Newly discovered genetic associations in primary Sjögren’s syndrome implicate disturbances in both the adaptive and the innate immune pathways ^{13, 14} . Genome-wide association and candidate gene studies have identified susceptibility loci in BLK and CXCR5, genes encoding proteins involved in B cell activation, trafficking, and spatial localization, and in STAT4, IL-12A, and IRF5, genes regulating both adaptive and innate immunity. The finding of risk variants in the IRF5 gene may explain in part the activation of the type 1 interferon (IFN) pathway in primary Sjögren’s syndrome ¹⁵ , as IRF5 encodes a transcription factor important for the production of type 1 IFN ¹⁶ . A risk variant of IRF5, a CGGGG indel, has been associated with increased levels of IRF5 transcripts, implying a functional role in the regulation of this pathway ¹⁷ . STAT4 is triggered by type 1 IFN, interleukin (IL)-12, and IL-23 and when activated leads to the induction of T helper type 1 (TH1) cells and up-regulation of IFN-γ.

Genome-wide association and candidate gene studies have also identified risk loci in the TNFAIP3 and TNIP genes, which regulate nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signaling. The potential importance of the TNFAIP3 locus in the pathogenesis of primary Sjögren’s syndrome is underscored by the finding that TNFAIP3 knockout mice develop an autoimmune phenotype and have high rates of lymphoma ¹⁸ . Moreover, a study of patients with primary Sjögren’s syndrome from a French cohort has recently found germline mutations in the coding region of TNFAIP3 (A20) that are associated with an increased risk for lymphoma ¹⁹ . Interestingly, in some instances, the samples of lymphoma tissue harbored the same germline mutations in TNFAIP3 that were associated with an increased risk for lymphoma development, while in other cases without the germline mutation, the lymphoma tissue had somatic mutations in TNFAIP3. Some of these TNFAIP3 variants impaired control of NFκB activation, suggesting a possible escape mechanism for transition into lymphoma ¹⁹ .

The epigenetic regulation of gene expression in primary Sjögren’s syndrome has not been studied until recently. Patients with primary Sjögren’s syndrome have an increase in hypomethylation of the STAT4, IL-12A, IRF5, BLK, CXCR5, and TNIP genes ^{20, 21} , suggesting a possible role for epigenetic regulation in the pathogenesis of this disease.

The role of B cells in the pathogenesis of primary Sjögren’s syndrome is strongly implied by the association of this disease with autoantibody production, B cell hyperactivity, germinal center formation in the target tissue, and lymphomagenesis. In primary Sjögren’s syndrome, it is not known if pathologic B cell activation occurs in the spleen and lymph nodes or the germinal center-like structures (tertiary lymphoid tissue) of the target tissues, or both. T-cell-dependent antigens that activate B cells rely on T follicular helper (TFH) cells, which stimulate the formation and maintenance of germinal centers through the expression of CD154 (CD40 ligand) on the cell surface and the secretion of IL-4 and IL-21; they mediate the selection and survival of B cells that differentiate into antibody-secreting plasma cells and memory B cells. TFH cells have been identified in labial salivary gland tissue, predominantly within organized structures ²² . The differentiation of CD4+ naïve T cells into TFH cells may be promoted by the glandular epithelium. Salivary gland epithelial cells expressing IL-6 and inducible T cell co-stimulator ligand (ICOS-L) have been shown in culture to stimulate the differentiation of CD4+ naïve T cells into IL-21-secreting TFH cells ²³ .

Controlling TFH cell-mediated B cell activation may be a useful therapeutic strategy in primary Sjögren’s syndrome. Two experimental therapies, abatacept and rituximab, have been shown to reduce the absolute number of circulating TFH cells ²⁴ , but the significance of these findings is unclear, since neither of these agents has been proven effective in randomized, controlled trials involving patients with primary Sjögren’s syndrome. In the future, inhibiting the differentiation of TFH cells by neutralizing IL-21 or IL-6 or blocking the interaction between ICOS and ICOS-L, either alone or in combination, may also be therapeutic strategies worth pursuing.

B-cell-activating factor (BAFF), a member of the TNF receptor family, is a potent activator of B cells. BAFF is the natural ligand of three receptors, BAFF-R (BR3), a transmembrane activator and calcium modulator, as well as transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA). Several lines of evidence implicate BAFF in the pathogenesis of primary Sjögren’s syndrome. First, compared with healthy controls, patients with primary Sjögren’s syndrome have higher serum levels of BAFF and show increased expression of BAFF in labial salivary glands ²⁵ . Second, BAFF transgenic mice exhibit a Sjögren’s syndrome phenotype characterized by infiltration of lacrimal and salivary glands with lymphocytes that include a unique population of marginal zone B cells ²⁶ . Third, salivary gland epithelial cells from patients with primary Sjögren’s syndrome also express BAFF; in addition, type 1 IFN, which is up-regulated in both blood and salivary gland tissue from patients with primary Sjögren’s syndrome, is a potent stimulator of BAFF expression ^{25, 27} . BAFF engagement with its receptor, BR3, has been shown to activate the phosphatidylinositol 3-kinase delta isoform (PI3kδ), a signaling molecule involved in the regulation of cell growth, proliferation, differentiation, motility, survival, and intracellular trafficking of B cells, as well as lymphomagenesis ²⁸ . For these reasons, the BAFF/BR3 pathway is a promising therapeutic target in primary Sjögren’s syndrome.

The lymphotoxin β receptor (LTβR) is part of the signaling system that directs stromal cells to differentiate into specialized vasculature and regulates certain reticular networks that guide and position cells for optimal encounters with antigen ²⁹ . Blockade of the LTβR system in mice reduces addressin expression on high endothelial venules (HEVs) and thereby inhibits entry of lymphocytes into lymph nodes and mucosal environments, resulting in a circulating lymphocytosis ³⁰ . In male NOD mice, LTβR-Ig treatment reduces glandular inflammation, blocks HEV formation, and partially restores salivary flow ³¹ . In a phase II trial, baminercept (LTβR-Ig) treatment was ineffective in decreasing the signs and symptoms of RA; however, it was shown to increase the blood lymphocyte counts and reduce the whole blood IFN signature ³² . Although inhibiting the lymphotoxin pathway has a sound theoretical basis for treating primary Sjögren’s syndrome, preliminary results from a randomized, placebo-controlled 24-week trial showed no significant differences between the baminercept and placebo groups in the change in stimulated salivary flow or ESSDAI ³³ .

Viruses have long been considered etiologic factors in primary Sjögren’s syndrome. They have been receiving more attention of late as a possible disease trigger because of the association between primary Sjögren’s syndrome and up-regulation of the type 1 IFN pathway, an innate immune mechanism of antiviral host defense. An IFN signature has been detected in the peripheral blood of 65% of patients with primary Sjögren’s syndrome ³⁴ and can be observed in labial salivary gland tissue from patients with this disease ³⁴ . The majority of the IFN inducible genes are up-regulated by both IFN-α and IFN-γ, while some are uniquely due to the effects of IFN-α or IFN-γ. In the labial salivary glands, there is heterogeneity among patients in the expression of IFN-α and IFN-γ inducible genes, with higher focus scores associated with predominant IFN-γ effects ^{34, 35} .

To protect against viral infection, the host innate immune system has evolved sensors of nucleic acids. The same nucleic acid sensors that defend against viruses may also contribute to the pathogenesis of autoimmune diseases. The two main systems for detecting nucleic acids are the Toll-like receptors (TLRs) and the cytosolic RNA and DNA sensors. The endosomal TLRs mainly detect nucleic acids, such as double-stranded RNA (TLR3), single-stranded RNA (TLR7), and double-stranded DNA (TLR9) ³⁶ . Through an autoantibody-dependent, FcγR-mediated process, nucleic acids gain access to the cytosol and in turn ligate endosomal TLRs, which activate dendritic cells and macrophages to produce type 1 IFN ³⁷ . Two other key intracellular sensors of RNA and DNA may also play a role in the pathogenesis of autoimmune diseases such as primary Sjögren’s syndrome, namely the retinoic acid-inducible gene I (RIG-I)-like receptors RIG-I and Mda5 and cyclic GMP-AMP synthase/STING, respectively, which also activate the production of type 1 IFN ³⁸ . It has been theorized that the evolutionary pressures to produce a more robust antiviral response may also tip the balance towards developing an autoimmune disease.

Epstein-Barr virus (EBV) is a promising candidate for triggering autoimmune disease owing to its ability to infect B cells and promote the survival of autoreactive B cell clones ³⁹ . A human DNA γ-herpesvirus, it establishes an asymptomatic latent infection in B cells and epithelial cells. EBV-encoded small RNA (EBER), the most abundant transcripts in latently infected cells, contribute to EBV-mediated pathogenesis by activating RIG-I and stimulating innate immune signaling through TLR3 ⁴⁰ . EBERs also bind to the La protein and are secreted outside the cell in exosomes, where they may bind to TLR3 and activate dendritic cells ⁴¹ . However, studies comparing the expression of EBV nucleic acids and proteins in the salivary glands between patients with primary Sjögren’s syndrome and healthy controls have produced conflicting results.

Croia and colleagues recently attempted to settle this controversy by focusing their search for latent EBV infection on labial salivary gland tissue with germinal center-like structures expressing the mRNA for activation-induced cytidine deaminase (AID) and CD21 ⁴² . AID is involved in somatic hypermutation, gene conversion, and class-switch recombination of immunoglobulin genes, while CD21 is a marker of follicular dendritic cells. They found that EBERs were preferentially expressed in the AID ⁺CD21 ⁺ labial salivary gland biopsies compared to AID ^-CD21 ^- samples from the disease patients and healthy controls, with EBERs localized mostly to the follicular B cells and plasma cells. Further evidence of EBV latency was obtained by detection of the latent EBV antigen LMP-2 in the B cells within the ectopic follicles. Notably, the lytic-phase antigen BFRF1 was expressed in the perifollicular plasma cells with Ro52 autoreactivity, a prominent autoantigen in primary Sjögren’s syndrome. Whether EBER transcripts from latent EBV infection are contributing to the inflammatory response in primary Sjögren’s syndrome is an open question, but the results of this study draw more attention to this possibility.

Retrotransposons comprise approximately 40% of the human genome and include the retrovirus long-terminal repeats (LTRs), members of the ‘long interspersed nuclear element 1’ (LINE-1, or L1) family, and ‘short interspersed nuclear elements’ (SINEs) ³⁹ . Most human SINEs are ‘Alu’ elements, which are capable of replicating themselves by hijacking the L1 reverse transcriptase ³⁹ . A recent study found that anti-Ro60–positive SLE immune complexes containing Alu RNAs and Alu transcripts were up-regulated in whole blood samples from patients with SLE relative to control samples and established that aberrant expression of endogenous Alu RNAs stimulated a TLR7-dependent response that enhances the secretion of IFN-α, IL-6, and TNF-α ⁴¹ . The results from these studies suggest that retroelements may stimulate autoimmunity by amplifying immune responses through endogenous RNA sensors ⁴¹ . There is also evidence that endogenous retroviruses and innate sensing pathways are integral to T cell-independent B cell activation ⁴³ . Thus, if retroelements play a significant role in the pathogenesis of primary Sjögren’s syndrome, then decreasing the abundance of nucleic acids inside cells or increasing the threshold of their intracellular sensors may represent a new strategy for treating this disease.

Natural killer (NK) cells are innate lymphoid cells that play an important role in antiviral host defense and are increasingly implicated in the pathogenesis of autoimmune disease ⁴⁴ . Human NK cells are CD3 ^- and separated into two major groups by their expression of CD16 and CD56. The CD56 ^dim NK cells that comprise approximately 90% of the circulating NK cells express high levels of CD16, inhibitory killer-like receptors (KIRs), and perforin; CD56 ^bright NK cells express low levels of CD16, KIRs, and perforin and are abundant in secondary lymphoid tissues where they modulate immune responses through their secretion of cytokines and chemokines ⁴⁴ .

NKp30, a NK cell-specific activating receptor, regulates cross-talk between NK and dendritic cells and mediates secretion of IL-12 and IFN-γ. In a large cohort of patients with primary Sjögren’s syndrome, genetic polymorphisms within the promoter region of the NKp30 gene were found to be associated with decreased disease susceptibility and reduced levels of gene expression and function of the NKp30 protein ⁴⁵ . In this study, circulating NK cells from the patients with primary Sjögren’s syndrome expressed higher levels of NKp30 than controls and showed increased secretion of IFN-γ ⁴⁵ . Moreover, excessive numbers of NK cells were found to be present in the labial salivary gland tissue from patients with primary Sjögren’s syndrome, localizing outside inflammatory foci, and nearby glandular epithelial cells expressing B7H6, the ligand for NKp30 ⁴⁵ . It is possible that NKp30 ⁺ cells may be an important source of IFN-γ in the target organ of primary Sjögren’s syndrome through the engagement of B7H6 on epithelial cells.

IL-22, a member of the IL-1 family, is produced by a variety of cell types, including TH17 cells, γδ T cells, NK T cells, and innate lymphoid cells (ILCs); it mainly acts on nonhematopoietic cells such as epithelial cells ⁴⁶ . Increased expression of IL-22 has been linked to the pathogenesis of tissue inflammation and regeneration of epithelial tissues following injury ⁴⁶ . Therefore, IL-22 exerts both pro-inflammatory and protective effects and its function has been shown to be tissue and context dependent. IL-22, IL-23, and IL-17 mRNA and protein levels are up-regulated in labial salivary gland tissue from patients with primary Sjögren’s syndrome compared to healthy controls ⁴⁷ .

In labial salivary glands from patients with primary Sjögren’s syndrome, IL-22 has been shown to be produced by activated dendritic cells, T cells, and the NKp44+ subset of NK cells ⁴⁷ . Further insights into the possible role of IL-22 have been investigated in a virus-induced mouse model of Sjögren’s syndrome, where IL-22 was found to be mainly produced in the early stages of infection by γδ T cells and then later by αβ T cells, with lesser amounts coming from ILCs and NK cells ⁴⁸ . In this study, IL-22/IL-22R engagement was needed for enhanced expression of CXCL12 and CXCL13, recruitment of B cells, and organization of tertiary lymphoid tissue. Although IL-22 may be a potential therapeutic target in primary Sjögren’s syndrome, its role in promoting tissue regeneration and restoring barrier function after tissue injury may preclude systemic IL-22 blockade over long periods of time.