Fig 2: IgG-Induced IL-1ß Production by Human Macrophages(A) Venn diagram of significant co-expressed genes between UC and Ova-IC-stimulated colonic macrophages (left) and IC-induced UC-associated cytokines and chemokines (right), as determined by sdef. Human UC data are from GEO: GSE38713 (UC = 15, HC = 13). For macrophage stimulation, n = 4 per condition.(B) GSEA of inflammasome genes (left) and heatmap of selected core enrichment genes (right) in flow-sorted murine intestinal CX3CR1+Ly6CloMHC-IIhi macrophages stimulated with Ova and Ova-IC for 4 h (n = 4 per condition).(C) IL-1ß production by WT and Nlrp3-/- BMDMs primed with Ova and Ova-IC for 4 h followed by 30 min of ATP stimulation (****, WT versus Nlrp3-/- Ova-IC; ####, WT Ova versus Ova-IC) (n = 3 per condition). Means ± SEM are indicated. Data are representative of three independent experiments. ND, not detected.(D) IL-1ß ELISA of human MDMs stimulated with plate-coated IgG (c-IgG) and intestinal commensals (Comm.) for 24 h ± anti-Fc?RIIA IgG-blocking antibody (n = 3 per condition). Means ± SEM are indicated. Data are representative of two independent experiments.(E) qPCR of IL1B mRNA (left, n = 3 per condition) and western blot of IL-1ß in cell lysates and cell-free supernatants (right) from human MDMs as stimulated in (D). For qPCR (left), means ± SEM are indicated. Data are representative of two independent experiments.(F) The effect of NLRP3 inhibition on IgG-induced MDM IL-1ß production. Western blot (left) and IL-1ß production (left, ELISA) by human MDMs stimulated as in (D) after NLRP3 “Trim-Away” or ± NLRP3 inhibitor MCC950 (right, ELISA) (n = 3 per condition). Means ± SEM are indicated.(G) IL-1ß production by human MDMs stimulated as in (D) ± mitochondrial ROS inhibitor mitoTEMPO (n = 3 per condition). Means ± SEM are indicated. Data are representative of 2 independent experiments.(H) Fold change in IL1B expression in Ova-IC-stimulated MDMs, normalized to R/R fold change (n = 6 per group). Data are pooled from 3 independent experiments. Medians are indicated.(I) Schematic of Fc?R A:I ratios with FCGR2A SNP (dbSNP: rs1801274) and murine transgenic models.(J) Il1b induction in WT, Fcgr2b-/-, and M-TG BMDMs stimulated with Ova and Ova-IC for 3 h (n = 3 per condition). Means ± SEM are indicated. Data are representative of 3 independent experiments.p values were calculated via the standard DESeq2 method with multiple correction using the BH procedure (A and B), two-way ANOVA with Tukey’s multiple comparisons test (C, F, and J), one-way ANOVA with Tukey’s multiple-comparisons test (D, E, and G), or parametric Student’s t test (H). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S4.

Fig 3: Activating Fc?R Signaling in Intestinal Inflammation(A) Analysis of the top significantly differentially expressed genes (log2 FC > 2) in human active UC mucosal biopsies compared with controls. Data are from GEO: GSE38713.(B) FCGR enrichment in inflamed active UC (n = 15) compared with healthy control (n = 13), remission (n = 8), and non-inflamed mucosal biopsies (n = 7). Data are from GEO: GSE38713.(C) FCGR expression in colonic biopsies from UC patients refractory (n = 16) or responsive (n = 8) to infliximab treatment and healthy controls prior to treatment initiation (n = 6). Data are from GEO: GSE16879. Minimum to maximum box-and-whisker plots are shown in (B) and (C).(D) Area under the receiver operator curve (AUROC) analysis for FCGR2A expression in infliximab-refractory mucosal biopsies compared with infliximab-responsive biopsies. Data were derived as in (C).(E) Gene-set enrichment analysis (GSEA) of the KEGG Fc?R-mediated phagocytosis pathway in UC and healthy colonic biopsies (left) and cDSS-inflamed and healthy murine colons (bottom). Data are from GEO: GSE38713 (UC = 15, HC = 13) and GEO: GSE42768 (cDSS = 5, H2O = 5).(F) Fc?R expression by murine colonic leukocytes at day 7 after aDSS. Data are representative of 3 independent experiments.(G) qPCR of Fc?R mRNA expression in flow-sorted murine colonic CX3CR1+Ly6CloMHC-IIhi macrophages, as identified in (F). Data are representative of 2 independent experiments.(H) Single-cell RNA-seq of immune cell subsets in healthy human colon. Data are from GEO: GSE81861. The heatmap illustrates the top 50 cell-type-specific markers ranked by AUROC score.(I) qPCR of Fc?R mRNA expression in flow-sorted human ileal CD14+CX3CR1+SSCint macrophages.(J) Colonic “monocyte waterfall” subset quantification by flow cytometry at day 7 after aDSS versus controls (n = 3 per group). Flow plots of CD11b+CX3CR1+ waterfall subsets (left) and quantification of absolute cell counts for the indicated subsets (right) are shown. For absolute cell counts, medians are indicated. Data are representative of 3 independent experiments.(K) Fc?R quantification of CX3CR1+Ly6CloMHC-IIhi macrophages, as shown in (J), by flow cytometry. Means ± SEM are indicated and normalized to the A:I ratio of control-treated mice.Data are representative of two independent experiments. p values were calculated via limma with multiple correction using the BH procedure (A–C), AUROC analysis (D and H), or Student’s two-tailed t test (J and K). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. See also Figure S2 and Table S2.

Fig 4: Expression of markers on macrophages cultured in serum and M-CSF.PBMC were cultured in SFM at 5×105 cells/ml in 8-well glass slides for 7 days in the presence of 10% FCS and 10 ng/ml M-CSF. Cells were then air-dried, fixed and stained with 5 µg/ml mouse IgG1 monoclonal antibodies against CD13, CD14, CD32, CD163, CD90, mouse IgG1, proyly-4-hydroxylase, 25F9, S100A8/A9, PM-2K, TE-7, and collagen-I. Cells were then counterstained with hematoxylin to identify nuclei. Inserts indicate staining at higher magnification. Bar is 50 µm.

Fig 5: Effect of SAP on markers expressed by macrophages.PBMC were cultured in 8-well glass slides in SFM for 7 days, in the presence or absence of SAP. Cells were then air-dried, fixed, and stained with antibodies. A–B) CD14, C–D) CD32a, E–F) CD32b, G–H) CD68, I–J) CD163, and K–L) CD172a. Cells were counterstained with hematoxylin to identify nuclei. Bar is 50 µm.