The Interaction Between Gut Microbiota And The Host Immune System

Symbiotic bacteria,the “external organ”,is known as an important regulator of the host immune system.However the role of gut microbiota in the autoimmune diseases and inflammations is still unclear.In the present study,taking advantage of model animals(pigs and mice),we showed several novel interactions between commensal bactirea and host immune systems,opening new approaches to the disease-resistance and bacteriostasis fields.Experiment 1 Gut microbiota differs in pig breeds and carry out immunophenotypes to GF mice through fecal microbiota transplantation(FMT)The aim of the present study was to compare the intestinal microbiota communities and immunogenetic features in two distinctive porcine model: the Yorkshire and the Tibetan,and to further investigate the influence of FMT on germ-free(GF)mice.The pigs took the same diet without antibiotics in the same environment for 56 days.Then feces was collected from Yorkshire and the Tibetan pigs(each n=5)for 16 S r RNA sequence-based microbiome analysis,the leftover was stored at-80℃ for FMT.Then the pigs were sacrificed,spleens,large intestines were collected for calculation of the splenic and large intestinal indices.Colonic samples were collected for cytokines measurments.Then we colonized the fecal microbiota of two pig breeds to germ-free mice by FMT to investigate whether commensal bacteira carry and transmit certain phenotypes from donors to receptor animals.Sixteen 1-day-old BALB/C mice were used in this study and randomly assigned to 2 groups(each n=8): Yorkshire lora-associated mice(YM)and Tibetan flora-associated mice(TM).At day 28 after colonization,the mice were sacrificed for spleen and large intestine collections.Splenic and large intestinal indices were measured and calculated,the colonic cytokines were analyzed.The results are as follows:1)16S r RNA sequence-based microbiome analysis indicated that the fecal microbiota communities in Yorkshire and Tibetan pigs were significantly different.Moreover the gut microbial communities of mice received pig feces were partly similar to the donors.2)Both spleen index and the large intestine index were higher in the Tibetan pigs,and spleen index was also higher in Tibetan flora-associated mice and their Yorkshire counterparts.3)Pro-inflammatory cytokines such as TNF-α,IL-1,IL-6 and IL-17 were found at higher level in the intestinal tissues of Tibetan pigs rather than in the Yorkshires,and same tendency had been found in the mice received Tibetan feces compared with those received Yorkshire feces.These results indicated that commensal microbial structure is specific to pig breeds.Gut microbiota may carry certain immunological characteristics,which may be transmitted between animal species by microbita transplantation.Experiment 2 The effects of early intestinal microbiota intervention on the immune system of piglets and mechanisms involvedTo further investigate the immunological effect of FMT on recipient animals,in this study,the fecal microbiota of Yorkshire and Tibetan pigs was transplanted to healthy commercial hybrid newborn piglets to establish “Tibetan/Yorkshire-intervened” porcine models.Then acute colitis was induced by dextran sulphate sodium(DSS)to test whether the Tibetan-intervened piglets had higher resistance to DSS-induced colitis.30 3-day-old DLY piglets were used in this study and randomly assigned to 5 groups(each n=6): Yorkshire pig fecal microbiota interfered(Y-int);Tibetan pig fecal microbiota interfered(T-int);Yorkshire pig fecal microbiota interfered with DSS(Y-int-DSS);Tibetan pig fecal microbiota interfered with DSS(T-int-DSS)and the negative control(CTL).The whole trial lasted for 56 d.The results are as follows:1)The DSS-treated groups showed disease phenotypes compared to control group,indicating experimental colitis was successfully induced.2)The weight change rate,diarrhea index and bloody stool index,as well as the morphology togather suggested that T-int-DSS group had stronger resistance to DSS-induced colitis compared with Y-int-DSS group.3)DSS administration promoted multiple proinflammatory cytokines in Y-int group,but not T-int group.4)The CD4/CD8 ratio,Ig A+ plasma cells and MAC387+ macrophages in the Y-int group were significantly increased by DSS-induced colitis,while those in the T-int piglets remained unchanged.5)DSS administration activated Toll/NOD-like receptor(TLR/NLR)signals in the colonic tissues of Yorkshire fecal microbiota-intervened piglets,while it had little influence on the “Tibetan-intervened” piglets.6)Compared with the Yorkshire fencal microbiota,the Tibetan fecal microbiota interference increased the levels of propionic acid,butyric acid and Ig A in the intestinal lumen of piglets.These results indicate that pigs inoculated with Tibetan microbiota acquired relatively strong resistance to experimental colitis,mechanically related to intestinal immune cells,PRR signaling pathways and SCFAs regulated by gut microbiota.Experiment 3 The effects of short chain fatty acids(SCFAs)on host immune responses and the mechanisms involvedThis study was conducted to answer the question remained in Experiment 2:whether the increasing of antibodies like Ig A is correlated with the increasing of SCFAs.(1)Twenty 8-week-old healthy wildtype(WT)C57BL/6 mice were randomly assighed to 4 groups: SCFAs treated(500m M acetate and 500 m M butyrate);Antibiotics(Abx,ampicillin,gentamycin,metronidazole and vancomycin,1 mg/ml each)treated;SCFAs and Abx treated and control(CTL).The whole trial lasted for 28 d.(2)Meanwhile,anther 5 WT and 5 GPR43-/-mice were used to study the role of GPR43 in the induction of antibodies by SCFAs.All mice were immunized with ovalbumin(OVA)and cholera toxin(CT)at d 0 and 14.(3)Then,Ig D+ na?ve B cells were isolated from the spleen of WT mice and treated with SCFAs in vitro in multiple conditions to investigate the non-speicific antibodies-induction capability of SCFAs on B cells.In addition,the hydrogen isotope was used to mark the na?ve T cells isolated from the spleens of both WT and GPR43-/-mice to investigate whether SCFAs increase T cell proliferation under immunization conditions.The results are as follows:1)Gut microbiota metabolites SCFAs promoted intestinal and peripheral antigen-specific antibodies(OVA/CTb-speicific Ig A and Ig G)responses in WT mice.2)Antibiotics treatment largely decreased the levels of antigen-specific antibodies,which could be partly recruited by supplementing with SCFAs.3)The levels of antigen-specific antibodies in GPR43-/-mice were lower that those in the WT mice.4)SCFAs induced Ig A in B cells in both T cell-dependent condition and T cell-independent condition,and induced Ig G only in T cell-independent condition.5)SCFAs improved T cell proliferation,which was slower in GPR43-/-mice than their WT counterparts.By these results,we identified a new function by which microbiota promotes intestinal antigen-specific and non-speicific Ig A/Ig G,as well as T cell proliferation through “metabolite-sensing” GPR43 pathway by producing SCFAs in immunization conditions.Experiment 4 The role of IL-33 in the interaction between host and gut microbiotaIn this study,we investigated whether and how IL-33,an alarmin produced by IEC in response to injures,regulates antimicrobial protein(AMP)expressions in IEC,thus,contributing to intestinal homeostasis by several associated in vivo and in vitro studies.(1)We first compared the gut microbial structures of wild type(WT)and IL-33-/-mice by 16 S r RNA sequence-based microbiome analysis.(2)Then,the expression levels of REG3γ and sevral β-defensins in the intestinal epithelial cells(IECs)of WT and IL-33-/-mice were measured.(3)Then we studied the mechanism by which IL-33 induced REG3γ using mice epithelial cell line MSIE cells in vitro.The in vitro antibacterial properties of REG3γ was further tested by treating gram-positive Clostridium difficile and gram-negative Citrobacter rodentium with the crude extracts of IL-33 treated and untreated normal/ REG3γ-deficient MSIE cells.(4)Afterwards experimental colitis was induced by DSS in both WT and IL-33 deficient mice to explore the role of IL-33 in the inflammation resistance.The results are as follows:1)The microbial communities of WT and IL-33-/-mice showd significant difference,mainly reflect in Lachnospiraceae and Desulfovibrio.Compared with WT mice,IL-33-/-mice showed higher abundance of total Bacteria,and weaker scavenging activity to C.rodentium.2)The expression of REG3γ in IEC of IL-33-/-mice was significantly lower than wild-type(WT)mice.3)IL-33 treatment induced the expression of REG3γ,but notβ-defensins in MSIE cells in vitro.Mechanistically,IL-33 activated STAT3,m TOR,and ERK1/2 in IEC.Inhibition of those pathways abrogated IL-33-induction of REG3γ.4)IL-33-/-mice had stronger resistance to DSS-induced colitis compared with the WT mice.In conclusion,this study demonstrated that IL-33 promotes IEC expression of AMP REG3γ,and controls the gut microbiota of the host through “m TOR-STAT3” and “MEKERK-STAT3” pathways.Experiment 5 IL-33 inhibits Treg cells in the presence of transforming growth factorbetaIn experiment 4,surprisingly,IL-33-/-mice showed stronger resistance to DSS-induced colitis.To find out the reason,in this study,(1)we first compared the CD4+ Foxp3+ Treg cells in the lamina propria lymphocytes(LPL)and intestinal epithelial lymphocytes(IEL)of both mice lines in healthy and colitis status.(2)Then,CD4+ CD62L+ na?ve T cells were isolated from CBir1 Flagellin-specific TCR transgenic mice,the T cell receptor of which can be activated only by Cbir1 flagellin,and treated with IL-33 in vitro in different transforming growth factor-beta(TGF-β)conditions to study the effect of TGF-β in the induction of Treg by IL-33.The results are as follows:1)The content of Treg cells was higher in the LPL of IL-33-/-mice than in WT mice in both healthy and inflammatory status.2)IL-33 promoted the differentiation of Treg cells from na?ve T cells only in the absence of TGF-β,otherwise IL-33 inhibited the differentiation of Treg cells from na?ve T cells.3)ST2,the receptor of IL-33,was found to express at the surfaces of CD4+ CD62L+ na?ve T cells.IL-33 promotes its receptor,ST2 only in the absence of TGF-β.These results indicated that endogenous IL-33 inhibits Treg in both inflammatory and healthy conditions because of the existence of TGF-β,and this might be reason that IL-33-/-mice had stronger resistance to DSS-induced colitis.In conclusion,gut microbial communities are different between pig breeds,and are able to carry and transmit certain immunological characteristics from one host to another.Gut microbiota targets intestinal mucosa,regulating cellular and humoral immunity,promote CD4+ T cell proliferation and induce the synthesis of antigen-specific and non-specific immunoglobulin through PRR signaling pathways and SCFAs-GPR43 pathway.Thus,under such circumstance,gut microbiota protects intestinal mucosa,suppresses the degree of enteritis and diarrhea occurred under stress conditions.On the other hand,hosts controls their microbiota by antimicrobial proteins like REG3γ,which could be mediated by IL-33 through m TOR-STAT3 and MEK-ERK-STAT3 pathways.Moreover,IL-33 inhibits Treg in in the presence of TGF-β.Taken togather,the present study preliminary revealed some of the functions and mechanisms of intestinal microbiota-host immunity interactions.