Background
Chikungunya virus (CHIKV) is a mosquito-transmitted pathogen responsible for over 10 million infections globally in the past two decades. It is associated with chronic arthralgia, polyarthritis, and neurological complications, yet no licensed antivirals or vaccines exist. Although CHIKV and O’nyong’nyong virus (ONNV) share similar nucleotide identity1,2, they induce distinct joint pathology3,4, suggesting that divergent host immune responses drive disease outcomes. Defining these differential immune mechanisms is critical for therapeutic development.
Methods
Four-week-old female C57BC6J mice were infected subcutaneously in the footpad with CHIKV or ONNV. Footpads (n = 10) and popliteal lymph nodes (n = 3) were harvested at 6 days post-infection (dpi) for immunophenotyping and at 1 dpi for single cell transcriptomics, respectively. Cell types were assigned using reference-based annotation from the Cellxgene database. Differential gene expression was assessed with edgeR, and pathway enrichment with clusterProfiler. Human primary peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured into an ex vivo lymphoid-organ-like model (LOM) and analyzed by immunofluorescence imaging and flow cytometry.
Results
At 6 dpi in murine footpads, ONNV infection resulted in elevated macrophage and CD4+ T cell accumulation compared to CHIKV. CHIKV induced higher T helper type 1 (Th1) polarization, whereas ONNV favored Th2 responses. The human LOM supported robust viral infectivity comparable to in vivo infection, unlike conventional 2D cultures. By 2 dpi, lymphoid follicle-like structures containing CD35+ follicular dendritic cells formed, suggesting functional germinal center formation and antigen presentation. The LOM recapitulated in vivo murine observations, with CHIKV promoting Th1 dominance and ONNV favoring Th2 polarization.
Single cell transcriptomics identified monocytes and macrophages (MoMacs) as the key infected immune cell populations at 1 dpi. CHIKV-infected MoMacs exhibited enhanced apoptosis, translational arrest, and cytokine unresponsiveness compared to ONNV. Subcluster analysis revealed an elevated frequency of interferon (IFN)-responsive regulatory monocytes and Gzmb+ Ccr7+ activated macrophages in CHIKV infection, whereas ONNV infection was associated with more M2-like reparative macrophages.
Conclusions
Elevated macrophage cell death, impaired cytokine responsiveness, and skewing toward pro-inflammatory migratory macrophage subsets may contribute to more severe joint pathology seen in CHIKV infection. The human LOM recapitulates adaptive immune responses from a naïve state, providing a scalable and physiologically relevant preclinical platform to dissect infection-induced immunity. Modulating macrophage apoptosis, IFN signaling, and CD4+ T cell Th1/Th2 polarization may represent therapeutic strategies to reduce CHIKV-induced joint pathology.