Prevention of lymphocyte apoptosis in septic mice with cancer increases mortality

Lymphocyte apoptosis is thought to have a major role in the pathophysiology of sepsis. However, there is a disconnect between animal models of sepsis and patients with the disease, because the former use subjects that were healthy prior to the onset of infection while most patients have underlying comorbidities. The purpose of this study was to determine whether lymphocyte apoptosis prevention is effective in preventing mortality in septic mice with preexisting cancer. Mice with lymphocyte Bcl-2 overexpression (Bcl-2-Ig) and wild type (WT) mice were injected with a transplantable pancreatic adenocarcinoma cell line. Three weeks later, after development of palpable tumors, all animals received an intratracheal injection of Pseudomonas aeruginosa. Despite having decreased sepsis-induced T and B lymphocyte apoptosis, Bcl-2-Ig mice had markedly increased mortality compared with WT mice following P. aeruginosa pneumonia (85 versus 44% 7-d mortality; p = 0.004). The worsened survival in Bcl-2-Ig mice was associated with increases in Th1 cytokines TNF-α and IFN-γ in bronchoalveolar lavage fluid and decreased production of the Th2 cytokine IL-10 in stimulated splenocytes. There were no differences in tumor size or pulmonary pathology between Bcl-2-Ig and WT mice. To verify that the mortality difference was not specific to Bcl-2 overexpression, similar experiments were performed in Bim(-/-) mice. Septic Bim(-/-) mice with cancer also had increased mortality compared with septic WT mice with cancer. These data demonstrate that, despite overwhelming evidence that prevention of lymphocyte apoptosis is beneficial in septic hosts without comorbidities, the same strategy worsens survival in mice with cancer that are given pneumonia.     

Akt decreases lymphocyte apoptosis and improves survival in sepsis

Sepsis induces extensive death of lymphocytes that may contribute to the immunosuppression and mortality of the disorder. The serine/threonine kinase Akt is a key regulator of cell proliferation and death. The purpose of this study was to determine whether overexpression of Akt would prevent lymphocyte apoptosis and improve survival in sepsis. In addition, given the important role of Akt in cell signaling, T cell Th1 and Th2 cytokine production was determined. Mice that overexpress a constitutively active Akt in lymphocytes were made septic, and survival was recorded. Lymphocyte apoptosis and cytokine production were determined at 24 h after surgery. Mice with overexpression of Akt had a marked improvement in survival compared with wild-type littermates, i.e., 94 and 47% survival, respectively, p < 0.01. In wild-type littermates, sepsis caused a marked decrease in IFN-gamma production, while increasing IL-4 production >2-fold. In contrast, T cells from Akt transgenic mice had an elevated production of IFN-gamma at baseline that was maintained during sepsis, while IL-4 had little change. Akt overexpression also decreased sepsis-induced lymphocyte apoptosis via a non-Bcl-2 mechanism. In conclusion, Akt overexpression in lymphocytes prevents sepsis-induced apoptosis, causes a Th1 cytokine propensity, and improves survival. Findings from this study strengthen the concept that a major defect in sepsis is impairment of the adaptive immune system, and suggest that strategies to prevent lymphocyte apoptosis represent a potential important new therapy.     

TAT-BH4 and TAT-Bcl-xL peptides protect against sepsis-induced lymphocyte apoptosis in vivo

Apoptosis is a key pathogenic mechanism in sepsis that induces extensive death of lymphocytes and dendritic cells, thereby contributing to the immunosuppression that characterizes the septic disorder. Numerous animal studies indicate that prevention of apoptosis in sepsis improves survival and may represent a potential therapy for this highly lethal disorder. Recently, novel cell-penetrating peptide constructs such as HIV-1 TAT basic domain and related peptides have been developed to deliver bioactive cargoes and peptides into cells. In the present study, we investigated the effects of sepsis-induced apoptosis in Bcl-x(L) transgenic mice and in wild-type mice treated with an antiapoptotic TAT-Bcl-x(L) fusion protein and TAT-BH4 peptide. Lymphocytes from Bcl-x(L) transgenic mice were resistant to sepsis-induced apoptosis, and these mice had a approximately 3-fold improvement in survival. TAT-Bcl-x(L) and TAT-BH4 prevented Escherichia coli-induced human lymphocyte apoptosis ex vivo and markedly decreased lymphocyte apoptosis in an in vivo mouse model of sepsis. In conclusion, TAT-conjugated antiapoptotic Bcl-2-like peptides may offer a novel therapy to prevent apoptosis in sepsis and improve survival.     

Agonistic monoclonal antibody against CD40 receptor decreases lymphocyte apoptosis and improves survival in sepsis

Sepsis causes a marked apoptosis-induced depletion of lymphocytes. The degree of lymphocyte apoptosis during sepsis strongly correlates with survival. CD40, a member of the TNFR family, is expressed on APCs and has potent antiapoptotic activity. In this study we determined whether an agonistic Ab against CD40 could protect lymphocytes from sepsis-induced apoptosis. Secondly, we examined potential antiapoptotic mechanisms of the putative protection. Lastly, we aimed to determine whether anti-CD40 treatment could improve survival in sepsis. CD1 mice were made septic by the cecal ligation and puncture method and treated postoperatively with anti-CD40 Ab. Treatment with anti-CD40 completely abrogated sepsis-induced splenic B cell death and, surprisingly, decreased splenic and thymic T cell death as well (p < 0.001). To investigate the mechanism of protection of anti-CD40 therapy on T cells, CD40 receptor expression was examined. As anticipated, the CD40 receptor was constitutively expressed on B cells, but, unexpectedly, splenic and thymic T cells were found to express CD40 receptor during sepsis. Furthermore, CD4+CD8- T cells were the predominant subtype of T cells expressing CD40 receptor during sepsis. Additionally, the antiapoptotic protein Bcl-x(L) was found to be markedly increased in splenic B and T cells as well as in thymic T cells after treatment with anti-CD40 Ab (p < 0.0025). Lastly, mice that were made septic in a double injury model of sepsis had improved survival after treatment with anti-CD40 as compared with controls (p = 0.05). In conclusion, anti-CD40 treatment increases Bcl-x(L), provides nearly complete protection against sepsis-induced lymphocyte apoptosis, and improves survival in sepsis.     

Increased apoptosis in lamina propria B cells during polymicrobial sepsis is FasL but not endotoxin mediated

Recent studies from our laboratory demonstrated that mucosal lymphoid tissue such as Peyer's patch cells and lamina propria (LP) B lymphocytes from mice shows evidence of increased apoptosis after sepsis that is associated with localized inflammation/activation. The mechanism for this is poorly understood. Endotoxin as well as Fas/Fas ligand (FasL) have been shown to augment lymphocyte apoptosis; however, their contribution to the increase of apoptosis in LP B-cells during sepsis is not known. To study this, sepsis was induced by cecal ligation and puncture (CLP) in endotoxin-tolerant C3H/HeJ or FasL-deficient C3H/HeJ-FasL(gld) (FasL(-)) mice and LP lymphocytes were isolated 24 h later. Phenotypic, apoptotic, and functional indexes were assessed. The number of LP B cells decreased markedly in C3H/HeJ mice but not in FasL-deficient animals at 24 h after CLP. This was associated with comparable alteration in apoptosis and Fas antigen expression in the B cells of these mice. Septic LP lymphocytes also showed increased IgA production, which was absent in the FasL-deficient CLP mice. Furthermore, Fas ligand deficiency appeared to improve survival of septic challenge. These data suggest that the increase in B cell apoptosis in septic animals is partially due to a Fas/FasL-mediated process but not endotoxin.     

p53-dependent and -independent pathways of apoptotic cell death in sepsis

Sepsis induces extensive apoptosis of lymphocytes, which may be responsible for the profound immune suppression of the disorder. Two potential pathways of sepsis-induced lymphocyte apoptosis, Fas and p53, were investigated. Lymphocyte apoptosis was evaluated 20-22 h after sepsis by annexin V or DNA nick-end labeling. Fas receptor-deficient mice had no protection against sepsis-induced apoptosis in thymocytes or splenocytes. p53 knockout mice (p53-/-) had complete protection against thymocyte apoptosis but, surprisingly, had no protection in splenocytes. p53-/- mice had no improvement in sepsis survival compared with appropriately matched control mice with sepsis. We conclude that both p53-dependent and p53-independent pathways of cell death exist in sepsis. This differential apoptotic response of thymocytes vs splenocytes in p53-/- mice suggests that either the cellular response or the death-inducing signal is cell-type specific in sepsis. The fact that p53-/- lymphocytes of an identical subtype (CD8-CD4+) were protected in thymi but not in spleens indicates that cell susceptibility to apoptosis differs depending upon other unidentified factors.     

Lymphocyte development and function in the absence of retinoic acid-related orphan receptor alpha

The orphan nuclear receptor, retinoid acid-related orphan receptor (ROR)alpha, is essential for the development of cerebellar Purkinje cells and bone tissue. RORalpha may also play a critical role in lymphocyte development and function because staggerer mice, a natural mutant strain with a disrupted expression of RORalpha, have reduced thymic and splenic cellularity. In this report, we analyzed the role of RORalpha in lymphocyte development by examining lymphoid compartments in RORalpha(-/-) mice and Rag-2(-/-) mice reconstituted with RORalpha(-/-) bone marrow. We found that T and B cell development was severely defective in RORalpha(-/-) mice, but not in Rag-2(-/-)/RORalpha(-/-) chimeric mice. We also analyzed cellular and humoral immune responses in Rag-2(-/-)/RORalpha(-/-) chimeric mice. Our results show that serum IgG levels were elevated in Rag-2(-/-)/RORalpha(-/-) chimeric mice after immunization with a T-dependent Ag compared with control chimeras. IFN-gamma production by RORalpha(-/-) CD8(+) T cells after TCR stimulation was also increased. Furthermore, RORalpha(-/-) mast cells and macrophages produced an increased amount of TNF-alpha and IL-6 upon activation. These results indicate that RORalpha indirectly regulates lymphocyte development by providing an appropriate microenvironment and controls immune responses by negatively regulating cytokine production in innate immune cells and lymphocytes.     

Deficiency of gammadelta T lymphocytes contributes to mortality and immunosuppression in sepsis

Studies have indicated that gammadelta T lymphocytes play an important role in the regulation of immune function and the clearance of intracellular pathogens. We have recently reported that intraepithelial lymphocytes (IEL), which are rich in gammadelta T cells, within the small intestine illustrated a significant increase in apoptosis and immune dysfunction in mice subjected to sepsis. However, the contribution of gammadelta T cells to the host response to polymicrobial sepsis remains unclear. In this study, we initially observed that after sepsis induced by cecal ligation and puncture (CLP), there was an increase in small intestinal IEL CD8+gammadelta+ T cells in control gammadelta+/+ mice. Importantly, we subsequently found an increased early mortality in mice lacking gammadelta T cells (gammadelta-/- mice) after sepsis. This was associated with decreases in plasma TNF-alpha, IL-6, and IL-12 levels in gammadelta-/- mice compared with gammadelta+/+ mice after sepsis. In addition, even though in vitro LPS-stimulated peritoneal macrophages showed a reduction in IL-6 and IL-12 release after CLP, these cytokines were less suppressed in macrophages isolated from gammadelta-/- mice. Alternatively, IL-10 release was not different between septic gammadelta+/+ and gammadelta-/- mice. Whereas T helper (Th)1 cytokine release by anti-CD3-stimulated splenocytes was significantly depressed in septic gammadelta+/+ mice, there was no such depression in gammadelta-/- mice. However, gammadelta T cell deficiency had no effect on Th2 cytokine release. These findings suggest that gammadelta T cells may play a critical role in regulating the host immune response and survival to sepsis, in part by alteration of the level of IEL CD8+gammadelta+ T cells and through the development of the Th1 response.     

Modulation of the phosphoinositide 3-kinase pathway alters innate resistance to polymicrobial sepsis

We examined the effect of modulating phosphoinositide 3-kinase (PI3K) activity in a murine model of cecal ligation and puncture-induced polymicrobial sepsis. Inhibition of PI3K activity with wortmannin increased serum cytokine levels and decreased survival time in septic mice. We have reported that an immunomodulator, glucan phosphate, induces protection in murine polymicrobial sepsis. We observed that glucan stimulated tissue PI3K activity, which positively correlated with increased survival in septic mice. We investigated the effect of PI3K inhibition on survival in septic mice treated with glucan. Treatment of mice with the PI3K inhibitors, wortmannin and LY294002, completely eliminated the protective effect of glucan, indicating that protection against septic mortality was mediated through PI3K. Inhibition of PI3K resulted in increased serum levels of IL1-beta, IL-2, IL-6, IL-10, IL-12, and TNF-alpha in septic mice. Apoptosis is thought to play a central role in the response to septic injury. We observed that inhibition of PI3K activity in septic mice resulted in increased splenocyte apoptosis and a change in the anatomic distribution of splenocyte apoptosis. We conclude that PI3K is a compensatory mechanism that suppresses proinflammatory and apoptotic processes in response to sepsis and/or inflammatory injury. Thus, PI3K may play a pivotal role in the maintenance of homeostasis and the integrity of the immune response during sepsis. We also observed that glucan phosphate decreased septic morbidity and mortality through a PI3K-dependent mechanism. This suggests that stimulation of the PI3K pathway may be an effective approach for preventing or treating sepsis and/or septic shock.     

The role of TCR engagement and activation-induced cell death in sepsis-induced T cell apoptosis

Sepsis induces extensive apoptosis in T and B cells suggesting that the loss of immune effector cells could be one explanation for the profound immunosuppression observed in this disorder. Unfortunately, the mechanisms responsible for lymphocyte apoptosis in sepsis remain unknown. In T cells, apoptosis can occur through activation-induced cell death (AICD) in which engagement of the Ag receptors by cognate Ag or polyclonal activators such as bacteria-derived superantigens induces activation, proliferation, and apoptosis. We examined whether proliferation and AICD are necessary for apoptotic cell death in sepsis using normal and TCR transgenic mice. Results show that although sepsis resulted in activation of a small percentage of T cells, no proliferation was detected during the first 48 h following onset, a time when extensive apoptosis is observed. We also observed that T cells do not enter the cell cycle, and stimulation via the TCR in TCR transgenic animals does not enhance or decrease cell death in sepsis. Interestingly, T cells recovered from septic mice retained their ability to proliferate and synthesize cytokines albeit at reduced levels. With the exception of IL-10, which was increased in lymphocytes from mice with sepsis, sepsis caused a decrease in the production of both proinflammatory and anti-inflammatory cytokines. We conclude that lymphocyte apoptosis in sepsis does not require proliferation, TCR engagement, or AICD. Thus the immunosuppression observed in sepsis cannot be the result of T cell deletion via the TCR.     

Accelerated lymphocyte death in sepsis occurs by both the death receptor and mitochondrial pathways

Patients with sepsis are immune compromised, as evidenced by their failure to clear their primary infection and their propensity to develop secondary infections with pathogens that are often not particularly virulent in normal healthy individuals. A potential mechanism for immunosuppression in sepsis is lymphocyte apoptosis, which may occur by either a death receptor or a mitochondrial-mediated pathway. A prospective study of blood samples from 71 patients with sepsis, 55 nonseptic patients, and 6 healthy volunteers was undertaken to quantitate lymphocyte apoptosis and determine cell death pathways and mechanisms of apoptosis. Apoptosis was evaluated by flow cytometry and Western blotting. Lymphocyte apoptosis was increased in CD4 and CD8 T cells, B cells (CD20), and NK cells (CD56) in septic vs nonseptic patients. Samples taken sequentially from 10 patients with sepsis showed that the degree of CD3 T cell apoptosis correlated with the activity of his/her sepsis. In septic patients, apoptotic lymphocytes were positive for active caspases 8 and 9, consistent with death occurring by both mitochondrial-mediated and receptor-mediated pathways. In support of the concept that both death pathways were operative, lymphocyte apoptosis occurred in cells with markedly decreased Bcl-2 (an inhibitor of mitochondrial-mediated apoptosis) as well as cells with normal concentrations of Bcl-2. In conclusion, apoptosis occurs in a broad range of lymphocyte subsets in patients with sepsis and correlates with the activity of the disease. Lymphocyte loss occurs by both death receptor and mitochondrial-mediated apoptosis, suggesting that there may be multiple triggers for lymphocyte apoptosis.     

Dual role of TLR2 and myeloid differentiation factor 88 in a mouse model of invasive group B streptococcal disease

Toll-like receptors (TLRs) are involved in pathogen recognition by the innate immune system. Different TLRs and the adaptor molecule myeloid differentiation factor 88 (MyD88) were previously shown to mediate in vitro cell activation induced by group B streptococcus (GBS). The present study examined the potential in vivo roles of TLR2 and MyD88 during infection with GBS. When pups were infected locally with a low bacterial dose, none of the TLR2- or MyD88-deficient mice, but all of the wild-type ones, were able to prevent systemic spread of GBS from the initial focus. Bacterial burden was higher in MyD88- than in TLR2-deficient mice, indicating a more profound defect of host defense in the former animals. In contrast, a high bacterial dose induced high level bacteremia in both mutant and wild-type mice. Under these conditions, however, TLR2 or MyD88 deficiency significantly protected mice from lethality, concomitantly with decreased circulating levels of TNF-alpha and IL-6. Administration of anti-TNF-alpha Abs to wild-type mice could mimic the effects of TLR2 or MyD88 deficiency and was detrimental in the low dose model, but protective in the high dose model. In conclusion, these data highlight a dual role of TLR2 and MyD88 in the host defense against GBS sepsis and strongly suggest TNF-alpha as the molecular mediator of bacterial clearance and septic shock.     

Intestinal permeability is reduced and IL-10 levels are increased in septic IL-6 knockout mice

Sepsis is associated with increased intestinal permeability, but mediators and mechanisms are not fully understood. We examined the role of interleukin (IL)-6 and IL-10 in sepsis-induced increase in intestinal permeability. Intestinal permeability was measured in IL-6 knockout (IL-6 -/-) and wild-type (IL-6 +/+) mice 16 h after induction of sepsis by cecal ligation and puncture or sham operation. In other experiments, mice or intestinal segments incubated in Ussing chambers were treated with IL-6 or IL-10. Intestinal permeability was assessed by determining the transmucosal transport of the 4.4-kDa marker fluorescein isothiocyanate conjugated dextran and the 40-kDa horseradish peroxidase. Intestinal permeability for both markers was increased in septic IL-6 +/+ mice but not in septic IL-6 -/- mice. Treatment of nonseptic mice or of intestinal segments in Ussing chambers with IL-6 did not influence intestinal permeability. Plasma IL-10 levels were increased in septic IL-6 -/- mice, and treatment of septic mice with IL-10 resulted in reduced intestinal permeability. Increased intestinal permeability during sepsis may be regulated by an interaction between IL-6 and IL-10. Treatment with IL-10 may prevent the increase in mucosal permeability during sepsis.     

Cholinergic regulatory lymphocytes re-establish neuromodulation of innate immune responses in sepsis

Many anti-inflammatory strategies that are successful in treating sepsis in healthy animals fail in clinical trials, in part because sepsis normally involves immunocompromised patients, and massive lymphocyte apoptosis prevents immunomodulation. In this article, we report a new set of regulatory lymphocytes that are able to re-establish the cholinergic anti-inflammatory modulation and to provide therapeutic advantages in sepsis. The vagus nerve controls inflammation in healthy, but not in septic, mice. Likewise, vagus nerve and cholinergic agonists fail to control inflammation in splenectomized and nude animals. Unlike typical suppressor CD25(+) cells, CD4(+)CD25(-) lymphocytes re-establish the anti-inflammatory potential of the vagus nerve and cholinergic agonists in immunocompromised and septic animals. These cholinergic lymphocytes re-establish splenic protection and the potential of cholinergic agonists to rescue immunocompromised animals from established sepsis. The study results revealed these new regulatory lymphocytes as, to our knowledge, the first known physiological target for neuromodulation of the innate immune responses and a potential therapeutic target for sepsis.     

Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis.

Activation of the cell surface receptor Fas/APO-1 (CD95) induces apoptosis in lymphocytes and regulates immune responses. The cytoplasmic membrane protein Bcl-2 inhibits lymphocyte killing by diverse cytotoxic agents, but we found it provided little protection against Fas/APO-1-transduced apoptosis in B lymphoid cell lines, thymocytes and activated T cells. In contrast, the cowpox virus protease inhibitor CrmA blocked Fas/APO-1-transduced apoptosis, but did not affect cell death induced by gamma-radiation or serum deprivation. Signalling through Fas/APO-1 did not down-regulate Bcl-2 or induce its antagonists Bax and Bcl-xS. In Fas/APO-1-deficient lpr mice, Bcl-2 transgenes markedly augmented the survival of antigen-activated T cells and the abnormal accumulation of lymphocytes (although they did not interfere with deletion of auto-reactive cells in the thymus). These data raise the possibility that Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis.     

CD28, IL-2-independent costimulatory pathways for CD8 T lymphocyte activation.

We investigate, here, the mechanism of the costimulatory signals for CD8 T cell activation and confirm that costimulation signals via CD28 do not appear to be required to initiate proliferation, but provide survival signals for CD8 T cells activated by TCR ligation. We show also that IL-6 and TNF-alpha can provide alternative costimulatory survival signals. IL-6 and TNF-alpha costimulate naive CD8 T cells cultured on plate-bound anti-CD3 in the absence of CD28 ligation. They act directly on sorted CD8-positive T cells. They also costimulate naive CD8 T cells from Rag-2-deficient mice, bearing transgenic TCRs for HY, which lack memory cells, a potential source of IL-2 secretion upon activation. IL-6 and TNF-alpha provide costimulation to naive CD8 T cells from CD28, IL-2, or IL-2Ralpha-deficient mice, and thus function in the absence of the B7-CD28 and IL-2 costimulatory pathways. The CD8 T cell generated via the anti-CD3 plus IL-6 and TNF-alpha pathway have effector function in that they express strong cytolytic activity on Ag-specific targets. They secrete only very small amounts of any of the cytokines tested upon restimulation with peptide-loaded APC. The ability of the naive CD8 T cells to respond to TCR ligation and costimulatory signals from IL-6 and TNF-alpha provides a novel pathway that can substitute for signals from CD4 helper cells or professional APC. This may be significant in the response to viral Ags, which can be potentially expressed on the surface of any class I MHC-expressing cell.     

Immune unresponsiveness to secondary heterologous bacterial infection after sepsis induction is TRAIL dependent

Sepsis is the leading cause of death in most intensive care units, and patients who survive the hyperinflammation that develops early during sepsis later display severely compromised immunity. Not only is there apoptosis of lymphoid and myeloid cells during sepsis that depletes these critical cellular components of the immune system, but also the remaining immune cells show decreased function. Using a cecal-ligation and puncture (CLP) model to induce intra-abdominal polymicrobial peritonitis, we recently established a link between the apoptotic cells generated during sepsis and induction of sepsis-induced suppression of delayed-type hypersensitivity. The present study extends this earlier work to include a secondary heterologous bacterial infection (OVA(257)-expressing Listeria monocytogenes [LM-OVA]) subsequent to sepsis initiation to investigate sepsis-induced alterations in the control of this secondary infection and the associated naive Ag-specific CD8 T cell response. We found that CLP-treated wild-type (WT) mice had a reduced ability to control the LM-OVA infection, which was paralleled by suppressed T cell responses, versus sham-treated WT mice. In contrast, CLP-treated Trail(-/-) and Dr5(-/-) mice were better able to control the secondary bacterial infection, and the Ag-specific CD8 T cell response was similar to that seen in sham-treated mice. Importantly, administration of a blocking anti-TRAIL mAb to CLP-treated WT mice was able to restore the ability to control the LM-OVA infection and generate Ag-specific CD8 T cell responses like those seen in sham-treated mice. These data further implicate TRAIL-dependent immune suppression during sepsis and suggest TRAIL neutralization may be a potential therapeutic target to restore cellular immunity in septic patients.     

T-Cell Autophagy Deficiency Increases Mortality and Suppresses Immune Responses after Sepsis

Background

Although the role of autophagy in sepsis has been characterized in several organs, its role in the adaptive immune system remains to be ascertained. This study aimed to investigate the role of autophagy in sepsis-induced T cell apoptosis and immunosuppression, using knockout mice with T cell specific deletion of autophagy essential gene Atg7.

Methods and Results

Sepsis was induced in a cecal ligation and puncture (CLP) model, with T-cell-specific Atg7-knockout mice compared to control mice. Autophagic vacuoles examined by electron microscopy were decreased in the spleen after CLP. Autophagy proteins LC3-II and ATG7, and autophagosomes and autolysosomes stained by Cyto-ID Green and acridine orange were decreased in CD4⁺ and CD8⁺ splenocytes at 18 h and 24 h after CLP. This decrease in autophagy was associated with increased apoptosis of CD4⁺ and CD8⁺ after CLP. Moreover, mice lacking Atg7 in T lymphocytes showed an increase in sepsis-induced mortality, T cell apoptosis and loss of CD4⁺ and CD8⁺ T cells, in comparison to control mice. This was accompanied by suppressed cytokine production of Th1/Th2/Th17 by CD4⁺ T cells, reduced phagocytosis in macrophages and decreased bacterial clearance in the spleen after sepsis.

Conclusion

These results indicated that sepsis led to down-regulation of autophagy in T lymphocytes, which may result in enhanced apoptosis induction and decreased survival in sepsis. Autophagy may therefore play a protective role against sepsis-induced T lymphocyte apoptosis and immunosuppression.     

Neutralisation of Peritoneal IL-17A Markedly Improves the Prognosis of Severe Septic Mice by Decreasing Neutrophil Infiltration and Proinflammatory Cytokines

Purpose

The current study aimed to elucidate the role of peritoneal fluid IL-17A in septic mice, and the effects of intraperitoneal or intravenous blockade of the IL-17A pathway by anti-IL17A antibody on survival, plasma, and peritoneal cavity cytokine profile in a murine caecal ligation and puncture (CLP) sepsis model. The main source of peritoneal fluid IL-17A in septic mice was identified.

Methods

Male C57BL/6 mice that underwent severe CLP or sham surgery were intraperitoneally or intravenously administered anti-IL17A antibodies or isotype antibodies. The survival rates were observed. IL-17A, TNF-α, and IL-6 cytokine levels were measured by ELISA. Surface and intracellular IL-17A immunofluorescence stains were detected by flow cytometry to identify the IL-17A–producing cells.

Results

The IL-17A level was elevated much higher and earlier in peritoneal fluid than in the blood of the CLP mice. The intraperitoneal IL-17A blockade more significantly protects against CLP-induced mortality than intravenous blockade because of decreased TNF-α and IL-6 levels both in peritoneal fluid and blood, neutrophil infiltration in the peritoneal cavity, and lung injury. γδ T lymphocytes were identified to be the main source of IL-17A in the peritoneal fluid of septic mice.

Conclusions

The earlier and higher elevated IL-17A derived from γδ T cells in peritoneal fluid plays a critical role during polymicrobial severe sepsis and effect of intraperitoneal IL-17A antibody administration superior to intravenous administration on survival of severe CLP-induced septic mice. The intraperitoneal blockade of IL-17A decreases proinflammatory cytokine production, neutrophil infiltration, and lung injury, thereby improving septic mice survival, which provides a new potential therapy target for sepsis.