Inhibition of MicroRNA-23b Attenuates Immunosuppression During Late Sepsis Through NIK, TRAF1, and XIAP.
ABSTRACT: Background:microRNA-23b (miR-23b) is a multiple functional miRNA. We hypothesize that miR-23b plays a role in the pathogenesis of sepsis. Our study investigated the effect of miR-23b on sepsis-induced immunosuppression. Methods:Mice were treated with miR-23b inhibitors by tail vein injection 2 days after cecal ligation puncture (CLP)-induced sepsis. Apoptosis in spleens and apoptotic signals were investigated, and survival was monitored. T-cell immunoreactivities were examined during late sepsis. Nuclear factor κB (NF-κB)-inducing kinase (NIK), tumor necrosis factor (TNF)-receptor associated factor 1 (TRAF1), and X-linked inhibitor of apoptosis protein (XIAP), the putative targets of miR-23b, were identified by a dual-luciferase reporter assay. Results:miR-23b expression is upregulated and sustained during sepsis. The activation of the TLR4/TLR9/p38 MAPK/STAT3 signal pathway contributes to the production of miR-23b in CLP-induced sepsis. miR-23b inhibitor decreased the number of spleen cells positive by terminal deoxynucleotidyl transferase dUTP nick-end labeling and improved survival. miR-23b inhibitor restored the immunoreactivity by alleviating the development of T-cell exhaustion and producing smaller amounts of immunosuppressive interleukin 10 and interleukin 4 during late sepsis. We demonstrated that miR-23b mediated immunosuppression during late sepsis by inhibiting the noncanonical NF-κB signal and promoting the proapoptotic signal pathway by targeting NIK, TRAF1, and XIAP. Conclusions:Inhibition of miR-23b reduces late-sepsis-induced immunosuppression and improves survival. miR-23b might be a target for immunosuppression.
Project description:Cardiac dysfunction is correlated with detrimental prognosis of sepsis and contributes to a high risk of mortality. After an initial hyperinflammatory reaction, most patients enter a protracted state of immunosuppression (late sepsis) that alters both innate and adaptive immunity. The changes of cardiac function in late sepsis are not yet known. MicroRNA-155 (miR-155) is previously found to play important roles in both regulations of immune activation and cardiac function. In this study, C57BL/6 mice were operated to develop into early and late sepsis phases, and miR-155 mimic was injected through the tail vein 48 h after cecal ligation and puncture (CLP). The effect of miR-155 on CLP-induced cardiac dysfunction was explored in late sepsis. We found that increased expression of miR-155 in the myocardium protected against cardiac dysfunction in late sepsis evidenced by attenuating sepsis-reduced cardiac output and enhancing left ventricular systolic function. We also observed that miR-155 markedly reduced the infiltration of macrophages and neutrophils into the myocardium and attenuated the inflammatory response via suppression of JNK signaling pathway. Moreover, overexpression of β-arrestin 2 (Arrb2) exacerbated the mice mortality and immunosuppression in late sepsis. Furthermore, transfection of miR-155 mimic reduced Arrb2 expression, and then restored immunocompetence and improved survival in late septic mice. We conclude that increased miR-155 expression through systemic administration of miR-155 mimic attenuates cardiac dysfunction and improves late sepsis survival by targeting JNK associated inflammatory signaling and Arrb2 mediated immunosuppression.
Project description:Myeloid-derived suppressor cells (MDSC) expand during sepsis, suppress both innate and adaptive immunity, and promote chronic immunosuppression, which characterizes the late/chronic phase of sepsis. We previously reported that the transcription factors Stat3 and C/EBP? synergize to induces the expression of microRNA (miR)-21 and miR-181b to promote MDSC expansion in a mouse model of polymicrobial sepsis that progresses from an early/acute proinflammatory phase to a late/chronic immunosuppressive stage. We also showed that Gr1+CD11b+ cells, the precursors of MDSCs, from mice genetically deficient in the inflammatory protein S100A9 lack miR-21 or miR-181b in late sepsis, and are not immunosuppressive. In the present study, we show that S100A9 induces miR-21 and miR-181b during the late sepsis phase. We find that S100A9 associates with and stabilizes the Stat3-C/EBP? protein complex that activates the miRNA promoters. Reconstituting Gr1+CD11b+ cells from S100A9 knockout mice with late sepsis with S100A9 protein restores the Stat3-C/EBP? protein complex and miRNA expressions, and switches the Gr1+CD11b+ cells into the immunosuppressive, MDSC phenotype. Importantly, we find that this process requires IL-10 mediated signaling, which induces S100A9 translocation from the cytosol to the nucleus. These results demonstrate that S100A9 promotes MDSC expansion and immunosuppression in late/chronic sepsis by inducing the expression of miR-21 and miR-181b.
Project description:Sepsis is a prevalent severe syndrome in clinic. Vascular leakage and lung injury are important pathophysiological processes during sepsis, but the mechanism remains obscure. Microvesicles (MVs) play an essential role in many diseases, while whether MVs participate in vascular leakage and lung injury during sepsis is unknown. Using cecal ligation and puncture induced sepsis rats and lipopolysaccharide stimulated vascular endothelial cells (VECs), the role and the underlying mechanism of endothelial microvesicles (EMVs) in pulmonary vascular leakage and lung injury were observed. The role of MVs from sepsis patients was verified. The results showed that the concentration of MVs in blood was significantly increased after sepsis. MVs from sepsis rats and patients induced apparent pulmonary vascular leakage and lung injury, among which EMVs played the dominant role, in which miR-23b was the key inducing factor in vascular leakage. Furthermore, downregulation and upregulation of miR-23b in EMVs showed that miR-23b mainly targeted on ZO-1 to induce vascular leakage. MVs from sepsis patients induced pulmonary vascular leakage and lung injury in normal rats. Application of classic antidepressants amitriptyline reduced the secretion of EMVs, and alleviated vascular leakage and lung injury. The study suggests that EMVs play an important role in pulmonary vascular leakage and lung injury during sepsis by transferring functional miR-23b. Antagonizing the secretion of EMVs and the miR-23b might be a potential target for the treatment of severe sepsis.
Project description:This study examined the effect of microRNA-125b (miR-125b) on sepsis-induced cardiac dysfunction.?Mouse hearts were transfected with lentivirus expressing miR-125b (LmiR-125b) 7 days before cecal ligation and puncture (CLP)-induced sepsis. Cardiac function was examined by echocardiography before and 6 hours after CLP (n = 6/group). Survival was monitored following CLP-induced sepsis (n = 12/group).?LmiR-125b transfection significantly attenuated cardiac dysfunction due to CLP-induced sepsis. Fractional shortening and ejection fraction values were significantly (P < .05) higher in the LmiR-125b-treated CLP group than in the untreated CLP group. Survival outcome in LmiR-125b-transfected septic mice was markedly improved, compared with mice with CLP-induced sepsis. Transfection of LmiR-125b into the heart significantly suppressed the expression of ICAM-1 and VCAM-1, decreased the accumulation of macrophages and neutrophils in the myocardium, and decreased serum levels of tumor necrosis factor ? and interleukin 1? by targeting tumor necrosis factor receptor-associated factor 6 (TRAF6)-mediated nuclear factor ?B (NF-?B) activation. In addition, sepsis-induced myocardial apoptosis was markedly attenuated by LmiR-125b transfection through suppression of p53, Bax, and Bak1 expression. In vitro transfection of endothelial cells with miR-125b mimics attenuate LPS-induced ICAM-1 and VCAM-1 expression by suppressing TRAF6 and NF-?B activation.?Increased myocardial miR-125b expression attenuates sepsis-induced cardiac dysfunction and improves survival. miR-125b may be a target for septic cardiomyopathy.
Project description:Immunosuppression is one hallmark of sepsis, decreasing the host response to the primary septic pathogens and/or secondary nosocomial infections. CD4 T cells and B cells are among the array of immune cells that experience reductions in number and function during sepsis. "Help" from follicular helper (Tfh) CD4 T cells to B cells is needed for productive and protective humoral immunity, but there is a paucity of data defining the effect of sepsis on a primary CD4 T cell-dependent B cell response. Using the cecal ligation and puncture (CLP) mouse model of sepsis induction, we observed reduced antibody production in mice challenged with influenza A virus or TNP-KLH in alum early (2 days) and late (30 days) after CLP surgery compared to mice subjected to sham surgery. To better understand how these CD4 T cell-dependent B cell responses were altered by a septic event, we immunized mice with a Complete Freund's Adjuvant emulsion containing the MHC II-restricted peptide 2W1S56-68 coupled to the fluorochrome phycoerythrin (PE). Immunization with 2W1S-PE/CFA results in T cell-dependent B cell activation, giving us the ability to track defined populations of antigen-specific CD4 T cells and B cells responding to the same immunogen in the same mouse. Compared to sham mice, differentiation and class switching in PE-specific B cells were blunted in mice subjected to CLP surgery. Similarly, mice subjected to CLP had reduced expansion of 2W1S-specific T cells and Tfh differentiation after immunization. Our data suggest CLP-induced sepsis impacts humoral immunity by affecting the number and function of both antigen-specific B cells and CD4 Tfh cells, further defining the period of chronic immunoparalysis after sepsis induction.
Project description:Myeloid-derived suppressor cells (MDSCs) increase late sepsis immunosuppression and mortality in mice. We reported that microRNA (miR) 21 and miR-181b expression in Gr1+CD11b+ myeloid progenitors increase septic MDSCs in mice by arresting macrophage and dendritic cell differentiation. Here, we report how sepsis regulates miR-21 and miR-181b transcription. In vivo and in vitro binding studies have shown that C/EBP? transcription factor, which promotes normal myeloid cell differentiation, binds both miRNA promoters in Gr1+CD11b+ cells from sham mice. In contrast, in sepsis Gr1+CD11b+ MDSCs miR-21 and miR-181b promoters bind both transcription factors Stat3 and C/EBP?, which co-imunoprecipitate as a single complex. Mechanistically, transcription factor Rb phosphorylation supports Stat3 and C/EBP? accumulation at both miRNA promoters, and C/EBP? or Stat3 depletion by siRNA in sepsis Gr1+CD11b+ MDSCs inhibits miR-21 and miR-181b expression. To further support this molecular path for MDSC accumulation, we found that Stat3 and C/EBP binding at miR-21 or miR-181b promoter was induced by IL-6, using a luciferase reporter gene transfection into naive Gr1+CD11b+ cells. Identifying how sepsis MDSCs are generated may inform new treatments to reverse sepsis immunosuppression.
Project description:BACKGROUND Immunosuppression is regarded as the main cause of death induced by sepsis. Anti-programmed death-ligand 1 (PD-L1) therapy is promising in reversing sepsis-induced immunosuppression but no evidence is available on use of commercially available anti-PD-L1 medications for this indication. The present preclinical study was performed to investigate the therapeutic effect of an anti-PD-L1 nanobody (KN035) in sepsis. MATERIAL AND METHODS The level of expression of PD-L1 in PD-L1 humanized mice was confirmed with flow cytometry. Plasma concentrations of KN035 at different dosages at different time points were detected using an enzyme-linked immunosorbent assay. PD-L1 humanized mice were allocated into 4 groups: sham, cecal ligation and puncture (CLP), isotype (isotype+CLP), and PD-L1 (KN035+CLP). The 7-day survival rate was observed to investigate outcomes in CLP mice. Disease severity was assessed with histopathological scoring of mice lungs and livers. Immune status was assessed based on cell apoptosis in the spleen and bacterial clearance. RESULTS PD-L1 levels were significantly elevated in peripheral lymphocytes, monocytes, and neutrophils after CLP surgery. Blood concentrations of KN035 showed that 2.5 mg/kg had potential to be an ideal dosage for KN035 therapy. Survival analysis demonstrated that KN035 was associated with significantly reduced mortality on Day 7 after surgery (P=0.0083). The histopathological tests showed that KN035 alleviated sepsis-induced injury in the lungs and liver. KN035 reduced the number of apoptotic cells in the spleen and almost eliminated bacterial colonies in the peritoneal lavage fluid from the CLP mice. CONCLUSIONS KN035, an anti-PD-L1 antibody, can improve the rate of survival in CLP mice and alleviate sepsis-induced apoptosis in the spleen.
Project description:Mesenchymal stem cells (MSCs) have been shown to elicit cardio-protective effects in sepsis. However, the underlying mechanism remains obscure. While recent studies have indicated that miR-223 is highly enriched in MSC-derived exosomes, whether exosomal miR-223 contributes to MSC-mediated cardio-protection in sepsis is unknown. In this study, loss-of-function approach was utilized, and sepsis was induced by cecal ligation and puncture (CLP). We observed that injection of miR-223-KO MSCs at 1?h post-CLP did not confer protection against CLP-triggered cardiac dysfunction, apoptosis and inflammatory response. However, WT-MSCs were able to provide protection which was associated with exosome release. Next, treatment of CLP mice with exosomes released from miR-223-KO MSCs significantly exaggerated sepsis-induced injury. Conversely, WT-MSC-derived-exosomes displayed protective effects. Mechanistically, we identified that miR-223-KO exosomes contained higher levels of Sema3A and Stat3, two known targets of miR-223 (5p &3p), than WT-exosomes. Accordingly, these exosomal proteins were transferred to cardiomyocytes, leading to increased inflammation and cell death. By contrast, WT-exosomes encased higher levels of miR-223, which could be delivered to cardiomyocytes, resulting in down-regulation of Sema3A and Stat3. These data for the first time indicate that exosomal miR-223 plays an essential role for MSC-induced cardio-protection in sepsis.
Project description:Sepsis is the leading cause of death in critically ill patients. While myocardial dysfunction has been recognized as a major manifestation in severe sepsis, the underlying molecular mechanisms associated with septic cardiomyopathy remain unclear. In this study, we performed a miRNA array analysis in hearts collected from a severe septic mouse model induced by cecal ligation and puncture (CLP). Among the 19 miRNAs that were dys-regulated in CLP-mouse hearts, miR-223(3p) and miR-223*(5p) were most significantly downregulated, compared with sham-operated mouse hearts. To test whether a drop of miR-223 duplex plays any roles in sepsis-induced cardiac dysfunction and inflammation, a knockout (KO) mouse model with a deletion of the miR-223 gene locus and wild-type (WT) mice were subjected to CLP or sham surgery. We observed that sepsis-induced cardiac dysfunction, inflammatory response and mortality were remarkably aggravated in CLP-treated KO mice, compared with control WTs. Using Western-blotting and luciferase reporter assays, we identified Sema3A, an activator of cytokine storm and a neural chemorepellent for sympathetic axons, as an authentic target of miR-223* in the myocardium. In addition, we validated that miR-223 negatively regulated the expression of STAT-3 and IL-6 in mouse hearts. Furthermore, injection of Sema3A protein into WT mice revealed an exacerbation of sepsis-triggered inflammatory response and myocardial depression, compared with control IgG1 protein-treated WT mice following CLP surgery. Taken together, these data indicate that loss of miR-223/-223* causes an aggravation of sepsis-induced inflammation, myocardial dysfunction and mortality. Our study uncovers a previously unrecognized mechanism underlying septic cardiomyopathy and thereby, may provide a new strategy to treat sepsis.
Project description:The non-canonical NF-κB pathway is an important arm of NF-κB signaling that predominantly targets activation of the p52/RelB NF-κB complex. This pathway depends on the inducible processing of p100, a molecule functioning as both the precursor of p52 and a RelB-specific inhibitor. A central signaling component of the non-canonical pathway is NF-κB-inducing kinase (NIK), which integrates signals from a subset of TNF receptor family members and activates a downstream kinase, IκB kinase-α (IKKα), for triggering p100 phosphorylation and processing. A unique mechanism of NIK regulation is through its fate control: the basal level of NIK is kept low by a TRAF-cIAP destruction complex and signal-induced non-canonical NF-κB signaling involves NIK stabilization. Tight control of the fate of NIK is important, since deregulated NIK accumulation is associated with lymphoid malignancies.