Fecal microbiota transplantation stimulates type 2 and tolerogenic immune responses in a mouse model

Abstract

Objectives: Clostridioides difficile infection (CDI) is the leading hospital-acquired infection in North America. While previous work on fecal microbiota transplantation (FMT), a highly effective treatment for CDI, has focused on colonization resistance mounted against C. difficile by FMT-delivered commensals, the effects of FMT on host gene expression are relatively unexplored. This study aims to identify transcriptional changes associated with FMT, particularly changes associated with protective immune responses.

Methods: Gene expression was assessed on day 2 and day 7 after FMT in mice after antibiotic-induced dysbiosis. Flow cytometry was also performed on colon and mesenteric lymph nodes at day 7 to investigate changes in immune cell populations.

Results: FMT administration after antibiotic-induced dysbiosis successfully restored microbial alpha diversity to levels of donor mice by day 7 post-FMT. Bulk RNA sequencing of cecal tissue at day 2 identified immune genes, including both pro-inflammatory and Type 2 immune pathways as upregulated after FMT. RNA sequencing was repeated on day 7 post-FMT, and expression of these immune genes was decreased along with upregulation of genes associated with restoration of intestinal homeostasis. Immunoprofiling on day 7 identified increased colonic CD45⁺ immune cells that exhibited dampened Type 1 and heightened regulatory and Type 2 responses. These include an increased abundance of eosinophils, alternatively activated macrophages, Th2, and T regulatory cell populations.

Conclusion: These results highlight the impact of FMT on host gene expression, providing evidence that FMT restores intestinal homeostasis after antibiotic treatment and facilitates tolerogenic and Type 2 immune responses.

Keywords: Clostridioides difficile; Fecal microbiota transplantation; Intestinal dysbiosis; Microbiota signaling; Tolerogenic immunity; Type 2 immunity.

Figure 1:. FMT restores the intestinal microbiome by day 7.

A) Overview of experimental design. C57BL6/6J mice were given an antibiotic cocktail in drinking water one week prior to treatment, followed by an IP injection of Clindamycin one day pre-FMT. On Day 0 and Day 1, mice were administered either FMT or vehicle (PBS) by oral gavage. In separate experiments, mice were sacrificed on either Day 2 or Day 7, at which time cecal contents, cecal tissue, and colon / MLN (Day 7 only) were harvested for downstream analysis. B,D) Alpha diversity (Total number of ASVs) on B) Day 2 or D) Day 7 for FMT donor (blue), FMT recipient (red), and PBS control (gray) groups. Statistics were calculated using a pairwise Wilcoxon Rank test with Bonferroni correction for multiple comparisons. C,E) Non-metric multidimensional scaling (NMDS) plot of Bray-Curtis Dissimilarity on C) Day 2 or E) Day 7 from FMT donor, FMT recipient, and PBS control groups. All groups were significantly different from each other at both timepoints using a PERMANOVA with pairwise comparison for multiple comparisons. Statistical significance is indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 2:. FMT treatment is associated with activation of immune genes within one week post-FMT.

A) Principal component analysis (PCA) of log transformed count data from bulk RNA sequencing of control (gray) or FMT-treated (red) mouse cecal tissue at day 2. B) Gene Set Enrichment Analysis (GSEA) ranking of significantly upregulated or downregulated Hallmark gene sets in the FMT-treated conditions according to Normalized Enrichment Score (NES) at day 2. C) GSEA enrichment plot for the Reactome “Interleukin-4 and Interleukin-13 Signaling” gene set. Lines along the x axis represent the position of genes in this gene set within a ranked list of RNAseq-identified genes. D-F) Boxplots of normalized counts for leading edge genes associated with enrichment of D) immune activation, E) tissue remodeling, or F) Type 2 immunity gene sets. P values represent the results of a Student’s t-test between normalized counts across groups. Statistical significance is indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 3:. FMT promotes transcriptional changes in immune and intestinal homeostasis genes at day 7.

A) Principal component analysis (PCA) of log transformed count data from bulk RNA sequencing of control (gray) or FMT-treated (red) mouse cecal tissue at day 7. B) Gene Set Enrichment Analysis (GSEA) ranking of significantly upregulated or downregulated Hallmark gene sets in the FMT-treated conditions according to Normalized Enrichment Score (NES) at day 7. C) Boxplots of normalized counts for leading edge genes associated with immune activation pathways. P values represent the results of a Student’s t test between normalized counts across groups. D) Ranking of the Top 10 most enriched gene sets in the FMT-treated group by GSEA using Gene Ontology: Biological Processes gene sets. E-G) Boxplots of normalized counts in control (gray) or FMT-treated (red) samples for leading edge genes associated with E) intestinal homeostasis, F) synapse assembly, and G) neuropeptide signaling. P values represent the results of a Student’s t-test between normalized counts across groups. Statistical significance is indicated as *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 4:. Fecal microbiota transplantation induces Type 2 and tolerogenic immune responses in the colon and MLN.

Colonic lamina propria A-J) and mesenteric lymph node (MLN) K-O) immune cells were analyzed by flow cytometry on day 7. A) Cell cluster visualization using Uniform Manifold Approximation and Projection (UMAP) B-O) Cell counts per 100,000 live cells of B) CD45+ immune cells, C) eosinophils D) alternatively activated macrophages, E) dendritic cells (CD11b− CD11C+ MHCII+), F) γδT cells, G) Th2 CD4+ T cells, H) Th17 cells, I) FoxP3+ Treg cells, J) RORγt+ Treg cell, K) Th2 cells, L) ILC3s, M) Th17 cells, N) FoxP3+ Treg cells, O) RORγt+ Treg cell cells. Statistical significance is demarked as *P < 0.05, **P < 0.01, and ***P < 0.001. The error bar indicates SEM.