Lachnospiraceae-mediated bile acid metabolism promotes ileal epithelial regeneration following graft-versus-host disease damage Free

In allogeneic hematopoietic stem cell transplantation (allo-HSCT), complications such as graft-versus-host disease (GVHD) continue to limit therapeutic success. Microbiome disruption has been identified as a risk factor for the development of intestinal GVHD. To date, microbiome analyses in the allo-HSCT setting have mainly been performed on fecal samples, while specimens from the small intestine are rarely studied, especially in patients. Interestingly, results of a recent study in a murine GVHD model suggest that the ileal microbiota is a critical factor influencing GVHD severity by regulating MHC class II expression in the ileal epithelium. Clinically, severe histological GVHD damage of the ileum, not the colon, is associated with refractoriness to treatment. Thus, understanding the role of the ileal microbiota is urgently needed.

Recently, we found that microbiota depletion using an antibiotic cocktail aggravates histological damage of the ileum in allo-HSCT mice. We hypothesize that disruption of the ileal microbiota impairs epithelial regeneration, leading to worsened GVHD. To investigate this, we established a novel murine GVHD model using in vivo splenic T cell depletion (SpTCD). In this model, we administered congenically labeled (Thy-1.1⁺) splenocytes from B6 Thy1.1 mice and Thy-1.2⁺ bone marrow (BM) cells from B6 Thy1.2 mice to B6D2F1 recipients after lethal irradiation on day 0 and then depleted allogeneic splenic T cells in vivo, with administration of a Thy-1.1-depleting antibody on day 7. These allogeneic hosts developed mild GVHD induced by BM graft-derived T cells. Given that the number of ileal Paneth cells was significantly correlated with both GVHD clinical and histological scores of the ileum in this model, we utilized Paneth cell numbers as a marker of impaired epithelial regeneration and GVHD severity.

To investigate the impact of the ileal microbiota on ileal epithelial damage, we depleted the ileal microbiota using an antibiotic cocktail (ampicillin, enrofloxacin, metronidazole, and vancomycin) in allogeneic mice after SpTCD. Following this, we reintroduced ileal bacteria in the form of 100 mg of murine ileal contents. Depletion of the ileal microbiota significantly reduced Paneth cell numbers, whereas reintroduction of ileal bacteria led to a marked restoration of Paneth cells. These findings indicate that the ileal microbiotaplay an important role in reducing GVHD severity. Using 16S rRNA gene sequencing, we found that the abundances of Lachnospiraceae species (Lachno spp.) were significantly associated with Paneth cell recovery. To determine whether Lachno spp. are sufficient to restore Paneth cells, we orally administered ethanol-treated feces (Lachno subset), which depletes nearly all subsets of intestinal bacteria with the exception of spore-forming species, including many Lachno spp., to decontaminated allogeneic mice with SpTCD. We found that administration of the Lachno subset significantly increased abundances of Lachno spp. as well as numbers of Paneth cells.

We then explored how Lachno spp.promote ileal epithelial regeneration following GVHD damage. In our bulk RNA sequencing of ileal tissues, we found that the CYP3A family genes, which are typically induced by the ligands of bile acid receptors, were downregulated in the setting of microbiota depletion and were significantly upregulated by reintroduction of ileal bacteria or Lachno subset into decontaminated allogeneic mice with SpTCD. Lachno spp. are known to serve in bile acid (BA) metabolism, particularly in the production of secondary BAs (SBAs) through 7α-dehydroxylation of primary BAs. Metabolomic analysis of the ileal luminal contents revealed that ileal microbiota depletion significantly reduced concentrations of SBAs, such as deoxycholic acid. In contrast, administration of Lachno spp. significantly increased concentrations of SBAs and promoted epithelial regeneration after GVHD damage. Importantly, this regenerative effect by Lachno spp. was abrogated by cholestyramine, which binds and sequesters BAs broadly.

The use of antibiotics is often necessary in allo-HSCT patients to treat bacterial infections. However, it is frequently accompanied by disruption of the microbiome. Preserving the abundance of Lachno spp. or their BA metabolic activity may represent a novel therapeutic strategy to enhance epithelial regeneration and reduce the severity of GVHD.