TRAPS mutations in Tnfrsf1a decrease the responsiveness to TNFα via reduced cell surface expression of TNFR1

Abstract

Tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is an autoinflammatory periodic fever syndrome associated with heterozygous mutations in TNFRSF1A, which encodes TNF receptor type I (TNFR1). Although possible proinflammatory mechanisms have been proposed, most previous studies were performed using in vitro overexpression models, which could lead to undesirable inflammatory responses due to artificial overexpression. It is crucial to reproduce heterozygous mutations at physiological expression levels; however, such studies remain limited. In this study, we generated TRAPS mutant mice and analyzed their phenotypes. Three Tnfrsf1a mutant strains were generated by introducing T79M, G87V, or T90I mutation. T79M is a known mutation responsible for TRAPS, whereas G87V is a TRAPS mutation that we have reported, and T90I is a variant of unknown significance. Using these murine models, we investigated whether TRAPS mutations could affect the inflammatory responses in vivo and in vitro. We found that none of the mutant mice exhibited detectable inflammatory phenotypes under standard housing conditions for 1 year. Interestingly, TRAPS mutant (T79M and G87V) mice had reduced mortality rates after the administration of lipopolysaccharide (LPS) and D-galactosamine, which induce TNFα-dependent lethal hepatitis. Moreover, TRAPS mutations strongly suppressed the development of TNFα-mediated arthritis when crossed with human TNFα transgenic mice. In in vitro primary bone marrow-derived macrophage cultures, the T79M and G87V mutations attenuated the inflammatory responses to TNFα compared with the wild-type, whereas these mutations did not alter the responsiveness of these cells to LPS. The T90I mutant macrophages behaved similarly to wild type in response to LPS and TNFα. The TNFR1 levels were increased in whole-cell lysates of TRAPS mutant macrophages, whereas the cell surface expression of TNFR1 was significantly decreased in TRAPS mutant macrophages. Taken together, TRAPS mutations did not augment the inflammatory responses to TNFα and LPS; instead, they suppressed the response to TNFα via decreased cell surface expression of TNFR1. The stimulation of lymphotoxin-α, adenosine triphosphate, and norepinephrine in primary macrophages or various stimuli in murine splenocytes did not induce detectable inflammatory responses. In conclusion, TRAPS mutations suppressed responsiveness to TNFα, and TRAPS-associated inflammation is likely induced by unconfirmed disease-specific proinflammatory factors.

Keywords: TNF receptor 1; TNF receptor-associated periodic syndrome; autoinflammatory disease; murine model; tumor necrosis factor-α.

Figure 1

No detectable inflammatory phenotypes in G87V TRAPS mutant mice under standard housing conditions. (A-D) G87V TRAPS mutant mice were observed until the age of 33 weeks under standard housing conditions. (A) Body weights. (B) Representative histological images of stained tissue sections at 33 weeks of age. The liver, lung, spleen, and inguinal lymph node were stained with hematoxylin and eosin. (C) mRNA expression in the whole blood of the G87V mutant mice at 33 weeks of age (n = 3 each). Whole blood RNA samples were collected by using PAXgene RNA collection tubes. Gene expression levels relative to Hprt were calculated and normalized to the expression level of the WT mice. (D) Serum concentrations of IL-1β were determined by ELISA at 18 and 33 weeks of age. Each dot denotes an individual mouse. Values are presented as means ± standard deviation. TRAPS, Tumor necrosis factor (TNF) receptor-associated periodic syndrome; IL, interleukin; WT, wild-type; Het, heterozygote; Hom, homozygote; n.s., not significant.

Figure 2

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.

Figure 3

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 TNFα and IL-1β in the culture supernatants. Culture supernatants were collected 6 h after LPS stimulation, and the concentrations of TNFα and IL-1β were determined by ELISA. Levels were calculated relative to those of the WT in each strain. TNFα, Tumor necrosis factor α; 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.

Figure 4

No substantial changes in LPS-induced activation of MAPK and NF-κB pathways by the TRAPS mutations. Primary murine bone marrow-derived macrophages with the indicated mutations were stimulated with 100 ng/mL LPS). Protein samples were collected before and 15 min after stimulation. Cell culture experiments were performed separately for each strain. Phospho- or total-JNK, ERK, p38, and NF-κB p65 levels were detected using specific antibodies. Actin was used as a loading control. TRAPS, TNF receptor-associated periodic syndrome; LPS, lipopolysaccharide; NF-κB, nuclear factor-κB; ERK, extracellular-signal-regulated kinase; JNK, jun N-terminal kinase; WT, wild-type; Het, heterozygote; Hom, homozygote.

Figure 5

Decreased responsiveness to TNFα in the TRAPS mutations. Primary murine bone marrow-derived macrophages were stimulated with 100 ng/mL TNFα. RNA samples were collected at indicated time points. Cell culture experiments were performed separately for each strain. The mRNA expression levels of Tnf and Il1b were determined using qPCR. The WT levels at 0 h were set to 1. Gray, orange, blue, and dark gray lines indicate WT, heterozygous, homozygous, and TNFR1KO mice, respectively. TNFα, Tumor necrosis factor α; TRAPS, TNF receptor-associated periodic syndrome; WT, wild-type; Het, heterozygote; Hom, homozygote.

Figure 6

Reduced activation of MPAK and NF-κB signaling pathways in response to TNFα by the TRAPS mutations. Primary murine bone marrow-derived macrophages were stimulated with 20 ng/mL TNFα. Protein samples were collected before and 15 min after stimulation. Cell culture experiments were performed separately for each strain. Phospho- or total-JNK, ERK, p38, and NF-κB p65 levels were detected using specific antibodies. Actin was used as a loading control. TNFα, Tumor necrosis factor α; TRAPS, TNF receptor-associated periodic syndrome; NF-κB, nuclear factor-κB; WT, wild-type; Het, heterozygote; Hom, homozygote.

Figure 7

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.

Figure 8

No substantial differences in the responses to inflammatory stimuli in TRAPS mutant macrophages and splenocytes. T79M TRAPS mutant bone marrow-derived macrophages were stimulated with 100 ng/mL LTα (A) or 10 mM norepinephrine (B). RNA samples were collected before and 6 h after the stimulation. The mRNA expression levels of Tnf and Il1b were determined using qPCR. The cell culture experiments were performed separately for each strain. (C) Murine splenocytes were collected from indicated mice. The splenocytes were treated with LPS, TNFα, PMA plus ionomycin, ConA, and anti-CD3ϵ/CD28 antibodies. mRNA expression levels of proinflammatory cytokines were determined using qPCR. The WT levels at 0 h were set to 1. *p<0.01 vs. T79M WT. **p<0.01 vs. T79M WT and Het 6 h after stimulation of LTα or norepinephrine. TRAPS, TNF receptor-associated periodic syndrome; LTα, lymphotoxin α; LPS, lipopolysaccharide; PMA, phorbol 12-myristate 13-acetate; ConA, concanavalin A; qPCR, real-time quantitative polymerase chain reaction; WT, wild-type; Het, heterozygote; Hom, homozygote.