| Sepsis is a life-threatening illness in clinic with high morbidity and mortality that can primarily occur after trauma,surgery,and severe infectious diseases.For a long time,sepsis was considered as a systemic inflammatory disease because an elevated expression of many pro-inflammtory cytokines such as TNF-α and IL-1β were detected in sepsis patients.These inflammatory cytokines,by a cytokine cascade mechanism,are prone to induce multiple organ injury,which constitutes an important pathologic mechanism of sepsis mortality.However,many therapies that block TNF-a or IL-1β function in clinic trials did not improve,in some cases even worsen the patients survival,indicating that sepsis is a more than inflammation.Lines of evidence suggest that sepsis is characterized by host immune response to pathogenic infection.In the early stage of sepsis,many pro-inflammatory cytokines and anti-inflammatory cytokines were concurrently produced in patients.These anti-inflammatory cytokines,including IL-10 and TGFβ act as a feedback mechanism to control the state of hyperinflammtion and prevent organ injury by inflammatory cytokines,however,the excessive production of anti-inflammatory cytokines are also prone to induce immunosuppression.It has been increasingly appreciated that immunosuppression is an important pathologic mechanism of sepsis.After therapies with antibiotics,inflammation in sepsis patients can be effectively contolled,but some of them will die of a secondary nosocomial infections because of immunosuppression.This immunosuppression,also termed as immunoparalysis,is characterized by the decrease in antigen-presenting ability of monocytes or other antigen-presenting cells,increases in T lymphocyte and B lymphocyte apoptosis,impaired cytokine production capacity of immune cells inhibition,a shift from Thl cytokines to Th2 cytokines,Treg cell activation,production of anti-inflammatory cytokines such as IL-10,TGFp.Given the impaired immunity in sepsis,immunoadjuvant-based therapy is increasingly appreciated as an effective approach.For example,a small-scale clinic trials showed that GM-CSF and IFN-y can reverse immunoparalysis.Although GM-CSF and IFN-y cytokine therapies have some advantages,there are many shortcomings including side effects,high dosage used,short half-life.Therefore,small molecule holds promise for development of new drugs in the treatment of sepsis.In this study,we established a clinically relevant two hit model,e.g.mouse cecal ligation and puncture(CLP)followed by Salmonella secondary infection to identify small molecules that can be effective against immunoparalysis.Unexpectedly,we found that one of sodium pump inhibitors,ouabain can reverse this experimental immunoparalysis,as characterized by the improved survival,enhanced bacterial clearance and suppression of CD4+/CD8+ T cells reductions in spleens.Besides animal experiments,sepsis-induced HLA-DR ablation in human monocytes was also up-regulated by ouabain.Further study found that the reversing effect of ouabain on immunoparalysis was dependent on TNF-a.Notably,both endotoxin and ouabain can induce TNF-a expression in a variety of immune cells and non-immune cells,but they produced totally opposing results in CLP-induced sepsis,endotoxin induced immunoparalysis,while ouabain reversed immunoparalysis.These contradictory results indicate that mechanisms underlying the regulation of TNF-a expression by ouabain and endotoxin may be different.To understand why both endotoxin and ouabain can rapidly increase TNF-a gene transcription,we found NF-κB is a shared transcriptional factor by endotoxin and ouabain.In addition,both endotoxin and ouabain induced miR-181 expression via transcription factor EGR1.miR-181 served as a negative feedback mechanism to promote the degradation of TNF-a mRNA.In vivo,it can significantly alleviate the TNBS-induced mouse colitis.However,miR-181 is a "double-edged" sword,which promoted endotoxin tolerance and CLP-induced septic mice into immunoparalysis,as characterized by increased mortality,decreases in pathogenic microorganisms clearance,and emhancement in the reduction of CD4+/CD8+ T cell in spleens and lymph nodes.Obviously,the shared mechanisms involving NF-κB-mediated TNF-a gene transcription and miR-181-mediated TNF-a mRNA degradation can not explain the different modulatory effects of endotoxin and ouabain on immunoparalysis.Bioinformatics studies show that TNF-α 3’-UTR has multiple AUUUA motif sequences,which are binding sites of RNA binding protein HuR.Interestingly,miR-181 binding sequences in TNF-a 3’-UTR locate exactly between two adjacent AUUUA sequences.We found that ouabain stimulated HuR phosphorylation and its translocation from nucleus to cytoplasm,but endotoxin failed to do so.After releasing from nucleus,HuR was found to bind with TNF-a 3’-UTR which leads to TNF-a mRNA stabilization.Animal experiment demonstrated that HuR has a pro-inflammatory effect because silencing HuR in vivo can alleviate the acute lung injury induced by LPS,However,HuR.can also protect CLP animals from immunoparalysis because HuR siRNA in vivo administration significantly increased mortality,decreased pathogenic microorganisms clearance and enhanced the CD4+/CD8+ T cell reduction in spleens and lymph nodes.The close proximity between miR-181 and HuR binding sites within TNF-α3’-UTR suggest the functional competition between them.By using a luciferase reporter assay,we found these two cis-elements,miR-181 binding sites and HuR binding sites depended on each other to functionalize.However,when miR-181 and HuR are co-present,HuR was able to antagonize the destabilizing effect of miR-181 on TNF-a mRNA.Therefore,ouabain-induced HuR export was able to compete with miR-181 to post-transcriptionally regulate TNF-a mRNA stability,thereby maintaining TNF-a level at a high level in the state of immunoparalysis,activating the immune system,and increasing the body resistance to pathogen invasion.The underlying competition mechanisms includes:(1)suppressing the binding of miR-181 to TNF-a 3 ’-UTR to degrade TNF-a mRNA;(2)increasing the binding affinity of HuR with TNF-a mRNA;(3)triggering stress granules formation to protect TNF-a mRNA from degradation by miR-181.In conclusion,miR-181 has anti-inflammatory effect but also can induce immunoparalysis;HuR have both pro-inflammatory and immunoparalysis-suppressive effects;HuR utilizes various multiple mechanisms to functionally compete with miR-181,and subsequently suppressed miR-181-mediated immunoparalysis;by a competitive mechanism between HuR and miR-181,ouabain precisely controlled TNF-a expression and reversesd immunoparalysis.In this study,we propose a novel pathological mechanism of sepsis and provide multiple possible interventions for sepsis therapy,such as inhibiting the expression of miR-181,stimulating HuR nuclear export,using sodium pump inhibitor to precisely control TNF-α expression.Meanwhile,we further confirmed the immunoadjuvant-based therapy in sepsis-induced immnuoparalysis,and propose a new mode of cytokine modulation at post-transcriptional level by integrating multiple mechanisms involving RNA binding protein,miRNA and stress granules.Undoubtedly,this study will lead to the discovery of more small molecules for the treatment of sepsis,especially during the state of immunoparalysis. |
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