Study On Mechanism Of IL-17B Regulating Listeria Monocytogenes Infection In Mice

Background:Listeria monocytogenes（L.monocytogenes）,a foodbome,pathogenic gram-positive bacteria,mainly affect pregnant women,fetuses,newborns,elderly and other people with low immunity.Listeria monocytogenes can cause systemic infection after invading human body,and its clinical manifestations include fever,headache,liver abscess,gastroenteritis,meningitis,encephalitis,abortion,septicemia,etc.At present,the clinical treatment of Listeria monocytogenes infection is mainly antibiotic treatment,but due to poor prognosis and high mortality,it still needs extensive attention from all sectors of society.Therefore,it is of great significance to study and explore the pathogenesis and new treatment of Listeria monocytogenes infection.IL-17,a new type of cytokine,first discovered and named in 1993,which is closely related to the occurrence and development of inflammation,autoimmune diseases and cancer.Study has found that IL-17 can participate in the host’s resistance to bacterial,fungal and other microbial infections.The IL-17 family includes six family members from IL-17A to IL-17F.It has been reported that in Listeria monocytogenes infection of mice,IL-17A up-regulates MHC-I molecules by stimulating dendritic cells,promotes the proliferation and activation of primitive T cells,and enhances cytotoxic T cell response.But the role of IL-17B in Listeria monocytogenes infection is unknown.Therefore,study the role and mechanism of IL-17B in mice with Listeria monocytogenes infection is expected to provide a new theoretical basis for the treatment of Listeria monocytogenes infection.Objective:To study the role and mechanism of IL-17B in Listeria monocytogenes infection in mice,and to explore the relevant mechanism of IL-17B regulating host immune response,and provide new theoretical basis for the treatment of Listeria monocytogenes infection.Method:1.Establish an acute mouse model of Listeria monocytogenes infection.（1）Determine the quantity of Listeria monocytogenes.Listeria monocytogenes which stored at-80℃ was cultured in the brain heart infusion at a ratio of 1:1000.After incubating for 16 hours at 37℃,the bacterial number was determined by plate count method.（2）Establish an acute mouse model of Listeria monocytogenes infection.C57BL/6（wild type）mice and IL-17B deficient(Il17b^-/-)mice were injected intravenously with 2×104 colony forming unit（CFU）of Listeria monocytogenes 1 9 115.2.To explore the role of IL-17B in Listeria monocytogenes infection in mice,the model of Listeria monocytogenes infection in mice was established.The mice were killed at 12 hours,24 hours and 48 hours after infection,and the corresponding tissues of the mice were taken for detection.（1）The livers and spleens of mice were collected,and the expression levels of IL-17B and IL-17RB in mice infected with Listeria monocytogenes and non-infected mice were detected by qRT-PCR.（2）The livers and spleens of mice were collected.Then the bacterial colonization in the spleen and liver of wild type mice and Il17b^-/-mice were detected by plate count method.（3）Collect the livers and spleens of mice,H&E staining was used to evaluate histopathological damage.（4）The peripheral blood and spleen of wild type and Il17b^-/-mice were collected,and the proportion of macrophages,NK cells,B cells,DC,neutrophils,CD4+T cells and CD8+T cells were detected by flow cytometry.（5）Collect the serum and spleen of wild type and Il17b^-/-mice,and detect the levels of IL-6,IL-12p40 and TNF-α in corresponding tissues by ELISA.（6）Collect the spleen of wild type and Il17b^-/-mice,and detect the levels of IL-1β,IL-6,IL-12,TNF-α,IFN-γ,iNOS in spleen by qRT-PCR.3.To study the effect of IL-17B on macrophage differentiation.Bone marrow-derived macrophages（BMDMs）of wild type and Il17b^-/-mice were induced in vitro and infected with Listeria monocytogenes.The expression of CD86 on the surface of macrophages was detected by flow cytometry.4.To study the effect of IL-17B on phagocytosis of macrophages.BMDMs of wild type and Il17b^-/-mice were induced in vitro and infected with Listeria monocytogenes.After 1 hour of infection,the phagocytosis of macrophages was measured by plate count method.RESULTS:1.The mRNA expression of IL-17B in the spleen of Listeria monocytogenes infected mice increased.And the expression of IL-17B has tissue differences.2.（1）Compared with wild type mice,bacterial colonization in the spleens of Il17b^-/-mice reduced,but there was no significant difference in the livers;（2）There was no significant difference in pathological damage of spleens and livers between wild type and Il17b^-/-mice;（3）The proportion of macrophages,M1 macrophages in the spleens of Il17b^-/-mice increased;（4）IL6 expression were significantly increased in serum and spleens of Il17b^-/-mice;（5）The mRNA expression of IL-1β,IL-6,IL-12,TNF-α,IFN-y,and iNOS were significantly increased in the spleens of Il17b^-/-mice.3.In vitro studies have shown that IL-17B doesn’t affect the differentiation of BMDMs into M1 macrophages,nor affect the ability of BMDMs to phagocytic Listeria monocytogenes.Conclusion:IL-17B can regulate the host immune response induced by Listeria monocytogenes infection by inhibiting the infiltration of macrophages,and inhibit the clearance of Listeria This process may be related to the inhibition of IL-6 secretion by IL-17B.