Responses And Mechanisms Of Mucosal Immunity Regulated By Resident Intestinal Bacteria In Genetically Engineered Mouse And The Regulation Of Herbal Medicine

Mucosal homeostasis requires interactions between enteric microbiota, genetic susceptibility, effector and regulatory immune responses, and environmental triggers.Gnteric microbiota might break the homestasis in certain conditions, and might induce T cell mediated immune responses, which highly threat both human and animal health.Intestinal immune responses is the key factor in maintain mucosal homeostasis. To explore how resident intestinal bacteria regulate mucosal homestasis and the mechamisms is very attractive.Escherichia coli NC 101 and Enterococcus faecalis are enteric microbiota, which could be found in both human and animal gastrointestinal tract. They could not cause any disease in the healthy host, however, there would induced intestinal mucosal immune responses if hosts' immune system are dysfuction, and then followed with diarrhea and colitis. IL-10 is a key mediator of mucosal homeostasis. IL-10 deficient mice are well established model to study intestinal immune responses, which because germ free IL-10 deficient mice had no evidence of immune systrm activation, however, it develops T cell regulated mucosal immune responses when be kept in SPF condition.In the present study, E. coli NC 101 monoassociated 129 background wildtype and IL-10 deficient mice were used to investigate how resident intestinal bacteria stimulated IL-10- mediated regulation of mucosal immune responses. C57B6 background IL-10 deficient mice monoassociated with E. coli NC 101 were used to explore the hosts' immune responses after colonization and also the histologial injury. We also evaluated how ATG16L1, MyD88, and NOD2 gene influent the function of Antigen Presenting Cells while mice were monoassociated with E. coli NC 101. To attempt study if Matrine is a potential medicine for spontaneously developed colitis,in vivo and in vitro studies were performed, and also studied the mucosal immunity mechanisms.1. Resident intestinal bacteria E. coli NC 101 stimulate IL-10- mediated regulation of mucosal immune responses129 background Germ Free wild type mice and IL-10 deficient mice were used in this study to investigate resident intestinal bacteria activate host immune responses and cytokine production. Germ free wild type mice and IL-10 deficient mice monoassociated with E. coli NC 101 were examined before colonization, 4 days, 7 days and 30 days post colonization. IFN-y secreted by unseparated MLN cells stimulated with E. coli NC101 lysate at 10 ?g/mL were measured by ELISA. Proinflammatory cytokines IFN-y and IL-17 secreted by unseparated splenocyte stimulated with E. coli NC101 lysate at 10 ?g/mL were measured, and also the spontaneous secretion of IL-12/23p40 in colon fragment culture supernatants were measured by ELISA. Then, we did a time course study, wild type mice was monoassociated with E. coli NC 101 and was sacrificed before monoassociation,post infection by 4d, 7d, 14d and 30d, respectively. IFN-y and IL-10 secretion in the supernatants of unseparated MLN cells stimulated with E. coli NC 101 lysates at 10 ?g/mL and 1 ?g/mL were measured through ELISA. IFN-y and IL-10 mRNA expression in cecal tissue from monoassociated mice were assessed using real-time PCR.To better understanding the regulation of mucosal effector immune responses by IL-10 induced by resident microbiota. Unfractionated MLN cells harvested from 4 day monoassociated mice were treated with additional recombinant IL-10 at 250 pg/mL, 500 pg/mL, 1000 pg/mL and 2000 pg/mL, respectively. And, anti-IL-10R antibody was exposed to unfractionated MLN cells from day 7 and 14 monoassociated mice. IFN-y and IL-10 secretion were measured through ELISA. In vivo, mice were treated with I.P. anti-IL-10R or isotype control antibody one day before, two days and five days after colonization with E.coli NC 101, then IFN-y and IL-10 secreted by MLNs activated by E. coli NC 101 lysates and cecal mRNA expression were measured.Our results showed that IFN-y secretion in the supernatants of E. coli- stimulated MLN cells dramatically increased by day 4 after monoassociation compared to Germ Free mice, then decreased in later time points, while that of the IL-10 deficient mice consistently high. IL-10 secretion showed a progressive but not significant increase over this time course, but no statistical difference. Likewise, IFN-? mRNA expression demonstrated transiently enhanced expression at day 4 with a subsequent decrease,while IL-10 expression slightly increased from GF to day 14, then decreased at day 30. Recombinant IL-10 added to MLN cell culture showed dose dependent decreased IFN-y secretion. On both day 7 and day 14, IL-10 receptor neutralization stimulated higher IFN-y secretion in the supernatants of MLN culture than did the isotype control antibody. In vivo,IFN-?secretion was stimulated by neutralizing anti-IL-10R antibody compared with isotype control antibody, with no change in IL-10 secretion. In parallel, IFN-? mRNA expression in cecal tips of the anti-IL-10R antibody injection group was significant higher than that of the isotype control antibody group. Our results suggest that intestinal bacteria stimulate MLN cells from 129 mice to produce IFN-? soon after bacterial colonization, but is downregulated by intestinal IL-10 stimulated by endogenous microbiota. Bacterial-stimulated IL-10 is a key mediator of mucosal homeostasis via its ability to regulate IFN-y production.2. Host immune response to E. coli NC 101 monoassociation in C57B6 IL-10 deficient miceGerm Free C57B6 background IL-10 deficient mice were monoassociated with E. coli NC101 by gavaged with 0.2 mL, OD600 = 0.9-1.0, overnight bacterial culture. In order to investigate how enteric microbiota cause host's immune responses, we sacrificed mice by before monoassociation, 5 w, 10 w, 11 w and 12w after initial bacterial colonization,respectively. We tested the histological score,cytokine secretion by unseparated splenocyte and unseparated mesenteric lymph nodes cells with E. coli NC 101 lysates at 10 ?g/mL, 1?g/mL and 0.1 pg/mL; we also tested spontaneous secretion of IL-12/23p40 in colon fragment culture supernatants from E. coli NC 101 monoassociated C57B6 IL-10 deficient mice, and IFN-? and IL-17 production from co-cultured supernatants of CD4+ T cells with antigen presenting cells.The results showed that there was mild inflammation in histological slides at the time course study, and the IL-12/23p40 secretion was quite similar in different time points after monoassication which was significant higher than that in the GF condition. Unseparated splenocyte and unseparated MLN cells producted extremely high amount of proimflamatory cytokines when stimulated with E. coli NC 101 lysate. The results presented herein indicated that monoassociation with E. coli NC 101 would activate immune responses and mucosal immune responses in C57B6 background IL-10 deficient mice, lymphocytes were activated sufficiently at 5 weeks post infection. And E. coli NC 101 monoassociation did not cause histological severe injury in the intestine of C57B6 background IL-10 deficient mice.3. Regulation of Antigen Presenting Cells function by ATG16L1, MyD88, and NOD2 geneIn this study we used C57B6 background IL-10 knockout mice, ATG16L1 hypermorph mice, MyD88 knockout mice and NOD2 knockout mice, attend to compare if and how ATG16L1, MyD88, and NOD2 gene would influence the antigen presenting cells'function. First, we used alive bacteria E. coli NC 101 and E. coli K12 to stimulate macrophages which isolated from spleen cells of ATG16L1 hypermorph mice, MyD88 knockout mice and NOD2 knockout mice, respectively, to evaluate bacterial uptaken by macrophages and survival ratio in 24 h and 48 h within macrophages. The results demonstrated that survival ratio of E. coli NC 101 was significant different as compared to E.coli K12 in the macrophages coming from spleen of ATG16L1 hypermorph mice and MyD88 knockout mice, while, there was no difference be found in NOD2 knockout mice.And E. coli NC 101 got higher survival ratio in spleen macrophages coming from ATG16L1 hypermorph mice as compared to that from wild type mice. Then, we isolated CD4+ T cell from MLN of the monoassociated C57B6 background IL-10 knockout mice which we developed in our former research, co-cultured with APCs coming from these three gene deficient mice. IFN-y and IL-17 production from co-cultured supernatants of CD4+T cells with APCs were measured via ELISA, to investigate how ATG16L1,MyD88,and NOD2 gene affect presenting functions. The results showed that IFN-y and IL-17 secretion from CD4+ T cells and APCs co-culture showed no difference. At last, in order to examining how macrophages and dendritic cells presenting alive resident intestinal bacteria to host, we used ATG16L1 hypermorph mice to isolate bone marrow derived macrophages and dendritic cells. IL-10, IL-12 and TNF-a from the culture supernatants were measured through ELISA. The results demonstrated IL-10 secretion in culture supernatants from bone marrow derived macrophages of ATG16L1 hypermorph mice got peak after stimulated with alive bacteria and cultured 8 h, and IL-12 reached the maximum at 12 h after culture, while TNF-a secretion in 6 h after culture showed difference as compared to that in wild type mice only when the cell number was 10.0x105 cell/well. These results indicated that ATG16L1 gene would influence T cell activation and production of proinflammatory cytokines, macrophages and dendritic cells which involved in innate immune responses would produce more cytokine when ATG16L1 gene is down regulated.4. Effect and mechamisms of mucosal immunity of Matrine for spontaneously developed colitis in interleukin-10-deficient miceInterleukin-10 (IL-10)-deficient mice spontaneously develop T cell-mediated colitis.Previous reports have shown that Matrine may reduce the symptoms of acute colitis induced by trinitrobenzene sulfonic acid (TNBS). However, whether Matrine impacts chronic colitis remains unknown. In this study, we investigated whether Matrine could limit the symptoms of spontaneously developed colitis and its potential molecular mechanisms.IL-10 deficient mice were given Matrine or a PBS control by oral gavage daily for 4 weeks and were euthanized at week 2 or week 4. We measured body weight, colon length and weight, and histological scores. We also evaluated the spontaneous secretion of IL-12/23p40, IFN-? and IL-17 in colon explant cultures as well as IFN-? and IL-17 secretion in unseparated mesenteric lymph node (MLN) cells, and assessed IFN-?,IL-17, IL-1? and IL-6 mRNA expression in colon tissue. In addition, we analyzed the proportions of CD4-positive and CD8-positive cells in unseparated MLN cells. Our results show that Matrine-treated mice exhibited better body weight recovery than controls and that histological scores and spontaneously secreted IL-12/23p40,IFN-? and IL-17 in colon tissue were significantly decreased in treated mice compared with controls. The proportion of CD4-positive cells of MLNs in treated mice was significantly smaller than that in controls at week 4. Both cytokine production and mRNA expression of IFN-? and IL-17 were significantly reduced in treated mice compared with controls. Taken together, our results indicate that Matrine may ameliorate spontaneously developed chronic colitis and could be considered as a therapeutic alternative for chronic colitis.