| ObjectiveSepsis is a kind of well-known syndrome, which defines a systemic inflammatoryresponse to infection, and results in the death of more than210,000people in the UnitedStates annually. It remains the leading cause of death in critical ill patients. Becausecritical care treatment is not eutherapeutic and becoming more and more expensive, so,understanding the molecular mechanisms underlying the development of sepsis isimportant in identifying new therapeutic strategies. Lymphocyte activation gene3(LAG-3) is one of the major inhibitory regulators of T cell function and proliferation,similar to CTLA-4and PD-1. Anti-LAG-3therapy has been effective in enhancingimmunity and improving lymphocytes function in cancer and virus infection diseases.But the role of LAG-3in sepsis is not known. This study aimed to examine theexpression of LAG-3in major immune cells, the ability of anti-LAG-3antibody inlymphocytes apoptosis, function and prognosis in mice with cecal ligation and puncture(CLP) sepsis.Measurements1. Experimental sepsis was induced by cecal ligation and puncture (CLP).24C57BL/6mice were randomly divided into sham group (n=10) and CLP group (n=14). LAG-3expression was detected by flow cytometry on blood CD4~+T lymphocytes, CD8~+Tlymphocytes, CD19~+B lymphocytes and natural killer cells. LAG-3expression was alsodetermined on spleen CD4~+CD25~+Treg cells, and spleen dendritic cells24h after shamor CLP operation.2.81C57BL/6mice were randomly divided into sham group (n=9), CLP group (n=16),CLP+anti-LAG-3antibody intraperitoneally group (n=16), CLP+isotype antibodyintraperitoneally group (n=12), CLP+anti-LAG-3antibody intravenously group (n=16),and CLP+isotype antibody intravenously group (n=12). Anti-LAG-3antibody orisotype antibody (50μg/200μL per mouse) were administrated3h after operation, andsurvival was recorded for10days.3.55C57BL/6mice were randomly divided into sham group (n=7), CLP group (n=18),CLP+anti-LAG-3antibody group (n=18), CLP+isotype antibody group (n=12). Anti-LAG-3antibody or isotype antibody (50μg/200μL per mouse) were administratedintraperitoneally3h after operation. Mice lung, blood, and peritoneal lavage fluid wereharvested24h after operation to determinate lung wet-to-dry ratio, blood and peritonealcavity bacterial burden, respectively. Mice plasma were detached to measure TNF-α,IL-6and IL-10level by ELISA.4.33C57BL/6mice were randomly divided into sham group (n=8), CLP group (n=9),CLP+anti-LAG-3antibody group (n=9), CLP+isotype antibody group (n=7).Anti-LAG-3antibody or isotype antibody (50μg/200μL per mouse) were administratedintraperitoneally3h after operation. Mice peripheral blood, spleen and thymus wereharvested to detect blood, spleen CD4~+T lymphocytes, CD8~+T lymphocytes cell count,and thymocytes apoptosis.18mice underwent the same treatment as above sacrificed,spleen and thymus were used to measure apoptotic level by TUNEL.5. Mice spleens were harvested18h after CLP and splenocytes were detached. Thespleen cells were divided into control group, anti-LAG-3antibody group and isotypeantibody group. All cells were induced apoptosis by anti-CD3/anti-CD28antibody for72h or induced apoptosis by TNF-α for18h. Then CD4~+T lymphocytes and CD8~+Tlymphocytes apoptosis was analysed via flow cytometry.6. Mice spleens were harvested18h after CLP and splenocytes were detached. Thespleen cells were divided into control group, anti-LAG-3antibody group and isotypeantibody group. All cells were cultured with anti-CD3/anti-CD28antibody for67h, andthen stimulated cells with PMA, ionomycin and brefeldin A for5h. IFN-γ in CD4~+T andCD8~+T lymphocytes were detected by flow cytometry.7. Mice spleens were harvested18h after CLP and splenocytes were detached. Thespleen cells were divided into control group, anti-LAG-3antibody group and isotypeantibody group. Cells were staind with CFSE and then cultured with anti-CD3/anti-CD28antibody for5d. CD4~+T lymphocytes and CD8~+T lymphocytes proliferation wasdetected by flow cytometry.Results1. LAG-3expression on blood CD4~+T lymphocytes, CD8~+T lymphocytes, CD19+Blymphocytes, natural killer cells, spleen CD4~+CD25+Treg cells, and spleen dendritic cellswere significantly elevated24h after CLP operation (P<0.05). 2. CLP-operated C57BL/6mice treated with50μg/mouse of anti-LAG-3antibodyintraperitoneally and intravenously had a marked improvement in survival compared withCLP group, CLP treated with isotype antibody intraperitoneally and intravenously group(P<0.05). But there was no difference between CLP+anti-LAG-3antibody intraperitoneallygroup and CLP+anti-LAG-3antibody intravenously group, or between CLP+isotypeantibody intraperitoneally group and CLP+isotype antibody intravenously group (P>0.05).3. After treated with anti-LAG-3antibody, CLP mice lungs showed lower wet-to-dry ratiocompared with CLP group and CLP+isotype antibody group (P<0.01). CLP+anti-LAG-3antibody group also showed lower bacterial burden and cytokines level (TNF-α, IL-6andIL-10)(P<0.01).4. Blood, spleen CD4~+T lymphocytes and CD8~+T lymphocytes cell count were elevatedafter administration of anti-LAG-3antibody in CLP mice (P<0.05). Apoptosis of thymus andspleen were also ameliorated both in flow cytometry (P<0.05) and in TUNEL assays(P<0.01).5. Detached CLP mice spleen CD4~+T lymphocytes and CD8~+T lymphocytes treated withanti-LAG-3antibody showed lower apoptosis ratio compared with control and isotypeantibody group after anti-CD3/anti-CD28antibody induced apoptosis (P<0.05). But as forthe TNF-α induced apoptosis, it didn't show difference amoung the three groups (P>0.05).6. CLP mice spleen cells were detached and stimulated with PMA, ionomycin andbrefeldin A for5h, and flow cytometry analysis showed that IFN-γ+CD4~+T lymphocytesratio was higher in anti-LAG-3antibody group compared with control and isotype antibodygroup (P<0.01), but there was no difference in CD8~+T lymphocytes.7. Flow cytometry analysis showed that both CD4~+T lymphocytes and CD8~+Tlymphocytes proliferation improved after administration of anti-LAG-3antibody comparedwith control and isotype antibody group (P<0.01).ConclusionThe expression of LAG-3on CD4~+T lymphocytes, CD8~+T lymphocytes, CD19+Blymphocytes, natural killer cells, spleen CD4~+CD25+Treg cells, and spleen dendritic cellswere significantly elevated24h after CLP induced sepsis. Administration of anti-LAG-3antibody could improve experimental sepsis on mice both intraperitoneallyand intravenously, and there was no difference between the two administration pathway. Anti-LAG-3antibodycould protect septic mice against sepsis induced lung injury and increase bacterial clearance,decrease TNF-α, IL-6,IL-10cytokines level. After treated with anti-LAG-3antibody,apoptosis of lymphocytes in thymus, spleen and blood was also decreased afteradministration of anti-LAG-3antibody. Induced CD4~+and CD8~+T lymphocytes apoptosisby anti-CD3/anti-CD28antibody or TNF-α in vitro indicated that anti-LAG-3antibodycould reverse TCR but not TNF-α induced apoptosis after sepsis. Stimulation of detachedCLP spleen lymphocytes showed higher ratio of IFN-γ+CD4~+T lymphocytes in anti-LAG-3antibody group but didn't improve much more CD8~+T lymphocytes to produce IFN-γ. Atlast, anti-LAG-3antibody could improve cell function via improve CD4~+and CD8~+Tlymphocytes proliferation. |
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