T cell dysfunction during Staphylococcus aureus osteomyelitis in humanized mice 2257659

Abstract

Staphylococcus aureus is the leading cause of implant-associated osteomyelitis. It has a high recurrence rate, a low post-operative cure rate, and can lead to sepsis, multiorgan failure, and death. This indicates a need for future innovation in therapeutics and vaccines. With our humanized mouse model, we aimed to investigate the human T cell response during infection, as T cells play a crucial role in controlling bacterial growth during chronic infections. Using immunohistochemistry, we have found preliminary evidence of T cell dysfunction in the bone marrow niche. This has led to our hypothesis that CD4 T cells are becoming exhausted in the bone marrow due to chronic infection. Female humanized NSG-SGM3 BLT mice (20-24 weeks old) underwent transtibial implant-associated osteomyelitis using bioluminescent MRSA (USA300 LAC::lux) or sham surgery. At fourteen days post-infection, bone marrow cells were isolated and subjected to flow cytometry and single-cell RNA sequencing. In the bone, we observed an influx of Th1/Th17 cells through single-cell RNA sequencing. Using spectral flow cytometry, we observed increases in immune checkpoint proteins LAG-3, PD-1, and TIM-3 on CD4 T cells. Interestingly, we also discovered that TIM-3-positive CD4 T cells exhibited reduced Ki67 staining, suggesting potentially impaired functional capacity. To explore this further, we examined the effector profile (IFN-γ, IL-17A, and TNF-α) of these cells and observed diminished cytokine production in TIM-3-positive cells. Finally, we analyzed human serum from arthroplasty patients and found TIM-3 levels to be highly predictive of adverse outcomes. These results suggest CD4 T cells may be dysfunctional and contribute to the chronicity of infection in S. aureus osteomyelitis. Ultimately, this work will provide novel mechanistic insights into bacteria-T cell interactions during S. aureus bone infections, hoping to inform better diagnostics and therapeutics. NIAMS Microbial, Parasitic, and Fungal Immunology (MPF)