Moderate effects of the Arginine to Histidine R47H variant of the Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) on bone structure in four core genotypes gonadal male and female mice

Background The T riggering R eceptor E xpressed on M yeloid Cells 2 (TREM2) gene is expressed in cells of the hematopoietic lineage, such as microglia and osteoclasts found in the brain and bone, respectively. Previous studies showed that expression of the TREM2 gene variant known as TREM2-R47H leads to altered musculoskeletal phenotype in a sex-dependent manner. Further, gonadectomy ameliorates the skeletal phenotype of gonadal female TREM2 R47H/+ mice. We also showed that sex chromosomes play a key role on skeletal mass and bone strength. Due to the sex-dimorphic bone and skeletal muscle phenotype exhibited by mice expressing the TREM2 gene variant, we investigated the role of chromosomal (XX vs. XY) or gonadal (ovaries vs. testes) sex in the Four Core Genotypes (FCG) mice model. Methods FCG C57Bl/6J mice expressing the TREM2-R47H variant were mated to obtain TREM2 wildtype (TREM2 +/+ , WT) and TREM2 R47H/+ FCG mice. Four to 5.5-month-old gonadal male (XXT and XYT) and female (XXO and XYO) mice were analyzed. Body weight and bone mineral density were measured at baseline and at the endpoint (5.5 months of age) by DXA/Piximus.​ Micro-computed tomography, dynamic and static histomorphometry, 3-point bending test (mechanical properties), and bone turnover markers were measured at the endpoint. Two-way ANOVA analyses were performed using Prism 10 to identify the contributions of chromosome sex, the presence of the TREM2-R47H variant, and their interaction, separately for each gonadal sex. Results Gonadal males : chromosome sex (XX/XY) effects are found for several bone structural parameters in femur and lumbar vertebra 5, whereas there was an interaction between gonadal sex and chromosome sex for other structural measurements in both bones by µCT. This suggests that the effect of the TREM2 variant depends on the presence of the Y chromosome in gonadal males. Mechanical testing shows chromosome sex effects, with higher overall values for XXT mice regardless of the presence of the TREM2 variant. At the cellular level, chromosome sex influences osteoblast, but not osteoclast, number and surface similarly for wild type and TREM2 mutants. Bone formation on the femur cortex and serum markers of formation/resorption were unchanged, suggesting that structural changes result from bone modeling/remodeling at an earlier age. Gonadal females : Chromosome sex affects body weight gain (higher in XYO than XXO mice), but no bone mineral density accrual. Chromosome sex affects the percent change (comparing final and initial values) in total lean mass (XYO > XXO) with chromosome sex x TREM2 genotype interaction and differences in total/%fat mass (TREM2 R47H/+ < WT), and %lean mass (TREM2 R47H/+ > WT) only for XYO mice. Chromosome sex affects distal femur volumetric bone mass (XYO > XXO), but the TREM2 genotype influences lumbar vertebra trabecular number and separation, which trended higher in TREM2 R47H/+ vs. WT mice for sex complement. Chromosome sex influences femur cortical bone, with overall higher values in XXO mice, independent of TREM2 genotype. Mechanical testing parameters were also influenced by chromosome sex with XXO > XYO mice. Femur cortical bone formation is higher on the endocortical but lower on the periosteal surface in XXO vs. XYO (chromosome sex effect). The opposite effects on the bone surfaces might explain the unchanged serum bone formation marker, Procollagen Type 1 N-terminal propeptide (P1NP). Yet, chromosome sex affects the levels of the resorption marker, C-terminal telopeptide of type 1 collagen (CTX-1), which were lower in XXO mice compared to XYO. At the cellular level, there is an interaction between chromosome sex and TREM2 genotype for osteoclast, but not osteoblast, parameters in vertebral trabecular bone. Osteoclast number and surface are higher in TREM2 R47H/+ mice only for XYO mice, and, as for CTX-1, are lower in XXO mice compared to XYO only for the mutant mice. Conclusion Our findings suggest that chromosome sex partially affects the consequences of expression of the TREM2-R47H variant on bone structure in 5.5 month-old mice, whereas the outcomes of the gene variant depend on the mouse gonadal sex. Overall, the findings of the current studies add to our previous publications and overall suggest that the consequences of expression of the R47H variant of the TREM2 gene on the musculoskeletal system depend on the presence of sex hormones and the chromosome sex in a manner that depends on the bone site or skeletal muscle studied. Future studies will determine whether the consequences of the genetic manipulations at the cellular level (both number and activity) result in a stronger bone phenotype at an older age.