Mouse Adaptation of Human Inflammatory Bowel Diseases Microbiota Enhances Colonization Efficiency and Alters Microbiome Aggressiveness Depending on Recipient Colonic Inflammatory Environment

Abstract

Understanding the cause vs consequence relationship of gut inflammation and microbial dysbiosis in inflammatory bowel diseases (IBD) requires a reproducible mouse model of human-microbiota-driven experimental colitis. Our study demonstrated that human fecal microbiota transplant (FMT) transfer efficiency is an underappreciated source of experimental variability in human microbiota associated (HMA) mice. Pooled human IBD patient fecal microbiota engrafted germ-free (GF) mice with low amplicon sequence variant (ASV)-level transfer efficiency, resulting in high recipient-to-recipient variation of microbiota composition and colitis severity in HMA Il-10^-/- mice. In contrast, mouse-to-mouse transfer of mouse-adapted human IBD patient microbiota transferred with high efficiency and low compositional variability resulting in highly consistent and reproducible colitis phenotypes in recipient Il-10^-/- mice. Human-to-mouse FMT caused a population bottleneck with reassembly of microbiota composition that was host inflammatory environment specific. Mouse-adaptation in the inflamed Il-10^-/- host reassembled a more aggressive microbiota that induced more severe colitis in serial transplant to Il-10^-/- mice than the distinct microbiota reassembled in non-inflamed WT hosts. Our findings support a model of IBD pathogenesis in which host inflammation promotes aggressive resident bacteria, which further drives a feed-forward process of dysbiosis exacerbated gut inflammation. This model implies that effective management of IBD requires treating both the dysregulated host immune response and aggressive inflammation-driven microbiota. We propose that our mouse-adapted human microbiota model is an optimized, reproducible, and rigorous system to study human microbiome-driven disease phenotypes, which may be generalized to mouse models of other human microbiota-modulated diseases, including metabolic syndrome/obesity, diabetes, autoimmune diseases, and cancer.

Keywords: Inflammatory bowel diseases; experimental colitis; fecal microbiota transplant; human microbiota associated mice; interleukin-10 deficient; microbiota transfer efficiency; mouse-adapted.

Figure 1.. Mouse-adapted human microbiota induces more consistent and reproducible colitis than directly transplanted human microbiota.

A) Experimental design. Pooled feces from 3 humans with active IBD (2 CD, 1UC) were transplanted to non-inflamed WT or colitis-susceptible Il-10^−/− (IL-10KO, KO) GF recipient mice. Mouse-adapted microbiotas were serial transplanted to non-inflamed WT or colitis-susceptible Il-10^−/− GF recipient mice. B) Total colon and ileum histology score for WT mice at day 28 post-colonization. C) f-LCN2 level at day 28 post-colonization. D) TNFα mRNA levels in cecal tissue at day 28 post-colonization. E) Segment, total colon and ileum, and max segment histology score for Il-10^−/− mice at day 28 post-colonization. F) Segment, total colon and ileum, and max segment histology score for IMM-g1 colonized Il-10^−/− mice at day 28 post-colonization from 4 independent experiments. Data shown are representative of (C-D) or cumulative (B, E-F) from 2–4 independent experiments. n=7–9 (B-D), n=15–26 (E), n=5–8 (F) mice per group. Data are expressed as mean±SD or geometric mean ± geometric SD (C). Statistical significance calculated by unpaired t-test or Mann-Whitney test (C) with *p<0.05, **p<0.01, ***p<0.001.

Figure 2.. Recipient host environment influences engraftment composition of human-microbiome associated mice.

A) 16S Seq taxonomic bar plots show top 8 most abundant genera in FMT inputs and recipient mouse feces at day 28 post-colonization. For mouse recipient groups, bar plots are average of 16S seq data from n=7–18 mice/group.

Figure 3.. Human microbiome restructuring with transplant to GF mice is host inflammatory environment specific.

A) Principal coordinates analysis, PCoA, of 16S Seq data for human and mouse-adapted FMT inputs and FMT recipient WT and KO mouse groups. B) PCoA of FMT recipient WT and KO mouse groups. C) PCoA of FMT recipient KO mouse groups. D) PCoA of FMT recipient WT mouse groups. E) Shannon index at ASV level for FMT recipient WT and KO mouse groups. F) Pearson correlation coefficients (r) within group for FMT recipient WT and KO mouse groups quantify variability of microbiota composition between mice in the same group (microbiota engraftment consistency). Dots in PCoA plots represent individual mice for FMT recipient WT and Il-10^−/− (KO) mouse groups. For FMT inputs, a single input slurry was used in each experiment and input dots represent sequencing data from three 16S amplicon PCR technical replicates. Analysis conclusions did not change when using average input vs individual technical replicates, so technical replicates are displayed to demonstrate the high consistency of 16S amplicon PCR in our dataset.

Figure 4.. Mouse-adapted human IBD microbiota transfers with higher efficiency than human fecal transplant.

A) ASV level log₁₀-normalized relative abundance correlations for FMT input and WT recipient mice where each dot represents a unique ASV plotted in the input microbiome (x-axis) vs recipient mouse microbiome (y-axis). B) Transfer efficiency quantified by Pearson correlation coefficient (r) between FMT input and WT recipient mouse groups at the ASV level. C) ASV level log₁₀-normalized relative abundance correlations for FMT input and KO recipient mice. D) Transfer efficiency quantified by Pearson correlation coefficient (r) between FMT input and KO recipient mouse groups at the ASV level. E-J) Representative histograms of non-transferring ASVs (red, representing y=0 ASVs in above dot plots) and newly detected in vivo ASVs (blue, representing x=0 ASVs in above dot plots) binned by log₁₀-normalized relative abundance for (E) HM1->WT, (F) NIMM-g1->WT, (G) NIMM-g2->WT, (H) HM1->KO, (I) IMM-g1->KO, and (J) IMM-g2->KO FMT recipient mouse groups.

Figure 5.. Transfer efficiency varies between taxa.

A) Genus-level and B) phylum-level log₁₀-normalized relative abundance correlations comparing HM1 input to HM1->KO, C-D) Pearson correlation coefficient (r) between input and inflamed (KO) recipient at the (C) genus- and (D) phylum-level. E) Genus-level and F) phylum-level log₁₀-normalized relative abundance correlations comparing HM1 input to HM1->WT, G-H) Pearson correlation coefficient (r) between input and non-inflamed (WT) recipient at the (G) genus- and (H) phylum-level. I-K) Pearson correlation coefficient (r) between input and non-inflamed (WT) recipients by phylum. L-N) Pearson correlation coefficient (r) between input and inflamed (KO) recipients by phylum.

Figure 6.. Inflamed mouse-adapted microbiome more rapidly induces severe colitis than non-inflamed mouse adapted microbiome.

A). Experimental design. Human IBD patient microbiota (HM1) was adapted in the inflamed (IMM-g1) or non-inflamed (NIMM-g1) host, then transplanted to Il-10^−/− (KO) GF recipient mice. B) Segment and total colon + ileum histology score for KO mice at day 14 post-colonization. C) Segment, total colon + ileum, and max segment histology score for KO mice at day 28 post-colonization. D) TNFα mRNA levels in cecal tissue at day 28 post-colonization. E) PCoA of FMT recipient WT and KO mouse groups, including NIMM-g1->KO group. F) 16S Seq taxonomic barplots show top 8 most abundant genera in FMT inputs and recipient mouse feces at day 28 post-colonization. For mouse recipient groups, barplots are average of 16S seq data from n=7–18 mice/group. G) Shannon diversity index at ASV level for IMM-g1->WT, NIMM-g1->KO and NIMM-g1->WT groups. Data shown are representative of (D) or cumulative (B-C, E-F) from 2–4 independent experiments. n=15–16 (B-C), n=5–8 (D), n=7–16 (E-G) mice per group. Data are expressed as mean±SD. Statistical significance calculated by unpaired t-test (B-D, G) with *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.