Butyrate ameliorates Tet2-deficient clonal hematopoiesis phenotypes driven by high B12-induced gut dysbiosis.

Abstract Introduction: Recent studies have linked elevated vitamin B12 serum levels with the presence of clonal hematopoiesis (CH) and an increased risk of developing myeloid malignancy. Excess B12 supplementation increases serum levels, alters gut microbial composition, and reduces the production of short-chain fatty acids (SCFAs), such as butyrate, which help maintain gut barrier function and mucosal integrity. Additionally, TET2 mutation is a frequent driver of CH that progresses in a positive feedback loop in response to microbial signals suggesting that B12 may influence CH via the gut microbiome. Our previous preliminary studies demonstrated high B12 supplementation in a Tet2-deficient murine CH model leads to increased gut permeability, a myeloid-lineage bias, and increased systemic inflammation. Using metagenomic analysis of shotgun-sequenced fecal pellets, we found that butyrate metabolism was decreased across the gut microbiome in B12-supplemented mice. We hypothesized butyrate supplementation could ameliorate B12-induced CH phenotypes by counteracting the gut dysbiosis and reducing inflammation. Methods: To investigate the therapeutic effect of butyrate, we modeled CH with a murine competitive bone marrow transplantation using a 1:1 ratio of Tet2+/+ and Tet2+/- cells, established a four-arm treatment cohort of control or high B12 ± oral butyrate supplementation, and treated for seven months. We measured gut permeability by FITC-dextran gut translocation, examined endpoint hematopoietic organs for changes in Tet2-deficient clonal cell populations by flow cytometry, performed serum cytokine arrays to measure levels of circulating inflammatory mediators, and performed single-cell RNA sequencing on sorted Tet2+/- bone marrow cells to identify cellular compartments that showed population-level and gene expression changes in response to treatment. Results: Butyrate supplementation reversed the B12-induced myeloid-biased differentiation of Tet2+/- cells observed in endpoint peripheral blood, bone marrow, and splenic hematopoietic compartments but did not reduce competitiveness of Tet2+/- cells. Serum inflammatory cytokines, including IL-6 and CCL1/I-309 were elevated in B12-treated mice and significantly reduced in the B12+butyrate co-treatment cohort. Tet2+/- lineage-negative, Kit-positive (LK) and granulocyte and macrophage progenitor (GMP) cells were elevated in frequency in the high B12 treatment cohort and showed significant phenotypic reversal in the B12+butyrate group. Butyrate co-treatment also abrogated B12-induced intestinal permeability, evidenced by reduced FITC-dextran translocation. Finally, single-cell RNA-seq analysis showed high B12 supplementation created a transcription profile that was enriched for granulocytic, monocyte, and neutrophil gene signatures compared to control, and depleted for B cell gene signatures. Importantly, these transcriptional phenotypes were also reversed upon co-treatment with butyrate: bone marrow stem and progenitor cells and B cells regained enrichment of B cell marker expression and reduced expression of myeloid markers and genes associated with inflammatory signaling, including Itgam, S100a8, S100a9, and Ifitm1. Conclusion: These findings demonstrate B12-induced myeloid lineage biases and pro-inflammatory signaling in Tet2-deficient CH can be tempered or reversed with butyrate supplementation, opening the possibility for therapeutic intervention using an inexpensive, orally bioavailable small molecule to mitigate CH-associated sequelae.