Protein citrullination and NET formation do not contribute to the pathology of A20/TNFAIP3 mutant mice

Abstract

A20 serves as a critical brake on NF-κB-dependent inflammation. In humans, polymorphisms in or near the TNFAIP3/A20 gene have been linked to various inflammatory disorders, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Experimental gene knockout studies in mice have confirmed A20 as a susceptibility gene for SLE and RA. Here, we examine the significance of protein citrullination and NET formation in the autoimmune pathology of A20 mutant mice because autoimmunity directed against citrullinated antigens released by neutrophil extracellular traps (NETs) is central to the pathogenesis of RA and SLE. Furthermore, genetic variants impairing the deubiquitinase (DUB) function of A20 have been shown to contribute to autoimmune susceptibility. Our findings demonstrate that genetic disruption of A20 DUB function in A20 C103R knockin mice does not result in autoimmune pathology. Moreover, we show that PAD4 deficiency, which abolishes protein citrullination and NET formation, does not prevent the development of autoimmunity in A20 deficient mice. Collectively, these findings provide experimental confirmation that PAD4-dependent protein citrullination and NET formation do not serve as pathogenic mechanisms in the development of RA and SLE pathology in mice with A20 mutations.

Figure 1

A20 DUB mutation does not induce NET formation or autoimmune pathology. (A) Generation of A20^C103R knockin mutation in mice using CRISPR/Cas9 technology. A20 domain structure with indication of C103R mutation in OTU deubiquitinase (DUB) domain (upper). Sanger sequencing of wild-type (+ / +), heterozygous (C103R/ +) and homozygous (C103R/C103R) clone (lower). Structural analysis of A20 DUB carrying the C103R mutation (pink, C103R), and comparison with the wildtype A20 DUB structure (cyan, C103) versus acetamidylated A20 DUB at C103 (cyan, C103ace). (B, C) Body weight (B) and spleen weight (C) of 25–30 week old wild-type (A20^+/+), A20^C103R/+ and A20^C103R/C103R mice. Each dot represents one mouse. Data are expressed as mean ± s.e.m. (D) Total IgA, IgM, IgG, anti-cardiolipin-IgA, and anti-dsDNA-IgG and neutrophil extracellular trap (NET) concentrations in serum of 25–30-week old mice. (E–F) Histological images of haematoxylin and eosin-stained (E) and PAS-stained (F) kidney sections of 25–30-week old mice, showing normal glomerular architecture and cellularity, and absence of granulomas, tubulo-interstitial atrophy, or vascular changes. Scalebar, 50 µm. (G) Histological scores for mice with the indicated genotypes (25–30 weeks). The arthritis was scored at the Achilles tendon (infiltrate) and the synovio-entheseal complex (SEC, exudate), each ranging from 0 (normal) to 3 (severely inflamed). Dots in the graphs indicate individual mice and data are expressed as mean ± s.e.m. (H) Histological images of haematoxylin and eosin-stained ankle joints of mice with the indicated genotypes. No signs of an arthritis-like phenotype can be observed in A20^C103R/C103R mice. Pictures are representative for 4–5 biologically independent mice for each genotype. Scalebar, 1000 µm. (I) Neutrophil Extracellular Traps (NETs) in serum of wild-type (A20^+/+), A20^C103R/+ and A20^C103R/C103R mice. Each dot represents one mouse. Data are expressed as mean ± s.e.m. n.s., non-significant.

Figure 2

PAD4 deficiency does not rescue DC-specific A20 deficient mice from developing SLE pathology. (A) Breeding scheme to generate A20^DC-KO and A20^DC-KOPadi4^−/− mice. (B, C) Body weight (B) and spleen weight (C) of 25–30 week old wild-type, A20^DC-KO, Padi4^−/− and A20^DC-KOPadi4^−/− mice. Each dot represents one mouse. Data are expressed as mean ± s.e.m. (D) Serum levels of TNF, IL-6, IL-22 and BAFF in indicated genotypes. (E) Quantitative PCR results for interferon-stimulated genes on whole spleen of mice with indicated genotypes. (F) Serum auto-antibody concentrations on ELISA in mice with indicated genotypes. (G) Representative immunofluorescent image of glomerular IgA deposition per genotype. White dotted circles indicate the glomeruli. (H) Quantification of the glomerular mean fluorescent signal for IgA. (I) Representative hematoxylin and eosin-stained kidneys of mice with the indicated genotypes, showing extensive perivascular infiltrates in both A20^DC-KO and A20^DC-KOPadi4^−/− mice. Each dot represents one mouse. Data are expressed as mean. Each dot represents a biologically independent mouse. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Scalebar, 50 µm. CNRQ = Calibrated Normalized Relative Quantity. Results are representative of two independent experiments.

Figure 3

PAD4 deficiency does not rescue myeloid-specific A20 deficient mice from developing RA pathology. (A) Breeding scheme to generate A20^myel-KO and A20^myel-KOPadi4^−/− mice. (B) Spleen weight of 25–30 week old wild-type, A20^myel-KO, Padi4^−/− and A20^myel-KOPadi4^−/− mice, with representative picture. Each dot represents one mouse. Data are expressed as mean ± s.e.m. (C) Biweekly clinical arthritis scores of the ankles of wild-type, A20^myel-KO, Padi4^−/− and A20^myel-KOPadi4^−/− mice. Data are expressed as mean ± s.e.m. ***p < 0.001 (REML analysis). (D) Graphs depicting histological scores for mice with the indicated genotypes (25–30 weeks). The arthritis was scored at the Achilles tendon (infiltrate) and the synovio-entheseal complex (SEC, exudate), each ranging from 0 (normal) to 3 (severely inflamed). Total arthritis score is the sum of the 3 individual scores. Dots in the graphs indicate individual mice and data are expressed as mean ± s.e.m. (E) Histological images of hematoxylin and eosin-stained ankle joints of mice with the indicated genotypes, demonstrating periarticular inflammation and infiltration of mononuclear cells (insert), as well as the infiltrate in the SEC (dashed insert) in A20^myel-KO and A20^myel-KOPadi4^−/− mice. Pictures are representative for 4–5 biologically independent mice for each genotype. Scalebar, 1000 µm. (F) Levels of IL-6, TNF and IL-18 in serum of wild-type, A20^myel-KO, Padi4^−/− and A20^myel-KOPadi4^−/− mice. Each dot represents a biologically independent mouse. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (G) Line Immunoassay (LIA) of 25-week-old wild-type, A20^myel-KO, Padi4^−/− and A20^myel-KOPadi4^−/− mice. Each lane represents an individual mouse. (H) Neutrophil Extracellular Traps (NETs) in serum of wild-type, A20^myel-KO, Padi4^−/− and A20^myel-KOPadi4^−/− mice. Each dot represents a biologically independent mouse. n.s., non-significant.