Disclosure: H. Herzog: None. C. Fang: None. L. Lam: None. A. Zamarioli: None. A. Sharma: None. T. Moody: None. C. Lal Gupta: None. D. Dumlao: None. K.L. Wentworth: None. K. Yu: None. V. Lu: None. K. Pollard: None. C. Hernandez: None. D. Perrien: None. E.C. Hsiao: None. Heterotopic ossification (HO) is the formation of bone in soft tissues, caused by trauma, surgeries, or genetic mutations. HO leads to the loss of mobility due to joint fusion, resulting in pain, physical disabilities, and deformities. Treatments for HO are limited and focus on symptom and pain management; current available therapies are moderately effective at reducing new HO, no therapies exist to reverse HO formation. HO is difficult to study due to varied triggers and the poor ability to predict timing and location of lesions. Genetic forms of HO provide a consistent driver of the condition, creating an opportunity to study factors that influence lesion formation. Fibrodysplasia Ossificans Progressiva (FOP) is a severe genetic form of HO caused by the ACVR1R206H mutation, which enhances BMP signaling and creates an inappropriate signaling response to Activin A. FOP pathology manifests as flare-ups of ectopic bone formation driven by local and systemic inflammatory responses including elevated inflammatory cellular activity and cytokines that precede HO. Despite the highly conserved ACVR1R206H mutation, FOP patients exhibit disease variability not explained by genotype, suggesting a contribution of environmental factors to disease progression. The gut microbiome influences systemic inflammation through the release of microbial metabolites into the blood and modulation of the gut microbiome has shown promise for treatment of RA. Thus, we examined if the gut microbiome may be a regulator of HO formation using a murine model of FOP (PDGFRα-cre/ACVR1R206H).PDGFRα-Cre mice were crossed with ACVR1flR206H mice to induce the ACVR1R206H mutation in PDGFRα cells, generating a murine FOP model. FOP mice exhibit a significantly shortened lifespan, averaging ∼40 days, primarily due to HO formation. To test the role of the gut microbiome in HO formation, we ablated the gut microbiome (GMA) in FOP mice starting at weaning using a broad-spectrum antibiotic cocktail (1 g/L ampicillin, 0.5 g/L metronidazole, 0.5 g/L neomycin, and 0.5 g/L vancomycin). GMA significantly increased early survival in female FOP mice to an average of 80 days (p=0.001) but had little effect in males, which remained at 40 days (p=0.6). MicroCT scans after 4 weeks of GMA showed a 37.8% reduction in whole-body HO volume compared to Non-GMA mice (p = 0.20). Metagenomic and metabolomic analyses indicated gender-specific inflammatory responses and bile acid metabolism changes in GMA mice. Five candidate anti-inflammatory metabolites were differentially abundant in GMA vs. non-GMA mice, with higher levels in females and lower levels in males. These results suggest that microbially-derived metabolites may drive HO severity in FOP mice by a sex-specific mechanism, highlighting the potential role the gut microbiome or specific microbially derived products as therapeutic targets for FOP and potentially other HO conditions. Presentation: Sunday, July 13, 2025

ABSTRACT People living with HIV (PLWH) represent a vulnerable population to adverse musculoskeletal outcomes due to HIV infection, antiretroviral therapy (ART), and at‐risk alcohol use. Developing measures to prevent skeletal degeneration in this group requires a grasp of the relationship between alcohol use and low bone mass in both the PLWH population and its constituents as defined by sex, age, and race. We examined the association of alcohol use with serum biochemical markers of bone health in a diverse cohort of PLWH enrolled in the New Orleans Alcohol Use in HIV (NOAH) study. To explore the effects of alcohol on bone in the context of HIV and ART and the role of estrogen, we conducted a parallel, translational study using simian immunodeficiency virus (SIV) + /ART + female rhesus macaques divided into four groups: vehicle (Veh)/Sham; chronic binge alcohol (CBA)/Sham; Veh/ovariectomy (OVX); and CBA/OVX. Clinical data showed that both osteocalcin (Ocn) and procollagen type I N‐propeptide (PINP) levels were inversely associated with multiple measures of alcohol consumption. Age (>50 years) significantly increased susceptibility to alcohol‐associated suppression of bone formation in both female and male PLWH, with postmenopausal status appearing as an additional risk factor in females. Serum sclerostin (Scl) levels correlated positively with measures of alcohol use and negatively with Ocn. Micro‐CT analysis of the macaque tibias revealed that although both CBA and OVX independently decreased trabecular number and bone mineral density, only OVX decreased trabecular bone volume fraction and impacted cortical geometry. The clinical data implicate circulating Scl in the pathogenesis of alcohol‐induced osteopenia and suggest that bone morphology can be significantly altered in the absence of net change in osteoblast function as measured by serum markers. Inclusion of sophisticated tools to evaluate skeletal strength in clinical populations will be essential to understand the impact of alcohol‐induced changes in bone microarchitecture. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

IL-17A (IL-17), a driver of the inflammatory phase of fracture repair, is produced locally by several cell lineages including γδ T cells and Th17 cells. However, the origin of these T cells and their relevance for fracture repair are unknown. Here, we show that fractures rapidly expanded callus γδ T cells, which led to increased gut permeability by promoting systemic inflammation. When the microbiota contained the Th17 cell–inducing taxon segmented filamentous bacteria (SFB), activation of γδ T cells was followed by expansion of intestinal Th17 cells, their migration to the callus, and improved fracture repair. Mechanistically, fractures increased the S1P receptor 1–mediated (S1PR1-mediated) egress of Th17 cells from the intestine and enhanced their homing to the callus through a CCL20-mediated mechanism. Fracture repair was impaired by deletion of γδ T cells, depletion of the microbiome by antibiotics (Abx), blockade of Th17 cell egress from the gut, or Ab neutralization of Th17 cell influx into the callus. These findings demonstrate the relevance of the microbiome and T cell trafficking for fracture repair. Modifications of microbiome composition via Th17 cell–inducing bacteriotherapy and avoidance of broad-spectrum Abx may represent novel therapeutic strategies to optimize fracture healing.

Ciliogenic components, such as the family of intraflagellar transport (IFT) proteins, are recognized to play key roles in endochondral ossification, a critical process to form most bones. However, the unique functions and roles of each IFT during endochondral ossification remain unclear. Here, we show that IFT20 is required for endochondral ossification in mice. Utilizing osteo-chondrocyte lineage-specific Cre mice (Prx1-Cre and Col2-Cre), we deleted Ift20 to examine its function. Although chondrocyte-specific Ift20 deletion with Col2-Cre mice did not cause any overt skeletal defects, mesoderm-specific Ift20 deletion using Prx1-Cre (Ift20:Prx1-Cre) mice resulted in shortened limb outgrowth. Primary cilia were absent on chondrocytes of Ift20:Prx1-Cre mice, and ciliary-mediated Hedgehog signaling was attenuated in Ift20:Prx1-Cre mice. Interestingly, loss of Ift20 also increased Fgf18 expression in the perichondrium that sustained Sox9 expression, thus preventing endochondral ossification. Inhibition of enhanced phospho-ERK1/2 activation partially rescued defective chondrogenesis in Ift20 mutant cells, supporting an important role for FGF signaling. Our findings demonstrate that IFT20 is a critical regulator of temporospatial FGF signaling that is required for endochondral ossification.

Bone metastases are frequent complications of malignant melanoma leading to reduced quality of life and significant morbidity. Regulation of immune cells by the gut microbiome influences cancer progression, but the role of the microbiome in tumor growth in bone is unknown. Using intracardiac or intratibial injections of B16-F10 melanoma cells into mice, we showed that gut microbiome depletion by broad-spectrum antibiotics accelerated intraosseous tumor growth and osteolysis. Microbiome depletion blunted melanoma-induced expansion of intestinal NK cells and Th1 cells and their migration from the gut to tumor-bearing bones. Demonstrating the functional relevance of immune cell trafficking from the gut to the bone marrow (BM) in bone metastasis, blockade of S1P-mediated intestinal egress of NK and Th1 cells, or inhibition of their CXCR3/CXCL9-mediated influx into the BM, prevented the expansion of BM NK and Th1 cells and accelerated tumor growth and osteolysis. Using a mouse model, this study revealed mechanisms of microbiota-mediated gut-bone crosstalk that are relevant to the immunological restraint of melanoma metastasis and tumor growth in bone. Microbiome modifications induced by antibiotics might have negative clinical consequences in patients with melanoma.

Fibrodysplasia ossificans progressiva (FOP) is a debilitating rare disease known for episodic endochondral heterotopic ossification (HO) caused by gain-of-function mutations in ACVR1/ALK2. However, disease severity varies among patients with identical mutations suggesting disease-modifying factors, including diet, may have therapeutic implications. The roles of vitamin D3 in calcium metabolism and chondrogenesis are known, but its effects on BMP signaling and chondrogenesis are less studied. This review attempts to assess the possibility of vitamin D’s effects in FOP by exploring relevant intersections of VD3 with mechanisms of FOP flares. In vitro and in vivo studies suggest vitamin D suppresses inflammation, while clinical studies suggest that vitamin D3 protects against arteriosclerosis and inversely correlates with non-genetic intramuscular HO. However, the enhancement of chondrogenesis, BMP signaling, and possibly Activin A expression by vitamin D may be more relevant in FOP. There appears to be little potential for vitamin D to reduce HO in FOP, but testing the potential for excess vitamin D to promote HO may be warranted.

Ciliogenic components, such as the family of intraflagellar transport (IFT) proteins, are recognized to play key roles in endochondral ossification, a critical process to form most bones. However, it remains unclear how each IFT protein performs its unique function to regulate endochondral ossification. Here, we show that intraflagellar transport 20 (IFT20) is required for early chondrogenesis. Utilizing three osteo-chondrocyte lineage-specific Cre mice (Prx1-Cre, Col2-Cre and Aggrecan-CreERT2), we deleted Ift20 to examine its function. While chondrocyte-specific Ift20 deletion with Col2-Cre or Aggrecan-CreERT2 drivers did not cause overt skeletal defects, mesoderm-specific Ift20 deletion using Prx1-Cre (Ift20:Prx1-Cre) resulted in shortened limb outgrowth. Although primary cilia were not formed in Ift20:Prx1-Cre mice, ciliary Hedgehog signaling was only moderately affected. Interestingly, loss of Ift20 lead to upregulation of Fgf18 expression resulting in ERK1/2 activation and sustained Sox9 expression, thus preventing endochondral ossification. Inhibition of enhanced phospho-ERK1/2 activation partially rescued defective chondrogenesis in Ift20 mutant cells, supporting an important role for FGF signaling. Our findings demonstrate a novel mechanism of IFT20 in early chondrogenesis during endochondral ossification.

Excess inflammation and canonical BMP receptor (BMPR) signaling are coinciding hallmarks of the early stages of injury-induced endochondral heterotopic ossification (EHO), especially in the rare genetic disease fibrodysplasia ossificans progressiva (FOP). Multiple inflammatory signaling pathways can synergistically enhance BMP-induced Smad1/5/8 activity in multiple cell types, suggesting the importance of pathway crosstalk in EHO and FOP. Toll-like receptors (TLRs) and IL-1 receptors mediate many of the earliest injury-induced inflammatory signals largely via MyD88-dependent pathways. Thus, the hypothesis that MyD88-dependent signaling is required for EHO was tested in vitro and in vivo using global or Pdgfrα-conditional deletion of MyD88 in FOP mice. As expected, IL-1β or LPS synergistically increased Activin A (ActA)-induced phosphorylation of Smad 1/5 in fibroadipoprogenitors (FAPs) expressing Alk2R206H. However, conditional deletion of MyD88 in Pdgfrα-positive cells of FOP mice did not significantly alter the amount of muscle injury-induced EHO. Even more surprisingly, injury-induced EHO was not significantly affected by global deletion of MyD88. These studies demonstrate that MyD88-dependent signaling is dispensable for injury-induced EHO in FOP mice.

OBJECTIVES:Conditional Alk2Q207D-floxed (caALK2fl) mice have previously been used as a model of heterotopic ossification (HO). However, HO formation in this model can be highly variable, and it is unclear which methods reliably induce HO. Hence, these studies report validated methods for reproducibly inducing HO in caALK2fl mice.METHODS:Varying doses of Adex-cre and cardiotoxin (CTX) were injected into the calf muscles of 9, 14, or 28-day-old caALK2fl/- or caALK2fl/fl mice. HO was measured by planar radiography or microCT at 14-28 days post-injury.RESULTS:In 9-day-old caALK2fl/- or caALK2fl/fl mice, single injections of 109 PFU Adex-cre and 0.3 μg of CTX were sufficient to induce extensive HO within 14 days post-injury. In 28-day-old mice, the doses were increased to 5 x 109 PFU Adex-cre and 3.0 μg of CTX to achieve similar consistency, but at a slower rate versus younger mice. Using a crush injury, instead of CTX, also provided consistent induction of HO. Finally, the Type 1 BMPR inhibitor, DMH1, significantly reduced HO formation in 28-day-old caALK2fl/fl mice.CONCLUSIONS:These data illustrate multiple methods for reliable induction of localized HO in the caALK2flmouse that can serve as a starting point for new laboratories utilizing this model.

Non-alcoholic fatty liver disease (NAFLD) affects over 30% of adults in the United States. Bone morphogenetic protein (BMP) signaling is known to contribute to hepatic fibrosis, but the role of BMP signaling in the development of NAFLD is unclear. In this study, treatment with either of two BMP inhibitors reduced hepatic triglyceride content in diabetic (db/db) mice. BMP inhibitor-induced decrease in hepatic triglyceride levels was associated with decreased mRNA encoding Dgat2, an enzyme integral to triglyceride synthesis. Treatment of hepatoma cells with BMP2 induced DGAT2 expression and activity via intracellular SMAD signaling. In humans we identified a rare missense single nucleotide polymorphism in the BMP type 1 receptor ALK6 (rs34970181;R371Q) associated with a 2.1-fold increase in the prevalence of NAFLD. In vitro analyses revealed R371Q:ALK6 is a previously unknown constitutively active receptor. These data show that BMP signaling is an important determinant of NAFLD in a murine model and is associated with NAFLD in humans.