| Background:Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to an infection,which is a clinical syndrome with a high incidence and mortality rate.Even if patients with sepsis could survive,the recurrence rate and late fatality rate are significantly higher than non-septic patients,resulting in its survivors requiring readmission into hospitals frequently.The readmission is mainly due to recurrent sepsis.Recurrent sepsis or sepsis recidivism is defined as the sepsis survivors experience severe infections again,with a high morbidity and mortality,which greatly increases the health care burden.Because immune imbalance is a key pathophysiological mechanism of sepsis,the underlying mechanisms of recurrent sepsis might be closely related with immune dysfunction.However,there remains a gap in the understanding of immunological characteristics and underlying mechanisms of recurrent sepsis,while immune dysfunction might be one of the key mechanisms.Objective:The purpose of this study is to explore the differences of innate and adaptive immune cells in the immune status and the immune response to secondary infections between acute septic and recurrent septic mice.And to clarify the immunological characteristics and underlying mechanisms of recurrent sepsis.Methods:Acute sepsis(AS)and recurrent sepsis(RS)mice models were induced by intraperitoneally(i.p.)injecting different frequencies of LPS.The body weight and survival rate were evaluated.To compare the percentages,absolute numbers,and functional markers of innate and adaptive immune cells,we subjected single-cell suspensions off the spleen to flow cytometry.And then we subjected the two mouse models to a secondary influenza A virus(H1N1)infection and characterized the different immune responses against infections.Virus titers in the lung tissue were determined by plaque assays.Lung tissue sections were stained with hematoxylin and eosin solution(H&E)and histopathological score were performed to directly assess lung pathology.In addition,we compared the expression of CD4and CD8 of lung tissue sections using immunohistochemical staining.We further estimated the levels of typical inflammation and anti-inflammation cytokine in peripheral blood using the LEGENDplex Mouse Inflammation Panel.Subsequently,we explored the underlying mechanisms.The most significant immune cells in RS mice were obtained using magnetic cell sorting and then were sent for RNA sequencing.After filtering and cleaning and mapping of the original sequencing data,we analyzed the different expressed genes and explored the main biological functions of these genes using GO and gene set enrichment analysis.Results:Our results showed that the percentage and number of CD4~+T cells and the CD4~+/CD8~+T cell ratio decreased in the acute and recurrent sepsis mouse models,which were more prominent in recurrent sepsis mice.CD69 is an early activation marker of immune cells,and a moderately weaker accumulation of surface CD69 was observed in CD4~+T cells in RS mice.CD28,a costimulatory molecule that interacts with the CD80/CD86 molecules,is expressed on antigen presenting cells and is critically required for effective T cell activation.A significant decreased CD28 expression on CD4~+T subsets was noted in RS mice.Our results revealed that both PD-1 and Tim-3 expression significantly increased on CD4~+T cells in RS mice.In addition,both the percentage and absolute number of Tregs increased significantly in RS mice.These results suggested that CD4~+T cells presented exhausted phenotype in RS.Despite these intrinsic alterations in T cell subsets,the total MHC-II expression decreased significantly in RS mice,suggesting the impaired ability of antigen presenting cells to present antigens to CD4~+T cells,exacerbating CD4~+T cell exhaustion.Subsequently,we subjected AS and RS mice to secondary influenza A virus(H1N1,PR8strain)infections to explore their immune responses against infections.Our results showed that the viral titers in lung increased significantly in RS+PR8 mice,suggesting an impairment in the ability to clear virus in RS.In addition,lung pathology and pro-inflammatory cytokines levels,such as that of IL-6,TNF-α,and IFN-γ,were lower,and the level of anti-inflammatory cytokine IL-10 was higher in RS+PR8 mice,indicating a decrease in the inflammatory responses to clear PR8,and this is consistent with the increased viral titers.Furthermore,CD4~+T cell infiltration was decreased significantly in RS+PR8 mice.Therefore,increased viral titers,reduced leukocyte infiltration and alleviated pulmonary inflammation and pathology,reduced CD4~+T cell infiltration,reduced inflammatory cytokines,and increased anti-inflammatory cytokines indicate a decreased antiviral immune response,which may be the consequence of exacerbated CD4~+T cell exhaustion,in RS.In addition,we explored the underlying mechanisms of the exhausted phenotype of CD4~+T cells in RS mice.We found that the significant biological process among Con and AS and RS mice were closely related with cellular process,biological regulation,metabolic process,and immune system response.In the gene set enrichment analysis,we found that the majority of enriched GO terms were closely related with inflammation and immune.After the genes with significant differences and high expression were selected,we found that the expression of Nlrp3 and Casp4 were increased significantly in RS mice and these results were verified using RT-q PCR and Western blotting.Both Nlrp3 and Casp4 are key molecules in the pyroptosis signaling pathways,suggesting that the exacerbated exhausted phenotype of CD4~+T cells in RS may be closely related with their increased susceptibility to pyroptosis.Conclusions:CD4~+T cells exhibited exacerbated exhausted phenotype in recurrent sepsis mice,which was mainly manifested as decreased number of CD4~+T cells,decreased expression of CD28,increased expression of PD-1 and Tim-3 on CD4~+T cells,and increased number of Tregs.In addition to these internal alterations of CD4~+T cells,the total MHC-II expression on antigen-presenting cells decreased significantly in recurrent sepsis mice,further exacerbating the exhaustion of CD4~+T cells.Moreover,because of the exhausted phenotype of CD4~+T cells,the adaptive immune responses were severely impaired,contributing to reduced pathogen clearance and weakened inflammatory responses.The expression of Nlrp3and Casp4,which are key molecules in the pyroptosis signaling pathways,increased significantly in CD4~+T cells in RS,suggesting that the exacerbated exhausted phenotype of CD4~+T cells in RS may be closely related with their increased susceptibility to pyroptosis,which leads to the significant morbidity,increased late mortality,and increased health care burden. |
