Colitis susceptibility in p47(phox-/-) mice is mediated by the microbiome

Abstract

Background: Chronic granulomatous disease (CGD) is caused by defects in nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) complex subunits (gp91(phox) (a.k.a. Nox2), p47(phox), p67(phox), p22(phox), p40(phox)) leading to reduced phagocyte-derived reactive oxygen species production. Almost half of patients with CGD develop inflammatory bowel disease, and the involvement of the intestinal microbiome in relation to this predisposing immunodeficiency has not been explored.

Results: Although CGD mice do not spontaneously develop colitis, we demonstrate that p47(phox-/-) mice have increased susceptibility to dextran sodium sulfate colitis in association with a distinct colonic transcript and microbiome signature. Neither restoring NOX2 reactive oxygen species production nor normalizing the microbiome using cohoused adult p47(phox-/-) with B6Tac (wild type) mice reversed this phenotype. However, breeding p47(phox+/-) mice and standardizing the microflora between littermate p47(phox-/-) and B6Tac mice from birth significantly reduced dextran sodium sulfate colitis susceptibility in p47(phox-/-) mice. We found similarly decreased colitis susceptibility in littermate p47(phox-/-) and B6Tac mice treated with Citrobacter rodentium.

Conclusions: Our findings suggest that the microbiome signature established at birth may play a bigger role than phagocyte-derived reactive oxygen species in mediating colitis susceptibility in CGD mice. These data further support bacteria-related disease in CGD colitis.

Keywords: Chronic granulomatous disease; Colitis; Dextran sodium sulfate; Inflammatory bowel disease; Microbiome; NADPH; Reactive oxygen species; p47 phox.

Fig. 1

p47^phox−/− mice have increased susceptibility to DSS colitis. Changes in body weight (a), disease activity index (b), and survival (c) were assessed daily (B6Tac (n = 15); p47^phox−/− (n = 28)). Significance was determined using the Mann-Whitney U test (*p < 0.05) and log-rank test for survival (p = 0.0003). d Colonic tissue sections were blindly scored for inflammation, depth of injury, and crypt damage on day 9 (B6Tac (n = 15); p47^phox−/− (n = 17); (p = 0.04)). e Representative distal colon sections stained with H&E (original magnification X 10). f Bacterial translocation to MLNs was determined on day 9. The number of CFUs per MLN is shown (B6Tac (n = 5); p47^phox−/− (n = 3); (p = 0.04)). Pie charts represent the proportion of mice per group where the cultured MLN grew one or more of the listed bacterial species. For all panels, data are representative of 4 independent experiments (except f)

Fig. 2

p47^phox−/− mice have a distinct colonic transcript profile at baseline and during DSS colitis. RNA was isolated from distal colon segments. Normalized transcript counts from nCounter analysis used for heat map reflecting hierarchical clustering of subjects based on differentially regulated transcripts (p < 0.05) and the fold change for each transcript comparing p47^phox−/− versus B6Tac mice at baseline (a) and on DSS day 7 (c). 2-dimensional PCA of p47^phox−/− versus B6Tac mice at baseline (b) and on DSS day 7 (d)

Fig. 3

Increased DSS colitis susceptibility in p47^phox−/− mice is not reversed by restoration of NOX2-mediated ROS production. BM transplantation was performed in 7-week-old age- and gender- matched irradiated mice (B6Tac-CD45.1⁺ ➔ B6Tac-CD45.1⁺, p47^phox−/− ➔ p47^phox−/−, B6Tac-CD45.1⁺ ➔ p47^phox−/−, p47^phox−/− ➔ B6Tac-CD45.1⁺) prior to DSS colitis induction. Changes in body weight (a), disease activity index (b), and survival (c) were assessed (B6Tac-CD45.1⁺ ➔ B6Tac-CD45.1⁺ (n = 5); p47^phox−/− ➔ p47^phox−/− (n = 5); B6Tac-CD45.1⁺ ➔ p47^phox−/− (n = 10); p47^phox−/− ➔ B6Tac-CD45.1⁺ (n = 8); (p = 0.615)). d Colonic tissue sections were blindly scored for inflammation, depth of injury, and crypt damage on day 9

Fig. 4

p47^phox−/− mice have a distinct intestinal microbiome signature before and after DSS colitis. Bacterial taxonomic classifications show colonization with unique taxa and altered representation of diverse taxa in p47^phox−/− compared to B6Tac mice. a Relative abundances of 7 major phyla taxonomies in p47^phox−/− and B6Tac mice, as well as p values from P tests are reported for the group comparisons listed for DSS days 0 and 9. b PCoA and p values of the weighted UniFrac comparing p47^phox−/− to B6Tac mice on DSS days 0 and 9 are shown. c Heat map depicting average relative abundance by LEfSe of bacterial genera in fecal samples from p47^phox−/− and B6Tac mice on DSS day 0 and day 9. Top quartile of OTUs where comparisons between each experimental group were significant (p < 0.05) is shown. Alpha diversity was measured in p47^phox−/− and B6Tac mice before and after DSS colitis by inverse Simpson and Shannon diversity (d). Gen. classified as distinct but unnamed genus in Greengenes reference database, sp. designates a distinct species in Greengenes reference database

Fig. 5

p47^phox−/− mice cohoused with B6Tac mice maintain increased susceptibility to DSS colitis. Changes in body weight (a), disease activity index (b), and survival (c) were assessed daily (B6Tac cohoused with p47^phox−/− (n = 8); p47^phox−/− cohoused with B6Tac (n = 8), p47^phox−/− (n = 28); B6Tac (n = 20)). Significance for comparisons between cohoused mice was determined using the Mann-Whitney U test (*p < 0.05) and a log-rank test for survival (p = 0.06). d Colonic tissue sections were blindly scored for inflammation, depth of injury, and crypt damage on day 9 (B6Tac cohoused with p47^phox−/− (n = 8); p47^phox−/− cohoused with B6Tac (n = 8); (p = 0.49)). e Bacterial translocation to MLNs was determined on day 9. The number of CFUs per MLN is shown (B6Tac cohoused with p47^phox−/− (n = 4); p47^phox−/− cohoused with B6Tac (n = 4); p = 0.03)). Pie charts represent the proportion of mice per group where the cultured MLN grew one or more of the listed bacterial species. For all panels, data were generated from 2 independent experiments (except e)

Fig. 6

Cohousing homogenizes the intestinal microbiota between p47^phox−/− and B6Tac mice. a Relative abundances of 7 major phyla taxonomies in p47^phox−/− and B6Tac mice pre-cohousing and post-cohousing (DSS days 0 and 9) are shown. p values from P tests were used to indicate whether the samples from B6Tac and p47^phox−/− mice pre-cohousing, on DSS day 0, and DSS day 9, have the same community structure. b PCoA and p values from weighted UniFrac analyses of p47^phox−/− and B6Tac mice pre-cohousing and post-cohousing (DSS days 0 and 9) are shown. c Heat map depicting average relative abundance by LEfSe of bacterial genera in fecal samples from p47^phox−/− and B6Tac mice post-cohousing (DSS day 0 and DSS day 9). d Alpha diversity was measured in p47^phox−/− and B6Tac mice pre- and post-cohousing (DSS days 0 and 9) by inverse Simpson and Shannon diversity. Gen. classified as distinct but unnamed genus in Greengenes reference database, sp. designates a distinct species in Greengenes reference database

Fig. 7

Homogenizing the intestinal microbiome at birth decreases DSS colitis susceptibility in p47^phox−/− mice. p47^phox+/− mice were bred, and weanlings were placed in separate cages by gender and genotype (p47^phox−/− versus B6Tac) prior to DSS colitis induction. Changes in body weight (a), disease activity index (b), and survival (c) were assessed daily (B6Tac (n = 7); p47^phox−/− (n = 8)). Significance was determined using the Mann-Whitney U test (*p < 0.05) and a log-rank test for survival (p = 0.17). d Colonic tissue sections were blindly scored for inflammation, depth of injury, and crypt damage on day 9 (B6Tac (n = 7); p47^phox−/− (n = 6); (p = 0.619)). e Bacterial translocation to MLNs was determined on day 9. The number of CFUs per MLN is shown (B6Tac (n = 7); p47^phox−/− (n = 6)); (p = 0.346)). Pie charts represent the proportion of mice per group where the cultured MLN grew one or more of the listed bacterial species. For all panels, data were generated from 2 independent experiments (except E). f Plasma was collected on day 9 for cytokine measurement; p values <0.05 are indicated

Fig. 8

Heterozygous breeding of p47^phox+/− mice homogenizes the intestinal microbiome in littermates. a Relative abundances of 9 major phyla taxonomies in littermate p47^phox−/− and B6Tac mice on DSS days 0 and 9 are shown. p values are reported for the group comparisons listed. b PCoA and p values from weighted UniFrac analyses of samples from littermate p47^phox−/− and B6Tac mice on DSS days 0 and 9 are shown. Numbers listed next to each sample reflect the litter to which the mouse generating the sample belonged. c Alpha diversity plots measured in littermate p47^phox−/− and B6Tac mice on DSS days 0 and 9 by inverse Simpson and Shannon diversity are shown