Bone infection (osteomyelitis) can arise from direct inoculation during trauma or surgery, contiguous spread from adjacent tissues, or hematogenous dissemination from systemic bacteremia. Despite medical advances, it continues to pose a significant clinical and socioeconomic burden. In the United States alone, bone infection has an estimated annual incidence of 22 cases per 100,000 person-years, particularly increasing among older adults and people with diabetes (Kremers et al., 2015). Children, older individuals, and immunocompromised individuals are particularly vulnerable to bone infections, which often arise when bloodstream infections introduce pathogens into the bone, a risk heightened in individuals with diabetes (Waldvogel et al., 1970). Bone infections can lead to bone destruction and corresponding functional disorders, such as difficulty in walking, eating, or even loss of hearing, severely affecting the quality of life of patients (Usui et al., 2021).

Systemic antibiotics and surgical debridement are the cornerstones of bone infection treatment (Dong et al., 2024). However, therapeutic efficacy is often compromised by factors inherent to bone anatomy and microbial adaptation. Bacteria residing in biofilms or sequestered within osteocyte lacuno-canalicular networks—such as the Haversian and Volkmann canals—are poorly accessible to both antibiotics and immune cells (Toni et al., 2020). Incomplete debridement, suboptimal drug penetration, and poor vascularization of infected bone further limit eradication (Kudo et al., 2003). These challenges are exacerbated by the increasing incidence of antibiotic resistance, which is especially prevalent in chronic or recurrent osteomyelitis caused by pathogens such as Staphylococcus aureus or Pseudomonas aeruginosa (Jones et al., 2004, Qin et al., 2022). In such contexts, antibiotics often fail to eliminate the infection, highlighting the need for alternative or complementary therapeutic strategies that address not only the pathogen but also the disrupted host immune microenvironment.

The immune system is intricately linked to bone homeostasis, giving rise to the interdisciplinary field of osteoimmunology. During bone infection, immune cells—including macrophages, neutrophils, regulatory T cells, B cells, and others—migrate to the lesion site, where they secrete inflammatory cytokines and growth factors that modulate osteoclast and osteoblast activity (Gruber, 2019, Xiao et al., 2018, Araujo-Pires et al., 2015, Tsukasaki et al., 2018, Mahanonda et al., 2016). Macrophages are particularly important due to their plasticity and dual role in immune defense and tissue repair (Walsh et al., 2018). They secrete key regulators of bone formation such as bone morphogenetic protein-2 (BMP-2), oncostatin M (OSM), and prostaglandin E₂ (PGE2) (Pirraco et al., 2013, Guihard et al., 2012). However, chronic inflammation or persistent infection can alter macrophage function, skewing polarization toward a proinflammatory (M1) phenotype (Cutolo et al., 2022), impairing efferocytosis (Korns et al., 2011), and promoting unresolved inflammation (Rodríguez-Morales et al., 2023). Concurrently, mesenchymal stem cells (MSCs), which are essential for osteogenic regeneration, may undergo inflammatory cell death, such as PANoptosis—a programmed cell death pathway involving pyroptosis, apoptosis, and necroptosis—thereby further compromising bone repair. Yet, the cellular and transcriptional mechanisms driving this osteoimmune imbalance in antibiotic-resistant infections remain poorly defined.

Myeloid immune checkpoints, including the CD47–SIRPα axis and MerTK, play crucial roles in regulating macrophage activation, phagocytosis, and inflammation resolution. Dysregulation of these checkpoints is linked to chronic inflammation and tissue damage. Recent studies have shown that targeting the CD47–SIRPα pathway in combination with adoptively transferred T cells has been shown to reduce tissue damage and enhance repair by preventing macrophage-mediated clearance of T cells (Yamada-Hunter et al., 2024). Similarly, MerTK-mediated efferocytosis regulates inflammation resolution and tissue repair by reprogramming lipid metabolism in macrophages (Lantz et al., 2025). However, the role of these immune checkpoints in antibiotic-resistant bone infections remains poorly understood.

In this study, we employed single-cell RNA sequencing (scRNA-seq) to map the cellular architecture and transcriptional heterogeneity of bone tissue under antibiotic-resistant infection. Using a murine model of bone infection, we profiled over 100,000 cells to identify key immune and stromal populations affected by resistance. Particular attention was given to macrophage phenotypes and their interactions with MSCs, as revealed through ligand–receptor communication analysis. We further validated macrophage and MSC functional alterations in vitro, and aimed to elucidate the immune mechanisms underlying impaired efferocytosis and osteogenic regeneration in antibiotic-resistant bone infection. Our findings provide mechanistic insight into how antibiotic-resistant infection disrupts the osteoimmune microenvironment and suggest that targeting myeloid immune checkpoints may represent a promising therapeutic strategy to restore immune balance and promote bone healing.