Engineered Pseudomonas aeruginosa Ameliorates Rheumatoid Arthritis via CCL4-Dependent Macrophage Repolarization

Abstract Rheumatoid arthritis (RA) is driven by immune dysregulation, with macrophage polarization playing a central role in disease progression. A critical bottleneck in current rheumatoid arthritis (RA) management is that a subset of patients display inadequate responses to existing anti-inflammatory therapies (e.g., TNF-α inhibitors), leading to persistent inflammation that remains refractory to complete control. To identify new therapeutic targets and interventions, we analyzed single-cell RNA sequencing (scRNA-seq) data from 20 RA patients and identified a distinct M1-like pro-inflammatory macrophage population. Among 50 differentially expressed genes, CCL4 emerged as a novel pro-inflammatory target associated with M1 polarization. To counter this phenotype, we evaluated Pseudomonas aeruginosa mannose-sensitive hemagglutinin (PA-MSHA), an engineered bacterium, in in vitro, ex vivo, and in vivo models. PA-MSHA promoted M2 polarization, enhanced anti-inflammatory markers, and suppressed M1-associated cytokines. In an adjuvant-induced arthritis (AIA) rat model, PA-MSHA significantly reduced paw swelling, improved joint integrity, and decreased macrophage and T cell infiltration. Mechanistically, PA-MSHA inhibited NF-κB and STAT1 signaling and downregulated CCL4 expressions. In vivo overexpression of CCL4 aggravated RA symptoms and sustained M1 polarization, confirming its pathogenic role. These findings highlighted CCL4 as a novel therapeutic target and demonstrated PA-MSHA's potential to reprogram macrophage phenotypes and ameliorate RA pathology.