Modulação da respiração anaeróbica em táxons disbióticos por IgG durante doenças inflamatórias intestinais: Estudos in silico e em modelo experimental

Abstract

Inflammatory bowel diseases, such as ulcerative colitis, generate changes in the intestinal lumen. Among them there is an increase in nitric oxide (NO) concentrations and an increase in the abundance of Proteobacteria, especially from the Enterobacteriaceae family, such as Escherichia coli. These are capable of using nitrate, S-oxides and N-oxides as terminal electron acceptors in anaerobic respiration. Thus, the excess of NO produced during colitis may favor the expansion of this family. It was recently observed in studies by our group that IgG can regulate NO production during ulcerative colitis, via activation of FcγR receptors. Therefore, the objective of this work is to understand the role of IgG in controlling the production of nitric oxide and its impact on the dysbiotic process generated by ulcerative colitis. We aimed to test the hypothesis that IgG controls the expansion of Enterobacteriaceae during intestinal inflammatory responses by regulating NO production through multi-omic analysis of a database of patients with IBD, in vitro experiments with dysbiotic isolates and in vivo experiments in a murine model of ulcerative colitis, induced by DSS (Sodium dextran sulfate). In this last system, we used animals deficient for the FcγRIIB receptor, which present alterations in their microbiota-reactive IgG repertoire, and competition assays between wild-type Escherichia coli strains or mutants incapable of using nitrate in anaerobic respiration. The serological profile allowed grouping patients with different IBD diagnosis. One of these groups, characterized by high titers of ANCA, showed differences in its intestinal microbiota, with a higher relative content of Citrobacter, Escherichia coli and Proteus, among other alterations. In addition, these patients had transcriptional alterations, characterized by enrichment of pathways involved in the immune and inflammatory response, indicating that the serological response is related to transcriptional and microbiota alterations. Fcgr2b-/- animals showed a marked colitis after exposure to DSS, associated with increased production of the chemokine CXCL1 and bacterial translocation to the liver. Furthermore, our data suggest that FcγRIIB is important to "instruct" the activation of macrophages, repressing NO production and inducing an increase in CD11b. In the absence of the receptor, the activation pattern is reversed. We also observed that the use of nitrate as a final electron acceptor confers some competitive advantage to bacteria isolated from feces and liver of Fcgr2b-/- animals, presumptively identified as E. coli. Competition assays using wild-type bacteria and ΔnarGnapAnarZ strains suggest that the use of nitrate as the final electron acceptor confers an advantage to bacteria during colitis in Fcgr2b-/- animals. Consistent with this, inhibition of iNOS prevents the competitive advantage of the wild-type strain over the mutant and results in less translocation of the wild-type strain into the liver of Fcgr2b-/- animals. In addition, we saw that the use of nitrate in anaerobic growth generates a lower production of indole, indicating a phenotypic change related to the virulence of the bacterium. In conclusion, these data demonstrate that IgG interferes with the composition of the microbiota during IBD in part by modulating the production of NO by the host and consequent expansion and eventual translocation of bacterial strains that use nitrate in anaerobic respiration.