Galectin-3 in host murine models lessens the severity of graft-versus-host disease by preserving intestinal stem cells irrespective of gut microbiota composition

Abstract Introduction The gut microbiome has been studied in preclinical inflammatory disease models. Dysbiosis, where normal gut bacteria are replaced by aberrant organisms, can worsen inflammation. Altered microbiomes are associated with more severe graft-versus-host disease (GvHD) after allogeneic hematopoietic cell transplantation (allo-HCT). Microbiome regulation and its impact on GvHD is not fully understood. Galectin 3 (Gal-3), a Galectin family member, can alter inflammation. We showed how donor T cell expression of Gal-3 decreases GvHD severity (Mohammadpour et al Cell Rep 2023). We now examine how recipient Gal-3 expression during murine allo-HCT impacts the gut microbiome and GvHD. Methods We used the following murine allo-HCT models 1) (Donor BALB/c (H-2d)→Recipient C57BL/6 (H-2b)) (T cell depleted (TCD) BM+/-CD90.2+ spleen T cells); 2) C57BL/6 wild type (WT) and Gal-3 deleted (Gal-3-/-) recipients, 3) Four BM chimera types were generated by HCT post total body irradiation (TBI) in C57BL/6 mice: WT→WT, Gal-3⁻/⁻→WT, WT→Gal-3⁻/⁻, and Gal-3⁻/⁻→Gal-3⁻/⁻. 4)We made selective Gal-3-/- in LGR5+ Intestinal stem cells (ISCs) by crossing Gal-3fl/fl mice with LGR5Cre mice, leading to Gal-3fl/flLGR5Cre mice. To induce Gal-3-/- in LGR5+ ISCs, Gal-3fl/flLGR5Cre mice were given Tamoxifen. For all first HCTs, TBI was given at 11Gy. For the 2nd allo-HCTs (chimeras), 8 Gy was used. After allo-HCT, mice were followed for weight and survival. Fecal pellets were saved weekly for microbiome analysis. Intracellular inflammatory cell cytokine flow cytometry analyses were done on isolated tissue. Multicolor immunofluorescence imaging (MFI) detected Gal-3 and other intestinal cell markers. For Graft-versus Leukemia (GvL) studies, Gal-3-/- and WT mice received TBI followed by syngeneic- or allo-HCT with donor TCD-BM+/-lower T cell doses to decrease GvHD but preserve GvL. Luciferase expressing C1498 AML cells were injected 4 hours post allo-HCT with weekly bioluminescence imaging. Results After allo-HCT with donor TCD-BM+T cells, Gal-3-/- mice had inferior survival compared to WT by day 60 (25% vs 60%, p<0.0135). Survival for Gal-3-/- and WT donor TCD-BM only, was 100%. TNF-α, IFN-γ, GM-CSF and IL-17 levels in donor H2kd+CD3+CD4+ T cells were not different in spleen, liver and intestine in WT and Gal-3-/- recipients. Distinct microbiome profiles were seen in WT and Gal-3⁻/⁻ mice pre and post allo-HCT, denoting an inflammatory Gal-3⁻/⁻ gut environment, including the phyla Verrucomicrobia and Proteobacteria. Co-housing to normalize microbiomes did not change the impact of Gal-3⁻/⁻ on GvHD severity. Antibiotic treatment prior to allo-HCT decreased microbial diversity in WT and Gal-3⁻/⁻ recipients, increasing GvHD severity, with Gal-3⁻/⁻ mice having the worst survival. Chimeras received allo-HCT with donor TCD-BM+/-T cells, testing the impact of Gal-3 on hematopoietic and non-hematopoietic tissue. Chimeras with Gal-3-/- non-hematopoietic tissue had the worst survival when compared to WT non-hematopoietic tissue chimeras. MFI identified Gal-3 expression in the intestinal epithelial (Villin+) (28%), IS (LGR5+) (12%) and <5% in Paneth (Lysozyme+), Goblet (Muc2+), Enteroendocrine (CHG-A+) cells. We theorized that ISC Gal-3 expression would impact gut GvHD. MFI analysis of Gal-3fl/flLGR5Cre mice confirmed the absence of Gal-3 on LGR5+ ISCs. WT, Gal-3fl/fl and Gal-3fl/flLGR5Cre mice received allo-HCT with donor TCD-BM+/-T cells. The highest day 60 mortality was seen in Gal-3fl/flLGR5Cre mice (95%) followed by Gal-3fl/fl recipients (50%) (p=0.0154). Gal-3fl/fl and Gal-3fl/flLGR5Cre mice receiving TCD-BM only, had 100% survival. GvL studies showed no difference in survival between Gal-3-/- and WT mice receiving T cells, both with a strong GvL effect. Conclusion Gal-3 expression impacts the gut microbiome and allo-HCT outcome. The lack of Gal-3 in recipient non-hematopoietic tissue led to significant dysbiosis and increased GvHD mortality. Altering the microbiome in WT and Gal-3-/- recipients increased GvHD severity while not affecting the impact of Gal-3 on GvHD incidence and severity. Lack of Gal-3 on ISCs increases the severity and mortality of GvHD. This study showed that Gal-3 protects ISCs, sustaining gut integrity post allo-HCT. Future studies aimed at strategies to enhance the effect of Gal-3 in non-hematopoietic gut cells may improve preclinical GvHD with the potential to translate to clinical trials to decrease GvHD severity without affecting GvL.