Study On The Role And Mechanism Of MLNs CD169~+ Macrophage In Dss-induced Colitis

Research background:Inflammatory bowel disease(IBD)is a chronic inflammatory condition of the gastrointestinal tract,incident IBD cases were divided into two main clinical entities:Crohn’s disease(CD)and ulcerative colitis(UC),and characterized by recurrent episodes of bowel pain,discomfort,fatigue,and multiple putrid bloody stools.IBD is believed to develop as a result of a combination of environmental factors,genetic,gut microbiota and immune response,but the disease pathogenesis is not fully understood.The incidence of IBD is increasing with time in different regions around the world,indicating its emergence as a worldwide disease.Patients with long-standing extensive colitis are easy to develop colorectal cancer(CRC).Innate and adaptive immunity both play crucial roles in the pathogenesis of colitis,distinct macrophages populations of intestinal are responsible for their functional plasticity under different conditions.CD169 is first member of the sialic acid-binding IgG-like lectin(Siglec)family of proteins characterized by affinity to specifically sialylated ligands,and is expressed on subsets of macrophages in secondary lymphoid tissues in mice,such as lymph node subcapsular sinus(SCS),spleen marginal zone and tissues other than lymphoid,such as lamina propria(LP).CD 169+ macrophages are located at the entry sites of lymph or blood,present as gatekeepers at the sites where antigens enter and perform activation of T and B cells and anti-pathogen immune response.Mesenteric lymph nodes(mLNs)are crucial inductive sites,responsible for the induction phase of the mucosal immune responses,after antigen uptake,intestinal dendritic cells(DCs)mature and transported to the draining mLNs,where they encounter and prime naive T cells,determining the differentiation of naive T cells into specific effector cells,including Th1,Th2,or the recently identified Thl7cells.These colitogenic effector cells then migrate from the mLNs to the gut where they initiate intestinal inflammation.How the sinus macrophage subset expressing CD 169 in mLNs regulate the mucosal inflammation remains elusive,the aim of this study was to reveal the regulatory roles and the molecular mechanism of mLNs SCS CD169+ macrophage subset in mucosal inflammation.Research purposes:C57BL/6 WT and CD169-DTR female mice received 3.5%DSS treatment to induce experimental IBD models.To reveal the roles of CD169+ macrophage subset of mLNs in mucosal inflammation by using these models,and explore the etiology and pathogenic mechanism of IBD,thus provide theoretical and clinical application potential experimental basis for reveal the complex etiology and pathological mechanism of IBD.Research methods:WT mice and CD169-DTR mice received one cycle(3 days or 7 days)of 3.5%DSS treatment in drinking water,to induce experimental models.Body weight detection and prevalence situation observation was monitored daily.Firstly,flow cytometry analysis,immunofluorescence histochemistry and real-time PCR were performed to examine the change of CD169+ macrophages in mLNs from DSS-treated mice,to ensure that CD169+ macrophages in mLNs would be an important subset regulating mucosal inflammation in the pathogenesis of colitis.Next,the deletion effect of CDllb+CD169+macrophages in mLNs by DT administration was detected by flow cytometry,immunofluorescence histochemistry and real-time PCR,the histopathological changes of colitis between WT and CD169-DTR mice were compared,to reveal the important roles of mLNs in the pathogenesis of UC.Finally,the change of inflammatory cells and the expression levels of cytokines and chemokines in mLNs from colitic WT and CD169-DTR mice were analyzed by flow cytometry,real-time PCR and ELISA,to explore the molecular mechanism of CD169+ macrophages in mLNs regulating the inflammation.Results:Firstly,experimented colitis models were induced successfully by DSS administration,the result revealed that the number of infiltrated cells and mRNA levels of inflammatory cytokines such as IL-17,IL-21,IL-23,IL-6,IL-1β,TNF-α,IL-12,IL-18 and chemokines such as CCL8,CCL3 are much higher in mLNs tissues from DSS treated mice than those from non-treated mice,the expression of CCL22 was significantly decreased.The subset expressing CD169 increased significantly during the DSS-induced colitis,and the result of immunofluorescence histochemistry showed that CD11b+CD169+ macrophages were mainly localized in the sinus of the mLNs.However,CD 169+macrophages in mLNs could be effectively deleted by DT administration in CD 169-DTR mice,compared with severe colitis in WT mice,DSS treated CD169-DTR mice showed moderate symptoms,including loss of body weight,shortening of colon length and tissue injury.The results clearly showed typical colitis caused by DSS treatment did not be observed in CD169-DTR mice,which raised the possibility that CD169+ macrophage in mLNs would be an important subset regulating mucosal inflammation in the pathogenesis of colitis.On the contrary,the CCL22 expression level decreased in mLNs from colitic WT mice and its levels from CD 169-DTR mice was much higher than that from WT mice with DSS-induced colitis,the expression levels of Th17 related cytokines,such as IL-17,IL-23,IL-21 and the number of Th17 cells was significantly decreased;And,further study revealed that supernatant of mLNs and colons from colitic mice could induce inflammatory cytokines production by mLNs cells and colons in vitro,and CD169+sinus macrophages could secreted higher levels of inflammatory cytokines directly,such as IL-17,TNF-a,IL-1β.Conclusions:The present study reported that sinus macrophage expressing CD 169 in mesenteric lymph nodes(mLNs)played its roles as key subset in DSS induced colitis models,the number of mLNs CDllb+CD169+ macphages was increased in mLNs tissues from colitic mice compared to control mice,the severity of DSS induced colitis could be improved in CD169-DTR mice due to,at least partially,the depletion of mLNs CD169+ macrophages.Further study revealed that the expression of Th17 related cytokines and the proliferation of Th17 cells decreased significantly,moreover,Supernatant of mLNs and colons from colitic mice could induce inflammatory cytokines production by mLNs cells and colons in vitro.These results demonstrated that the development of mucosal inflammation is related to the presence of CD 169 cells or not.CD169+ macrophages in mLNs may play essential roles on regulating mucosal inflammation through expressing higher levels of inflammatory factors directly.