Abstract
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. 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. 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. 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.
Highlights
The Murphy Roths Large (MRL) mouse strain was originally selected for large body size in the 1960s; at the F12 generation, these mice developed a spontaneous mutation in the immune regulator Fas
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
Comparing B6-transplanted MRL to MRL-vehicle mice, we identified the transfer of B6-associated clades into recipient animals, including members of phylum Verrucomicrobiae (LDA-ES 4.40, P = 7.8E-4), whereas MRL-vehicle animals were again characterized by members of the phylum Firmicutes (LDA-ES 4.56, P = 7.8E-4), among others
Summary
The Murphy Roths Large (MRL) mouse strain was originally selected for large body size in the 1960s; at the F12 generation, these mice developed a spontaneous mutation in the immune regulator Fas. In experiments over the past decade, several backcrosses of LG/J and SM/J (a non-healer strain) have been generated to evaluate the heritability of ear hole closure and OA protection [11,12,13,14] Those backcrosses most genetically similar to LG/J mice have the greatest regenerative ear hole wound healing capacity [15], as well as the highest articular cartilage regeneration ability [11, 16]. Genetic analyses of these strains have been conducted [10, 12, 17, 18], with several genes and gene pathways known to be involved with wound healing [19], including DNA repair and Wnt signaling [12]. A 2015 report made an unexpected discovery: C57BL/ 6J mice did not improve either ear-wound or articular cartilage healing following an allogeneic bone marrow transplant from MRL/MpJ donors [20], and a recent study found the heritability of OA protection in LG/SM backcrosses to be only moderate (0.18 to 0.58) [21], suggesting that other environmental factors play a role in healing in MRL mice
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