammation and immunosuppression and multiple organ failure.¹⁰ Approximately one-third of sepsis mortality occurs during early sepsis, while the majority of the sepsis-related deaths occur late, during persistent inflammation and immunosuppression, without a timely return of homeostasis.^11–13

Innate immunity is the first line of defense in any pathogen invasion. Inflammation, a highly conserved process, is a protective mechanism against all pathogenic and nonpathogenic (traumatic, environmental exposures, etc.) insults to the organism. Acute and chronic ethanol exposure dysregulates innate and adaptive immunity in a dose and duration-dependent manner.¹⁴ The interplay between the two leads to further dysregulation of the immune response. While others have described the role of adaptive immunity in ethanol with sepsis, in this review we focus on this interplay of ethanol with all aspects of the innate immune response.^15–17 Table 1 provides an overview of the effects of ethanol exposure on different innate immune cell types and their potential mechanisms.

Table 1.

Phenotype and functions of different immune cell populations: effect of alcohol.^18–29

Cell type	Phenotype	Function	Reference
Neutrophil	N1: CD54high/CD95high/CD182low N2: CD54low/CD95low/CD182high High density (HDNs) Low density (LDNs)	Proinflammatory: induces TNFα and IP-10 Higher ability to produce ROS Induces IL-8, lesser ability to produce ROS Proinflammatory, phagocytosis intact Exhausted, phagocytosis impaired	Ohms et al.18 Fridlender et al.19 Ohms et al.18 Cho et al.20 Cho et al.21
Monocyte	CD14highCD16 low (classical) CD14highCD16internediate (intermediate) CD14 lowCD16high (nonclassical)	Increases early (2 h) following alcohol intoxication in human Decreases TLR4 expression Enriched population in AH patients Enhanced expression of CCR2/CD206 Significantly decreased in AH patients.	Janicova et al.22 Dhanda et al.23 Rasmussen et al.24
Macrophage	CD68+ (human) F4/80+ (mouse)	Ethanol mutes LPS-stimulated phagocytosis Ethanol decreases LPS-stimulated phagocytosis and glycolysis	Gandhirajan et al.25 Gandhirajan et al.25
DC	Myeloid DC: HLA-DRhigh/CD83high	Alcohol decreases circulating myeloid DCs CD11c surface expression decreases Reduces antigen presentation	Siggins et al.26
ILCs	ILC1 ILC1: NK1.1CD44high ILC3	Alcohol does not change the numbers but changes their ability to fight infection Alcohol increases the population in thymus Impaired secretion of IL-22	Ruiz-Cortes et al.27 Zhang et al.28 Ruiz-Cortes et al.27 Hendrikx et al.29

AH = alcoholic hepatitis; HDN = high-density neutrophil; LDN = low-density neutrophil; ROS = reactive oxygen species.

2. Ethanol exposure and modulation of pathogen-associated molecular patterns, damage-associated molecular patterns, and pattern recognition receptors

The pathogen response is initiated by a system of pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides (LPS) (produced by gram-negative bacteria), peptidoglycans and lipoteichoic acid (produced by gram-positive bacteria), lipoproteins, nucleic acids, etc. PAMPs are produced by all microorganisms, not necessarily by just the pathogens.³⁰ Immune cells recognize PAMPs using the pattern recognition receptors (PRRs). The PRRs are expressed on the cell surface and in the cytosol in order to screen for pathogen invasion continuously.³¹ The PRRs families of toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), and C-type lectin receptors (CLRs) detect a variety of pathogens and PAMPs.^31,32 PAMPs and their respective TLRs include LPS binding to TLR4, peptidoglycan to TLR2, flagellin binding to TLR5, TLR3 binding to double-stranded RNA ligand, and TLR7/8 binding to RNA fragments, and TLR9 binds to CpG motifs associated with bacterial DNA.^33–36 Upon binding, the intracellular TLR signaling and interleukin (IL)-1 receptor domains promote activation of immune response.³⁷ The family of TLRs (2, 4, 7, and 9) trigger receptors expressed on myeloid cells-1, to further carry out the immune response during pathogen invasion in sepsis.^38,39 Thrombocytopenia, a known marker of hematological dysfunction in multiple organ failure in sepsis, is known to be associated with increased mortality.⁴⁰ TLR7 was recently shown to be crucial for platelet activation and platelet-leukocyte aggregates and was implicated along with TLR2 in thrombocytopenia and coagulopathy in experimental model of sepsis.^41,42 Historically, sepsis-associated multiple organ failure and death were thought to be solely due to PAMPs.⁴³

Ethanol exposure modulates PRRs in the innate immune cells. Acute ethanol exposure suppresses TLR3 signaling pathway in vivo in mice.⁴⁴ In addition, evidence also supports that the acute ethanol exposure suppresses interferon (IFN)-related amplification loop and thereby other proinflammatory cytokine and chemokine expressions and increases susceptibility to viral infections.^45,46 Evidence implicates acute and chronic ethanol exposure–related interference with actin cytoskeleton that leads to disruption of TLR4-CD14 clustering and decreased proinflammatory cytokine expression in response to LPS.^47–49 The suppressive effect of acute ethanol exposure with ethanol levels equivalent to 300 mg/100 mL in male mice on TLR4 with LPS exposure in macrophages is also shown to be via p38-MAPK and extracellularly regulated kinases 1 and 2 (ERK1/2).^44,50 TLR4 responsive status is suggested to be crucial for survival during the binge drinking model of acute ethanol exposure with late phase sepsis in female mice with sepsis.^51,52 Upon activation, the TLR2 and TLR4 receptors associate with cholesterol-rich domains of the cell membrane. Ethanol exposure affects TLR2 and TLR4 differentially, in that it affects TLR4 but not the TLR2 association with the cholesterol-rich domain and thus differentially affecting membrane localization of these 2 TLRs.⁵³ Others have shown the effect of acute ethanol exposure on TLR7- and TLR9-induced pathways.^44,50 In communication between the TLRs, the NLR system of PRRs also modulates pathogen-induced and sterile inflammation, including neuroinflammation, via nuclear factor κB (NFκB) activation.⁵⁴

The PRR expression is tightly controlled in order to prevent an unrestricted inflammatory response. For example, upon activation by LPS on the cell membrane, TLR4 initiates 2 signaling cascades. The TIRAP–MYD88 induction is followed by the formation of a large protein complex known as “Mydodome,” ultimately culminating into activation of transcription factors like NFκB and transcription of proinflammatory genes. The second pathway involves a complex, which engages TRAM and TRIF, begins an endosome formation and endocytosis of the TLR4, mediated by CD14. Endocytosis of TLR4 essentially terminates MyD88-dependent signaling, preventing unrestricted inflammatory response.^32,55,56

It was not until the recognition of the inability of the host immune response to differentiate between “self” and “non-self,” and the “danger model,” that the concept of danger-associated molecular patterns (DAMPs) was tied to infectious and sterile inflammatory response.⁵⁷ During early sepsis, PAMPs recognized by PRRs stimulate an immune response and release DAMPs into the tissue environment and circulation. Several types of DAMPs such as high-mobility group box 1 (HMGB1), extracellular histones, extracellular cold-inducible RNA binding protein, and heat shock proteins are described in literature.⁵⁸ HMGB1 is an extensively studied DAMP in sepsis.^59–61 HMGB1 release is directly modulated by ethanol exposure via redox pathway, decreased HDAC, and other mechanisms.^62–64 HMGB1 is recognized by RAGE, leading to the release of tissue-type plasminogen activator, which ultimately leads to extracellular proteolysis and tissue injury.³¹ Several innate and adaptive immune cells also secrete HMGB1, which is detected by receptors such as TLR2, TLR4, RAGE, etc. to initiate further local and systemic tissue damage.³¹ These and other DAMPs known to play a critical role during sepsis, such as mitochondrial DNA, heat shock proteins, interleukins, etc. are reviewed extensively elsewhere.^31,43,61

In addition to a direct effect on the PRRs, ethanol exposure modulates release of DAMPs to further impact innate immunity. For example, complement C1q, C3, and C5 are shown to contribute to alcoholic liver injury in female mice with chronic ethanol drinking.⁶⁵ Chronic alcohol feeding in the mice increases expression of proinflammatory cytokines and DAMPs such as IL-1β, IL-6, and intercellular adhesion molecule-1 via modulation of macrophage chemoattractant protein (macrophage chemoattractant protein 1).⁶⁶ Similarly, the role of macrophage migration inhibitory factor is known to modulate innate immune response with chronic ethanol exposure in patients.⁶⁷ The role of ethanol in modulating these and several other DAMPs is extensively reviewed in the literature.^{34,65,68–70}

In addition to the direct effect of ethanol exposure on immune cells, alcoholic hepatitis and cirrhosis are independently associated with increased sepsis-related mortality.^6,15 In chronic ethanol exposure, TLR2, TLR4, TLR6, and TLR9 are implicated in hepatic injury via increased oxidative stress.⁷¹ Interestingly, TLR4- but not TLR2-dependent mechanism seems to be independent of MyD88.⁷² Widely used chronic ethanol exposure model, the Lieber–DeCarli liquid diet, uses ethanol with dietary fat, protein, and carbohydrates. Composition of the fatty acids, unsaturated vs saturated, modulates intestinal epithelial integrity, adding further to the ethanol-induced hepatic injury via increased blood endotoxin (LPS) levels.⁷³ Interestingly, dietary insulin feeding attenuates alcohol-induced liver injury via suppression of endotoxin and TLR4 in macrophages by modulation of gut microbiome.⁷⁴ While out of the scope of this review, a crucial role of ethanol in modulating the gut microbiome and innate immune response thereafter is reviewed extensively elsewhere.^75,76

Inflammasomes are large (∼700KD) multiprotein complexes in the immune cells, well known for their ability to control the activation of proteolytic enzyme caspase-1.⁷⁷ In addition to regulation of proinflammatory cytokines IL-1β and IL-18, Caspase-1 also regulates an inflammatory cell death, pyroptosis.⁷⁸ Inflammasomes are activated upon detection of DAMPs and PAMPs by using PRRs. A classical inflammasome contains sensor, which is usually a NLR or an AIM2-like (absent melanoma-2-like) receptor, an adaptor protein that is ASC (apoptosis-associated speck-like protein containing CARD), and an effector, caspase pro-caspase-1. The NLRs, such as the NLRPs, assemble their own inflammasomes. The NLRP3 inflammasome is the most studied of the inflammasomes, of particular importance in sepsis.⁷⁹ Evidence shows that the NLRP3 inflammasome mediates cognitive dysfunction in sepsis via p38-MAPK and ERK phosphorylation.⁸⁰ Similarly, NLRP3 inflammasome is involved in hepatic dysfunction⁸¹ in endotoxemia as well as cardiac⁸² and renal⁸³ dysfunction, and acute lung injury⁸⁴ in sepsis.

Ethanol-exposure perturbs NLRP3 inflammasome pathway in innate immune cells. A recent study implicated NLRP3 inflammasome pathway in neutrophil migration and macrophage dysfunction in alcohol-related hepatitis in a rodent model.⁸⁵ NLRP3 inflammasome pathway was also implicated in alcohol-associated neuroinflammation and atherosclerosis.^86,87 The effect of ethanol on inflammasome pathway in sepsis is not very well studied. Interestingly, the NLRP3 inflammasome pathway was implicated in acute ethanol-induced immunosuppression.⁸⁸ However, the effect of ethanol-induced inhibition of inflammasome activation on individual organ systems needs further evaluation.

3. Ethanol exposure and neutrophil function

Neutrophils play a critical role in responding to pathogen invasion. Although thought to be terminally differentiated cells in the past, a growing body of evidence suggests that neutrophils, like other innate cells, and just as macrophages, also differentiate into subphenotypes.^58,89 After production in the bone marrow, mature neutrophils are released into circulation. They are attracted to the site of inflammation/microbial invasion by chemoattractants to exert their proinflammatory and microbicidal activities. Senescent neutrophils return to the bone marrow, where they are engulfed by macrophages and destroyed.⁵⁸ Like M1/M2 macrophage subtypes, the N1 neutrophils are proinflammatory, and N2 neutrophils are associated with an anti-inflammatory phenotype.^58,90 Regulatory neutrophils are thought to be immunosuppressive.⁹¹ During sepsis, prolonged lifespan and senescence of neutrophils via antiapoptotic mechanisms are implicated in neutrophil dysfunction.^92,93 Classification of neutrophils based on density gradient into high-density neutrophils vs low-density neutrophils (LDNs) shows that the LDN subtype was associated with impaired immune function. An increased LDN population is observed in sepsis patients, and these neutrophils exhibit increased degranulation, decreased phagocytic capacity, and impaired immune function in sepsis.^94,95 Moreover, reduced immune function in the LDN subphenotype was found to be associated with increased expression of PD-1 and PD-L1 during sepsis.⁹⁶

Acute ethanol exposure perturbs neutrophil function. In an experimental model of endotoxemia, acute ethanol exposure muted the neutrophil function by inhibition of macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant.⁹⁷ Delayed neutrophil recruitment was implicated with acute ethanol exposure in a streptococcal pneumonia model via muted MIP-2 and cytokine-induced neutrophil chemoattractant.⁹⁸ Poor neutrophil accumulation with staphylococcal skin infection via disruption of IL-23/IL-17 axis was shown to be a novel mechanism for dysregulation of innate immune response in chronic alcohol abuse subjects, making them susceptible to sepsis.⁹⁹ Ethanol exposure is shown to increase the LDN population in alcohol-associated hepatitis patients. Interestingly, the LDNs are generated from high-density neutrophils, and after neutrophil extracellular trap (NET) formation in an alcoholic hepatitis model, LDNs exhibit an exhausted phenotype with decreased sensitivity to LPS stimulation.^20,21

In a classification of neutrophils based on senescence, they can be divided into (1) nonaged neutrophils with appropriate apoptosis mechanism; (2) aged neutrophils that undergo apoptosis, but over a prolonged period of time; and (3) aged neutrophils that do not undergo apoptosis.⁵⁸ PD-L1 delays neutrophil apoptosis, and these neutrophils modulate T cell responses during late sepsis.^91,100 Evidence also suggests that in face of pathogen invasion, the aged neutrophils do not return to bone marrow. Instead, they migrate to the lungs and serve as the first line of defense as well, and they are destroyed locally by efferocytosis in the tissue.¹⁰¹ Neutrophils can act as antigen-presenting cells to CD4+ T cells in a major histocompatibility complex class II (HLA-DR)–dependent manner, although less efficiently compared with monocytes and dendritic cells (DCs).^95,102 LDNs, a mixture of neutrophils at various stages of maturation, are increased in number in sepsis.

Neutrophils defend against an invading pathogen by phagocytosis followed by destruction, using antimicrobial mechanisms such as oxidant damage, or secretion of either an antimicrobial peptide or NETs.⁶¹ NETosis is a form of neutrophil death when the granule protein and chromatin bind together to kill the invading bacteria.¹⁰³ Recently, NETosis was linked with venous thromboembolism and disseminated intravascular coagulation in sepsis and septic shock.^104,105 Thus, while conceptually a protective mechanism, excessive NETosis can be harmful and be a target for potential therapeutic use.^61,106 NETosis is dysregulated with acute ethanol exposure via CXCL1. In fact, recombinant CXCL1 not only rescues NETosis, but also bacterial clearance and survival in male mice with ethanol with sepsis.¹⁰⁷ Thus, harnessing the power of DAMPs such as CXCL1 to regulate NETosis may be a potential therapeutic strategy.

However, the effect of acute or chronic ethanol exposure on neutrophil subphenotypes and their respective functional impact needs further study. Furthermore, the impact of ethanol in the setting of sepsis on neutrophil subphenotypes needs further evaluation. This area of study can lead to novel and targeted therapeutic strategies, much needed for sepsis with or without AUD patients.

4. Ethanol exposure and alteration of monocytes, macrophages, and DCs

4.1. Ethanol exposure and monocytes/macrophages

Monocytes and macrophages play a pivotal role in pathogen clearance during sepsis. Monocytes are generated in the bone marrow, released into the circulation, and differentiate into either macrophages or DCs depending on the tissue microenvironment.¹⁰⁸ Circulating monocytes are short-lived compared with tissue-resident macrophages. During early sepsis, monocytes and macrophages exhibit a proinflammatory phenotype characterized by excessive secretion of cytokines and chemokines including tumor necrosis factor α (TNF-α), macrophage chemoattractant protein 1, IL-6, and IFN-γ, and enhanced phagocytosis of the invading pathogens. However, the inflammatory and phagocytic abilities of these cells are dysregulated during late sepsis with persistent inflammation and immunosuppression.^109–111

AUD impairs bacterial clearance and dampens infection-induced inflammatory responses by modulating the plasticity and function of monocytes and macrophages. Ethanol exposure also suppresses the overproduction of inflammatory mediators by the monocytes during sepsis.¹¹² The ability of ethanol-exposed immune cells in humanized mice was studied using chimeras of peripheral blood mononuclear cells transferred from healthy volunteers or people with AUD. The humanized mice then received intratracheal Klebsiella pneumoniae or oral Enterococcus faecalis infection. The mice with chimeras from AUD subjects were unable to clear either K. pneumoniae or E. faecalis infection. However, mice receiving chimeras from healthy volunteers showed significantly fewer complications from these infections, suggesting that chronic alcohol abuse dampens immune resistance to bacterial infection.¹¹² A recent report indicates that short-term abstinence after chronic alcohol drinking improves antibacterial immunity against oral E. faecalis infection–induced sepsis in mice.¹¹³

Several monocyte subtypes are described in literature. Under normal physiological conditions, majority of the monocytes in the human blood are CD14⁺/CD16⁻ (classical monocytes), with a lower percentage of the CD14⁺/CD16⁺ (intermediate) and CD14⁺/CD16⁺⁺ (nonclassical monocytes).¹¹⁴ Following infection, the number of CD14⁺/CD16⁺⁺ monocytes increases in circulation. Historically, the monocytes were thought to exclusively exhibit a proinflammatory phenotype with alcohol drinking.^115,116 However, recent evidence suggest that chronic alcohol abuse increases the nonclassical monocytes resulting in downregulation of TNF-α, IL-6, HLA-DR, and IL-1β.^117,118

Ethanol exposure modulates monocytes to change subtypes over time. During the initial phase of ethanol exposure (acute ethanol exposure), the monocytes are proinflammatory; however, the phenotype gradually turns into an anti-inflammatory during chronic alcohol drinking.²² Chronic alcohol abuse increases the population of a specific subset of these intermediate CD14⁺/CD16⁺/CD163 monocytes in circulation, commonly termed as M2b.¹¹² However, recent evidence shows that CD163⁺ monocytes become highly proinflammatory in nature during sepsis.¹¹⁹ The exact role of M2b/CD163⁺ monocytes with chronic alcohol use in dampening immunity during ethanol exposure needs further investigation. Monocytes form aggregates with platelets following infection and sepsis.¹²⁰ These aggregates are linked to the proinflammatory phenotype of the monocytes, but the mechanisms are not well understood. Evidence suggests that in healthy volunteers, while the total leukocyte and granulocyte numbers increase early (within 6 h) of drinking alcohol, the monocyte count increases after 24 h. Interestingly, even if the monocyte numbers increase, the reactive oxygen species–producing monocytes remain low in male and female drinkers; however the phagocytosis capacity decreased only in female drinkers, suggesting deranged overall leukocyte and monocyte function.¹²¹

Macrophages, the key immune cell type for pathogen clearance, live longer than monocytes.¹²² Like monocytes, macrophages show hyperinflammatory phenotype, characterized by increased cytokine production and higher phagocytic ability during the early phase of sepsis, which gradually turns into an anti-inflammatory or immune-suppressive phenotype as identified by reduction in phagocytosis and increased production of anti-inflammatory cytokines. Specifically, ethanol exposure increases the expression of CD206 and IL-10, in addition to decreased expressions of IL-1β, HLA-DR, CD80, and CD86.¹²³ In an alcohol-related hepatitis model, ethanol exposure promotes quantitative expression of Arg1, Mrc1, and IL-10 genes and frequency of CD206+ CD163+ macrophages, suggesting M2 phenotype via KLF4 expression.¹²⁴ Evidence implicates extracellular vesicles containing miR-27a from ethanol-exposed macrophages in regulating naïve macrophage polarization toward the M2 phenotype.¹²⁵ Impaired macrophage function is implicated to organ damage and mortality in sepsis. Ethanol exposure mutes proinflammatory response in macrophages.¹²⁶ Recently, our laboratory has also found that alcohol perturbs the metabolic fitness of the bone marrow–derived macrophages through downregulation of glycolysis and phagocytosis.²⁵ Ethanol exposure induces the cytosolic deacetylase sirtuin 2, which deactivates a rate-limiting glycolytic enzyme, platelet isoform of phosphofructokinase. Increased glycolysis facilitates phagocytosis of the invading pathogens via increased intracellular acidification. Ethanol-driven reduction in glycolysis and its by-product lactate reduced phagocytosis.²⁵

Extracellular ATP plays a critical role in regulating macrophage function and bacterial clearance during sepsis, via binding to its receptors commonly known as P2XRs (inotropic) or P2YRs (metabotropic). Macrophage-specific deficiency in ATP receptor P2X4Rs perturbs bacterial clearance in mice with sepsis.¹²⁷ Interestingly, alcohol plays a dual role in P2X4R signaling. On one hand, it increases the surface expression of P2XRs,^128,129 while it also blocks the activity of these receptors, impairing the downstream signaling.¹³⁰ More work is needed to dissect the role of P2XRs in macrophages during alcohol-induced sepsis.

4.2. Ethanol exposure and DCs

DCs are the major antigen-presenting cells in the body, critical for pathogen recognition and clearance. DCs, generated from hematopoietic progenitor cells in the bone marrow, are classified as plasmacytoid/circulating DC conventional DCs (cDCs), which are further subdivided into cDC1 and cDC2.^131,132 Plasmacytoid DCs are generated from stem cells in the circulation, while the classical DCs are generated from the bone marrow–derived myeloid cells.¹³³ DCs act as a linker between the innate and adaptive immunity. Several studies have shown that the numbers of DCs decrease in both humans and mice during sepsis.¹³⁴ Ayala et al.¹³⁵ have shown that the numbers of both splenic and peritoneal DCs decrease 24 h post–cecal ligation puncture–induced polymicrobial sepsis.

Monocytes and DCs also play a critical role in antigen presentation following infection. These cells engulf the invading pathogens, process them by proteolysis in the cytosol, and present them on the plasma membrane in association with HLA-DR to elicit a CD4⁺ T cell–specific immune response.¹³⁶ During infection, pathogens try to evade clearance by inhibiting the antigen presentation machinery. Recent studies have demonstrated a correlation between reduced human leukocyte antigen (HLA-DR) expression in monocytes and disease severity (Sequential Organ Failure Assessment scores) in sepsis.^137,138 In addition to suppressing the production of inflammatory mediators, alcohol consumption also leads to impairment of antigen presentation and expression of HLA-DR in monocytes and DCs. Monocyte-derived DCs, from acute ethanol-exposed mice, exhibited much lower T cell proliferation capacity compared with controls when challenged with an allo-antigen,¹³⁹ suggesting that alcohol impairs antigen presentation capacity in DCs.

Mitochondrial DNA is a potent stimulator of DCs. Costimulatory molecules such as CD40, CD80, and CD86 are the most characterized costimulatory molecules expressed in DCs. During sepsis, due to ongoing cell death, the circulating levels of mitochondrial DNA increase. Evidence suggests that bone marrow–derived DCs engulf these circulating mitochondrial DNA, which subsequently inhibit the expression of CD40 and CD86 via increased production of IL-10 and activation of STING pathway leading to immunoparalysis in sepsis.¹⁴⁰

Making use of both in vivo and in vitro models, several groups have shown that acute and chronic ethanol exposure not only modulates the monocyte differentiation to DCs,¹⁴¹ but also skews the ability of these cells to produce inflammatory mediators including TNF-α, IL-6, and IL-12 in response to septic insults.^134,142 A combined insult of PAMPs (LPS) and DAMPs (poly DA/DT) induces the AIM2 inflammasome in bone marrow–derived DCs, as indicated by increased production of IL-1β and ASC proteins, which is inhibited by alcohol pre-exposure.⁸⁸ Recent studies have shown that alcohol exposure reduces the expression of CD40 and CD86 in resting bone marrow–derived DCs.¹⁴² Collectively, these results suggest that a defective antigen presentation machinery in DCs and monocytes could be one of the decisive factors for making AUD patients more vulnerable to sepsis.

5. Ethanol exposure and innate lymphoid cells

Innate lymphoid cells (ILCs) are innate immune cells play a crucial role during pathogen invasion during sepsis. Ethanol is known to perturb ILC function. The first described prototype are natural killer (NK) cells, and subsequently described lymphoid tissue inducer (LTi) cells have differential functions but are developmentally related.¹⁴³ Other ILC phenotypes such as NK22 cells, natural cytotoxicity receptor 22 cells or NK receptor positive (NKR+) LTi cells have been previously described. The NK cells secrete IFN-γ whereas the LTi and NKR + LTi-like cells produce IL-17 and/or IL-22.¹⁴³ IL-17 is crucial for immune response to infection, and IL-17–deficient mice show increased mortality due to decreased neutrophil recruitment to the site of infection.^144,145 While IL-22 is not very well studied in sepsis, a recent publication suggested increased IL-22 and IL-17 levels to be significant contributors to sepsis-induced acute lung injury.¹⁴⁶ In contrast, treatment with IL-22 was also shown to attenuate sepsis-induced hepatic function via JAK/STAT3 signaling pathway and reduction of hepatocyte apoptosis.¹⁴⁷ Thus, the role of IL-22 in sepsis remains controversial.

The 2 populations of NK cells are based mainly on CD56 and CD16 expressions. CD56_lo–CD16+ cells are known for pathogen-killing capacity, while CD56_hi–CD16– cells secrete large amounts of proinflammatory cytokines.¹⁴⁸ NK cells kill pathogens using 2 strategies: (1) the cytotoxic granule pathway in which perforin, a membrane-disrupting protein, and granzymes, the serine proteases secreted by exocytosis, activate apoptosis of target cells via caspase-dependent and -independent pathways^149,150; and (2) the death ligand pathway in which the cell death ligand such as FasL engages with death receptors such as Fas/CD95 to induce cell death using classical caspase-induced apoptosis.¹⁵⁰

NK cell activation is a double-edged weapon. The debate whether NK cell immune responses are friends or foe of the host is ongoing.^149,151,152 In sepsis, NK cells, like other innate immune cells, respond to PAMPs by expression of TLRs.¹⁵³ The signal transduction follows post-TLR stimulation. The cytokine secretion with other coexisting immune cells acts as potent stimulus for the activation of NK cells at the site of infection. Moreover, IL-12 is a potent IFN-γ inducer post-LPS challenge costimulated by IL-15 and IL-18. IL-10 inhibits IFN-γ production from NK cells.^149,154 The IFN-γ stimulus further feeds into the positive feedback loop of more NK cell activation and increased proinflammatory cytokine secretion, leading to tissue damage.^149,154 Several studies have established a role of NK cells in the pathogenesis of septic shock.^155–157 The roles of IL-12, IL-15, and IFN-γ in NK cell–induced systemic inflammation are well established.^158–162

The protective role of NK cells against pathogen invasion is also evident. NK cell depletion not only leads to defective defense against invading pathogen, but also is associated with an immunosuppressive phenotype.^163–166 A significant number of studies observed decreased NK cell numbers and function during early sepsis,^167,168 while others concluded detrimental role of NK cells in sepsis outcomes.¹⁶⁹ Thus, a context-dependent role of NK cells during sepsis remains controversial.

Ethanol exposure is known to influence NK cell activity. The suppressive effect of chronic ethanol exposure on basal NK cell function nearly 3 decades ago.¹⁷⁰ The role of ethanol in vitro vs in vivo suggested that the ethanol affects NK cell numbers rather than the activity.¹⁷¹ The role of beta-endorphin in ethanol-induced suppression of NK cells was reported subsequently.^172,173 Interestingly, chronic ethanol exposure suppresses basal activity of NK cells by disrupting granzyme B, perforin, and IFNγ.¹⁷⁴ The suppressive role of ethanol exposure on NK cell activation in response to poly I:C or IL-2 was shown to be due to decreased production and response to IFN-α in mice, at least partly via induction of endogenous corticosterone.^175,176 Low levels of ethanol exposure differentially affect immune cell activity via STAT5. Specifically, low levels of ethanol decrease IL-2–induced activation of NK cells via STAT5, while it increases STAT5 activation in T cells.¹⁷⁷

The antifibrotic function of NK cells in fibrotic liver diseases is shown to be inhibited by chronic ethanol consumption by the Gao group. Specifically, they showed that the chronic ethanol exposure abrogates antifibrotic effects of NK cells via downregulation of cytotoxic molecules secreted by NK cells, such as perforin; increased TGF-β secretion by hepatic stellate cells, which decreases NK cell killing activity; increased suppressor of cytokine 1 expression; and increased oxidative stress by hepatocytes and thereby inhibition of IFN-γ signaling.^178,179 More recently, NK T 10 cells were implicated in alcoholic steatohepatitis with chronic ethanol consumption.¹⁸⁰

Thus, evidence supports that the NK cell activity is perturbed in sepsis and septic shock, although whether NK cells are friend or foe in sepsis is an ongoing debate. Ethanol is known to suppress NK cell activity, as outlined previously and by others.¹⁷⁹ The effect of either acute or chronic ethanol exposure on NK cells during acute stress of sepsis is not well understood and needs further investigation.

5.1. Ethanol exposure and innate cell interactions

We describe the role of individual innate immune cell types on sepsis outcomes and the effect of ethanol exposure on each cell type individually in the previous sections. Intercellular communication and interactions are important in face of pathogen invasion, and ethanol is known to affect those as well.^26,141,181 For example, as the first responders, neutrophils and monocytes undergo adhesion and extravasation to gain access to subendothelial compartment. In addition to phagocytosis and NETosis, the neutrophils secrete proinflammatory cytokines, chemokines, other DAMPs as described previously to signal and attract innate immune cells such as additional neutrophils/monocytes/DCs etc. Monocytes differentiate into macrophages for effective phagocytosis and pathogen clearance. Circulating DCs, similarly, are attracted to the DAMPs in the interstitial space with an intention to kill pathogen. Acute ethanol exposure dampens while the chronic ethanol exposure increases the proinflammatory milieu, in general as previously described in individual cell types. Acute and chronic ethanol exposures are known to dampen the innate immune response and hence intercellular communications as well via various mechanisms, as summarized in Fig. 1.

Fig. 1.

Effect of ethanol exposure on innate immune cell interactions. Ethanol exposure affects interactions between different innate immune cell types in addition to affecting individual cell types. In general, acute ethanol dampens while chronic ethanol exposure increases the proinflammatory and pro-oxidant milieu.

In summary, a poised innate immune response is critical for pathogen invasion in sepsis. Every aspect of innate immunity, including different innate immune cell types and their function, are essential for early response to infection. Ethanol exposure dysregulates innate immune response. Unsurprisingly, the effect of ethanol exposure on innate immune response in sepsis is perturbed, leading to increased morbidity and mortality in patients with sepsis.⁶ Extensive research shows the effect of ethanol exposure on innate immune cells during sepsis. Several genetic, epigenetic, and metabolic mechanistic targets continue to be discovered in experimental sepsis with ethanol exposure. However, the targeted treatment strategies are elusive. Specific molecular targets in immune response to ethanol with sepsis need further investigation.

Funding

This work was supported by National Institutes of Health grants R01028763 (V.V.) and R01HL155064 (R.S.).