Noninvasive Low-Frequency Pulsed Focused Ultrasound Therapy for Rheumatoid Arthritis in Mice

Rheumatoid arthritis (RA) is a common autoimmune disease characterized by chronic and progressive inflammation of the synovium. Focused ultrasound therapy is an increasingly attractive alternative for treating RA owing to its noninvasiveness; however, it remains unclear which immune subsets respond to ultrasound stimulation. In this study, we showed that spleen-targeted low-frequency pulsed focused ultrasound (LFPFU) effectively improved the severity of arthritis in an arthritis mouse model established in DBA/1J mice. Additionally, we performed in-depth immune profiling of spleen samples from RA mice, RA mice that underwent ultrasound therapy, and healthy controls using mass cytometry along with extensive antibody panels and identified the immune composition of 14 cell populations, including CD4⁺/CD8⁺ T cells, B cells, natural killer cells, and dendritic cells. Moreover, multidimensional analysis according to cell-surface markers and phenotypes helped in identifying 4 and 5 cell subpopulations among T and myeloid cells, respectively, with 6 T cell subsets and 3 myeloid cell subsets responsive to ultrasound therapy among the 3 groups. Of these cell subsets, CD8⁺ T cell subsets showed a unique response to ultrasound stimulation in RA mice. Specifically, CD8⁺ T cells show a noticeable correlation with the degree of arthritis progression and could serve as an indicator for spleen-focused ultrasound-based therapy. Furthermore, single-cell RNA sequencing of spleen cells revealed the importance of T, B, and myeloid cell populations in the anti-inflammatory pathway. These results elucidated the unique cell subsets and transcriptome of splenic cells responsive to LFPFU and demonstrated the potential of spleen-focused ultrasound stimulation in the treatment of inflammatory diseases.