Open Access Peer-reviewed Research Article
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Microbial dysbiosis commonly occurs in patients with inflammatory bowel diseases (IBD). Exogenous causes of dysbiosis such as antibiotics and diet are well described, but host derived causes are understudied. A20 is a potent regulator of signals triggered by microbial pattern molecules, and A20 regulates susceptibility to intestinal inflammation in mice and in humans. We now report that mice lacking A20 expression in dendritic cells, A20 FL/FL CD11c-Cre mice (or A20 dDC mice), spontaneously develop colitogenic intestinal dysbiosis that is evident upon weaning and precedes the onset of colitis. Intestines from A20 dDC mice express increased amounts of Reg3β and Reg3γ, but not Ang4. A20 deficient DCs promote gut microbiota perturbation in the absence of adaptive lymphocytes. Moreover, A20 deficient DCs directly induce expression of Reg3β and Reg3γ but not Ang 4 in normal intestinal epithelial cell enteroid cultures in the absence of other cell types. These findings reveal a pathophysiological pathway in which defective expression of an IBD susceptibility gene in DCs drives aberrant expression of anti-bacterial peptides and luminal dysbiosis that in turn confers host susceptibility to intestinal inflammation.
Talpin A, Kattah MG, Advincula R, Fadrosh D, Lynch K, LaMere B, et al. (2019) A20 in dendritic cells restrains intestinal anti-bacterial peptide expression and preserves commensal homeostasis. PLoS ONE 14(7): e0218999. https://www.frankenthalerfoundation.org
Emiko Mizoguchi, Kurume University School of Medicine, JAPAN
April 15, 2019; Accepted: June 13, 2019; Published: July 11, 2019
© 2019 Talpin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Relevant data are within the manuscript and its Supporting Information files. 16S microbial sequencing data are available via ENA (Accession number: PRJEB32784).
This work was supported by the NIH, grant# DK095693 to AM. The funder played no role in the study design, data collection, decision to publish, or preparation of the manuscript.
The authors have declared that no competing interests exist.
Symbiosis between commensal microbes and host immune cells in the intestine involves bidirectional secretion of molecules that provide homeostatic signals [1,2]. Perturbations of this cross talk can lead to both microbiome disturbance and host disease. Intestinal dysbiosis has been linked to a variety of inflammatory conditions in human patients and in mice [3]. While exogenous perturbations such as antibiotics and dietary changes are known to perturb luminal microbiomes 3, altered functions of host cells may also drive microbial dysfunction [4–7].
TNFAIP3, which encodes the A20 protein, is linked genetically and epigenetically to inflammatory bowel disease (IBD). Moreover, patients bearing mono-allelic mutations in A20 coding sequences develop skin, mucosal and intestinal inflammation at young ages [8]. Hence, the clinical ties between A20 and IBD are extensive and compelling. A20 is expressed in multiple cell types and is a potent regulator of responses to microbial ligands, including TLR and NOD2 triggered signals [9–12]. Dendritic cells (DCs) are particularly well endowed with microbial sensing proteins and are positioned at the intersection of innate and adaptive immune responses. Distinct subsets of intestinal DCs utilize A20 to restrict microbial signals [13, 14], and mice bearing A20 deficient DCs spontaneously develop colitis and spondyloarthritis [13] or autoimmunity [15]. As A20 deficient DCs exert potent physiological influences upon intestinal immune homeostasis, we investigated whether these cells might regulate the composition of intestinal microbial communities.
We previously observed that mice lacking A20 expression in DCs (A20 FL/FL CD11c-Cre mice, or as termed herein, A20 dDC mice) spontaneously develop intestinal inflammation after 4 month of age [13]. Given the importance of DCs in regulating intestinal immune homeostasis, we hypothesized that aberrant DC function due to A20 deficiency might perturb luminal microbiota. To test this hypothesis, we explored the temporal evolution of microbial dysbiosis in these mice using 16S ribosomal RNA (16S rRNA) sequencing of samples collected between 2–7 months of life. These data indicated that reduced alpha diversity (Fig 1A and 1B) and distinct microbiota composition (Fig 1C) are consistent features of A20 dDC mice compared to control A20 FL/FL (Cre-) mice over the period of observation (Fig 1A–1C). A between-group comparison of taxon relative abundance (S1 Fig) across these repeated measures identified sporadic and age-dependent genus and taxon enrichments (S2 Fig). A large group of organisms including taxa belonging primarily to the Bacteroides, Parabacteroides and Desulfovibrio were consistently enriched throughout adulthood in A20 dDC mice compared with control animals (S1 Table). These data indicate a strong association between A20 ablation in DCs and distinct gut microbiota successional trajectories into adulthood, and suggest that A20 deficient DCs perturb luminal microbes before driving histological evidence of colitis.
Fig 1. A20 expression in dendritic cells preserves microbial homeostasis.
(A) Between-group and (B) cross-sectional analysis of Faith’s phylogenetic diversity of fecal samples collected longitudinally from 2 to 7 month old A20 dDC mice (red circles) and A20 FL/FL (Cre-) control mice (blue circles). (C) β-diversity analyses of microbial communities from 2 to 7 month-old A20 dDC mice (red circles) and A20 FL/FL (Cre-) mice (blue circles); *** = p<0.001, ** = p<0.01, * = p <0.05. (D) Faith’s Phylogenetic diversity and (E) β-diversity analyses of fecal microbiota from 1 week, 2 week and 4 week-old co-housed A20 dDC mice (red) and control A20 FL/FL (Cre-) mice (blue). No statistically significantly differences in microbiota from 1, 2, or 4 week-old pups were observed.
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Intestinal microbes are shared between littermate pups and their mothers prior to weaning. Emerging evidence from human studies indicates that early life gut microbiota dysbiosis precedes the development of chronic inflammatory disease in childhood [16–18]. We thus asked whether ablation of A20 from DCs plays a role in modulating the gut microbiota prior to weaning. We profiled the fecal bacterial microbiota of co-housed 1, 2 and 4 week old A20 dDC and A20 FL/FL (Cre–) littermate pups from the same A20 FL/FL (Cre–) dams. While a small number of taxa were modestly enriched in 2 or 4 week old A20 dDC pups compared with control (Cre-) littermates, these experiments revealed no significant differences in bacterial microbiota alpha or beta-diversity in 1, 2 or 4 week old A20 dDC mice compared to controls (Fig 1D and 1E and S1 Fig) (S2 Table). Collectively these data suggest taxonomic but not global differences in the gut microbiota of A20 dDC mice versus control mice prior to weaning, which probably reflects the early-life selective pressure of maternal breast milk on these communities at this developmental stage [19]. Differences in microbiota composition became more pronounced in A20 dDC pups between 4 and 8 weeks of age, as pups transition to eating solid food. Hence, microbiota perturbation evolves in A20 dDC pups as their microbiomes diversify in response to complex diets (S1 Fig).
The data above indicate that germline encoded genetic changes affecting host intestinal myeloid cells drive commensal microbiota perturbation soon after weaning. Such perturbations could in turn amplify host susceptibility to inflammation. To determine whether microbiota perturbation in A20 dDC mice contributes to intestinal inflammation in normal mice, we harvested stool pellets from 7 month old A20 dDC mice or control mice, gavaged bacteria from these fresh stool pellets into normal syngeneic C57BL/6J mice, and then tested the susceptibility of the recipient mice to dextran sulfate sodium (DSS). These studies re
