Fig 2: Schematic Model of GM-CSF-CCL22 Axis in RA Pathogenesis. A STAT5 Activation in RA Monocytes: Peripheral blood monocytes from RA patients exhibit enhanced STAT5 phosphorylation (p-STAT5), priming them for hyperactivation and differentiation into pro-inflammatory M1 macrophages; B GM-CSF Drives M1 Polarization: In the synovial microenvironment, GM-CSF (↑ in RA synovium) promotes monocyte differentiation into M1 macrophages; C Metabolic Reprogramming: RA GM-DMs display a hypermetabolic phenotype with enhanced oxidative phosphorylation but impaired glycolysis, favoring sustained inflammatory activity; D CCL22-Mediated T Cell Recruitment and Polarization: GM-DMs secrete CCL22, which: (a) recruits CD4 + T cells2; (b) promotes Th1/Th17 differentiation; E Inflammatory Cascade and Joint Destruction: Th1/Th17-derived cytokines synergize with macrophage-produced mediators to drive synovitis, cartilage erosion, and bone damage

Fig 3: Metabolic reprogramming in RA GM-CSF-polarized macrophages. A Flow cytometry quantification of phosphorylated STAT5 (p-STAT5) in RA monocytes (anti-human p-STAT5 Alexa Fluor 488); B KEGG/GO enrichment of DEGs in GM-DMs between RA and HC. The color represents the p-value, and the size of the circle indicates the count; C energetic panel of GM-DMs in HC and RA; open symbols represent baseline conditions and closed symbols the stressed ones. The metabolic potential was showed on the right; OCR (D) and ECAR (F) traces, expressed as pmol O2/min/mg proteins and mpH/min/mg proteins in HC and RA respectively. The broken lines indicate the time of addition of oligomycin (ATP synthase inhibitor), FCCP (mitochondrial uncoupler), rotenone/antimycin A (complex I/III inhibitors) and 2-DG (glycolysis inhibitor); key mitochondrial parameters (E) and glycolytic parameters (G). Bars = mean ± SD (n ≥ 3). P values: *<0.05, **<0.01, ***<0.001, ****<0.0001

Fig 4: A GM-CSF Drives Pro-inflammatory Macrophage Polarization in RAA Experimental design for GM-CSF/M-CSF-induced macrophage differentiation; B PCA of transcriptomes from GM-DMs and M-DMs (RA vs. HC); C Macrophage polarization analysis (M1/M2) in GM-DMs and M-DMs between HC and RA; D correlation between synovial GM-CSF/M-CSF levels and disease activity (DAS28-CRP); E Synovial GM-CSF and M-CSF expression levels among different groups through GSE89408 dataset. Bar range = minimum to maximum. P values: *<0.05, **<0.01, ***<0.001, ****<0.0001.

Fig 5: Inhibitors prevent polyP-induced survival of ingested E. coli and reduced phagosome acidification. (A–B) Viable E. coli in GM-CSF macrophages, in the absence (Control) or presence of 15 µg/mL polyP, without (No drug) or with 1,000 nM of the indicated inhibitor, was determined as CFU at 4 hours (A),or 48 hours (B).CFU in all treatment conditions were compared with the No drug condition for statistical significance. (C) A schematic showing macrophages with phagosomes containing pHrodo red-labeled yeast, in the presence or absence of polyP, with or without MRS2279 (P2Y1 inhibitor) or TNP (IP6K inhibitor). pHrodo red-labeled yeast have low fluorescence outside the cell but show red fluorescence in acidic phagosomes. (D) Human GM-CSF macrophages were incubated with yeast, in the absence (Control) or presence of 15 µg/mL polyP without (No drug) or with 1,000 nM of the indicated inhibitor (+ polyP) for 1 hour, fixed, and fluorescence images were taken. 100 nM of ConcanA is a positive control for inhibition of phagosome acidification. DIC merged with fluorescence images is at the left, and fluorescence images are at the right for each treatment condition. Bar is 20 µm. Images are representative of six independent experiments (three females and three males). (E–G) Images from D were used to measure fluorescence intensities of yeast (E), percent of macrophages with yeasts (F), and number of yeasts per macrophages (G). The fluorescence intensity of yeast in control was set to 100 for E. (H–K) Experiments from D–G were performed with M-CSF macrophages. Size bars are 20 µm. Male data points are shown in blue and female data points are shown in red. For each bar, there was no significant difference between male and female (unpaired t-test). All values are mean ± SEM of six (three females and three males) independent experiments. * or # P < 0.05; **P < 0.01; ### P < 0.001; #### P < 0.0001 (one-way analysis of variance with Dunnett’s test). * indicates compared to no drug; # indicates compared to control.