OA susceptibility in mice is partially mediated by the gut microbiome, is transferrable via microbiome transplantation and is associated with immunophenotype changes

Abstract

Objectives: The Murphy Roths Large (MRL)/MpJ 'superhealer' mouse strain is protected from post-traumatic osteoarthritis (OA), although no studies have evaluated the microbiome in the context of this protection. This study characterised microbiome differences between MRL and wild-type mice, evaluated microbiome transplantation and OA and investigated microbiome-associated immunophenotypes.

Methods: Cecal material from mixed sex C57BL6/J (B6) or female MRL/MpJ (MRL) was transplanted into B6 and MRL mice, then OA was induced by disruption of the medial meniscus surgery (DMM). In other experiments, transplantation was performed after DMM and transplantation was performed into germ-free mice. Transplanted mice were bred through F2. OARSI, synovitis and osteophyte scores were determined blindly 8 weeks after DMM. 16S microbiome sequencing was performed and metagenomic function was imputed. Immunophenotypes were determined using mass cytometry.

Results: MRL-into-B6 transplant prior to DMM showed reduced OA histopathology (OARSI score 70% lower transplant vs B6 control), synovitis (60% reduction) and osteophyte scores (30% reduction) 8 weeks after DMM. When performed 48 hours after DMM, MRL-into-B6 transplant improved OA outcomes but not when performed 1-2 weeks after DMM. Protection was seen in F1 (60% reduction) and F2 progeny (30% reduction). Several cecal microbiome clades were correlated with either better (eg, Lactobacillus, R=-0.32, p=0.02) or worse (eg, Rikenellaceae, R=0.43, p=0.001) OA outcomes. Baseline immunophenotypes associated with MRL-into-B6 transplants and MRL included reduced double-negative T cells and increased CD25+CD4+ T cells.

Conclusion: The gut microbiome is responsible in part for OA protection in MRL mice and is transferrable by microbiome transplantation. Transplantation induces resting systemic immunophenotyping changes that correlate with OA protection.

Keywords: Inflammation; Osteoarthritis; Osteoarthritis, Knee.

Figure 1:

OA outcomes in mice under various transplantation conditions, including OARSI score, histologic synovitis and histologic osteophyte scores. Horizontal line represents mean, bars represent SEM. (A) Results of mice transplanted 48 hours prior to DMM and B6/MRL controls; (B) Results of MRL-into-B6 mice transplanted at various times after DMM (48h, 1 week, and 2 weeks), along with transplant-before-DMM and B6 controls for comparison; (C) Results of progeny of MRL-into-B6 transplanted mice, with primary transplanted (F0) mice and B6 controls for comparison; (D) Results of MRL- and B6-transplantation into C57BL6/n germ-free mice, transplanted 48h prior to DMM, with B6 and MRL controls for comparison. P values were calculated using a non-FDR-corrected Student t-test (data confirmed to be normally distributed).

Figure 2:

Example histology images from various transplantation groups, modified coronal sections stained with Safranin-O. Medial knee compartment shown.

Figure 3:

Microbiome composition analysis by 16S deep sequencing. (A) Microbiome group comparisons. Numbers represent linear discriminant analysis effect size (LDA-ES); negative values indicate enrichment in mouse groups associated with worse OA outcome (B6, B6-into-MRL, B6-into-GF), whereas positive values indicate enrichment in mouse groups associated with better OA outcomes (MRL, MRL-into-B6, progeny of MRL-into-B6, MRL-into-GF). Only statistically significant clade difference (Kruskal-Wallis P≤0.05 followed by linear discriminant analysis) values are shown. (B) OARSI score correlation with individual-level gut microbiome data, top 3 negatively- and top 3 positively correlated microbiome clades presented. This correlation includes B6 controls, MRL controls, MRL-into-B6 transplants, and F1 and F2 progeny of MRL-into-B6 mice. Blue line indicates best-fit linear regression among all groups, red curved lines are 95% confidence intervals of linear regression. R values represent Pearson correlation coefficient, P values presented significance level of Pearson correlation coefficient.

Figure 4:

Mass cytometry immunophenotyping of a second group of animals post-transplantation but without DMM surgery, reflecting baseline immunophenotype changes induced by microbiome transplantation. (A) t-distributed stochastic neighbor embedding plot (tSNE) under various microbiome conditions (B) individual peripheral immune cell subset differences, only statistically significant cell subsets presented; (C) intracellular cytokine differences, only selected statistically significant cytokines presented; (D) serum lipopolysaccharide (LPS) levels among various transplant groups. P values calculated using a non-FDR-corrected Student t-test (data confirmed to be normally distributed).