Chikungunya Virus Infection of Monocytes Generates a Persistent Macrophage Reservoir That Promotes IFN-π/IL-27–STAT1- and NF-κB–Driven Chronic Arthritis

Abstract Chikungunya virus (CHIKV) infection causes acute febrile illness that frequently progresses to chronic arthralgia. Although monocytes are CHIKV targets, whether viral exposure drives their differentiation into macrophages to sustain persistent inflammation remains incompletely understood. To address this, we integrated transcriptomic profiling of pediatric CHIKV patients during acute and convalescent phases, in vitro differentiation of primary human monocytes into macrophages under CHIKV infection, and single-cell RNA-sequencing (scRNA-seq) of joint-associated macrophages from an immunocompetent mouse model of chronic CHIKV arthritis at 28 days post-infection. In acute patients, we observed a marked upregulation of monocyte/macrophage markers alongside robust, IFN-dependent JAK-STAT signaling. In vitro , CHIKV-infected monocytes differentiated into persistently infected macrophages (MDM-CHIKV) that closely aligned with an M1-polarized transcriptional profile. These cells exhibited STAT1/NF-κB-dependent inflammation, ISG induction, and enhanced antigen presentation capacity. Remarkably, MDM-CHIKV uniquely produced IFN-π/IL-27, revealing an alternative non-canonical axis where JAK-STAT activation persists despite the absence of classical type I IFNs. Functionally, MDM-CHIKV displayed an enhanced respiratory burst upon immune complex uptake, pointing to FcγR-mediated pathology, as well as complement activation and T-cell stimulatory capacity. However, upon TLR4 challenge, these cells retained partial regulatory responses via IL-10 and TGF-β; this hybrid immunophenotype offers a potential mechanistic explanation for relapsing arthralgia. Additionally, scRNA-seq of murine joints confirmed that persistent CHIKV replication was restricted to a distinct macrophage subset that resembles MDM-CHIKV, establishing these cells as the primary in vivo viral reservoir. Mirroring our human findings, joint-associated murine macrophage subsets exhibited elevated IFN-π / IL-27 expression alongside robust NF-κB/STAT1-dependent inflammatory signatures ( Tnf , Il1β , Il6, Ccl2 , Ccl5 , Cxcl9 , Cxcl10 , Cxcl1–3, and/or Ptgs2 ), underscoring their critical role in sustaining chronic arthritis. Collectively, our findings demonstrate that CHIKV actively drives monocyte-to-macrophage differentiation into a persistently infected, M1-skewed hybrid phenotype that serves as a viral reservoir and fuels pro-arthritic inflammation. Consequently, macrophage reprogramming, the non-canonical IFN-π/IL-27–STAT1 axis, FcγR-mediated phagocytosis, and complement activation emerge as central pathogenic nodes and promising therapeutic targets for mitigating chronic post-CHIKV arthropathy. Author Summary Chikungunya virus (CHIKV) is a mosquito-borne pathogen that causes acute fever and, in a substantial proportion of patients, progresses to chronic, debilitating joint pain that can last for months or even years. The mechanisms by which the virus establishes this persistent arthritic disease remain poorly understood, and effective treatments for chronic CHIKV arthritis are lacking. In this study, we investigated whether CHIKV infection of monocytes, immune cells that circulate in the blood, actively drives their transformation into macrophages, a cell type that resides in tissues and plays a central role in inflammation. By analyzing blood samples from children with acute and convalescent CHIKV disease, we found strong signs that monocyte-to-macrophage differentiation is actively occurring during natural infection. Using an in vitro model, we showed that CHIKV-infected monocytes differentiate into macrophages that become persistently infected, carrying the virus for extended periods. These virus-exposed macrophages adopt a mixed activation state, combining features of classical pro-inflammatory (M1) and regulatory (M2) macrophages. Notably, they produce a cytokine called IFN-π/IL-27, which drives chronic inflammation through a specific signaling pathway, while also showing enhanced capacity to activate T cells, trigger complement cascades, and mount strong oxidative responses upon encountering immune complexes, features that may explain the waxing and waning joint symptoms seen in patients. To confirm these findings in a living system, we analyzed joint tissues from a mouse model of chronic CHIKV arthritis using single-cell technology. We discovered that a specific subset of joint-resident macrophages serves as the primary viral reservoir, actively replicating the virus and producing IFN-π/IL-27 and other inflammatory molecules that perpetuate joint inflammation and damage. Collectively, our work reveals that CHIKV hijacks the normal differentiation process of monocytes to create a long-lived, virus-harboring macrophage population that fuels chronic joint inflammation. We identify the IFN-π/IL-27 signaling pathway, complement activation, and antibody-driven macrophage responses as key drivers of disease. These findings open new avenues for therapeutic intervention, suggesting that targeting these pathways could alleviate chronic arthritis in patients suffering from long-term CHIKV disease.