Composition of the Intestinal Microbiota Determines the Outcome of Virus-Triggered Colitis in Mice

Abstract

The intestinal microbiota is a complex ecosystem implicated in host health and disease. Inflammatory bowel disease (IBD) is a multifactorial chronic disorder of the gastrointestinal mucosa. Even though the exact mechanisms are still unknown, the intestinal microbiota is crucial in IBD development. We previously showed that murine norovirus (MNV) induces colitis in the Il10-deficient (Il10^-/-) mouse model of IBD in a microbiota-dependent manner. Thus, in this study we analyzed whether distinct minimal bacterial consortia influence the outcome of MNV-triggered colitis in Il10^-/- mice. Gnotobiotic Il10^-/- mice associated with Oligo-Mouse-Microbiota 12 (OMM¹²) or Altered Schaedler Flora (ASF) developed little to no inflammatory lesions in the colon and cecum. MNV infection exacerbated colitis severity only in ASF-colonized mice, but not in those associated with OMM¹². Four weeks after MNV infection, inflammatory lesions in ASF-colonized Il10^-/- mice were characterized by epithelial hyperplasia, infiltration of inflammatory cells, and increased barrier permeability. Co-colonization of ASF-colonized Il10^-/- mice with segmented filamentous bacteria (SFB) abolished MNV-induced colitis, whereas histopathological scores in SFB-OMM¹²-co-colonized mice stayed unchanged. Moreover, SFB only colonized mice associated with ASF. The SFB-mediated protective effects in ASF-colonized mice involved enhanced activation of intestinal barrier defense mechanisms and mucosal immune responses in the chronic and acute phase of MNV infection. SFB colonization strengthened intestinal barrier function by increasing expression of tight junction proteins, antimicrobial peptides and mucus. Furthermore, SFB colonization enhanced the expression of pro-inflammatory cytokines such as Tnfα, Il1β, and Il12a, as well as the expression of the regulatory cytokine Tgfβ. Altogether, our results showed that MNV-triggered colitis depends on the microbial context.

Keywords: ASF; Il10-deficient mice; MNV; Oligo-MM12; SFB; colitis; gnotobiotic models; intestinal microbiota.

Figure 1

Gnotobiotic mouse model of experimental IBD. (A) Experimental design: GF Il10^−/− mice were colonized at weaning with ASF or OMM¹² consortia by co-housing with minimal microbiota donor mice for 4 weeks. One week after co-housing commenced, mice were inoculated with SFB on 2 consecutive days. At the end of co-housing mice were infected with MNV for 4 weeks (chronic phase of MNV infection) or for 48 h (acute phase of MNV infection) and subsequently sacrificed. (B) Representative image of capillary gel electrophoresis showing ASF member-specific PCR products determined by strain-specific PCR assay in Il10^−/− mice after 4 weeks co-housing with ASF donor (Clostridium sp. ASF356, Lactobacillus acidophilus ASF360, Lactobacillus murinus ASF361, Mucispirillum schaedleri ASF457, Eubacterium plexicaudatum ASF492, Pseudoflavonifractor sp. ASF500, Clostridium sp. ASF502, and Parabacteroides goldsteinii ASF519); n = 15. (C) Representative OMM¹² composition (relative abundance) determined by strain-specific qPCR assay in Il10^−/− mice after co-housing with OMM¹² donor mouse for 4 weeks (Akkermansia muciniphila YL44, Bacteroides caecimuris I48, Muribaculum intestinale YL27, Turicimonas muris YL45, Bifidobacterium longum subsp. animalis YL2, Enterococcus faecalis KB1, Acutalibacter muris KB18, Clostridium clostridioforme YL32, Blautia coccoides YL58, Flavonifractor plautii YL31, Lactobacillus reuteri I49, and Clostridium innocuum I46); n = 16. GF, germ-free; ASF, Altered Schaedler Flora; OMM¹², Oligo-Mouse-Microbiota 12; SFB, segmented filamentous bacteria; MNV, murine norovirus; p.i., post infection. ^*Below detection limit.

Figure 2

Histological analysis of intestinal tissues of gnotobiotic B6-Il10^−/− mice 4 weeks after MNV infection. (A) Histological score quantifying alterations observed in the colon tissue 4 weeks after MNV infection in ASF- or OMM¹²-colonized B6-Il10^−/− mice with or without SFB co-colonization. (B) Histological score quantifying alterations observed in the ceca 4 weeks after MNV infection in ASF- or OMM¹²-colonized B6-Il10^−/− mice with or without SFB co-colonization. Data presented in box and whiskers plots are the medians with minimum, maximum, and individual values obtained from two to three independent experiments (n = 5–9). (C,D) Representative images of H&E stained proximal colon sections of (C) ASF-colonized and (D) OMM¹²-colonized B6-Il10^−/− mice infected with MNV for 4 weeks and/or co-colonized with SFB. Scale bars: 200 μm. Insets: magnification of areas outlined by black boxes in main images. (E) Histological score quantifying alterations observed in the colon of GF B6-Il10^−/− mice 4 weeks after MNV infection with or without SFB monocolonization. (F) Histological score quantifying alterations observed in the ceca of GF B6-Il10^−/− mice 4 weeks after MNV infection with or without SFB monocolonization. Data presented in box and whiskers plots are the medians with minimum, maximum, and individual values obtained from one to two independent experiments (n = 5–7). (G) Representative images of H&E stained proximal colon tissue of GF B6-Il10^−/− mice, MNV-infected GF B6-Il10^−/− mice, SFB monocolonized B6-Il10^−/− mice and SFB monocolonized and MNV-infected B6-Il10^−/− mice. Scale bars: 200 μm. Insets: magnification of areas outlined by black boxes in main images. GF, germ-free; ASF, Altered Schaedler Flora; OMM¹², Oligo-Mouse-Microbiota 12; SFB, segmented filamentous bacteria; MNV, murine norovirus; H&E, hematoxylin and eosin. Statistically significant diffrences are indicated as follows: ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001.

Figure 3

Histological analysis of intestinal tissues of gnotobiotic C3H-Il10^−/− mice 4 weeks after MNV infection. (A) Histological score quantifying alterations observed in the colon tissue 4 weeks after MNV infection in ASF- or OMM¹²-colonized C3H-Il10^−/− mice with or without SFB co-colonization. (B) Histological score quantifying alterations observed in the ceca 4 weeks after MNV infection in ASF- or OMM¹²-colonized C3H-Il10^−/− mice with or without SFB co-colonization. Data presented in box and whiskers plots are the medians with minimum, maximum, and individual values obtained from one to three independent experiments (n = 4–9). (C,D) Representative images of H&E stained proximal colon sections of (C) ASF-colonized and (D) OMM¹²-colonized C3H-Il10^−/− mice infected with MNV for 4 weeks, and/or co-colonized with SFB. Scale bars: 200 μm. Insets: magnification of areas outlined by black boxes in main images. (E) Histological score quantifying alterations observed in the colon of GF C3H-Il10^−/− mice 4 weeks after MNV infection with or without SFB monocolonization. (F) Histological score quantifying alterations observed in the ceca of GF C3H-Il10^−/− mice 4 weeks after MNV infection with or without SFB monocolonization. Data presented in box and whiskers plots are medians with minimum, maximum, and individual values obtained from one to three independent experiments (n = 4–8). (G) Representative images of H&E stained proximal colon tissue of GF C3H-Il10^−/− mice, MNV-infected GF C3H-Il10^−/− mice, SFB monocolonized C3H-Il10^−/− mice and SFB monocolonized and MNV-infected C3H-Il10^−/− mice. Scale bars: 200 μm. Insets: magnification of areas outlined by black boxes in main images. GF, germ-free; ASF, Altered Schaedler Flora; OMM¹², Oligo-Mouse-Microbiota 12; SFB, segmented filamentous bacteria; MNV, murine norovirus; H&E, hematoxylin and eosin. Statistically significant differences are indicated as follows: ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 and ^****P < 0.0001.

Figure 4

Different intestinal microbiota compositions distinctively modulate host response. (A–E) Gene expression of pro-inflammatory cytokines (A) Tnfα, (B) Ifnγ, (C) Il1β, (D) Il12a, and (E) Il17a measured by qPCR in total RNA isolated from proximal colon of ASF-colonized or OMM¹²-colonized B6-Il10^−/− mice 4 weeks after MNV infection with or without SFB co-colonization. Relative differences in gene expression were calculated by the comparative 2^−ΔCt method. Parametric data were shown as mean ± SEM (n = 5–7). Non-parametric data were shown as median ± interquartile range (n = 5–7). Data were obtained from one to two independent experiments. (F,G) Quantification of (F) CD3+ and (G) CD45R+ cells in the colon tissue of ASF- or OMM¹²-colonized B6-Il10^−/− mice with or without SFB co-colonization 4 weeks after MNV infection. Data presented in box and whiskers plots are medians with minimum, maximum, and individual values obtained from one to two independent experiments (n = 5–7). (H) Gene expression of tight junction genes claudin 8 and claudin 4 measured by qPCR in total RNA isolated from proximal colon of ASF- or OMM¹²-colonized B6-Il10^−/− mice with or without SFB co-colonization 4 weeks after MNV infection. Parametric data were shown as mean ± SEM (n = 5–7). Non-parametric data were shown as median ± interquartile range (n = 5–7). Data were obtained from one to two independent experiments. ASF, Altered Schaedler Flora; OMM¹², Oligo-Mouse-Microbiota 12; SFB, segmented filamentous bacteria; MNV, murine norovirus; Tnfα, Tumor necrosis factor alpha; Ifnγ, Interferon gamma; Il1β, Interleukin 1 beta; Il12a, Interleukin 12a; Il17a, Interleukin 17a. Statistically significant differences are indicated as follows: ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 and ^****P < 0.0001.

Figure 5

SFB co-colonization depends on the microbiota composition. (A) Using virus-specific qPCR, viral load was measured in total RNA isolated from the proximal colon of GF, ASF- and OMM¹²-colonized B6-Il10^−/− mice infected with MNV for 4 weeks with or without co-colonization with SFB. Data were shown as median ± interquartile range (n = 4–6). Data were collected from one to two independent experiments. (B) SFB copy numbers were measured in total DNA isolated from feces of 12 week old GF, ASF-, or OMM¹²-associated B6-Il10^−/− mice co-colonized with SFB with or without MNV infection for 4 weeks. Data were shown as median ± interquartile range and collected from three to nine independent experiments (n = 8–27). (C) Representative images of FISH staining of ileal and proximal colon tissue sections of GF and OMM¹²-associated B6-Il10^−/− mice co-colonized with SFB (n = 5). DNA was stained using DAPI (blue), SFB were detected using an SFB-specific probe (yellow) and all bacteria were detected by the EUB338 probe (red). Scale bars: 50 μm. (D) SFB colonization kinetic was determined by using an SFB-specific qPCR measuring SFB copy numbers in the total DNA isolated from feces of OMM¹²-colonized B6-Il10^−/− mice over a period of 20 days. OMM¹²-colonized B6-Il10^−/− mice were inoculated with the gut content of SFB monocolonized mice at day 0 and 1. Data were shown as median ± interquartile range (n = 7). Data were obtained from two independent experiments. (E) SFB colonization kinetics were determined by an SFB-specific qPCR measuring SFB copy numbers in the total DNA isolated from feces of OMM¹²-colonized B6-Rag2^−/− mice over a period of 39 days. OMM¹²-colonized B6-Rag2^−/− mice were inoculated with the gut content of SFB monocolonized mice at day 0 and 1. Data were shown as median ± interquartile range (n = 7). Data were obtained from two independent experiments. (F) SFB colonization kinetics were determined by an SFB-specific qPCR measuring SFB copy numbers in the total DNA isolated from feces of B6-Il10^−/− mice first monocolonized with SFB and subsequently colonized with OMM¹². GF B6-Il10^−/− mice were inoculated with the gut content of SFB monocolonized mice at day 0 and 1. OMM¹² consortium transfer started when stable SFB colonization was established. Data were shown as median ± interquartile range (n = 7). Data were obtained from two independent experiments. GF, germ-free; ASF, Altered Schaedler Flora; OMM¹², Oligo-Mouse-Microbiota 12; SFB, segmented filamentous bacteria; MNV, murine norovirus; FISH, fluorescent in situ hybridization. Dash-dotted line: detection limit. Statistically significant differences are indicated as follows: ^*P < 0.05, ^**P < 0.01 and ^****P < 0.0001.

Figure 6

The presence of SFB enhances expression of intestinal barrier defense and immune regulatory factors in the chronic phase of MNV infection. (A–C) Gene expression of (A) Muc2, (B) Reg3γ, and (C) Tgfβ measured by qPCR in total RNA isolated from the proximal colon of ASF-colonized B6-Il10^−/− mice infected with MNV for 4 weeks with or without SFB co-colonization. Relative differences in gene expression were calculated by the comparative 2^−ΔCt method. Parametric data were shown as mean ± SEM (n = 5–6). Data were obtained from one to two independent experiments. (D) Representative images of H&E stained ileum tissue sections of ASF-associated B6-Il10^−/− mice infected with MNV for 4 weeks with and without SFB co-colonization (n = 5). ASF, Altered Schaedler Flora; SFB, segmented filamentous bacteria; MNV, murine norovirus; H&E, hematoxylin and eosin; h, hours; Muc2, Mucin 2; Reg3γ, Regenerating islet-derived 3 gamma; Tgfβ, Transforming growth factor beta.1. Statistically significant differences are indicated as follows: ^*P < 0.05.

Figure 7

SFB-mediated protective effect in the acute phase of MNV infection enhances intestinal epithelial barrier defense mechanisms. (A) Representative images of immunofluorescence staining of MUC2 (red) on colon sections obtained from ASF-associated B6-Il10^−/− mice infected with MNV for 48 h with or without SFB co-colonization. Nuclei (blue) were counterstained with DAPI (n = 7). Scale bars: 50 μm. (B) Quantification of MUC2 immunofluorescent stained layer thickness measured in the colon of ASF-associated B6-Il10^−/− mice 48 h after MNV infection with or without SFB so-colonization. Colon tissue sections of seven mice per group were blindly analyzed. Six images per tissue section were taken and five measurements of layer thickness per image were recorded using Zeiss ZEN blue software. Subsequently one mean value of layer thickness per image was generated. In total six technical replicates per animal are shown. Bars represent the median. Data were obtained from one to two independent experiments. (C) Gene expression of Muc2 measured by qPCR in total RNA isolated from the proximal colon of ASF-colonized B6-Il10^−/− mice 48 h after MNV infection with and without SFB co-colonization. Relative differences in gene expression were calculated by the comparative 2^−ΔCt method (n = 6–7). (D,E) Gene expression of antimicrobial peptides (D) β-defensin 2 and (E) Reg3γ measured by qPCR in total RNA isolated from proximal colon of ASF-colonized B6-Il10^−/− mice 48 h after MNV infection with and without SFB co-colonization. Relative differences in gene expression were calculated by the comparative 2^−ΔCt method (n = 6–7). (F) Densitometric quantification of REG3γ protein expression measured in colon lysates of ASF-colonized B6-Il10^−/− mice 48 h post MNV infection with and without SFB co-colonization normalized to GAPDH (n = 6–7). Blot images were analyzed by Image Lab^TM Software. (G) Gene expression of claudin 4 measured by qPCR in total RNA isolated from proximal colon of ASF-colonized B6-Il10^−/− mice 48 h after MNV infection with and without SFB co-colonization. Relative differences in gene expression were calculated by the comparative 2^−ΔCt method (n = 6–7). (H) Densitometric quantification of claudin 4 protein expression measured in colon lysates of ASF-colonized B6-Il10^−/− mice 48 h post MNV infection with and without SFB co-colonization normalized to GAPDH (n = 6–7). Blot images were analyzed by Image Lab^TM Software. (I) Gene expression of claudin 8 measured by qPCR in total RNA isolated from proximal colon of ASF-colonized B6-Il10^−/− mice 48 h post MNV infection with and without SFB co-colonization. Relative differences in gene expression were calculated by the comparative 2^−ΔCt method (n = 6–7). (J) Densitometric quantification of claudin 8 protein expression measured in colon lysates of ASF-colonized B6-Il10^−/− mice 48 h after MNV infection with and without SFB co-colonization normalized to GAPDH (n = 6–7). Blot images were analyzed by Image Lab^TM Software. Parametric data were presented as mean ± SEM. Non-parametric data were presented as median ± interquartile range. Data were obtained from one to two independent experiments. GF, germ-free; ASF, Altered Schaedler Flora; SFB, segmented filamentous bacteria; MNV, murine norovirus; h, hours; MUC2, Mucin 2; REG3γ, Regenerating islet-derived 3 gamma. Statistically significant differences are indicated as follows: ^*P < 0.05, ^**P < 0.01 and ^****P < 0.0001.

Figure 8

SFB-mediated protective effect in the acute phase of MNV infection enhances host immune responses. (A–C) Gene expression of (A) pro-inflammatory cytokines Tnfα, Il1β, and Ifnγ, (B) regulatory cytokine Tgfβ, and (C) Foxp3 measured by qPCR in total RNA isolated from proximal colon of ASF-colonized B6-Il10^−/− mice 48 h after MNV infection with and without SFB co-colonization. Relative differences in gene expression were calculated by the comparative 2^−ΔCt method (n = 7). (D,E) Quantification of (D) CD3+ and (E) CD45R+ cells in the colon tissue of ASF-colonized B6-Il10^−/− mice 48 h post MNV infection with and without SFB co-colonization. Data presented in box and whiskers plots are medians with minimum, maximum, and individual values (n = 7). (F,G) Gene expression of (F) Ifnλ₂ and (G) Mmp7 measured by qPCR in total RNA isolated from proximal colon of ASF-colonized B6-Il10^−/− mice 48 h post MNV infection with and without SFB co-colonization. Relative differences in gene expression were calculated by the comparative 2^−ΔCt method (n = 6–7). Parametric data were presented as mean ± SEM and non-parametric data as median ± interquartile range. Data were obtained from one to two independent experiments. GF, germ-free; ASF, Altered Schaedler Flora; SFB, segmented filamentous bacteria; MNV, murine norovirus; h, hours; Tnfα, Tumor necrosis factor alpha; Il1β, Interleukin 1 beta; Ifnγ, Interferon gamma; Tgfβ, Transforming growth factor beta; Foxp 3, Forkhead box P3; Ifnλ₂, Interferon lambda 2; Mmp7, Matrix metallopeptidase 7. Statistically significant differences are indicated as follows: ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 and ^****P < 0.0001.