Targeted phagocytosis of TNF-α by CAR macrophages restores immune homeostasis in arthritis.

Abstract

Rationale: Dysregulated phagocytic clearance within the synovial microenvironment contributes to persistent inflammation and impairs immune homeostasis in rheumatoid arthritis (RA). Because MerTK-positive macrophages are essential for efferocytosis and inflammation resolution, but are functionally impaired in RA, this study aimed to engineer macrophages capable of enhancing tumor necrosis factor-α (TNF-α) clearance and restoring inflammation-resolving macrophage function.

Methods: We generated anti-TNFα chimeric antigen receptor macrophages (CAR-M) by replacing the MerTK antigen-binding domain with a TNF-R1 fragment. The phagocytic and degradative capacity of CAR-M toward soluble TNF-α was assessed, together with activation of Rho GTPases Rac1, Cdc42, and RhoA. Macrophage inflammatory mediator production, phenotypic polarization, SOCS1/3, NF-κB, and MAPK signaling were analyzed. The effects of CAR-M cells were further compared with adalimumab and control treatments under synovial fluid stimulation from patients with arthritis and in mice with collagen-induced arthritis (CIA).

Results: Anti-TNFα CAR-M showed markedly enhanced phagocytosis and degradation of soluble TNF-α, accompanied by Rac1, Cdc42, and RhoA activation. CAR activation reduced inflammatory mediator production, including soluble TNF-α, interleukin-1 β (IL-1β), and interleukin-6 (IL-6), while increasing interleukin-10 (IL-10) secretion. This functional shift was associated with polarization toward an inflammation-resolving MerTK⁺CD206⁺ phenotype, SOCS1/3 upregulation, and NF-κB and MAPK pathway inhibition. Under stimulation with synovial fluid from arthritis patients, anti-TNFα CAR-M decreased TNF-α and IL-6 levels and drove macrophages toward an M2-like anti-inflammatory state. In CIA mice, local in situ CAR-M cell treatment produced stronger therapeutic effects than adalimumab or other controls, significantly alleviating inflammatory activation, clinical symptoms, and joint pathologies.

Conclusions: Anti-TNFα CAR-M exert therapeutic effects via a dual mechanism: direct TNF-α clearance and synovial macrophage reprogramming toward a reparative phenotype. This synthetic biology strategy highlights the potential of precise cell engineering to correct phagocytic deficits and resolve chronic inflammation in RA.