Mechanisms of sepsis‐induced immunosuppression and immunological modification therapies for sepsis

Abstract Surgical injury can be a life‐threatening complication, not only due to the injury itself, but also due to immune responses to the injury and subsequent development of infections, which readily result in sepsis. Sepsis remains the leading cause of death in most intensive care units. Unfavorable outcomes of several high‐profile trials in the treatment of sepsis have led researchers to state that sepsis studies need a new direction. The immune response that occurs during sepsis is characterized by a cytokine‐mediated hyper‐inflammatory phase, which most patients survive, and a subsequent immunosuppressive phase. Therefore, therapies that improve host immunity might increase the survival of patients with sepsis. Many mechanisms are responsible for sepsis‐induced immunosuppression, including apoptosis of immune cells, increased regulatory T cells and expression of programmed cell death 1 on CD4+ T cells, and cellular exhaustion. Immunomodulatory molecules that were recently identified include interleukin‐7, interleukin‐15, and anti‐programmed cell death 1. Recent studies suggest that immunoadjuvant therapy is the next major advance in sepsis treatment.

definition of sepsis. Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Organ dysfunction was identified as an acute change in total Sequential Organ Failure Assessment score 2 (SOFA) of ≥2 as a consequence of the infection (Table 1).

| ME CH ANISM OF SEPSIS -INDUCED IMMUNOSUPPRESSION
This initial immune recognition response is mediated by pathogenassociated molecular patterns and damage-associated molecular patterns originating from bacterial or fungal organisms that blind pattern recognition receptors expressed on innate immune cells. 3 The activation of pattern recognition receptors results in the production of numerous proinflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-8, and interferon (IFN)-γ and antiinflammatory cytokines that induce excessive hyper-inflammatory responses and counter-responses. These responses include chemotaxis of leukocytes to sites of infection/inflammation, vascular endothelial injury with capillary leak, and activation of the coagulation system. 4 Until recently, most research on sepsis was focused on blocking the initial hyper-inflammatory response. Initially, the proinflammatory response was believed to be the major cause of mortality in patients with sepsis and was frequently targeted for therapeutic intervention. 5 However, efforts to improve outcomes by targeting proinflammatory cytokines and mediators, such as TNF and IL-1β antagonists, endotoxin antagonists, Toll-like receptor (TLR) blockers, and platelet activating factor inhibitors, have been unsuccessful. 6 This profound proinflammatory state, which occurs during the early onset of sepsis, is rapidly counterbalanced by an anti-inflammatory response, which may adversely affect immune functions. 7 This was initially referred to as compensatory anti-inflammatory response syndrome. 8 The vast majority of patients with sepsis survive the initial insult. Sepsis-induced immunosuppression is increasingly recognized as the overriding immune dysfunction in these vulnerable patients 7 (Figure 1). Immunosuppression in sepsis thus provides a novel understanding of the disorder as well as a new therapeutic approach. 9

| Host immune response in sepsis
Recent studies show that the activation of both proinflammatory and anti-inflammatory immune responses occurs promptly after the onset of sepsis. 10 Cells of the innate immune system, including monocytes and neutrophils, release high levels of proinflammatory cytokines. The rapid deaths of patients with sepsis are typically owing to a hyper-inflammatory "cytokine storm" response. If sepsis persists, the failure of crucial elements of both the innate and the adaptive immune system occurs, such that patients enter a marked immunosuppressive state. 10 We have shown that patients who die of sepsis have marked immunosuppression 11 ( Figure 1

| Apoptosis and immunosuppression
Apoptosis is an irreversible reaction in which the immune system maintains homeostasis by eliminating activated cells. 12 Central to apoptosis are caspases, which are cysteine proteases that degrade cellular proteins and nuclear factor-kappa B (NF-κB), a transcription factor that activates the transcription of both proapoptotic and prosurvival genes. Whereas hyper-inflammatory responses of sepsis require NF-κB for the production of proinflammatory cytokines and the activation by caspase cleavage, both NF-κB and caspases concurrently induce the apoptosis of immune cells. 3 Consistent with this, a concurrent apoptotic response has been shown to be present in sepsis in association with the proinflammatory response. 13 Although the deletion of adaptive immune cells is recognized as an important part of the pathology of sepsis, the mechanisms responsible for this are not fully understood. 14  infections. The detrimental effects of apoptosis are not only associated with the severe loss of immune cells but also with the effect that apoptotic cell uptake has on surviving immune cells. 16 The uptake of apoptotic cells by monocytes, macrophages, and DC results in immune tolerance by inducing anergy or a T helper 2 cellassociated immune phenotype with increased IL-10 production. 17

| Monocytes and macrophages
We have already reported that patients with sepsis have monocytes with a decreased capacity to release proinflammatory cytokines in response to endotoxin. 18  by Treg cells and Th2-type cells and suppresses the Th1 response, further potentiating an anti-inflammatory environment. 23 We have reported that blocking IL-10 can reverse sepsis-induced immunosuppression and improve survival in a mouse model of sepsis. 24 Low levels of monocyte human leukocyte antigen (HLA)-DR expression function as a surrogate marker of monocyte unresponsiveness. 25 We previously reported that expression of the human leukocyte antigen HLA-DR by peripheral blood monocytes was decreased in septic patients, particularly in patients with septic shock or severe sepsis. 26 Several studies showed an association of reduced monocyte HLA-DR expression with impaired monocyte function.
These data show that monocyte unresponsiveness and immunosuppression independently contribute to the increased risk of adverse events in sepsis.

| T cells
In sepsis, T cells become hypo-responsive in terms of proliferation and turn toward a type 2 profile with an increased production of IL-4 and IL-10, and suppression of IL-12 and IFN-γ. We previously reported that serum IFN-γ levels and IFN-γ production by PBMC were significantly decreased in patients with sepsis compared with healthy volunteers. 28,30 In the late 1990s, Sakaguchi et al 31 showed for the first time that the suppression mediated by CD4 + T cells appeared to result from the function of a small subset of T cells that expressed CD4 + CD25 + . These CD4 + CD25 + T cells were reported to act on T cells through a cell-contact mechanism involving cytotoxic T-lymphocyte antigen, 32 and are also thought to produce IL-10 and transforming growth factor (TGF)-β, and to suppress IFN-γ production. 32 Thereafter, Forkhead box protein 3 (Foxp3) was found to be expressed in CD4 + CD25 + T cells, and these cells were subsequently named Treg cells. Treg cells are central to the maintenance of immunological homeostasis and tolerance. 33,34 In septic patients, the percentages of circulating Treg cells are markedly increased, which presumably contributes to the occurrence of sepsis-induced immunosuppression. 35 Our data showed that the total CD4 + T-cell count and the percentage of CD4 + T cells in lymphocytes were significantly lower in patients with septic shock than in patients without septic shock. 36 The percentage of Treg cells in the CD4 + T-cell population, and serum IL-10 and IL-6 levels were significantly higher in patients with septic shock than in patients without septic shock. These clinical data indicate that IL-10 may contribute to the increased percent- A recent study provided insight into the molecular mechanisms that underlie immune depression following sustained inflammation.
This study showed a crucial monocyte-macrophage protein known as PD-1, which is found in patients infected with the human immunodeficiency virus (HIV). PD-1, which is a negative costimulatory molecule expressed on immune effector cells, is upregulated along with its cognate ligand PD-L1 during chronic HIV infection. PD-1 impairs immunity by inducing apoptosis, increasing the production of IL-10, preventing T-cell proliferation, and causing T cells to become nonresponsive. 11  present and may be dysregulated in human sepsis. 11,48 In oncology, anti-PD-1 and anti-PD-L antibody therapy have been used successfully to treat various tumors. 49 These data indicate that blocking the PD-1/PD-L1 axis is a promising target for restoring immune function in human sepsis.

| Polymyxin B direct hemoperfusion therapy for septic immunoparalysis
Polymyxin B (PMX) has long been known to neutralize the various biological activities of endotoxins. 50 In 1990, after the biocompatibility of F I G U R E 3 Immunostimulation therapies for sepsis. Targets of potential immunotherapeutic approaches include agents that block apoptosis, block negative costimulatory molecules, decrease the level of anti-inflammatory cytokines, increase human leukocyte antigen (HLA)-DR expression, and reactivate "exhausted" or anergic T cells. G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFN, interferon; IL, interleukin; NK, natural killer; PAMP, pathogen-associated molecular patterns; PD-1, programmed cell death-1; PD-L1, programmed cell death 1 ligand 1; PMX-DHP, polymyxin B direct hemoperfusion; TCR, T cell receptor; TGF, transforming growth factor; TLR, Toll-like receptor sterilized PMX-F (polymyxin B covalently immobilized on fibers) was demonstrated, the Critical Network Group in Japan obtained permission from the Japanese Ministry of Health and Welfare to clinically test hemoperfusion with PMX-F, and subsequently reported that it was safe and effective for patients with septic shock. 51,52 The main objective of therapeutic apheresis is the removal of toxic substances, although the method can also be applied for immunomodulation. It was reported that the absorption of anandamide by polymyxin B might abolish the diverse negative effects of anandamide, such as hypotension, immunosuppression, and cytotoxicity, 53 and that the reduction of serum cytokine levels 54 or monocyte mRNA expression, 55 and the proapoptotic activity of plasma 56 from septic patients might contribute to the efficacy of hemoperfusion with PMX-F. We previously reported that the expression of HLA-DR surface antigen on monocytes is decreased in patients with septic shock, and that PMX-F therapy is effective for increasing its expression. 26 However, the molecular mechanism underlying these effects, and the potential of this treatment have not yet been fully analyzed. We found that there was an increase in the percentages of Treg cells in peripheral blood circulating CD4 + T cells from patients with sepsis, particularly those with septic shock, and that hemoperfusion with PMX-F could remove Treg cells. 36 Furthermore improved survival rates in septic mice. 25 We also found that PMX F I G U R E 4 Changes in CD4 + T cells, Treg cells, and percentage of Treg cells among the CD4 + T-cell population and serum cytokine levels before and after polymyxin B covalently immobilized on fibers (PMX-F) therapy. Number of Treg cells was significantly decreased immediately after PMX-F therapy, and the percentage of Treg cells in the CD4 + T-cell population was significantly decreased immediately and 24 h after PMX-F therapy compared with that before PMX-F therapy. Both serum interleukin (IL)-10 and IL-6 levels were significantly decreased immediately and 24 h after PMX-F therapy compared with before PMX-F therapy (data from ref. (36)). TGF, transforming growth factor septic shock. The removal of Treg cells by hemoperfusion with PMX-F might represent a novel strategy for inducing recovery from immunosuppressive conditions that arise during sepsis.

| FUTURE ASPECTS
Immunotherapy is expected to be used in individual patients on the basis of specific laboratory or clinical findings. For example, a recent trial of GM-CSF to treat sepsis tested the effect only on patients in whom monocyte HLA-DR expression was significantly suppressed.
Flow-cytometry data regarding the expression levels of negative costimulatory molecules, such as PD-1 and PD-L1, on leukocytes may be useful as a guide for deciding on immunotherapies.

Conflicts of interest:
Authors declare no conflicts of interest for this article.