The immunology of sepsis

Sepsis has been described as the quintessential medical disorder of our time because it is not only a leading cause of morbidity and mortality in hospitalized patients but often also the direct result of the improvements in the medical care for patients with various disorders for which, until recently, no treatments were available (). Over the course of thousands of years, the meaning of the term sepsis has evolved. Historical definitions from the time of Hippocrates onward are depicted in Figure 1. Now, sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection (). Septic shock is a subset of sepsis with profound circulatory, cellular, and metabolic abnormalities associated with a greater risk of mortality than with sepsis alone (). In the current sepsis definition, the terms dysregulated and host response are not explicitly defined but conceptualized as maladapted responses within the immune and non-immune systems that are in the causal pathway to organ dysfunction and death.

A recent Global Burden of Diseases report highlights that sepsis is common, with nearly 50 million cases globally per year (). Sepsis affects all ages. While the site of infection and causative pathogen in sepsis vary by geographic location and age, bacterial infections of the respiratory system and gastrointestinal systems are most common. Sepsis had an estimated mortality of 11 million in 2017, which equates to an age-standardized mortality of 148 per 100,000 population, representing nearly 20% of all deaths globally (). When patients with sepsis require admission to critical care units, one-in-three patients do not survive 30 days (; ) and mortality varies by the age, comorbid status, number, and type of organ dysfunctions (; ; ). Further, patients who survive sepsis have a longer-term risk of rehospitalization and death (; ). Nearly 50% of sepsis survivors are re-hospitalized at least once within a year, and one-in-six sepsis survivors do not survive the first year (; ; ).

Despite more than three decades of research and more than 200 randomized controlled trials, we do not have a single treatment that consistently saves lives in sepsis patients (). Treatment of sepsis remains largely supportive with simple measures such as source control, timely antibiotics, resuscitation, and supportive care for organ dysfunction (). In a hope for more successful clinical trials and better outcomes, the critical care community now considers the value of subgrouping sepsis patients either on measurable characteristics that inform treatment response (predictive enrichment) or outcome (prognostic enrichment) or to identify two or more homogeneous subgroups with common clinical and laboratory features (subphenotypes) or specific pathobiological abnormalities that could be targeted (endotypes) (; ).

Here, we review current knowledge of immune dysregulations in sepsis. We describe mechanisms that contribute to (a return to) homeostasis during and after bacterial encounters, with subsequent attention for immune imbalance characterized by concurrent proinflammatory and immune suppressive aberrations. Our review focuses on the immune alterations in sepsis of bacterial origin. We note that sepsis could occur from viral, fungal, or parasitic infections and that non-immune alterations form part of the dysregulated host responses concept in sepsis. At present, sepsis remains an ill-defined syndrome, and we argue that successful implementation of additional technologies and bedside computational support may enable real-time immunological profiling of individual patients and inform patient selection for clinical trials of targeted therapies.

The immune system is equipped with a range of cell membrane associated and intracellular pattern recognition receptors (PRRs) that can detect pathogens through their ability to recognize pathogen-associated molecular patterns (PAMPs), conserved motifs expressed by microbes (; ). Main PRR classes include Toll-like receptors (TLRs), nucleotide-binding oligomerization domain and leucine-rich repeat containing gene family (NLRs), retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), C-type lectin receptors (CLRs), and DNA-sensing molecules (). Induction of innate immunity further occurs through inflammasomes—cytosolic multiprotein oligomers that, upon activation and assembly, promote a number of downstream events, including caspase-1 mediated cleavage of the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18 (). A balanced immune reaction to a pathogen typically is localized and characterized by inflammatory, anti-inflammatory, and repair responses, with elimination of microorganisms and return to normal homeostasis (). In such a protective immune response, different cell types become activated through an interaction between PAMPs and PRRs, triggering intracellular signal transduction pathways and activation of key transcription factors such as nuclear factor κB (NF-κB) and activator protein (AP)-1, which coordinate inflammatory responses. Soluble components of expected responses to infection include proinflammatory cytokines, chemokines, proteins released from activated neutrophils and platelets, complement products, and coagulation factors (). The vascular endothelium supports a protective immune response by increasing its expression of adhesion molecules and widening its gap junctions, enabling the adherence of immune cells and their migration to sites of infection. Moreover, the adaptive immune system is triggered by presenting antigen via dendritic and other cells to B and T lymphocytes, resulting in the production of pathogen-specific antibodies and memory for subsequent infection by the same pathogen ().

The trillions of commensal bacteria that colonize the gut also play an important role in homeostasis and host defense against invasion by pathogens. A healthy microbiota provides resistance against colonization and invasion by harmful microorganisms by utilizing both direct and indirect mechanisms (; ). The microbiota directly competes for nutrients, maintains epithelial barrier function, and produces antibacterial peptides. The microbiota can also regulate the production of antimicrobial proteins by host cells. An example is the regeneration of islet-derived protein IIIγ (REGIIIγ), which is produced by Paneth cells upon TLR-mediated stimulation of epithelial cells and dendritic cells by commensal-microbe-associated molecular patterns such as lipopolysaccharide (LPS) and flagellin (; ). Among the pleiotropic effects of short-chain fatty acids (SCFAs), which are the main metabolites produced by the microbiota, is their ability to drive monocyte-to-macrophage differentiation and inhibition of histone deacetylase 3. Through this inhibition, butyrate mediates macrophage metabolism and further induces the production of antimicrobial peptides to enhance antimicrobial activity in murine models of infection (). Other gut bacteria interact with gut epithelial cells in order to enhance IgA production by B cells and induce T helper 17 (Th17) cell differentiation.

Disruption of the gut microbiome can lead to a transition from homeostasis to disease. Large observational patient studies have provided indirect evidence that gut microbiome disruption predisposes to sepsis (; ; ). An altered gut microbiota can increase gut barrier permeability and translocation of pathobionts toward distant organs (; ; ). Disruption of the intestinal microbiota can additionally impact distant organs such as the bone marrow and the lung, leading to diminished effectiveness of host defense against infection. By using an Escherichia (E.) coli sepsis model in neonatal mice, Deshmukh and colleagues have shown that the microbiota regulates neutrophil homeostasis (). Commensal Gammaproteobacteria, Gram-negative bacteria, which express cell-surface LPS, activate IL-17A production by innate lymphoid cells via a TLR4-induced signaling cascade, which triggers an increase in plasma granulocyte colony-stimulating factor. This could subsequently stimulate neutrophil recruitment from the bone marrow into the bloodstream in order to combat invading pathogens such as E. coli (). Gut-derived SCFAs can influence the immunological environment in the lung (; ). Mice in which the gut microbiota is disrupted by antibiotics show increased bacterial dissemination, inflammation, and mortality during pulmonary infection with Streptococcus (S.) pneumoniae, Klebsiella pneumoniae, Burkholderia pseudomallei, or Mycobacterium tuberculosis when compared with controls (; ; ; ). Moreover, alveolar macrophages derived from gut microbiota-depleted mice show upregulation of metabolic pathways and altered cellular responses, leading to a diminished capacity to phagocytose S. pneumoniae, hence resulting in a less pronounced immunomodulatory response (). Dysbiosis has been associated with poor outcome in patients with severe infections (; ; ). The gut microbiome of the septic patient is characterized by a decrease in diversity, a lower relative abundance of Firmicutes and Bacteroidetes with decreased numbers of commensals such as Faecalibacterium, Blautia, and Ruminococcus spp., and an overgrowth of opportunistic pathogens such as Enterobacter, Enterococcus, and Staphylococcus (; ; ). Most recent data have not only shown the disruptive impact of the septic response, including the effects of treatment, on the composition of the bacterial microbiome, but also on the other kingdoms, such as viruses, fungi, and protozoa (). The impact of these altered gut kingdoms on immune pathways and the host defense against invading pathogens remains undefined.

Sepsis is regarded as an unbalanced immune response, wherein pathogens have evaded protective defense mechanisms and continue to multiply, resulting in persistent stimulation of host cells and injury, and associated with a failure to return to homeostasis (). In this unbalanced response, many of the immune mechanisms initially activated to provide protection have become detrimental, both related to excessive inflammation and immune suppression. Longitudinal analyses of immune reactions from early pathogen-host interactions to clinically manifested sepsis in humans are lacking, making a time-dependent reconstruction of sequential proinflammatory and immune-suppressive reactions during the pathophysiological path toward the “septic host response” speculative. Once in the hospital, the host response in patients with sepsis shows signs of concurrent hyperinflammation and immune suppression, involving partially different cell types and organ systems () (Figure 2). The mechanistic underpinnings of the concurrent hyperinflammation and immune suppression and the persistent longitudinal immune system changes in critically ill sepsis patients have yet to be clarified. Persistent immune stimulation in sepsis is not only caused by invading pathogens but also by the release of “damage-associated molecular patterns” (DAMPs, or alarmins), endogenous molecules liberated from injured cells. DAMPs activate PRRs that oftentimes also sense PAMPs, initiating a vicious cycle with sustained immune activation and dysfunction (; ). Patients who remain dependent on intensive care after primary therapeutic measures frequently develop a chronic critical illness termed “persistent inflammation, immunosuppression, and catabolism syndrome” or PICS, which involve many different cell types, organ systems, and pathophysiological mechanisms, with prolonged hyperinflammation, immune suppression, dysregulated myelopoiesis, and catabolism represented by muscle wasting and cachexia as main features (; ).

The “dysregulated host response to an infection” captured in the present Sepsis 3.0 definition () relates to concurrent unbalanced hyperinflammation and immune suppression. Among the many different cell types and mediator networks implicated in sepsis-associated excessive inflammation, leukocytes (neutrophils, macrophages, natural killer cells), endothelial cells, cytokines, complement products, and the coagulation system are prominently featured (Figure 3). Early preclinical studies have introduced the term “cytokine storm” to indicate the strong systemic release of proinflammatory cytokines in experimental animals exposed to intravenous challenges of viable bacteria or their products; these animal models have demonstrated that elimination of proinflammatory cytokines such as tumor necrosis factor (TNF), IL-1β, IL-12, and IL-18 provides strong protection against organ damage and mortality (). While there is consensus now that acute systemic challenge models have little relevance for human sepsis, uncontrolled activity of proinflammatory cytokines still is considered to contribute to injury in sepsis.

Neutrophils can contribute to hyperinflammation in sepsis through the release of proteases and reactive oxygen species. Neutrophils can release neutrophil extracellular traps (NETs) composed of a network of chromatin fibers containing antimicrobial peptides and proteases including myeloperoxidase, elastase, and cathepsin G (). NETs contribute to antibacterial defense mechanisms by trapping and subsequently killing bacteria (), and inhibition of NET formation by DNase leads to an increased bacterial burden in blood and a decreased survival of mice with abdominal sepsis (). However, like many components of innate immunity, NETs act as double-edged swords in infection. Excessive NETosis during sepsis can be detrimental through various mechanisms, including induction of intravascular thrombosis and multiple organ failure (). Histones are abundant in NETs, and NETs can adhere and activate the endothelium during sepsis, resulting in vascular damage in a histone-dependent manner (). NETs and histones can directly damage endothelial and epithelial cells (), and cell-free histones mediate lethality induced by high dose LPS or TNF, as well as cecal ligation and puncture (CLP)-induced sepsis in mouse models (). Macrophages can also produce extracellular traps; their potential role in sepsis has not been studied in detail ().

Close links exist between the coagulation and complement systems, which can be viewed upon as two evolutionary cascades originating from shared ancestral pathways. Complement activation results in the release of the anaphylatoxins C3a and C5a, which exert potent proinflammatory activities, including recruitment and activation of leukocytes, endothelial cells, and platelets (). While complement activation is a vital component of protective innate immunity, uncontrolled activation can injure tissues and cause organ failure (). Activation of the coagulation system can be considered part of the innate immune response to an invading pathogen and the term “immunothrombosis” was introduced to support this concept (). In agreement, elements of the coagulation system can trigger important innate defense mechanisms and inhibition of coagulation has been shown to impair antibacterial defense in several infection models (). In sepsis, activation of the coagulation system becomes unbalanced, resulting in a tendency toward thrombosis in the microvasculature (). The most severe manifestation of sepsis-associated coagulopathy is disseminated intravascular coagulation (DIC), which apart from thrombosis, can be associated with bleeding due to consumption of clotting factors, anticoagulant proteins, and platelets (). Tissue factor is the primary initiator of blood coagulation by forming a complex with clotting factor (F)VIIa, thereby inciting blood coagulation by activating FX and FIX (). Tissue factor is constitutively expressed by perivascular cells, such as fibroblasts, pericytes, and epithelial cells, which is important for hemostasis and vascular integrity. Tissue factor is induced on endothelial cells, monocytes, and macrophages by microbial agents and a variety of inflammatory mediators, including cytokines and complement factors (; ). Under pathological conditions, high quantities of bioactive tissue factor are present in microvesicles derived from several cellular sources, which can bind to other cells, such as activated platelets, neutrophils, and endothelial cells, thereby amplifying coagulation. Early studies have documented the crucial role of tissue factor-FVIIa in models with relevance for sepsis: inhibition of this pathway in humans and non-human primates strongly diminishes coagulation activation after infusion of LPS or bacteria respectively (). The prothrombotic state in sepsis is aggravated by concomitantly compromised activity of three main anticoagulant pathways, i.e., antithrombin, tissue factor pathway inhibitor (TFPI), and the protein C system (). Herein, inflammation-driven disruption of the glycocalyx, a glycoprotein-polysaccharide layer covering the endothelium and essential for maintaining its physiological anticoagulant properties, plays a key role, together with decreased expression of thrombomodulin, an endothelial cell receptor that catalyzes the production of the natural anticoagulant activated protein C after forming a complex with thrombin. Platelets further contribute to coagulation and inflammation, both directly through cell-to-cell contact (e.g., complex formation with leukocytes) and indirectly through the release of proteases and other mediators (). Platelets increase endothelial cell adhesion and leukocyte extravasation at sites of inflammation and enhance activation of neutrophils (). Platelets and platelet-derived microparticles express phospholipids (including phosphatidylserine), which increase the activity of tissue factor, FVa and FXa, thereby facilitating coagulation ().

Bimodal interactions exist between coagulation and innate immunity. Neutrophil elastase and cathepsin G, together with externalized nucleosomes, stimulate coagulation and intravascular thrombus growth in vivo by mechanisms that involve enhanced tissue factor and FXII-dependent coagulation and local proteolysis of the coagulation inhibitor TFPI (). Platelets contribute to hyperinflammation and thrombosis in sepsis by mechanisms that partially involve neutrophils. Activated platelets can attract leukocytes to sites of infection, form complexes with neutrophils, reduce their threshold to release NETs, and enhance their ability to kill pathogens (; ). NETs in turn promote platelet adhesion, activation, and aggregation (). In mouse models of sepsis platelet aggregation, thrombin activation and fibrin clot formation occur within NETs in vivo, which is crucial for the development of sepsis-induced intravascular coagulation (). Intravascular coagulation induced by NETs is dependent on a cooperative interaction between histone H4 in NETs, platelets, and the release of inorganic polyphosphate (). The extent of NET formation is predictive of the development of DIC and mortality in patients with sepsis, further pointing at a role for NETs in sepsis associated coagulopathy ().

Tissue factor and the clotting factors FVIIa, FXa, thrombin, and fibrin can induce proinflammatory signaling, particularly by activation of members of the G-protein-coupled protease-activated receptor family (). Complement factors can activate coagulation proteases and vice versa. For example, FIXa, FXa, FXIa, and thrombin can convert C3 and C5 into C3a and C5a, respectively, whereas C5a and the membrane attack complex (C5b-9) can stimulate expression of tissue factor on endothelial cells (). C5a can facilitate clotting further by disturbing the endothelial glycocalyx function. Recent studies have implicated inflammasomes and gasdermin D (GSDMD) in the interaction between inflammation and coagulation. Triggering of inflammasomes results in the activation of inflammatory caspases (). Among these, caspase-1 can be activated by the bacterial products flagellin and type III secretion system (T3SS) proteins, while caspase-11 can be triggered by cytoplasmatic LPS (; ; ). Both caspase-1 and caspase-11 cleave GSDMD, which can form pores in plasma membranes, thereby facilitating IL-1β release, IL-18 release, and pyroptosis (; ; ). Recent research has implicated GSDMD-mediated pyroptosis of monocytes and macrophages, mediated by caspase-1 or caspase-11, in activation of the coagulation system by permitting the release of tissue factor containing microvesicles from pyroptotic cells (). Tissue-factor-mediated DIC elicited by intravenous injection of the E. coli T3SS inner rod protein EprJ is abolished in Casp1^−/− and Gsdmd^−/− mice; likewise, infection with viable E. coli and administration of rod proteins from other bacterial strains (Burkholderia BsaK and Salmonella PrgJ) trigger severe coagulation activation in wild-type but not Gsdmd^−/− mice (). Genetic ablation of the IL-1β or IL-18 receptor does not affect EprJ-induced coagulation, confirming the role of pyroptosis herein (). Pyroptosis also was crucial for coagulation activation induced by LPS, as demonstrated by systemic coagulopathy in LPS-challenged wild-type mice primed with poly I:C, but not in Casp11^−/− or Gsdmd^−/− mice (; ). Similar observations have been made after infection with E. coli or during polymicrobial sepsis induced by CLP (). GSDMD enhanced tissue factor activity by inducing calcium release and subsequent phosphatidylserine exposure on leukocytes (). Of note, some data suggest that GSDMD-mediated increased phosphatidylserine exposure is sufficient for tissue factor release, not requiring pyroptosis (). Caspases other than caspase-1 and caspase-11 can also contribute to coagulation activation through GSDMD cleavage. Transmembrane protein 173 (TMEM173) is an endoplasmic protein that acts as an amplifier of inflammation (). Recently, myeloid cell TMEM173 (STING) has been shown to drive tissue factor release and blood coagulation via GSDMD activation in experimental sepsis in mice induced by CLP, E. coli, or S. pneumoniae (). Caspases involved in TMEM173-mediated GSDMD cleavage depend on the inciting pathogen: caspase-11 for E. coli and caspase-8 for S. pneumoniae (). An additional mechanism of coagulation activation in sepsis may be mediated by high mobility group box 1 (HMGB1). This nuclear protein acts as a DAMP once released into the extracellular environment and inhibition of HMGB1 exerts protective effects in preclinical sepsis models (; ). Exogenous HMGB1 can form molecular complexes with LPS that can be transported into the lysosomes of macrophages and endothelial cells via the receptor for advanced glycosylation end-products (RAGE), resulting in HMGB1-mediated destabilization of lysosomal membranes with subsequent release of LPS into the cytosol and activation of caspase 11-dependent pyroptosis and NLRP3 inflammasome activation and lethal coagulation activation (). HMGB1-mediated GSDMD activation and pore formation is necessary to activate tissue factor through phosphatidylserine exposure, rather than via GSDMD-mediated pyroptosis (). Together, these data place caspase-mediated GSDMD cleavage, resulting in tissue factor release, at the center stage of DIC and expose inflammasome activation as an important link between inflammation and coagulation.

Immune suppression in patients with sepsis involves different cell types and characteristics and is related to enhanced apoptosis of immune cells, T cell exhaustion, reprogramming of cells through epigenetic changes, and reduced expression of activating cell surface molecules (; ). Immune suppressive changes have been implicated in the increased susceptibility of sepsis patients to secondary infections, often caused by opportunistic pathogens, and viral reactivation (; ). Apoptosis especially occurs in CD4⁺ T cells, CD8⁺ T cells, B cells, natural killer (NK) cells, and follicular dendritic cells, and both death receptor- and mitochondrial-mediated pathways are involved (; ; ; ). Sepsis-associated B cell depletion is related to enhanced apoptosis or deficient helper T cell support and occurs post-activation, affects the memory B cell subsets the most (), and remaining B lymphocytes have an exhausted phenotype characterized by decreased major histocompatibility complex class II (MHC class II) expression and increased production of the anti-inflammatory cytokine IL-10 (). Interventions that inhibit or prevent apoptosis confer protection in several preclinical sepsis models, signifying the functional importance of enhanced apoptosis (). Impaired autophagy, a process that removes redundant or dysfunctional cellular components, may also contribute to immunosuppression, i.e., mice with a reduced autophagy capacity in lymphocytes by cell-specific loss of Atg5 or Atg7 show an increased mortality together with immune dysfunction in abdominal sepsis, and Atg5 deficiency in T cells resulted in higher release of IL-10 by these cells during sepsis ().

T cells from blood, as well as spleen and lung, from sepsis patients have a reduced capacity to produce cytokines (; ; ). CD8⁺ T cells show attenuated cell proliferation, impaired cytotoxic function, and attenuated IL-2 and interferon (IFN)-γ production (; ). The anti-inflammatory milieu in sepsis is further shaped by increased numbers of regulatory T (Treg) cells (; ) and myeloid-derived suppressor cells (MDSCs), which represent a mixed population of predominantly immature myeloid cells that suppress effector immune cells, particularly T cells (). MDSCs can impede immune functions through a number of mechanisms, including deprivation of L-arginine (essential for T cell functions), stimulation of Treg cell expansion and inhibition of macrophage and dendritic cell functions (). Expansion of MDSCs is associated with an increased risk of secondary infections in critically ill patients with sepsis (; ). Neutrophils show several immune compromised features in sepsis, including migration to a variety of chemoattractants, decreased intracellular myeloperoxidase and lactoferrin content, and reduced oxidative burst capacity (). Kinome profiling of neutrophils have uncovered impaired kinase activity in neutrophils from sepsis patients when compared with critically ill patients without infection, further suggesting an immune-suppressed neutrophil phenotype ().

Recent studies have implicated checkpoint regulators in sepsis-induced immune suppression. Checkpoint regulators are membrane-bound proteins, which function as a second signal to direct the immune response (either inhibitory or stimulatory) to a specific antigen (). A checkpoint regulator extensively studied in the field of sepsis is programmed cell death-1 (PD-1). Triggering of PD-1 on T cells results in release of immunosuppressive molecules and may culminate in apoptosis. Enhanced peripheral blood T cell expression of PD-1 was associated with attenuated T cell proliferative capacity, increased incidence of nosocomial infections, and increased mortality in patients with sepsis (). In addition, sepsis patients show enhanced expression of PD-1 on blood monocytes and granulocytes (), and a postmortem study reports increased PD-1 expression on T cells and increased PD-L1 and PD-L2 expression on dendritic cells (). The expression of PD-1 on T cells and PD-L1 on antigen presenting cells show associations with lymphopenia, T cell apoptosis, and mortality in patients with sepsis (; ; ). The functional relevance of these observational reports is supported by the finding of reduced apoptosis and enhanced IFN-γ production upon ex vivo treatment of CD8⁺ T cells from sepsis patients with an anti-PD-1 antibody (). Moreover, increased PD-L1 expression on neutrophils and monocytes correlates with an impaired phagocytic capacity of these cells, and ex vivo treatment with an anti-PD-1 antibody improved the phagocytic capacity of blood leukocytes harvested from sepsis patients (). The pathobiological significance of the PD-1 pathway is further supported by reports of improved survival of septic mice with blocked or genetically eliminated PD-1 (; ). These results have raised the hypothesis that PD-1 and/or PD-L1 pathway inhibitors may reverse sepsis-induced immune suppression and several phase I and phase II clinical trials with an anti-PD-L1 antibody have been done in sepsis patients; anti-PD-L1 treatment has been well tolerated (; ; ) and induced an increase in absolute lymphocyte counts and monocyte human leukocyte antigen-DR isotype (HLA-DR) expression (). Although abundant literature hints at possible roles of other checkpoint regulators in immune suppression, studies directly addressing their role in sepsis are scarce (). Among these cytotoxic T lymphocyte antigen (CTLA)-4 is a negative checkpoint regulator expressed on T cells that binds B cell activation antigens B7-1 (CD80) and B7-2 (CD86). CD4⁺ T cells, CD8⁺ T cells, and Treg cells show increased CTLA-4 expression after induction of abdominal sepsis in mice, and treatment with an anti-CTLA-4 antibody decreases sepsis-induced apoptosis in the spleen and improved survival (). Anti-CTLA-4 treatment also reduces mortality caused by fungal sepsis following bacterial sepsis from sub-lethal cecal ligation and puncture in mice, which is accompanied by increased IFN-γ production by splenocytes ().

A key feature of immune suppression is a reprogramming of monocytes and macrophages, with an impaired capacity to produce proinflammatory cytokines upon ex vivo stimulation with bacterial agonists (also referred to as “LPS tolerance”) and a reduced surface expression of MHC class II (; ; ). Blood leukocytes of septic patients have a diminished ability to release proinflammatory cytokines such as TNF, IL-1α, IL-6, and IL-12 following stimulation ex vivo, whereas their ability to release anti-inflammatory mediators such as IL-1 receptor antagonist and IL-10 is either unimpaired or enhanced (; ; ). Reduced proinflammatory responses upon restimulation of blood leukocytes may relate to impairments in cellular NF-κB phosphorylation capacity, as indicated by intracellular flow cytometry analyses of ex vivo stimulated monocytes, CD4⁺ T cells, CD8⁺ T cells, B cells, and neutrophils from patients with sepsis (). The anti-inflammatory phenotype observed in blood leukocytes in a LPS-tolerant state has also been demonstrated in organ-specific monocytes, including in the lungs in animal peritonitis and post-mortem septic patients (; ; ). Cross-tolerance refers to the finding that stimulation with one bacterial agonist can cause a reduced capacity to produce proinflammatory cytokines by multiple other agonists. For example, blood leukocytes obtained from human subjects injected with LPS in vivo displayed reduced ex vivo cytokine production in response to TLR2, TLR4, TLR5, and TLR7 ligands and to whole bacteria (). DAMPs also induce a cross-tolerization with LPS, indicating multiple mechanisms that can induce reprogramming of mononuclear cells in sepsis (; ). Of note, mouse studies have suggested that some cell types do not show this “tolerant” phenotype and even become primed, including alveolar macrophages, Kupffer cells, microglial cells, and lymphocytes in the intestinal epithelium and skin (). A characteristic feature of sepsis is diminished expression of HLA-DR on blood monocytes, which is a well-established surrogate marker for sepsis-induced immunosuppression that correlates with impaired outcomes such as a higher incidence of nosocomial infections and increased mortality (; ).

Epigenetic regulation of gene expression—in particular, through histone modifications and DNA methylation—contributes to the reprogramming of immune cells in sepsis (). Gene transcription is regulated by organization of gene loci on chromatin into transcriptionally active or silent states (). Histones shape DNA in a chromatin structure, whereby particular histone modifications can wind or unwind chromatin to make it inaccessible (heterochromatin) or accessible (euchromatin) for transcription, respectively. Histone acetylation of lysines typically endorses transcription, while methylation can support either active euchromatin or silent heterochromatin formation, depending on the lysine that is methylated (). Among different histone modifications, methylation of histone 3 lysine-4 (H3K4) and histone 3 lysine-27 (H3K27) is highly correlated with activation and repression of transcription, respectively. In a pivotal study, downregulation of marks of open chromatin, such as histone 3 lysine-4 trimethylation (H3K4me3), has been shown to underlie LPS-induced tolerance in monocytes (). LPS-tolerant macrophages display increased amounts of the repressive histone modification H3K9me2 at the promoter regions of the genes encoding IL-1β and TNF (; ). Similar results have been obtained in LPS-tolerant blood monocytes from septic patients (). Molecular mechanisms underlying LPS effects on epigenetic regulation of gene transcription include increased expression of the histone demethylase KDM6B (JMJD3) through NF-κB activation () and accumulation of the histone deacetylase sirtuin-1 at the promoters of TNF and IL1B, resulting in inhibition of gene transcription (). Single-cell transcriptomics of peripheral blood mononuclear cells and dendritic cells from sepsis patients have disclosed an expansion of a unique CD14⁺ monocyte population, named MS1, which (relative to other CD14⁺ monocytes) displayed an immunosuppressive phenotype, i.e., reduced MHC class II expression and a reduced capacity to activate NF-κB and produce TNF upon ex vivo stimulation with LPS (). This study indicates that single-cell analyses might be useful to gain insight into distinct cellular phenotypes (proinflammatory versus immune suppressive) in sepsis ().

Infection-induced epigenetic changes are long-lasting. Mice recovered from abdominal sepsis show a sustained depression of dendritic-cell-derived IL-12 that lasted for at least 6 weeks, caused by histone modifications affecting transcription of the genes encoding IL-12p35 and IL-12p40 (). Moreover, pneumonia in mice causes long-lasting epigenetic changes that tolerized macrophages and decreased their capacity to phagocytose antigen-nonspecific, unrelated bacteria (). Recent mouse studies documented that sepsis induces epigenetic alterations in cells in the bone marrow (). Bone-marrow-derived macrophages obtained from mice recovered from abdominal sepsis show decreased expression of mixed-lineage leukemia 1 (Mll1), an epigenetic enzyme, and impairs H3K4me3 (activation mark) at NF-κB-binding sites on inflammatory gene promoters (). Bone marrow transfer experiments have demonstrated that epigenetic modifications initiated in bone marrow progenitor stem cells following sepsis results in long-lasting impairment in peripheral macrophage function (). As such, these results suggest that immune-suppressive changes persist in subsequent generations of leukocytes, which may have long-term consequences related to morbidity and mortality.

The central nervous system (CNS) participates in the regulation of the immune response (). The CNS can modify peripheral immune functions through the neuro-immune inflammatory reflex, which comprises sensory input from peripheral tissues via the afferent vagus nerve to the CNS and stimulation of the efferent vagus nerve resulting in acetylcholine mediated inhibition of TNF production (; ). The contribution of disturbed neuro-immune interactions in sepsis-induced immune suppression remains to be established.