Enteral supplementation with a TGF-β-enriched polymeric diet mitigates gastrointestinal acute gvhd

Abstract Gastrointestinal acute graft-versus-host disease (GI-aGVHD) is a major contributor to non-relapse mortality after allogeneic hematopoietic cell transplantation (allo-HCT), particularly in steroid-refractory patients. While immunosuppressive therapies are standard, they risk compromising the beneficial graft-versus-leukemia effect. Nutritional interventions offer a non-immunosuppressive, tissue-protective adjunct. However, mechanistic preclinical evaluations of clinically administered nutritional formulas in aGVHD remain scarce. Addressing this gap, we evaluated whether Modulen® IBD (a casein-based formula enriched with transforming growth factor-beta (TGF-β) and approved for enteral use in Crohn's disease) could mitigate GI-aGVHD. Based on the metabolic signatures of aGVHD reported by Michonneau et al. (Nat. Commun., 2019), we assessed the effects of Modulen® IBD in a pilot clinical study. Allo-HCT recipients (n = 3 to 4) received daily prophylactic Modulen® IBD (1.5 kcal/ml), from the start of conditioning regimen until consent withdrawal, typically coinciding with conditioning-related toxicity. Serum samples were collected at two timepoints (start of treatment and post-transplant) and analyzed using targeted liquid chromatography-mass spectrometry (LC-MS) for the abundance of fatty acids (FAs) and polar metabolites. We observed increased levels of anti-inflammatory long-chain (LC) FAs like oleic and eicosatrienoic acids, as well as elevated kynurenic acid, a microbiota-derived aryl hydrocarbon receptor ligand implicated in intestinal barrier protection. These findings suggest a systemic metabolic shift toward reduced inflammation. In parallel, we employed a pre-clinical model to validate and expand upon the immune, microbial, and metabolic mechanisms of our clinical study. We used a well-established major histocompatibility complex-mismatched murine allo-HCT model (BALB/c → C57BL/6), administering Modulen® IBD or vehicle orally daily from day 0 to day 14 post-transplant (1kcal/ml). Modulen® IBD treatment significantly reduced histological aGVHD scores in the colon, small intestine and liver of mice. Kaplan-Meier analysis indicated a non-significant increase toward improved survival. Similar to patients, LC-MS analysis of serum from treated mice confirmed increased levels of oleic and eicosatrienoic acids. Additionally, there was a reduction in pro-inflammatory LCFAs (palmitic, stearic, and arachidonic acids) and GVHD-associated amino acids (arginine, ornithine, leucine, and aspartate) in the colon. Notably, butyrate, a short-chain FA known to promote epithelial TGF-β production and regulatory T cell induction, was significantly elevated in colon tissue and stool. Stool 16S rRNA gene sequencing revealed no changes in alpha diversity, but we found an enrichment of Lactococcus and Bacillus, genera that support butyrogenic bacteria and butyrate production. These microbial shifts were accompanied by increased TGF-β abundance in colonic epithelial cells and Ly6G⁺ neutrophils, increased IL-10 in CD4⁺ T cells and Ly6C⁺ monocytes, and reduced CD3⁺ T cell infiltration in the colon, suggesting attenuation of local alloreactivity. Beyond the gut, Modulen® IBD treatment enhanced IL-10 production in peripheral CD4⁺ T cells and reduced CD8⁺ T cell infiltration in the brain, indicating systemic immunoregulatory effects. Based on the three main mechanistic changes observed (cytokine-mediated immune modulation, microbial remodeling favoring beneficial commensals, and metabolic shifts away from pro-inflammatory lipids and amino acids), we hypothesize that enrichment of Lactococcus and Bacillus contributes to increased colonic butyrate levels. This may support epithelial and innate immune-derived TGF-β production, followed by IL-10 induction in CD4⁺ T cells. The resulting immunosuppressive environment is likely a key factor in mitigating local and systemic aGVHD. While our data demonstrate strong associations, further studies (for e.g. with gnotobiotic models) are needed to establish definitive causal links. There is no universal consensus on an optimal diet for aGVHD, partly due to the complexity and variability of dietary patterns across geographic, economic, and cultural contexts. This gap is compounded by limited preclinical evaluation of therapeutic nutritional formulas. Our findings support Modulen® IBD as a promising non-pharmacological adjunctive therapy, providing a rationale for integrating it into clinical GI-aGVHD management.