Sepsis-induced immunosuppression: From cellular dysfunctions to immunotherapy. and puncture using T cellCspecific autophagy-deficient mice. Dexpramipexole dihydrochloride Measurements and Main Results: We observed an increase of autophagosomes, which was assessed by flow cytometry. However, an autophagy process in CD4+ T cells during sepsis was insufficient including the accumulation of p62. On the other hand, a blockade of autophagy accelerated T cell apoptosis compared with the control mice, augmenting the gene expression of Bcl-2-like 11 and programmed cell death 1. Furthermore, mitochondrial accumulation in T cells occurred via a blockade of autophagy during sepsis. In addition, interleukin-10 production in CD4+ T cells from the cecal ligation and punctureCoperated knockout mice was markedly increased. Consequently, deficiency of autophagy in T cells significantly decreased the survival rate in the murine sepsis model. Conclusions: We exhibited that blocking autophagy accelerated apoptosis and increased mortality in concordance with the insufficient autophagy process in CD4+ T cells in Dexpramipexole dihydrochloride the murine sepsis model, suggesting that T cell autophagy plays a protective role against apoptosis and immunosuppression in sepsis. test for continuous data and used two-way analysis of variance among different categoric impartial variables. Statistical analyzes were conducted using the GraphPad Prism 6 (GraphPad Hhex Software, San Diego, CA). RESULTS Although Lymphocyte Autophagosomes are Increased by Septic Stimulation, the Process of Autophagy is usually Insufficient in a Murine Sepsis Model in CD4+ T Cells To evaluate the autophagy kinetics of lymphocytes in sepsis, we performed in vitro assay to replicate the condition at first. Lymphocytes from GFP-LC3 mice were stimulated with anti-CD3/CD28 or lipopolysaccharide (LPS) for 48 hours. Mean fluorescence intensity (MFI) of GFP-LC3, which represents autophagosomes, was measured Dexpramipexole dihydrochloride by flow cytometry. As shown in Figure ?Determine11= 3C6 mice in each group. Results are shown as mean sd in a bar graph. Data are analyzed by student test; #< 0.05. E, MFI Dexpramipexole dihydrochloride of Lysotracker staining lymphocytes from sham- and CLP-operated mice are shown. Each sample was stained with surface antigen markers. Data are expressed as mean and sd, and analyzed by two-way analysis of variance (ANOVA) and student test. = 3C4 mice in each group; #< 0.05. F, MFI of p62 protein conjugated with fluorescent second antibody in lymphocytes from wild-type mice. Mice underwent CLP and sham procedure, and were killed at the time of 24?hr after the operation. Harvested splenocytes were stained with p62 and surface antigen markers concomitantly, and then measured by flow cytometry. Data are expressed as mean and sd, and analyzed by two-way ANOVA and student test. = 4C6 mice in each group; #< 0.05. FACS = fluorescence activated cell sorting, GAPDH = glyceraldehyde-3-phosphate dehydrogenase. Based on the above results, we performed in vivo assay using a murine sepsis model. A CLP procedure was performed on GFP-LC3 mice and measured MFI of GFP-LC3 by flow cytometry. Autophagosomes, which were assessed by the MFI of GFP-LC3, were significantly increased in the CLP model over time, in CD4+ T cells (Fig. 1, and = 8C10 mice in each group; #< 0.05 was significance analyzed by two-way analysis of variance (ANOVA) and student test. E, Subpopulation of Annexin V and PI staining lymphocytes from peripheral blood were shown for early (Annexin V positive and PI negative) and late (Annexin V positive and PI positive) phase of apoptosis. Data are expressed as mean and sd; = 6C8 mice in each group; #< 0.05 was significance analyzed by student test between CLP-operated Atg5f/f mice and CLP-operated CD4-Cre/Atg5f/f mice. F, Relative RNA expression for apoptotic gene in sham, CLP-operated Atg5f/f mice and sham, and CLP-operated CD4-Cre/Atg5f/f mice. Total RNA in CD4+ splenic lymphocytes was extracted from experimental mice at 24?hr after the procedure, and then relative RNA expression for the several genes was analyzed. Data are expressed as mean and sd; = 8C10 mice in each group; #< 0.05 was significance analyzed by two-way ANOVA and student test. APC = allophycocyanin, Bcl-2 = B-cell leukemia/lymphoma 2, BIM = Bcl-2-like 11, Dexpramipexole dihydrochloride FACS = fluorescence activated cell sorting, PDCD1 = programmed cell death 1, PE = phycoerythrin, PI = propidium iodide. The expression of Bcl-2-like 11 (BIM) and programmed cell death 1 (PDCD1), which have roles of apoptosis induction, was increased in CD4+ T cells.