The Sjögren's syndrome-associated autoantigen Ro52/TRIM21 modulates follicular B cell homeostasis and immunoglobulin production

Abstract

Systemic rheumatic diseases are characterized by abnormal B cell activation with autoantibody production and hypergammaglobulinaemia. Ro52/SSA, also denoted tripartite motif (TRIM)21, is a major autoantigen in Sjögren's syndrome and systemic lupus erythematosus. Interestingly, TRIM21-deficient mice develop systemic autoimmunity with B cell-driven manifestations such as autoantibodies, hypergammaglobulinaemia and glomerulonephritis following tissue injury. The mechanisms by which TRIM21-deficiency leads to enhanced B cell activation and antibody production are, however, not well understood, and to further elucidate the role of TRIM21 in systemic autoimmunity, we investigated the B cell phenotype and antibody responses of Trim21^-/- mice following immunization with thymus-dependent (TD) and thymus-independent (TI) antigens. We found that TRIM21-deficient mice developed significantly higher specific antibody titres than their wild-type counterparts upon B cell receptor (BCR) engagement by TD and TI type II antigens, and this was accompanied by an altered B cell phenotype. Furthermore, BCR cross-linking, but not anti-CD40 stimulation, in vitro resulted in a significantly higher proliferation of Trim21^-/- cells. We also observed that splenic follicular B cells were expanded not only in immunized mice but also already in young, unmanipulated Trim21^-/- mice, and transcriptomic analysis of these cells revealed an up-regulation of genes associated with B cell differentiation, indicating a role for TRIM21 in their regulation. In conclusion, in this study we describe a link between the rheumatic autoantigen Ro52/TRIM21 and increased antibody production associated with follicular B cell expansion, implicating a potential role for Ro52/TRIM21 in the pathogenesis of systemic autoimmune diseases.

Keywords: B cell; Ro52; Sjögren’s syndrome; TRIM21; immunoglobulin.

Figure 1

T cell‐dependent activation induces higher antibody titres in Trim21‐deficient mice. Trim21 ^+/+ and ^–/– mice were immunized subcutaneously with 4‐hydroxy‐3‐nitrophenyl ovalbumin (NP‐OVA) in complete Freund’s adjuvant on day 0 and boosted with incomplete Freund’s adjuvant on day 14. Blood was collected for serological analysis at days 0, 5, 7, 14, 21 and at the end of the experiment at day 28 (Trim21 ^+/+ n = 9, Trim21 ^–/– n = 10). NP‐specific antibodies were measured by enzyme‐linked immunosorbent assay (ELISA) in sera. (a) Anti‐NP‐OVA immunoglobulin (Ig)M and total IgG titres. (b) Anti‐NP‐OVA IgG_1, IgG_2a, IgG_2b and IgG₃ subclass analysis. Data are presented as mean ± standard error of the mean (s.e.m.). *p < 0·05, **P < 0·01 (Mann–Whitney U‐test)

Figure 2

Increased antigen‐specific B cell response upon T cell‐dependent activation. Trim21 ^+/+ and ^–/– mice were immunized subcutaneously with 4‐hydroxy‐3‐nitrophenyl ovalbumin (NP‐OVA) in complete Freund’s adjuvant on day 0 and boosted with incomplete Freund’s adjuvant on day 14 (n = 9 and 10, respectively). Cellular composition of spleens and bone marrow was analysed at day 28. (a) NP‐specific immunoglobulin (Ig)M‐ and IgG‐producing B cells from spleen detected by enzyme‐linked immunospot (ELISPOT). Representative wells for each genotype are shown in the right panel. (b) Frequency of NP‐specific IgM‐ and IgG‐expressing B cells among total CD19⁺ cells in spleen. (c) Frequency of CD138⁺CD19^– plasma cells in bone marrow. (d) Absolute numbers of CD19⁺ B cells and (e) frequency of B cell subsets in spleens. (f) Absolute numbers of CD3⁺ T cells and CD11b⁺ cells. Data are presented as mean ± standard error of the mean (s.e.m.). *P < 0·05, **P < 0·01, ***P < 0·001 (Mann–Whitney U‐test)

Figure 3

T cell‐independent type II activation results in a stronger response in Trim21 ^–/– mice than T cell independent type I stimulation. Mice were immunized intraperitoneally with 4‐hydroxy‐3‐nitrophenyl‐lipopolysaccharide (NP‐LPS) or NP‐Ficoll in phosphate‐buffered saline (PBS) at day 0 and serum was collected at days 0, 5, 7 and 14. NP‐specific antibody levels were assessed by enzyme‐linked immunosorbent assay (ELISA). (a) Anti‐NP‐LPS immunoglobulin (Ig)M and IgG titres (Trim21 ^+/+ n = 9, Trim21 ^–/– n = 10). (b) Anti‐NP‐Ficoll IgM and IgG titres (Trim21 ^+/+ n = 9, Trim21 ^–/– n = 10) (data are presented as mean ± standard error of the mean (s.e.m.). *P < 0·05 (Mann–Whitney U‐test)

Figure 4

Trim21^–/– B cells proliferate more readily upon anti‐immunoglobulin (Ig)M stimulation. Spleen cells were cultured with either anti‐IgM, anti‐CD40 or without stimuli. Incorporation of thymidine was measured after 3 days. (a) anti‐IgM and (b) anti‐CD40 stimulation of total splenocytes from Trim21 ^+/+ and ^–/– (five per group). (c) Anti‐IgM stimulation of B cells purified from spleens (three per group). (d) Total splenocytes were cultured with 0·1 g/ml anti‐IgM for 4 days and expression of the proliferation marker Ki67 in CD19⁺ B cells was assessed by flow cytometry (six per group). (e) Activation‐induced cell death was assessed in anti‐IgM stimulated cells at day 5, 2 days after restimulation. Frequencies of live, annexin V⁺ apoptotic and annexin V⁺ 7‐aminoactinomycin D (7‐AAD)⁺ necrotic cells among Trim21 ^+/+ and ^–/– cells (six per group). Data are presented as mean ± standard error of the mean (s.e.m.). *P < 0·05 (Mann–Whitney I‐test)

Figure 5

Follicular B cells are expanded in unmanipulated Trim21‐deficient mice. The cellular composition of spleens of naive, unmanipulated mice was assessed by flow cytometry. (a) Frequency of CD19⁺ cells and (b) CD19⁺CD21⁺CD23⁺ follicular B cells, CD19⁺CD21⁺⁺CD23⁺ marginal zone B cells and CD19⁺CD21^–CD23^– T1 transitional B cells in mice aged 8–16 weeks (n = 17 per group, pooled data from four experiments). (c) Frequency of CD19⁺ cells and (d) CD19⁺CD21⁺CD23⁺ follicular B cells, CD19⁺CD21⁺⁺CD23⁺ marginal zone B cells and CD19⁺CD21^–CD23^– T1 transitional B cells in mice aged 52–58 weeks (Trim21 ^+/+ n = 25, Trim21 ^–/– n = 29, pooled data). Data are presented as mean ± standard error of the mean (s.e.m.). *P < 0·05, **P < 0·01 (Mann–Whitney U‐test)

Figure 6

Distinct transcriptomic profiles in Trim21 ^–/– follicular B cells. Total RNA was extracted from CD19⁺CD21⁺CD23⁺ follicular B cells isolated from Trim21 ^+/+ and Trim21 ^–/– mice (six per group), and the whole‐genome transcriptome was analysed using the Mouse Transcriptome Assay 1.0 microarray chip (Affymetrix). (a) Gene expression profiling showing top differentially regulated genes (P‐value < 0·01). (b) Pathway analysis based on GO search terms. (c) Top differentially expressed genes (P < 0·05) belonging to the GO term GO:0045577 ‘GO: regulation of B cell differentiation’ pathway.