Dysregulated Ca2+ signaling, fluid secretion, and mitochondrial function in a mouse model of early Sjögren's disease.

The molecular mechanisms leading to saliva secretion are largely established, but factors that underlie secretory hypofunction, specifically related to the autoimmune disease Sjögren's syndrome (SS) are not fully understood. A major conundrum is the lack of association between the severity of salivary gland immune cell infiltration and glandular hypofunction. SS-like disease was induced by treatment with DMXAA, a small molecule agonist of murine STING. We have previously shown that the extent of salivary secretion is correlated with the magnitude of intracellular Ca2+ signals (Takano et al., 2021). Contrary to our expectations, despite a significant reduction in fluid secretion, neural stimulation resulted in enhanced Ca2+ signals with altered spatiotemporal characteristics in vivo. Muscarinic stimulation resulted in reduced activation of the Ca2+-activated Cl- channel, TMEM16a, although there were no changes in channel abundance or absolute sensitivity to Ca2+. Super-resolution microscopy revealed a disruption in the colocalization of Inositol 1,4,5-trisphosphate receptor Ca2+ release channels with TMEM16a, and channel activation was reduced when intracellular Ca2+ buffering was increased. These data indicate altered local peripheral coupling between the channels. Appropriate Ca2+ signaling is also pivotal for mitochondrial morphology and bioenergetics. Disrupted mitochondrial morphology and reduced oxygen consumption rate were observed in DMXAA-treated animals. In summary, early in SS disease, dysregulated Ca2+ signals lead to decreased fluid secretion and disrupted mitochondrial function contributing to salivary gland hypofunction.

Deleting Mitochondrial Superoxide Dismutase 2 in Salivary Gland Ductal Epithelial Cells Recapitulates Non-Sjögren's Sicca Syndrome.

Elevated oxidative stress can play a pivotal role in autoimmune diseases by exacerbating inflammatory responses and tissue damage. In Sjögren's disease (SjD), the contribution of oxidative stress in the disease pathogenesis remains unclear. To address this question, we created mice with a tamoxifen-inducible conditional knockout (KO) of a critical antioxidant enzyme, superoxide dismutase 2 (Sod2), in the salivary glands (i-sg-Sod2 KO mice). Following tamoxifen treatment, Sod2 deletion occurred primarily in the ductal epithelium, and the salivary glands showed a significant downregulation of Sod2 expression. At twelve weeks post-treatment, salivary glands from the i-sg-Sod2 KO mice exhibited increased 3-Nitrotyrosine staining. Bulk RNA-seq revealed alterations in gene expression pathways related to ribosome biogenesis, mitochondrial function, and oxidative phosphorylation. Significant changes were noted in genes characteristic of salivary gland ionocytes. The i-sg-Sod2 KO mice developed reversible glandular hypofunction. However, this functional loss was not accompanied by glandular lymphocytic foci or circulating anti-nuclear antibodies. These data demonstrate that although localized oxidative stress in salivary gland ductal cells was insufficient for SjD development, it induced glandular dysfunction. The i-sg-Sod2 KO mouse resembles patients classified as non-Sjögren's sicca and will be a valuable model for deciphering oxidative-stress-mediated glandular dysfunction and recovery mechanisms.

Dysregulated Ca2+ signaling, fluid secretion, and mitochondrial function in a mouse model of early Sjögren's syndrome.

Saliva is essential for oral health. The molecular mechanisms leading to physiological fluid secretion are largely established, but factors that underlie secretory hypofunction, specifically related to the autoimmune disease Sjögren's syndrome (SS) are not fully understood. A major conundrum is the lack of association between the severity of inflammatory immune cell infiltration within the salivary glands and glandular hypofunction. In this study, we investigated in a mouse model system, mechanisms of glandular hypofunction caused by the activation of the stimulator of interferon genes (STING) pathway. Glandular hypofunction and SS-like disease were induced by treatment with 5,6-Dimethyl-9-oxo-9H-xanthene-4-acetic acid (DMXAA), a small molecule agonist of murine STING. Contrary to our expectations, despite a significant reduction in fluid secretion in DMXAA-treated mice, in vivo imaging demonstrated that neural stimulation resulted in greatly enhanced spatially averaged cytosolic Ca2+ levels. Notably, however, the spatiotemporal characteristics of the Ca2+ signals were altered to signals that propagated throughout the entire cytoplasm as opposed to largely apically confined Ca2+ rises observed without treatment. Despite the augmented Ca2+ signals, muscarinic stimulation resulted in reduced activation of TMEM16a, although there were no changes in channel abundance or absolute sensitivity to Ca2+. However, super-resolution microscopy revealed a disruption in the intimate colocalization of Inositol 1,4,5-trisphosphate receptor Ca2+ release channels in relation to TMEM16a. TMEM16a channel activation was also reduced when intracellular Ca2+ buffering was increased. These data are consistent with altered local coupling between the channels contributing to the reduced activation of TMEM16a. Appropriate Ca2+ signaling is also pivotal for mitochondrial morphology and bioenergetics and secretion is an energetically expensive process. Disrupted mitochondrial morphology, a depolarized mitochondrial membrane potential, and reduced oxygen consumption rate were observed in DMXAA-treated animals compared to control animals. We report that early in SS disease, dysregulated Ca2+ signals lead to decreased fluid secretion and disrupted mitochondrial function contributing to salivary gland hypofunction and likely the progression of SS disease.

Age-associated B cell infiltration in salivary glands represents a hallmark of Sjögren's-like disease in aging mice.

Sjögren's disease (SjD), characterized by circulating autoantibodies and exocrine gland inflammation, is typically diagnosed in women over 50 years of age. However, the contribution of age to SjD pathogenesis is unclear. C57BL/6 female mice at different ages were studied to investigate how aging influences the dynamics of salivary gland inflammation. Salivary glands were characterized for immune cell infiltration, inflammatory gene expression, and saliva production. At 8 months, gene expression of several chemokines involved in immune cell trafficking was significantly elevated. At this age, age-associated B cells (ABCs), a unique subset of B cells expressing the myeloid markers CD11b and/or CD11c, were preferentially enriched in the salivary glands compared to other organs like the spleen or liver. The salivary gland ABCs increased with age and positively correlated with increased CD4 T follicular helper cells. By 14 months, lymphocytic foci of well-organized T and B cells spontaneously developed in the salivary glands. In addition, the mice progressively developed high titers of serum autoantibodies. A subset of aged mice developed salivary gland dysfunction mimicking SjD patients. Our data demonstrates that aging is a significant confounding factor for SjD. Thus, aged female C57BL/6 mice are more appropriate and a valuable preclinical model for investigating SjD pathogenesis and novel therapeutic interventions.

Age-associated B cell infiltration in salivary glands represents a hallmark of Sjögren's-like disease in aging mice.

Sjögren's disease (SjD), characterized by circulating autoantibodies and exocrine gland inflammation, is typically diagnosed in women over 50 years of age. However, the contribution of age to SjD pathogenesis is unclear. C57BL/6 female mice at different ages were studied to investigate how aging influences the dynamics of salivary gland inflammation. Salivary glands were characterized for immune cell infiltration, inflammatory gene expression, oxidative stress, and saliva production. At 8 months, gene expression of several chemokines involved in immune cell trafficking was significantly elevated. At this age, Age-associated B cells (ABCs), a unique subset of B cells expressing the myeloid markers CD11b and/or CD11c, were preferentially enriched in the salivary glands compared to other organs like the spleen or liver. The salivary gland ABCs increased with age and positively correlated with increased CD4 T follicular helper cells. By 14 months, lymphocytic foci of well-organized T and B cells spontaneously developed in the salivary glands. In addition, the mice progressively developed high titers of serum autoantibodies. A subset of aged mice developed salivary gland dysfunction mimicking SjD patients. Our data demonstrates that aging is a significant confounding factor for SjD. Thus, aged female C57BL/6 mice are more appropriate and a valuable preclinical model for investigating SjD pathogenesis and novel therapeutic interventions.

STING Agonist-Induced Skin Inflammation Is Exacerbated with Prior Systemic Innate Immune Activation.

Activation of the Stimulator of Interferon Genes (STING) protein has paradoxical outcomes in skin disease. STING activation exacerbates psoriatic skin disease and delays wound healing in diabetic mice, yet it also facilitates wound healing in normal mice. To address the role of localized STING activation in the skin, mice were injected subcutaneously with a STING agonist, diamidobenzimidazole STING Agonist-1 (diAbZi). The effect of a prior inflammatory stimulus on STING activation was addressed by pre-treating mice intraperitoneally with poly (I:C). The skin at the injection site was evaluated for local inflammation, histopathology, immune cell infiltration, and gene expression. Serum cytokine levels were measured to assess systemic inflammatory responses. Localized diABZI injection induced severe skin inflammation with erythema, scaling, and induration. However, the lesions were self-limiting and resolved within 6 weeks. At the peak of inflammation, the skin showed epidermal thickening, hyperkeratosis, and dermal fibrosis. Neutrophils, CD3 T cells, and F4/80 macrophages were present in the dermis and subcutaneous layers. Gene expression was consistent with increased local interferon and cytokine signaling. Interestingly, the poly (I:C)-pre-treated mice showed higher serum cytokine responses and developed worse inflammation with delayed wound resolution. Our study demonstrates that prior systemic inflammation amplifies STING-mediated inflammatory responses and skin disease.

Anti-vimentin antibodies are associated with higher severity of Sjögren's disease.

Vimentin is a ubiquitously present Type III intermediate filament protein, often targeted by autoimmune responses in multiple rheumatic disorders. Although previous studies have reported anti-vimentin antibodies in Sjögren's disease (SjD) patients, the clinical significance of such antibodies is unknown. To address this issue, the presence of anti-vimentin antibodies was determined in serum samples from a well-characterized cohort of primary SjD patients, non-SjD Sicca, and healthy controls. The occurrence of anti-vimentin antibodies and their association with different clinical features of the disease were evaluated. Anti-vimentin antibodies were detected in 24% of primary SjD patients, compared to 4% in non-SjD sicca patients and 3% in healthy controls. In primary SjD patients, higher levels of anti-vimentin antibodies were significantly associated with reduced saliva and tear flow and severe ocular surface damage indicators. The anti-vimentin antibody levels did not show significant associations with the presence or absence of other autoantibodies like ANA, RF, and anti-Ro/La. Our data suggest that the anti-vimentin antibody specificity arises in a subset of primary SjD patients and is associated with oral and ocular features of the disease. Anti-vimentin can potentially serve as a novel biomarker for evaluating the severity of salivary and lacrimal gland dysfunction in primary SjD.

Antibody deposition on vascular endothelial cells contributes to localized inflammation in salivary glands.

BACKGROUND: Oral and ocular dryness due to reduced saliva and tear production, exocrine gland inflammation, and autoantibodies to multiple cellular proteins are the cardinal features of Sjögren's Disease. Among the autoantibody specificities, anti-Ro52 is linked with higher disease severity. We have previously reported that mice immunized with recombinant Ro52 developed IgG deposits in salivary and lacrimal glands and showed reduced saliva and tear production. Furthermore, passive transfer of sera from Ro52-immunized mice rapidly induced glandular dysfunction without immune cell infiltration in recipient mice. METHODS: To identify mechanisms driving antibody-mediated salivary gland dysfunction, hyperimmune rabbit antiserum to mouse Ro52 was passively transferred into NZM2758 female mice, pretreated with alum adjuvant. Alum-pretreated mice given hyperimmune rabbit antiserum to maltose-binding protein served as controls. Antibody deposition and its distribution in the salivary glands were studied by immunofluorescence staining for rabbit IgG, nerve fibers, and endothelial cells. The nCounter inflammation panel was used to determine differentially expressed genes in the salivary gland. RESULTS: Rabbit IgG deposits were detected in salivary glands of anti-Ro52 immune sera recipients. The rabbit IgG was present on the endothelial cells in small blood vessels, and it did not co-localize with nerve fibers. Ingenuity pathway analysis of the gene expression dataset predicted the canonical vascular endothelial growth factor (VEGF) pathway as the most activated and Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) as the most inhibited pathway in the salivary glands of anti-Ro52 sera recipients. CONCLUSION: Our study suggests that autoantibody deposition on salivary gland endothelial cells might play a critical role in the pathogenesis of Sjögren's Disease.

Systemic immune response to vimentin and granuloma formation in a model of pulmonary sarcoidosis.

A characteristic feature of sarcoidosis is a dysregulated immune response to persistent stimuli, often leading to the formation of non-necrotizing granulomas in various organs. Although genetic susceptibility is an essential factor in disease development, the etiology of sarcoidosis is not fully understood. Specifically, whether autoimmunity contributes to the initiation or progression of the disease is uncertain. In this study, we investigated systemic autoimmunity to vimentin in sarcoidosis. IgG antibodies to human vimentin were measured in sera from sarcoidosis patients and healthy controls. Mice immunized with recombinant murine vimentin were challenged intravenously with vimentin-coated beads to mimic pulmonary sarcoidosis. Lungs from treated mice were studied for cellular infiltration, granuloma formation, and gene expression. Immune cells in the bronchoalveolar lavage fluid were evaluated by flow cytometry. Compared to healthy controls, sarcoidosis patients had a higher frequency and levels of circulating anti-vimentin IgG. Vimentin-immunized mice developed lung granulomas following intravenous challenge with vimentin-coated beads. These sarcoidosis-like granulomas showed the presence of Langhans and foreign body multinucleated giant cells, CD4 T cells, and a heterogeneous collection of MHC II positive and arginase 1-expressing macrophages. The lungs showed upregulated pro-inflammatory gene expression, including Ifng, Il17, and Tnfa, reflecting TH1/TH17 responses typical of sarcoidosis. In addition, genes in the TH2 canonical pathway were also upregulated, congruent with increased numbers of ILC2 in the bronchoalveolar lavage. Overall, these results further validate vimentin as an autoantigen in sarcoidosis and provide evidence for an anti-vimentin immune response in disease pathogenesis. Our study also highlights the possible role of ILC2-driven TH2-like responses in the formation of lung granulomas in sarcoidosis.

Immune Response to Enterococcus gallinarum in Lupus Patients Is Associated With a Subset of Lupus-Associated Autoantibodies.

Interactions between gut microbes and the immune system influence autoimmune disorders like systemic lupus erythematosus (SLE). Recently, Enterococcus gallinarum, a gram-positive commensal gut bacterium, was implicated as a candidate pathobiont in SLE. The present study was undertaken to evaluate the influence of E. gallinarum exposure on clinical parameters of SLE. Since circulating IgG antibodies to whole bacteria have been established as a surrogate marker for bacterial exposure, anti-E. gallinarum IgG antibodies were measured in banked serum samples from SLE patients and healthy controls in the Oklahoma Cohort for Rheumatic Diseases. The associations between anti-E. gallinarum antibody titers and clinical indicators of lupus were studied. Antibodies to human RNA were studied in a subset of patients. Our results show that sera from both patients and healthy controls had IgG and IgA antibodies reactive with E. gallinarum. The antibody titers between the two groups were not different. However, SLE patients with Ribosomal P autoantibodies had higher anti-E. gallinarum IgG titers compared to healthy controls. In addition to anti-Ribosomal P, higher anti-E. gallinarum titers were also significantly associated with the presence of anti-dsDNA and anti-Sm autoantibodies. In the subset of patients with anti-Ribosomal P and anti-dsDNA, the anti-E. gallinarum titers correlated significantly with antibodies to human RNA. Our data show that both healthy individuals and SLE patients were sero-reactive to E. gallinarum. In SLE patients, the immune response to E. gallinarum was associated with antibody response to a specific subset of lupus autoantigens. These findings provide additional evidence that E. gallinarum may be a pathobiont for SLE in susceptible individuals.

Single Cell Transcriptomics Implicate Novel Monocyte and T Cell Immune Dysregulation in Sarcoidosis.

Sarcoidosis is a systemic inflammatory disease characterized by infiltration of immune cells into granulomas. Previous gene expression studies using heterogeneous cell mixtures lack insight into cell-type-specific immune dysregulation. We performed the first single-cell RNA-sequencing study of sarcoidosis in peripheral immune cells in 48 patients and controls. Following unbiased clustering, differentially expressed genes were identified for 18 cell types and bioinformatically assessed for function and pathway enrichment. Our results reveal persistent activation of circulating classical monocytes with subsequent upregulation of trafficking molecules. Specifically, classical monocytes upregulated distinct markers of activation including adhesion molecules, pattern recognition receptors, and chemokine receptors, as well as enrichment of immunoregulatory pathways HMGB1, mTOR, and ephrin receptor signaling. Predictive modeling implicated TGFß and mTOR signaling as drivers of persistent monocyte activation. Additionally, sarcoidosis T cell subsets displayed patterns of dysregulation. CD4 naïve T cells were enriched for markers of apoptosis and Th17/Treg differentiation, while effector T cells showed enrichment of anergy-related pathways. Differentially expressed genes in regulatory T cells suggested dysfunctional p53, cell death, and TNFR2 signaling. Using more sensitive technology and more precise units of measure, we identify cell-type specific, novel inflammatory and regulatory pathways. Based on our findings, we suggest a novel model involving four convergent arms of dysregulation: persistent hyperactivation of innate and adaptive immunity via classical monocytes and CD4 naïve T cells, regulatory T cell dysfunction, and effector T cell anergy. We further our understanding of the immunopathology of sarcoidosis and point to novel therapeutic targets.

The NZB/W F1 mouse model for Sjögren's syndrome: A historical perspective and lessons learned.

Sjögren's syndrome (SS) is a chronic rheumatic autoimmune disorder affecting multiple organ systems. The clinical findings in SS patients show considerable heterogeneity and overlap with other autoimmune diseases. In addition, the autoimmune response in SS initiates several years before the appearance of clinical symptoms. Thus, understanding the pathogenic mechanisms involved in the disease process have been a challenge. Several animal model systems of SS-like disease have been developed to overcome these issues. The New Zealand Black (NZB) x New Zealand White (NZW) F1 (NZB/W F1) mouse represents the first spontaneous mouse model of SS. In this review, we provide a historical perspective and detailed description of this mouse model focusing on exocrine gland histopathology, autoantibody populations, and glandular dysfunction. Considering that NZB/W F1 mice also develop a systemic lupus erythematosus (SLE)-like disease, this mouse model mimics the clinical presentation of polyautoimmunity seen in a sizable subset of SS patients. It is plausible that such patients will require distinct therapeutic interventions necessary to treat both SLE and SS. Therefore, the NZB/W F1 mouse is a powerful tool to decipher pathogenic mechanisms involved in SS related polyautoimmunity and develop appropriate therapeutic strategies.

Protocols for Experimental Sjögren's Syndrome.

Sjögren's syndrome (SS) is a systemic autoimmune disease affecting multiple organ systems. Salivary and lacrimal gland involvement cause dry mouth and dry eye and are the most common clinical presentations of the disease. Patients with SS also have autoantibodies targeting multiple nuclear and cytoplasmic antigens. Innate immune activation plays a critical role in SS pathogenesis. This article describes the activation of specific innate immune pathways in mice to study SS salivary gland manifestations. Methodologies for evaluating salivary gland inflammation and salivary function are described. This article also describes protocols for in-house assays to measure autoantibody titers in serum. © 2020 Wiley Periodicals LLC Basic Protocol 1: Acceleration of Sjögren's syndrome by activating the toll-like receptor 3 pathway Basic Protocol 2: Induction of Sjögren's syndrome by activating the stimulator of interferon genes pathway Alternate Protocol: Acceleration of Sjögren's syndrome by the administration of Freund's incomplete adjuvant Support Protocol 1: Evaluating salivary gland function Support Protocol 2: Evaluating salivary gland inflammation Support Protocol 3: Measuring autoantibody titers by indirect immunofluorescence.

Pulmonary Involvement in a Mouse Model of Sjögren's Syndrome Induced by STING Activation.

Sjögren's Syndrome (SS), a chronic autoimmune disorder affecting multiple organ systems, is characterized by an elevated type I interferon (IFN) response. Activation of Stimulator of Interferon Genes (STING) protein induces type I IFN and in mice, several features of SS, including anti-nuclear antibodies, sialadenitis, and salivary gland dysfunction. Since lung involvement occurs in one-fifth of SS patients, we investigated whether systemic activation of STING also leads to lung inflammation. Lungs from female C57BL/6 mice injected with the STING agonist 5, 6-Dimethylxanthenone-4-acetic acid (DMXAA), were evaluated for acute and chronic inflammatory responses. Within 4h of DMXAA injection, the expression of Ifnb1, Il6, Tnf, Ifng, and Mx1 was significantly upregulated. At 1 and 2 months post-treatment, lungs showed lymphocytic infiltration in the peri-bronchial regions. The lungs from DMXAA treated mice showed an increased expression of multiple chemokines and an increase in lymphatic endothelial cells. Despite STING expression in bronchial epithelium and cells lining the alveolar wall, bone marrow chimeras between STING knockout and wild type mice showed that STING expression in hematopoietic cells was critical for lung inflammation. Our results suggest that activation of the STING pathway might be involved in SS patients with concomitant salivary gland and lung disease.

Hyperglycemia and Salivary Gland Dysfunction in the Non-obese Diabetic Mouse: Caveats for Preclinical Studies in Sjögren's Syndrome.

The Non-obese Diabetic (NOD) mouse model for type I diabetes also develops some features of Sjögren's syndrome (SS). Since the source of the mice and the environment exert a strong influence on diabetes, this study investigated SS development in NOD mice obtained from two vendors. Female NOD mice from The Jackson Laboratory (JAX) and Taconic Biosciences were monitored for blood glucose and pilocarpine-induced salivation. The gut microbiome was analyzed by 16S rRNA sequencing of stool DNA. At euthanasia, serum cytokines and sialoadenitis severity were evaluated. The onset of diabetes was significantly accelerated in JAX mice compared to Taconic mice. Although the gut microbiome between the two groups was distinct, both groups developed sialoadenitis. There was no correlation between the severity of sialoadenitis and reduced saliva production. Instead, salivary gland dysfunction was associated with hyperglycemia and elevation of serum IL1ß, IL16, and CXCL13. Our data suggest that inflammatory pathways linked with hyperglycemia are confounding factors for salivary gland dysfunction in female NOD mice, and might not be representative of the mechanisms operative in SS patients. Considering that NOD mice have been used to test numerous experimental therapies for SS, caution needs to be exerted before advancing these therapeutics for human trials.

IRF4-dependent dendritic cells regulate CD8+ T-cell differentiation and memory responses in influenza infection.

Acute respiratory disease caused by influenza viruses is imperfectly mitigated by annual vaccination to select strains. Development of vaccines that elicit lung-resident memory CD8+ T cells (TRM) would offer more universal protection to seasonal and emerging pandemic viruses. Understanding how lung-resident dendritic cells (DCs) regulate TRM differentiation would be an important step in this process. Here, we used CD11c-cre-Irf4f/f (KO) mice, which lack lung-resident IRF4-dependent CD11b+CD24hi DCs and show IRF4 deficiency in other lung cDC subsets, to determine if IRF4-expressing DCs regulate CD8+ memory precursor cells and TRM during influenza A virus (IAV) infection. KO mice showed defective CD8+ T-cell memory, stemming from a deficit of T regulatory cells and memory precursor cells with decreased Foxo1 expression. Transfer of wild-type CD11b+CD24hi DCs into KO mice restored CD8+ memory precursor cell numbers to wild-type levels. KO mice recovered from a primary infection harbored reduced numbers of CD8+ TRM and showed deficient expansion of IFNγ+CD8+ T cells and increased lung pathology upon challenge with heterosubtypic IAV. Thus, vaccination strategies that harness the function of IRF4-dependent DCs could promote the differentiation of CD8+ TRM during IAV infection.

Antibodies to periodontogenic bacteria are associated with higher disease activity in lupus patients.

OBJECTIVES: Microbial infections and mucosal dysbiosis influence morbidity in patients with systemic lupus erythematosus (SLE). In the oral cavity, periodontal bacteria and subgingival plaque dysbiosis provide persistent inflammatory stimuli at the mucosal surface. This study was undertaken to evaluate whether exposure to periodontal bacteria influences disease parameters in SLE patients. METHODS: Circulating antibodies to specific periodontal bacteria have been used as surrogate markers to determine an ongoing bacterial burden, or as indicators of past exposure to the bacteria. Banked serum samples from SLE patients in the Oklahoma Lupus Cohort were used to measure antibody titres against periodontal pathogens (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Treponema denticola) and commensals (Capnocytophaga ochracea, and Streptococcus gordonii) by ELISA. Correlations between anti-bacterial antibodies and different clinicalparameters of SLE including, autoantibodies (anti-dsDNA, anti-SmRNP, anti-SSA/Ro and anti-SSB/La), complement, and disease activity (SLEDAI and BILAG) were studied. RESULTS: SLE patients had varying amounts of antibodies to different oral bacteria. The antibody titres against A. actinomycetemcomitans, P. gingivalis, T. denticola, and C. ochracea were higher in patients positive for anti-dsDNA antibodies, and they showed significant correlations with anti-dsDNA titres and reduced levels of complement. Among the periodontal pathogens, only antibodies to A. actinomycetemcomitans were associated with higher disease activity. CONCLUSIONS: Our results suggest that exposure to specific pathogenic periodontal bacteria influences disease activity in SLE patients. These findings provide a rationale for assessing and improving periodontal health in SLE patients, as an adjunct to lupus therapies.

Immune Response Targeting Sjögren's Syndrome Antigen Ro52 Suppresses Tear Production in Female Mice.

Autoantibodies reactive against Ro52 are present in 70% of Sjögren's syndrome patients and are associated with higher disease severity. However, their role in causing aqueous deficient dry eye, a major cause for morbidity in Sjögren's syndrome, is unclear. To investigate whether immune responses targeting Ro52 contribute towards the dry eye, male and female NZM2758 mice were immunized with recombinant Ro52. Tear production was measured by the phenol red thread test. Sera were analyzed for anti-Ro52 levels by immunoprecipitation. Lacrimal glands were evaluated for inflammatory foci and IgG deposits. Our results showed that, although all mice generated anti-Ro52 antibodies, only females developed a significant drop in tear production. None of the mice developed severe lacrimal gland inflammation, and female mice with anti-Ro52 showed higher levels of IgG deposits within their glands. Passive transfer of anti-Ro52 sera caused reduced tear production in female mice, but not in males. This study demonstrates for the first time that immune responses initiated by Ro52 induce aqueous dry eye, and this may be driven by anti-Ro52 antibodies. Furthermore, the sexual dimorphism in glandular dysfunction suggests that the lacrimal glands in females are more susceptible to autoantibody-mediated injury.

Immune response against the coiled coil domain of Sjögren's syndrome associated autoantigen Ro52 induces salivary gland dysfunction.

OBJECTIVES: The structural domains of Ro52, termed the RING, B-box, coiled coil (CC) and B30.2/SPRY are targets of anti-Ro52 in multiple autoimmune disorders. In Sjögren's syndrome patients, the presence of anti-Ro52 is associated with higher disease severity, and in mice, they induce salivary gland hypofunction. This study was undertaken to investigate whether immune responses against different domains of Ro52, influences salivary gland disease in mice. METHODS: Female NZM2758 mice were immunised with Ro52 domains expressed as recombinant fusion proteins with maltose binding protein (MBP) [MBP-RING-B-box, MBP-CC, MBP-CC(ΔC19), MBP-B30.2/SPRY]. Sera from immunised mice were studied for IgG antibodies to Ro52 by immunoprecipitation, and to salivary gland cells by immunofluorescence. Pilocarpine-induced saliva production was measured to evaluate salivary gland function. Submandibular glands were investigated by histopathology for inflammation and by immune-histochemistry for IgG deposition. RESULTS: Mice immunised with different Ro52-domains had comparable reactivity to Ro52 and to salivary gland cells. However, only mice immunised with the CC domain and its C-terminal truncated version CC(ΔC19) showed a significant drop in saliva production. None of the mice developed severe salivary gland inflammation. The salivary gland hypofunction significantly correlated with increased intra-lobar IgG deposits in the submandibular salivary glands. CONCLUSIONS: Our data demonstrate that epitope specificity of anti-Ro52 antibodies plays a critical role in the induction of glandular dysfunction. Clearly, screening Sjögren's syndrome patients for relative levels of Ro52 domain specific antibodies will be more informative for associating anti-Ro52 with clinical measures of the disorder.