Fig 2: No substantial changes in the responsiveness to LPS in the TRAPS mutant macrophages. Bone marrow-derived macrophages were stimulated with LPS (100 ng/mL). After stimulation, the RNA and supernatant samples were collected at the indicated time points. Cell culture experiments were performed separately for each strain. (A) qPCR analysis. The mRNA expression levels of Tnf and Il1b were determined. Gray, orange, blue, and dark gray lines indicate WT, heterozygous, homozygous, and TNFR1KO mice, respectively. (B) Relative protein levels of TNFa and IL-1ß in the culture supernatants. Culture supernatants were collected 6 h after LPS stimulation, and the concentrations of TNFa and IL-1ß were determined by ELISA. Levels were calculated relative to those of the WT in each strain. TNFa, Tumor necrosis factor a; TRAPS, TNF receptor-associated periodic syndrome; IL, interleukin; LPS, lipopolysaccharide; TNFR1, TNF receptor type I; KO, knockout; qPCR, real-time quantitative polymerase chain reaction; WT, wild-type; Het, heterozygote; Hom, homozygote.

Fig 3: TNFa Neutralization Disrupts the Efficacy of CXCR2 Blockade in Syngeneic Prostate Tumors In VivoFor the allograft experiments, 2.5 × 106 TRAMP-C1 cells were injected subcutaneously into the flank of male C57BL/6 mice. When tumors were approximately 200 mm3, mice were randomized to the treatment groups. Tumor growth was monitored daily by measuring the tumor size with caliper. Mice were treated with aCXCR2 (AZD5069 100 mg/kg) daily for 3 weeks.(A) Bar graph showing the relative tumor volume (mm3) at days 10, 20, and 30 after injection in mice bearing TRAMPTRAMP-C1C1 Pten-/-; Trp53-/- allografts treated with aCXCR2 with or without combination with a monoclonal antibody for TNFa. The graph is also showing the tumor size of Pten-/-; Trp53-/- TNFR1-/- TRAMP-C1 cells in the presence or absence of aCXCR2 treatment, at the same time points. CRISPR/Cas9 was performed on TRAMP-C1 cells to delete TNFR1.(B and C) Representative images of IHC staining (B) and quantification (C) of p16 in all the conditions.(D) Schematic model showing the mechanism by which CXCR2 blockade reprograms TAMs in prostate tumors.Error bars are mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001.

Fig 4: TRAPS mutations strongly suppressed lethal response by LPS and D-galactosamine and diminished TNFa-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, TNFa transgenic; TNFa, Tumor necrosis factor a; TRAPS, TNF receptor-associated periodic syndrome; IL, interleukin; LPS, lipopolysaccharide; TNFR1, TNF receptor type I; TNFtg, Human TNFa-transgenic mice; Het, heterozygote; Hom, homozygote. *p<0.01; n.s., not significant.

Fig 5: zVAD does not alter the TNFR1 protein expression level in rat brain cortex. (A) S group rats at 6, 12, 24 and 72 h. (B) IR group. (C) IR+I group. (D) Quantification of TNFR1 positive cells in the three groups. Magnification, ×100. Values (n=6 animals/group) are presented as the mean ± standard deviation. *P<0.05 vs. S group; **P<0.01 vs. S group. zVAD, z-fluoromethylketone; TNFR1, tumor necrosis factor-a receptor 1; S, sham; IR, ischemia-reperfusion; IR+I, ischemia-reperfusion + zVAD.