• Sjøgren’s syndrome
• autoantibodies
• B cells
• DIORA-1
• FAM167A

Sjögren’s syndrome (SS) is characterised by B cell abnormalities and immune-mediated destruction of exocrine glands, primarily the salivary and lacrimal glands.1 2 Among the reported genetic polymorphisms associated with primary SS (pSS), the FAM167A-BLK locus distinguishes itself as an interesting candidate for further analysis based on the strong expression quantitative locus effect of pSS-associated polymorphisms on FAM167A (member A of the Family with sequence similarity 167), contrasted with only moderate or no effect on BLK.3 4 Little is known about the FAM167A gene and its relevance to rheumatic disease pathogenesis. We recently explored FAM167A and its encoded protein Disordered autoimmunity-1 (DIORA-1),4 and reported that DIORA-1 is conserved in vertebrates, has an intracellular, cytoplasmic localisation and in mice is predominantly expressed in lung and spleen—two organs with a high content of immune cells. In the present study, we investigated the expression of DIORA-1 in human immune cells and in salivary glands of patients with pSS, and assessed DIORA-1 expression in relation to pSS clinical manifestations.

Notably, we observed expression of DIORA-1 in CD19⁺ B cells, but little or no expression in monocytes or T cells (figure 1A). DIORA-1 expression in CD19⁺ B cells was similar in patients with pSS and healthy donors (figure 1B). To further define the expression pattern in B cells, we analysed DIORA-1 expression in cell lines representing discrete differentiation stages of B cells. Interestingly, we observed a graded expression of DIORA-1 in these cell lines, with the highest expression found in the two plasma cell myeloma lines and intermediate expression in other B cell lines, and little or no expression in T cells and other investigated cell types (figure 1C).

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Figure 1

Expression of DIORA-1 and clinical associations in Sjögren’s syndrome (SS). (A) Expression of DIORA-1 in CD19⁺ B cells, CD14⁺ monocytes and CD3⁺ T cells sorted from buffy coats of healthy donors (n=10) assessed by real-time PCR. (B) Expression of DIORA-1 in CD19⁺ B cells sorted from peripheral blood of healthy donors (n=16) and patients with primary SS (pSS) (n=17) assessed by real-time PCR. (C) Expression of DIORA-1 in panel of cell lines: HeLa (cervical cancer), A375 (malignant melanoma), HEK293 (human embryonic kidney), Jurkat (acute T cell leukaemia), Nalm6 (B cell precursor leukaemia), Raji (B lymphoblast, Burkitt lymphoma), Daudi (B lymphoblast, Burkitt lymphoma), KM3 (plasma cell myeloma), LP-1 (plasma cell myeloma) assessed by real-time PCR. (D) Representative minor salivary gland biopsies of a patient with pSS and a non-SS patient stained for DIORA-1 by immunohistochemistry. (E) Semiquantitative assessment of DIORA-1 staining of inflammatory foci expressed as per cent DIORA-1⁺ cells of the infiltrate in patients with pSS and non-SS. (F) Semiquantitative assessment of DIORA-1 staining of interstitial cells expressed as per cent stained cells in patients with pSS and non-SS. (G) Representative double immunofluorescence staining of a salivary gland from a patient with pSS by anti-DIORA-1 (green) and anti-CD138 plasma cell surface marker (red). DAPI stain visualising cell nuclei is represented by blue colour. In the merged image white arrows indicate examples of CD138⁺ cells with intracellular DIORA-1 expression. Correlation between interstitial cells expressing DIORA-1 and discrete pSS parameters: focus score (H), serum IgG levels (I) and presence of Ro/SSA autoantibodies in serum (J). ***P<0.001. Scale bar represents 200 µm.

We next assessed the expression of DIORA-1 in salivary glands, which constitute the main target organs in pSS. Minor salivary gland biopsies from 38 patients taken at the time of diagnosis and from 20 individuals investigated for SS due to sicca symptoms but not fulfilling the classification criteria (non-SS)5 were analysed by immunohistochemistry. The included individuals and their characteristics are summarised in online supplementary table 1. In patients with pSS, we observed expression of DIORA-1 in focal infiltrates and in discrete cells interstitially (figure 1D). Few or no positive cells were observed in biopsies from non-SS individuals (online supplementary figure 1). Assessment of the percentage of cells in the focal infiltrates expressing DIORA-1 demonstrated a significant difference between pSS and non-SS individuals (figure 1E, online supplementary figure 2). Quantification of DIORA-1-expressing cells as the percentage of positive interstitial cells also revealed the presence of significantly more DIORA-1⁺ cells in biopsies of patients with pSS compared with non-SS controls (figure 1F). The interstitial DIORA-1-expressing cells had a plasma cell-like morphology, being large cells with a considerable cytoplasm-to-nucleus ratio and an eccentrically positioned nucleus. Double immunofluorescence staining using CD138 or CD38 as plasma cell surface markers confirmed that plasma cells in salivary gland tissue from patients with pSS express DIORA-1 (figure 1G, online supplementary figure 3). DIORA-1 expression in other B cell populations was confirmed by CD20 staining (online supplementary figure 3).

Analysing expression of DIORA-1 in relation to clinical manifestations of pSS, we found that the percentage of DIORA-1⁺ cells positively correlated with salivary gland focus score as well as serum IgG levels and the presence of Ro/SSA autoantibodies (p<0.001) (figure 1H–J). Detailed information on patients and all methods are available in online supplementary methods. We observed no correlation with patient gender, age, salivary flow rate, extraglandular manifestations or adverse predictors of lymphoma (salivary gland enlargement, rheumatoid factor and lymphopenia) (data not shown). Interestingly, however, a recent study reported a blood plasmablast signature correlating with glandular inflammation in Ro/SSA⁺ patients with pSS.6 Limitations of the study relate to the fact that mechanistic studies were not performed and that additional patient groups were not investigated.

In summary, our study reveals that the novel protein DIORA-1, encoded by the FAM167A gene linked to pSS, is expressed in B cells and plasma cells in the target organs of patients with pSS and positively correlates with focus score, serum IgG levels and the presence of Ro/SSA autoantibodies. Our findings indicate a role for DIORA-1 in distinct B cell subsets, and suggest that DIORA-1 may contribute to the inflammatory process and disease pathogenesis in pSS through B cell involvement.