Mechanism Of Th17 Differentiation Regulated By Prevotella And Its Protective Effect On Mycobacterium Tuberculosis Infection

Tuberculosis（TB）is the tenth leading cause of death and has the highest death rate caused by a single pathogen.The WHO’s End TB Strategy aims to reduce TB-related deaths by 95per cent and TB incidence by 90 per cent from 2015 to 2035.According to the Global Tuberculosis Report 2020,there were about 10 million new TB cases and 1.4 million TB-related deaths worldwide in 2019.There are about 1.7 billion latent TB infected（LTBI）individuals worldwide.Preventing and stopping the progression of LTBI to active TB（ATB）is therefore key to achieving the goals of the End TB Strategy.Studies have shown that Th17 plays a protective role against TB infection and may help maintain LTBI state.For example,Th17 can promote the formation of Tertiary Lymphoid Structures（TLS）,enhance the contact between antigen-presenting Cells（APC）and lymphocytes,and enhance the Th1-type immune response.Th17 also induces stromal cells to express chemokines to recruit Polymorphonuclear（PMN）cells to the site of TB infection.It is reported that the gut microbiome was associated with immunity against TB infection.For example,the proportion of Prevotella in the intestinal tract of ATB patients was significantly lower than that of healthy controls,and the number of CD4⁺T cells in the peripheral blood of ATB patients was positively correlated with the relative abundance of Prevotella in the intestinal tract.This suggests that intestinal Prevotella may contribute to the host immune system’s resistance to TB infection.Interestingly,in vitro studies have proved that Prevotella can induce Th17 differentiation.For example,when Prevotella Melaninogenica was co-incubated with T cells isolated from lymph nodes,the proportion of Th17 and the expression level of IL-17A gene in T cells were significantly increased.Based on this,we hypothesized that intestinal Prevotella may regulate intestinal and pulmonary mucosal immunity by inducing intestinal Th17 differentiation,and play a protective role in the immunity of tuberculosis infection.The purpose of our study was:（1）To investigate the relationship between intestinal Prevotella and TB infection;（2）To verify the effect of intestinal Prevotella on the differentiation of mouse colon Th17 cells and explore the mechanisms;（3）To clarify the protective effect of IL-17A in immunity against TB infection;（4）To verify the protective effect of intestinal Prevotella and its induced Th17 in the immune response to TB infection.Part 1.The relative abundance of intestinal Prevotella correlates with the latent infection status of Mycobacterium tuberculosisObject:To investigate the characteristics of intestinal microflora in patients infected with tuberculosis at different pathophysiological stages.And to find out the intestinal bacteria related to the immunity of latent Mycobacterium tuberculosis infection.Methods:The intestinal microbiota composition of 19 patients with Active Tuberculosis（ATB）,21 with Latent Tuberculosis Infection（LTBI）and 20 Healthy controls（HC）were determined by 16S r DNA sequencing.Spearman correlation analysis was used to test the correlation between the relative abundance of specific intestinal bacteria and IFN-γproduction in IGRA experiment.Results:The diversity of intestinal microbiota in LTBI and ATB group was significantly decreased compared with HC group.The relative abundance of Clostridium in M.tuberculosis infected individuals（LTBI group and ATB group）decreased significantly compared with the HC group.Specifically enriched species of the three groups were as follows:（1）Enterobacteriaceae,Enterobacteriales,Gammaproteobacteria,Proteobacteria and Erysipelatoclostridium in ATB group;（2）Prevotella and Roseburia in LTBI group.（3）Clostridia,Clostridiales,Lachnospiraceae,Ruminococcaceae,Faecalibacterium,Agathobacter,Subdoligranulum,Ruminococcus and Fusicatenibacter in HC group.The relative abundance of Prevotella（enriched in the LTBI group）was positively correlated with the production of IFN-γfrom peripheral blood after incubation with the antigens of M.tuberculosis.While the relative abundance of Clostridium,Clostridium,Agathobacter and Subdoligranulum（enriched in the HC group）was negatively correlated with the production of IFN-γby peripheral blood after incubation with the antigens of M.tuberculosis.Conclusion:The intestinal microbial diversity decreased significantly after M.tuberculosis infection.Prevotella is specifically enriched in gut microbiota of LTBI and is associated with anti-tuberculosis immunity.Part 2.Prevotella induce the production of Th17 cells in the colon of miceObject:To investigate whether intestinal Prevotella can induce Th17 cell differentiation and to explore the mechanism.Methods:Mice were treated with a combination of broad-spectrum antibiotics（including ampicillin,neomycin sulfate,vancomycin hydrochloride and metronidazole）in their drinking water for 4 weeks and then gavaged with Prevotella for 4weeks.After inoculation,16S r DNA sequencing was used to verify the colonization of Prevotella in the colon of mice.The IL-17A as well as IL-17A-expressing T cells was localized and quantified by immunofluorescence assay（IFA）of colon sections.Th17 cells in the mesenteric lymph nodes of mice were counted by flow cytometry.Systemic immune response to Prevotella colonization was evaluated based on the serum levels of IL-6,TNF-α,IL-1β,IL-17A,IL-10,IL-4,IFN-γ,and IL-2.Th17-polarizing cytokines（IL-6,TNF-α,IL-1β,and IL-2）induced by Prevotella were evaluated by stimulation of bone marrow-derived dendritic cells（BMDCs）.Results:After inoculation,Prevotella successfully colonized in the intestine of mice and induced the production and accumulation of colonic Th17 cells in the colon.Moreover,Prevotella elevated some of the Th17-related cytokines in the serum of mice.And Th17-polarizing cytokines（IL-6 and IL-1β）produced by BMDCs were mediated mainly through the interaction between Prevotella and Toll-like receptor 2（TLR2）.Conclusion:Prevotella induce the production of Th17 cells in the colon of mice,and the cytokines related to Th17 differentiation produced by BMDCs are mainly mediated by TLR2.Part 3.Protective role of IL-17A in anti-TB immunityObject:To investigate the role of IL-17A in anti-infection immunity of tuberculosis.Methods:Il-17a^-/-mice were infected with the standard strain of Mycobacterium tuberculosis（MTB）H37RV through respiratory tract.And the load of Mycobacterium in the organs and serum cytokine level was detected.Lung histomathology evaluation was done to describe the damage of lung tissue in H37RV infected mice.Results:The level of serum IL-17A increased after H37RV infection.Compared with C57BL/6 mice,Il-17a^-/-mice infected with H37RV had significantly increased pulmonary bacterial load and more severe pulmonary pathological injury.The level of serum IFN-γin Il-17a^-/-mice infected with H37RV was lower than that in C57BL/6 mice,while IL-1βand IL-4 were significantly increased.For Il-17a^-/-mice infected with H37RV after BCG-immunization,the pulmonary bacterial load decreased significantly,and the pulmonary pathological injury was alleviated.Serum IL-1βand IL-4 level of Il-17a^-/-mice infected with H37RV after BCG-immunization,was significantly lower than those of unimmunized Il-17a^-/-mice.Conclusion:IL-17A has a protective effect on immunity against tuberculosis infection in mice,and IL-17A is dispensable for BCG-induced immune protection against tuberculosis.Part 4.Protective effect of Th17 regulated by intestinal Prevotella on immunity against mycobacterium infectionObject:To investigate whether the immune protection of intestinal Prevotella in early tuberculosis infection is related to the regulation of intestinal Th17 cell differentiation.Methods:4-6 week-old female C57BL/6 mice were divided into 3 groups（5 mice in each group）:（1）After intragastric colonization of P.copri,mice were infected with BCG（10⁶cfu）by nasal drip（PCP+BCG）;（2）After blank medium（BM）gavage,mice were infected with BCG（10⁶cfu）by nasal drip（BM+BCG）;（3）No gavage or intranasal infection（BLANK）;Four weeks after infection,the mice were sacrificed for the analysis of the load of bacteria in the organs,for the evaluation of lung histopathology,for the analysis of immune cells by flow cytometry in the spleen and peripheral blood,and for the detection of plasma cytokines.Results:Compared with BM+BCG and BLANK groups,the secretion and distribution of colonic IL-17A in PCP+BCG group was significantly increased,and colonic IL-17A was mainly distributed in intestinal epithelial cells.Compared with BM+BCG group,pulmonary BCG bacterial load of mice in PCP+BCG group was significantly decreased（P=0.0059）,and the pulmonary pathological injury was alleviated.Compared with the BM+BCG and BLANK groups,the distribution of IL-17A in lung of mice in PCP+BCG group was significantly increased.No tertiary lymphoid structure（TLS）was found in the BLANK group.While improved TLS formation with enhanced B-cell follicle formation was found in the PCP+BCG group than in the BM+BCG group.Conclusion:Th17 induced by intestinal Prevotella contributes to the immune protection of Mycobacterium infection.Based on the above studies,we concluded that the relative abundance of intestinal Prevotella is related to LTBI,intestinal Prevotella can induce colon Th17 differentiation through the TLR2 signaling pathway in DC cells,and IL-17A can reduce the pulmonary bacterial load and alleviate pulmonary pathological injury.Thus,in BCG infected mice with intestinal colonization of Prevotella by gavage,intestinal Prevotella can protect the host by regulating intestinal Th17 cells.Through"Gut-Lung"axis,intestinal Th17 cells transfer to pulmonary mycobacterial infection site,and promote the protective immunity by inducing the formation of TLS.Our experiment confirmed the protective role of intestinal Prevotella regulated Th17 in the immunity of tuberculosis infection,laying a foundation for clarifying the mechanism of specific intestinal bacteria regulating immunity to TB infection.