Ear wound healing in MRL/MpJ mice is associated with gut microbiome composition and is transferable to non-healer mice via microbiome transplantation

Abstract

Objective Adult elastic cartilage has limited repair capacity. MRL/MpJ (MRL) mice, by contrast, are capable of spontaneously healing ear punctures. This study was undertaken to characterize microbiome differences between healer and non-healer mice and to evaluate whether this healing phenotype can be transferred via gut microbiome transplantation. Methods We orally transplanted C57BL/6J (B6) mice with MRL/MpJ cecal contents at weaning and as adults (n = 57) and measured ear hole closure 4 weeks after a 2.0mm punch and compared to vehicle-transplanted MRL and B6 (n = 25) and B6-transplanted MRL (n = 20) mice. Sex effects, timing of transplant relative to earpunch, and transgenerational heritability were evaluated. In a subset (n = 58), cecal microbiomes were profiled by 16S sequencing and compared to ear hole closure. Microbial metagenomes were imputed using PICRUSt. Results Transplantation of B6 mice with MRL microbiota, either in weanlings or adults, improved ear hole closure. B6-vehicle mice healed ear hole punches poorly (0.25±0.03mm, mm ear hole healing 4 weeks after a 2mm ear hole punch [2.0mm—final ear hole size], mean±SEM), whereas MRL-vehicle mice healed well (1.4±0.1mm). MRL-transplanted B6 mice healed roughly three times as well as B6-vehicle mice, and half as well as MRL-vehicle mice (0.74±0.05mm, P = 6.9E-10 vs. B6-vehicle, P = 5.2E-12 vs. MRL-vehicle). Transplantation of MRL mice with B6 cecal material did not reduce MRL healing (B6-transplanted MRL 1.3±0.1 vs. MRL-vehicle 1.4±0.1, p = 0.36). Transplantation prior to ear punch was associated with the greatest ear hole closure. Offspring of transplanted mice healed significantly better than non-transplanted control mice (offspring:0.63±0.03mm, mean±SEM vs. B6-vehicle control:0.25±0.03mm, n = 39 offspring, P = 4.6E-11). Several microbiome clades were correlated with healing, including Firmicutes (R = 0.84, P = 8.0E-7), Lactobacillales (R = 0.65, P = 1.1E-3), and Verrucomicrobia (R = -0.80, P = 9.2E-6). Females of all groups tended to heal better than males (B6-vehicle P = 0.059, MRL-transplanted B6 P = 0.096, offspring of MRL-transplanted B6 P = 0.0038, B6-transplanted MRL P = 1.6E-6, MRL-vehicle P = 0.0031). Many clades characteristic of female mouse cecal microbiota vs. males were the same as clades characteristic of MRL and MRL-transplanted B6 mice vs. B6 controls, including including increases in Clostridia and reductions in Verrucomicrobia in female mice. Conclusion In this study, we found an association between the microbiome and tissue regeneration in MRL mice and demonstrate that this trait can be transferred to non-healer mice via microbiome transplantation. We identified several microbiome clades associated with healing.