Fig 2: OPCs upregulate and activate TNF receptors following inflammatory stimulation. (A) Experimental workflow for assessing intracellular signaling activation in WT and TNFR2−/− OPCs (co-expressing Olig2 and PDGFRα) following stimulation with cytokine cocktail (TNF/IFNγ/IL1β, 25 ng/mL each) at 3 days in vitro (DIV); scale bar: 20 μm. (B) Quantification of TNF receptor gene expression (Tnfrsf1b and Tnfrsf1a) in WT and TNFR2−/− OPCs at 3DIV in non-stimulated conditions; n = 3, * p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001, two-way ANOVA, Holms–Sidak multiple comparison test. (C) Western blot analysis of PI3K/AKT signaling activation in WT and TNFR2−/− OPCs at 3DIV after 10 and 20 min of cytokine stimulation; active phosphorylated AKT (pAKT) normalized to total AKT is plotted as fold of non-stimulated (NS) WT OPCs. Results represent mean ± SEM of 3 independent experiments run in duplicate, * p ≤ 0.05, *** p ≤ 0.001, two-way ANOVA, Holms–Sidak multiple comparison test. (D) Western blot analysis of canonical NF-κB signaling activation PI3K/AKT signaling activation in WT and TNFR2−/− OPCs at 3DIV after 10 and 20 min of cytokine stimulation; active phosphorylated p65 (p-p65) normalized to total p65 is plotted as fold of non-stimulated (NS) WT OPCs. Results represent mean ± SEM of 3 independent experiments run in duplicate, ** p ≤ 0.01, two-way ANOVA, Holms–Sidak multiple comparison test.

Fig 3: Decreased cell surface expression of TNFR1 in the TRAPS mutant cells. The expression of TNFR1 and TNFR2 were determined in primary murine bone marrow-derived macrophages and peritoneal macrophages. (A) mRNA expression levels of Tnfrsf1a and Tnfrsf1b in bone marrow-derived macrophages. (B) Immunoblot analysis of TNFR1 and TNFR2 expression in the bone marrow-derived macrophages. Two different antibodies against TNFR1 were used: Ab#1 (13377, Cell Signaling Technology) recognizes aa 29–43 (extracellular domain), and Ab#2 (AF-425-PB, R&D Systems) recognizes the C-terminal intracellular region. (C–E) Flow cytometric analysis of TNFR1 and TNFR2 expression. Peritoneal exudate cells were collected from the indicated mice 3 days after the intraperitoneal administration of thioglycolate. (C) Representative histograms of the cell surface expression of TNFR1 and TNFR2. The expression levels of TNFR1 (D) and TNFR2 (E) on the surface of CD11b-positive cells were determined by flow cytometry. The cells within the horizontal line on each histogram were recognized as TNFR1- or TNFR2-positive cells. The proportions of the positive cells were calculated relative to those of the WT in each strain. The following antibodies were used: TNFR1 (113005, BioLegend) and TNFR2 (113405, BioLegend). Both antibodies recognize the extracellular domains. (F, G) ELISA for soluble TNFR1 (sTNFR1) in the culture supernatant of bone marrow-derived macrophages. Culture supernatant was collected before (F) and 24 h after (G) LPS stimulation. Concentrations of sTNFR1 in the culture supernatant were measured by ELISA, and the levels were calculated relative to that of the WT in each strain. TRAPS, TNF receptor-associated periodic syndrome; LPS, lipopolysaccharide; ELISA, enzyme-linked immunosorbent assay; WT, wild-type; Het, heterozygote; Hom, homozygote.

Fig 4: TRAPS mutations strongly suppressed lethal response by LPS and D-galactosamine and diminished TNFα-mediated arthritis. (A-C) T79M and G87V TRAPS mutant mice and TNFR1 KO mice were intraperitoneally administrated with LPS (100 μg/kg body weight) and D-galactosamine (400 mg/kg body weight). Survival rates of the mice after the administration of LPS and D-galactosamine. Survival rates of WT (n = 23), T79M strain (A); Het (n =18), Hom (n = 9), G87V strain (B); Het (n =13), Hom (n = 13), and TNFR1 KO strain (C); Het (n =11), Hom (n = 9). Gray, orange, and blue lines indicate WT, heterozygotes, and homozygotes, respectively. p-values for the differences between subgroups of mice were calculated by log-rank test using stratified analysis. (D, E) T79M or G87V mutant mice were crossed with TNFtg mice, and the severity of arthritis was evaluated. WT (n = 5), T79M Het (n = 4), T79M Hom (n = 3), G87V Het (n = 3), G87V Hom (n = 3), TNFtg (n = 5), TNFtg/T79M Het (n = 4), TNFtg/T79M Hom (n = 3), TNFtg/G87V Het (n = 4), TNFtg/G87V Hom (n = 2). (D) Representative images of the hind paw of TNFtg T79M heterozygous mouse and TNFtg mouse. (E) Arthritis scores of the mice at the age of 17 weeks. **p<0.01 vs. any other groups. WT, wild-type; TNFtg, TNFα transgenic; TNFα, Tumor necrosis factor α; TRAPS, TNF receptor-associated periodic syndrome; IL, interleukin; LPS, lipopolysaccharide; TNFR1, TNF receptor type I; TNFtg, Human TNFα-transgenic mice; Het, heterozygote; Hom, homozygote. *p<0.01; n.s., not significant.

Fig 5: IL-33 Promotes TNF-a Secretion and TNFR2 Expression on ILC2s(A) BALB/cByJ mice were challenged i.n. on days 1–3 with 0.5 µg rmIL-33 or PBS.(B) On day 4, BAL fluid was collected, and supernatant TNF-a was measured by ELISA.(C) Number of TNF-a-producing CD45+ lung cells on day 4, cultured for 4 h with GolgiPlug.(D–F) Representative flow cytometry plots in naive (D) and activated (E) lungs of CD45+ TNF-a+ cells backgated for AMs (CD45+ SiglecF+ CD11b-, CD11c+, and F4/80+) and SiglecF-CD11b+ and SiglecF-CD11b- populations and (F) corresponding quantitation presented as mean frequency ± SEM.(G) AMs were FACS sorted from PBS- or rmIL-33-challenged mice on day 4 and cultured for 24 h in vitro (106 AMs/well), and supernatant TNF-a was measured by ELISA.(H and I) Gating strategy for murine ILC2s showing (H) lineage- staining and (I) lineage-CD45+ CD127+ ST2+ GATA-3hi ILC2s.(J) nILC2s were FACS sorted from naive BALB/cByJ mice and cultured (50 × 104/mL) ex vivo in the presence of rmIL-2 (10 ng/mL), rmIL-7 (10 ng/mL), and rmIL-33 (50 ng/mL) for the indicated times. nILC2s were gated as lineage- CD45+ ST2+ CD127+.(K and L) Representative flow cytometry plot of TNFR1 (K) and TNFR2 (L) expression and corresponding quantitation, presented as Mean Fluorescence Intensity (MFI) ± SEM.(M) BALB/cByJ mice were challenged i.n. on days 1–3 with 0.5 µg rmIL-33 or PBS.(N and O) Representative flow cytometry plot of lung ILC2 expression of TNFR1 (N) and TNFR2 (O) expression on day 4 and corresponding quantitation, presented as MFI ± SEM.Data are representative of 4 individual experiments (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001; ns, non-significant. See also Figure S1.