Fig 2: Tissue-resident macrophages show distinct phagocytic speeds and inflammatory gene expression upon stimulation in vitroMG-LCs are shown in yellow, KC-LCs in green, PM-LCs in pink, and AM-LCs in blue. One of three to eight independent experiments is shown. Cells were gated as depicted in Figure S7.(A) Representative histograms of PE-pHrodo labeling after 5, 10, 15, and 30 min of TRM-LCs phagocytosis of PE-pHrodo-coupled zymosan beads in vitro.(B) Quantification of percentage of TRM-LCs labeled with PE-pHrodo after 5, 10, 15, and 30 min. Mean ± SEM is shown; n = 3–8; *p < 0.05, **p < 0.01, and ***p < 0.001.(C) Quantification of percentage of differently cultured BMDMs labeled with PE-pHrodo after 5, 10, 15, and 30 min. BMDMs in normoxia + GM-CSF are shown in white, BMDMs in normoxia + M-CSF are shown in gray, BMDMs in hypoxia + M-CSF are shown in dark gray. Mean ± SEM is shown; n = 3–6; *p < 0.05, **p < 0.01, and ***p < 0.001.(D–F) Levels of secreted cytokines are measured in mean fluorescence intensity (MFI). Control is shown in blue, LPS in red, poly(I:C) in green, and zymosan in violet. Released cytokine levels of IL-1ß are shown in (D), IL-6 in (E), and TNF-a in (F). Mean ± SEM is shown; n = 3/group; *p < 0.05, **p < 0.01, and ***p < 0.001.See also Figures S6 and S7.

Fig 3: Tendon-derived mEPs can induce macrophage infiltration and cytokine release.a Chemotaxis assay for RAW 264.7 cells toward tendon-derived mEPs and (b) corresponding quantitative colorimetric measurements are presented as three biological replicates from three independent experiments. RAW 264.7 cells that migrated to the bottom of the insert microporous membrane in the Boyden chamber were stained. The mEPs from tendon constructs receiving 0%, 3%, 6%, or 9% strain were used as chemoattractants in the lower chamber. Scale bar, 50 μm. c xCell analysis of bulk RNA sequencing data from healthy tendons and tendons with tendinopathy showing macrophage infiltration in diseased human tendons. The dots in orange indicate that P < 0.2. d Gating strategy for identification of different cytokines by a bead-based immunoassay. A total of 13 bead populations were distinguished by size and allophycocyanin (APC) fluorescence. e Bead-based immunoassay showed that the mEPs from tendon constructs that received 0%, 3%, 6%, or 9% strain induced macrophages to secrete different cytokines (n = 3 per group). Lipopolysaccharide (LPS) (500 ng/mL) was used as the positive control to induce cytokine release from RAW 264.7 cells. Phosphate-buffered saline (PBS) as the carrier was used as another control. f The bead-based immunoassay showed that mEPs mediated RAW 264.7 cells to release IL-6, CXCL1 and IL-18 in a dose-dependent manner (n = 3 each group). Two-way ANOVA with Tukey’s multiple comparisons test was used for statistical analysis of the main concentration effect at different doses. Two-way ANOVA with Tukey’s multiple comparisons test was used to compare the effects of different doses within mEP9% for simple effect analysis. The data are presented as the means ± SEMs. One-way ANOVA with Tukey’s multiple comparisons test was used for statistical analysis. ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant.

Fig 4: Itaconate differentially modulates CpG ODN 1826-induced cytokine and chemokine production.a IL-1β and IL-6 levels in supernatants of bone marrow-derived macrophages (BMDMs) pretreated with itaconate for 2 h (indicated concentrations), followed by CpG (1 μM) stimulation for 6 h (n = 3). For measurement of IL-1β, 5 mM ATP was added 30 min prior to supernatant collection (also applicable to other figures exhibiting secreted IL-1β). b IL-1β and IL-6 levels in supernatants of WT or Acod1−/− BMDM after CpG (1 μM, 6 h) stimulation (n = 3). c Gene-expression analysis of Cxcl10, Cxcl1 and Ccl2 in BMDMs after CpG (1 μM, 3 h) stimulation with itaconate pre-treatment (2 h) (n = 3). d CXCL10, CXCL1 and CCL2 levels in supernatants of BMDMs pretreated with itaconate for 2 h, followed by CpG (1 μM) stimulation for 18 h (n = 3). e, f CXCL10 mRNA in human peripheral monocytes (e) or mouse spleen monocytes/macrophages (f) pretreated with itaconate (10 mM, 2 h), followed by CpG (1 μM, 3 h) stimulation (n = 3). g, h Cxcl10 mRNA in WT or Acod1−/− BMDMs (g) or mouse spleen monocytes/macrophages (h) after CpG (1 μM, 3 h) stimulation (n = 3). Figures are representative of three independent experiments. The bar plots were presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 5: Elevated CXCL10 levels and CD8+ T cell percentage in peripheral blood of patients with Stillʼs disease with macrophage activation syndrome.a IL-1β, IL-6, IL-8, IL-10, TNF-α and sIL-2R in peripheral blood of patients with Stillʼs disease (SD) without macrophage activation syndrome (MAS) (n = 34) vs those with MAS (n = 25). b CXCL10 in peripheral blood of patients with SD without MAS (n = 34) vs those with MAS (n = 25). c Neutrophil, monocyte, CD4+ T, CD8+ T, CD19+ B and NK cell percentage in peripheral blood of patients with SD without MAS (n = 34) or those with MAS (n = 25). d Correlations between CXCL10 levels and IL-10, sIL-2R, PLT, ALT or AST in patients with SD with MAS (n = 25). e The correlation between CXCL10 levels and peripheral blood CD8+ T cell percentage in patients with SD with MAS (n = 25). Data in a–c are presented as violin plots (median line, kernel density estimation) with overlaid individual points. *P < 0.05; **P < 0.01; ***P < 0.001. ALT, alanine transaminase; AST, aspartate transaminase; sIL-2R, soluble IL-2 receptor.