We have applied microarray technology to define global gene expression profiles in pSS and identified several key pathways that are dysregulated in cases versus normal controls. Our study is the first to demonstrate that upregulation of IFN-inducible gene expression is prominent in peripheral blood cells of many SS cases, and correlates with high titers of anti-Ro/SSA and anti-La/SSB. In addition, analysis of two independent cohorts revealed evidence for dysregulation of signaling through the B cell/T cell receptors, IGF-1, GM-CSF, PPARα/RXRα, and several cytokine pathways that appear to be consistent across all SS cases.
Microarray-based studies in human pSS have previously focused on the identification of disease associated pathways in saliva or in minor salivary gland tissue from relatively small cohorts (10 or fewer cases plus controls)16; 17; 20; 21
. A common finding across the four studies reported to date is upregulation of IFN-inducible genes. Genes overexpressed in our data generated using peripheral blood that have also been reported as upregulated in minor salivary glands and/or saliva from SS cases include interferon-induced transmembrane proteins 1 (9–27, IFITM1) and 3 (1-8U, IFITM3), promyelocytic leukaemia (PML), transporter 2 ATP-binding cassette (TAP2), spleen tyrosine kinase (SYK), guanylate binding protein, 2 (GBP2), and interferon-induced protein 44 (IFI44)16; 17; 20
. These genes and others that show similar consistency across multiple sample types underscore both the local and systemic nature of IFN pathway dysregulation. Furthermore, these genes may serve as especially attractive targets for development of clinically useful biomarkers. Disease markers that are both central to pathology in target tissues (e.g. salivary glands) and potentially more feasible to assay through saliva or serum-based diagnostic tests would provide a significant improvement over the current approaches to classification of SS cases.
In recent years, upregulation of IFN pathway signaling has been noted in a growing list of autoimmune disorders, including psoriasis, multiple sclerosis, rheumatoid arthritis, dermatomyositis, primary biliary cirrhosis, and insulin-dependent diabetes mellitus 18
. The IFN-inducible gene expression profile we report in SS is remarkably similar to the “IFN signature” that has been observed in similar studies of peripheral blood in SLE, present in a majority of cases22
(Moser KL, unpublished observations). In addition to overlap of certain clinical features in both SLE and SS, production of anti-Ro/SSA and anti-La/SSB autoantibodies are common in both disorders. In our study, the IFN signature in SS was significantly correlated with high titers of anti-Ro/SSA and anti-La/SSB. Although the precise underlying disease mechanism connecting IFN pathway activation and autoantibody production is unclear, these results provide further support to link both innate and adaptive immune responses to the pathogenesis of disease.
Activation and control of IFN-inducible genes may be dysregulated due to abnormal levels or activity of a class of transcription factors known as interferon regulatory factors (IRFs). For example, IRF-1 and IRF-2 are structurally similar DNA-binding factors which were originally identified as regulators of the type I IFN system; IRF-1 functions as a transcriptional activator, and IRF-2 represses IRF-1 function by competing for the same cis elements23
. Evidence from our data sets suggests IRF-1 is upregulated and IRF-2 is downregulated in SS cases. Such an imbalance is consistent with upregulation of IFN-inducible genes. Furthermore, IRF-5 and IRF-7, both upregulated in our data, play a crucial role in the expression of type I IFN genes, cytokines and some chemokines24; 25
. Interestingly, EBV regulates and uses IRF-7 as a secondary mediator for several target genes involved in latency and immune regulation. In addition, Ning et al.
have demonstrated that the virus activated factor of Sendie virus binds to IRF7 IFN stimulating element and can directly activate IRF7 transcription independent of IFN-triggered JAK-STAT pathway 26
. Finally, genetic association of polymorphisms in IRF5 and STAT4, directly involved in IFN pathway signaling, with both SLE and SS has been reported12; 27–30
Collectively, these observations indicate that overexpression of IFN responding genes in SS may result not from overexpression of IFN genes themselves but rather from effects mediated more directly by viral infection and/or genetic variants in IRFs and other IFN pathway mediators that contribute to altered signaling. The potential role of the Type I interferon system in SS was recently reviewed by Nordmark et al31
. Current data supports a mechanism of disease in which an initial viral infection induces Type I interferon production in salivary glands, leading to apoptosis or necrosis of glandular epithelial cells and exposure of autoantigens such as anti-Ro/SSA and anti-La/SSB followed by activation of adaptive immune responses (both locally and systemically). Production of autoantibodies (including anti-Ro/SSA and anti-La/SSB) that form immune complexes with nucleic acids may trigger prolonged activation of IFN pathways through Toll-like receptor-medicated stimulation of plamacytoid dendritic cells31
. Additional production of IFNs, as well as cytokines known to be relevant to SS including, IL-12, IL-6, TNF, CXCL10, and CCL3, can be produced by pDCs, leading to recruitment and perpetuation of a continuous cycle if not properly downregulated32
. Consequently, this process leads to impaired function of affected exocrine glands and potential systemic manifestations commonly seen in SS patients. Our results showing correlations between IFN pathway activation and autoantibodies bring up important considerations for the development of improved diagnostic and therapeutic strategies. We propose that development of biomarkers which reflect the IFN signature and therapies directed against IFN pathway activation are most likely to be successful in the subset of patients with high-titers of anti-Ro/SSA and/or anti-La/SSB.
IPA identified 59 functional categories associated with the list of differentially expressed genes identified in Cohort 1. We found these categories to be too broad for the development of hypotheses of disease mechanisms, and as a result, have focused our attention on canonical pathways. In addition to upregulation of an IFN-inducible gene expression pattern, we identified over 40 additional canonical pathways that were differentially expressed in our PBMC dataset using IPA. However, these pathways do not appear to be independent of each other. Close examination of the genes included in these pathways revealed a significant amount of overlap, most likely reflecting the extensive “crosstalk” that occurs among closely related biological pathways. These results suggest that certain pathways, such as those initiated through B or T cell receptor signaling, account for the seemingly large number of the pathways identified by using approaches such as IPA.
Several of the canonical pathways and dysregulated genes (outside of the “IFN signature”) represent interesting and potentially important new avenues for further investigation. For example, B cell/T cell receptor signaling was significantly dysregulated in this study. One of the genes in these pathways, PTPRC or protein-tyrosine phosphatase, receptor-type, C (also known as CD45, CD45R, and Ly5), is a major leukocyte cell surface molecule that suppresses JAK kinase and negatively regulates cytokine receptor signaling 33
. PTPRC is essential for activation of T cells and B cells, and important for integrin-mediated adhesion and migration of immune cells. In our data, PTPRC was overexpressed in cases versus controls, consistent with enhanced downregulation of other B/T cell pathway genes observed. Targeted disruption of PTPRC has been shown to enhance cytokine and interferon receptor-mediated activation of JAK and STAT proteins33
. Furthermore, genetic associations of variants in PTPRC have been reported with multiple sclerosis, Grave’s disease and Hashimoto’s thyroiditis34
. In murine models, genetic variants in PTPRC lead to lymphoproliferation and severe autoimmune nephritis with autoantibody production and alterations in cytokine production. Thus, evaluation of PTPRC and other related genes in lymphocyte signaling pathways may be informative in further defining autoimmune responses in SS.
The insulin-like growth factor 1 receptor (IGF1R) was underexpressed in our study, consistent with a study of SS minor salivary glands by Katz et al35
. Low levels of IGF1R have also been shown in the non-obese diabetic mouse model of experimental autoimmune sialadenitis36
. Dysregulation of this pathway may result in the inability of IGF-1 to exert its homeostatic, protective effect in salivary tissue and lead to glandular atrophy and disfunction35
Altered signaling through PPARα/RXRα pathways also offers intriguing clues to SS pathogenesis. PPARs (peroxisome proliferator-activated receptors) are nuclear receptors that when activated by ligand, form a functional transcriptional unit upon heterodimerization with retinoid X receptors (RXRs) 37
. PPARα and related family members are critical modulators of environmental and dietary stimuli, and play a key role in downregulating inflammatory responses37; 38
. In immune cells, PPARα inhibits inflammatory pathways through sequestration and repression of c-jun and NF-κB transcription factors38; 39
. Underexpression of PPARα in SS cases relative to controls, as observed in our study, is thus consistent with a pro-inflammatory process. Interestingly, studies in experimental autoimmune encephalitis, a murine model of multiple sclerosis (MS), have demonstrated baseline lower expression levels of PPARα in CD4+ T cells from females relative to males, resulting in increased NF-κB and c-jun activity, higher production of IFN γ and tumor necrosis factor and thus, differential regulation of PPARα between genders may contribute to increase risk of disease in females with MS and other autoimmune diseases40
. Agonists of PPARα have been proposed as a potential therapeutic approach in MS and several other autoimmune and inflammatory disorders associated with decreased PPARα expression such as psoriasis and atopic dermatitis41
. Furthermore, PPARα agonists have been proposed as an effective therapeutic intervention for treatment of dry eye in SS42
. Thus, further studies should be considered to explore the potential application of PPARα agonists as novel therapeutic agents.
In summary, using varying peripheral blood cell populations (mononuclear cells and whole blood), two independently collected cohorts of cases and controls, and two different versions of Affymetrix GeneChips (U95A and U133A), we have shown a consistent upregulation of IFN inducible genes in SS cases. Our results further show that this pattern is most prominent in the subset of cases serologically defined by increased titers of anti-Ro/SSA and anti-La/SSB autoantibodies. We also identified numerous additional signaling pathways that collectively support a significant role for both innate and adaptive immune dysregulation in SS. These results should foster multiple lines of further investigation including genetic and functional studies that will hopefully lead to new insights into pathogenesis of this complex autoimmune disorder.