Effects of faecal microbiota transplantation on the small intestinal mucosa in systemic sclerosis

Abstract

Objectives: In SSc, gastrointestinal tract (GIT) involvement is a major concern, with no disease-modifying and limited symptomatic therapies available. Faecal microbiota transplantation (FMT) represents a new therapeutic option for GIT-affliction in SSc, showing clinical promise in a recent controlled pilot trial. Here, we aim to investigate effects of FMT on duodenal biopsies collected from SSc patients by immunohistochemistry and transcriptome profiling.

Methods: We analysed duodenal biopsies obtained pre-intervention (week 0) and post-intervention (weeks 2 and 16) from nine SSc patients receiving an intestinal infusion of FMT (n = 5) or placebo (n = 4). The analysis included immunohistochemistry (IHC) with a selected immune function and fibrosis markers, and whole biopsy transcriptome profiling.

Results: In patients receiving FMT, the number of podoplanin- and CD64-expressing cells in the mucosa were lower at week 2 compared with baseline. This decline in podoplanin- (r = 0.94) and CD64-positive (r = 0.89) cells correlated with improved patient-reported lower GIT symptoms. Whole biopsy transcriptome profiling from week 2 showed significant enrichment of pathways critical for cellular and endoplasmic reticulum stress responses, microvillus and secretory vesicles, vascular and sodium-dependent transport, and circadian rhythm. At week 16, we found enrichment of pathways mandatory for binding activity of immunoglobulin receptors, T cell receptor complexes, and chemokine receptors, as well as response to zinc-ions. We found that 25 genes, including Matrix metalloproteinase-1 were upregulated at both week 2 and week 16.

Conclusion: Combining selective IHC and unbiased gene expression analyses, this exploratory study highlights the potential for disease-relevant organ effects of FMT in SSc patients with GIT involvement.

Trial registration: ClinicalTrials.gov, http://clinicaltrials.gov, NCT03444220.

Keywords: RNA-sequencing; SSc; faecal microbiota transplantations; gastrointestinal tract; immunohistochemistry.

Figure 1.

Immunohistochemical analysis and representative pictures of duodenal samples of ReSScue patients (FMT n = 5, Placebo n = 4). Duodenal biopsies were stained with antibodies for podoplanin, VEGFR3, CD64, CD38, CD3, CD8, and αSMA, and Sirius Red. (A–G) Staining/area or nuclei depicted as fold change between weeks 0 and 2 (fold change 1) and between weeks 0 and 16, respectively, for patients who received FMT (left) and placebo (right) (unpaired t test). (A–E) Representative pictures at weeks 0 (left) and 2 (right) in magnification ×20. All pictures are from patients who received FMT. FMT: faecal microbiota transplantation

Figure 2.

Immunohistochemical analysis and clinical correlation of unselected SSc patients (n = 17) and the ReSScue patients (n = 9). (A) Mean ratios of unselected SSc patients (circle) and ReSScue patients (square). ReSScue patient data are depicted from week 0; therefore, FMT and placebo patients are not marked separately. The values of CD3 and CD8 were divided by 10, as the ratios were higher due to higher staining intensity. The bar indicates standard deviation, and P was calculated with an unpaired t test. (B) Mean staining ratios and standard deviations for individual markers of SSc control patients with disease duration ≤3 years (circle) and >3 years (square). (C) Mean staining ratios and standard deviations for individual markers of SSc control patients having lcSSc (circle) and dcSSc (square) disease. FMT: faecal microbiota transplantation

Figure 3.

Transcriptomic analysis of duodenal samples from SSc patients 2 weeks after treatment with FMT or placebo, Part 1. Bulk RNA sequencing was performed on duodenal biopsies from SSc placebo (4) vs FMT (5) patients. (A) The heat map and (B–D) gene ontology (GO) analysis of significantly upregulated genes in duodenal biopsies from patients 2 weeks after treatment with FMT or placebo. The EnrichR software performed pathway enrichment analysis of differentially expressed genes (P ≤ 0.05, log2 ratio ≥ 0.5). FMT: faecal microbiota transplantation

Figure 4.

Transcriptomic analysis of duodenal samples from SSc patients 2 weeks after treatment with FMT or placebo, Part 2. Pathway analysis of significantly upregulated genes in duodenal biopsies from SSc patients after treatment with FMT or placebo at week 2 (P ≤ 0.05, log2 ratio ≥ 0.05). The analysis was performed with the comprehensive gene set enrichment web server EnrichR for Pathways [database: (A) Elsevier Pathway Collection, (B) KEGG2021 Human, (C) NCI-Nature 2016] of significantly upregulated genes in samples from SSc patients treated with FMT or placebo at week 2. Arrows indicate upregulated expression of NR1D1, PER1 and BHLME41. FMT: faecal microbiota transplantation

Figure 5.

Transcriptomic comparative analysis of duodenal samples from SSc patients treated with FMT or placebo between weeks 2 and 16. Bulk RNA sequencing was performed on duodenal biopsies from SSc placebo (n = 4) vs FMT (n = 5) patients. (A) The heat map and (B–D) pathway analysis of significantly upregulated and downregulated genes. The analysis was performed with the comprehensive gene set enrichment web server EnrichR for Pathways [database: (A) Reactome 2016, (B) Human Phenotype Oncology, (C) Jensen DISEASES] of significantly upregulated genes in samples from SSc patients treated with FMT at weeks 2 and 16 (P ≤ 0.05, log2 ratio ≥ 0.5). Arrows indicate upregulated expression of MMP1. FMT: faecal microbiota transplantation