Signalling pathways identified in salivary glands from primary Sjögren's syndrome patients reveal enhanced adipose tissue development.

A characteristic feature of primary Sjögren's syndrome (pSS) is the destruction of salivary and lacrimal glands mediated by mononuclear cell infiltration. Adipocytes can also occupy a large portion of the salivary gland (SG) tissue area, although little is known about their significance in pSS. We have previously investigated adipose tissue infiltration in SG biopsies from pSS patients and non-SS sicca controls. Our findings indicated the distinct incidence of adipose tissue replacement in pSS patients, where adipocytes were detected in interleukin (IL) 6 rich regions. We now aimed to examine the development of adipocytes in the SG microenvironment, and delineate their possible involvement in immune reactions. A microarray analysis was performed on SG from 6 pSS patients and 6 non-SS controls, where the expression levels of genes involved in adipose tissue development, inflammatory responses, and lymphoma development were assessed. Real-time PCR was carried out on SG from 14 pSS patients and 15 non-SS controls to account for IL6, IL10, and IL17 mRNA levels. Immunohistochemical staining of frozen SG tissue using IL17 was also conducted. Our results indicate signalling pathways identified in SG of pSS patients displayed genes leading to prominent adipose tissue development and reduced mitochondrial fatty acid beta-oxidation (ARID5B, OXCT1, BDH1, SOX8, HMGCS2, FTO, ECHS1, PCCA, ACADL and ACADVL), inflammatory responses (IL1R1, IL7R, IL10RA, IL15, IL18RAP, CCL2, CCL5, CCL22, CXCR6, CD14, and CD48), and lymphoma development via JAK-STAT signalling (STAT2, TYK2, EBI3, FAS, TNFRSF1B, MAP3K8, HMOX1, LTB, TNF, STAT1, and BAK1). Genes involved in interferon production and signalling were also detected (IRF1, IRF9, and IRF7), in addition to IL6, IL10, and IL17. Higher mRNA levels of IL6, IL17 and IL10 were observed in the SG of pSS patients compared to controls. Moreover, IL17 positive cells were detected mostly interstitially in the SG and around adipocytes, also within the focal infiltrates. In conclusion, adipocyte development seems to be more prominent in the SG of pSS patients, where adipose tissue replacement is also evident. Whether this is due to disease progression, or the repair process, remains to be investigated. Detection of IL17 positive adipocytes in the target organ suggests their involvement in immune reactions.

Adipose tissue is prominent in salivary glands of Sjögren's syndrome patients and appears to influence the microenvironment in these organs.

A minor salivary gland (SG) biopsy with focal lymphocytic sialadenitis and a focus score of ≥1 is today's widely accepted pathological finding confirming the SG component of Sjögren's syndrome (SS). Adipocytes can occupy a large percentage of the SG area although little is known about their significance in SS lesions. This study aimed to characterise adipose tissue infiltration in labial SG biopsies from 27 SS patients and 28 non-SS sicca controls. Biopsies were evaluated by one oral pathologist and assessed for focus score, acinar atrophy, fatty replacement and non-specific chronic inflammation. Moreover, to explore the SG microenvironment, immunohistochemical staining of paraffin-embedded SG tissue was performed using interleukin-6 (IL-6). The fatty replacement was evident in all SS patients possessing autoantibodies (Ro/SSA and/or La/SSB) as well as a positive SG biopsy (focus score ≥1). Additionally, 62% of SS patients having autoantibodies but a negative biopsy showed fatty infiltration (FI) while non-SS controls demonstrated fatty replacement in only 32% of the cases. Overall, the SS group (mean age 53.0 years) had a significantly higher incidence (p value 0.005) of FI than the non-SS controls (mean age 54.8 years). Interestingly, adipocytes were located in IL-6 rich areas, and IL-6 positive adipocytes were detected. As fat deposition seems to be more recurrent in SGs affected by SS, we propose the assessment of adipose tissue replacement as a helpful tool for diagnostic evaluation in SS. Detection of IL-6 positive adipocytes suggests their involvement in immune reactions. Still, functional studies are needed to investigate the SG microenvironment further.

Salivary glands of primary Sjögren's syndrome patients express factors vital for plasma cell survival.

INTRODUCTION: The presence of circulating Ro/SSA and La/SSB autoantibodies has become an important marker in the classification criteria for primary Sjögren's syndrome (pSS). Plasma cells producing these autoantibodies are mainly high affinity plasma cells originating from germinal centre reactions. When exposed to the right microenvironment these autoimmune plasma cells become long-lived and resistant to immunosuppressive treatment. Since autoimmune plasma cells have been detected in the salivary glands of SS patients, we wanted to investigate if the glandular microenvironment is suitable for plasma cell survival and if glandular residing plasma cells are the long-lived plasma cell subset. METHODS: Single, double and triple immunohistochemistry as well as immunofluorescence staining was performed on minor salivary gland tissue retrieved from pSS, chronically inflamed and normal subjects. RESULTS: We detected significant numbers of CD138+, non-proliferating, Bcl-2 expressing plasma cells in the salivary glands of pSS patients with high focus score (FS). Furthermore, we demonstrated that CXCL12 and interleukin (IL)-6 survival factors were highly expressed in pSS salivary gland epithelium and by focal mononuclear infiltrating cells. Notably, adipocytes when present in the salivary gland tissue were an important source of CXCL12. We clearly demonstrate that plasma cells are localised in close proximity to CXCL12 and IL-6 expressing cells and thus that the environment of salivary glands with high FS provide factors vital for plasma cell survival. CONCLUSIONS: Plasma cells residing in the salivary glands of pSS patients with high FS showed phenotypic characteristics of the long-lived plasma cell subtype. Furthermore, the pSS salivary gland microenvironment provided niches rich in factors vital for plasma cell survival.

Levels of plasmacytoid dendritic cells and type-2 myeloid dendritic cells are reduced in peripheral blood of patients with primary Sjogren's syndrome.

OBJECTIVE: Sjögren's syndrome (SS) is a lymphoproliferative autoimmune disease, characterised by dryness of the mouth and eyes. Dendritic cells (DC) are potent antigen-presenting cells crucial for initiating and maintaining primary immune responses. This study quantified interferon-producing plasmacytoid DC (pDC) and two myeloid DC subsets (mDC1 and mDC2) in peripheral blood (PB) from primary SS (pSS) patients and healthy controls. METHODS: Blood samples from 31 pSS patients and 28 gender and age-matched healthy controls were analysed by flow cytometry using the Miltenyi Blood DC enumeration kit. The presence of pDC in salivary glands (SG) from pSS patients was analysed by immunohistochemistry. RESULTS: Patients with pSS had significantly less pDC and mDC2 in PB compared with healthy controls. Moreover, pDC are present in SG from patients with pSS. CONCLUSION: Patients with pSS have alterations among DC populations in PB, and pDC are present in the SG, suggesting a potential role of these cells in SS.

Nuclear BCL10 in primary Sjögren's syndrome.

BACKGROUND: The events following triggering of antigen receptors and subsequent activation of the transcription factor nuclear factor kappa B (NFkappaB) need to be carefully controlled to prevent abnormal immune responses. BCL10 links the antigen receptor to NFkappaB. The aim of this study was to determine the expression pattern of BCL10 and NFkappaB in minor salivary gland infiltrates of patients with primary Sjögren's syndrome (pSS). METHODS: Minor salivary glands from patients with primary SS (n = 17) and sicca controls (n = 4) were evaluated by single and double immunohistochemistry and immunofluorescence for confocal microscopy. BCL10 and NFkappaB-p65 expression were evaluated in the infiltrating lymphocytes. Ectopic germinal centers (GCs) were investigated by CD21. Tonsil, lymph node and lymphoma tissue were used as positive controls. RESULTS: BCL10 nuclear positive cells were observed in focal lymphocytic infiltrates in the investigated minor salivary glands and were not restricted to patients with ectopic GCs. By double-staining, some of the BCL10 nuclear positive cells were identified as B cells. There was, however, no constitutive activation of NFkappaB as depicted by the exclusive cytoplasmic expression of p65 in the infiltrating lymphocytes in the pSS. CONCLUSION: Nuclear expression of BCL10 in infiltrating lymphocytes was a common occurrence in pSS minor salivary glands indicating it as a possible marker of autoimmune induced chronic inflammation. There was, however, no constitutive activation of NFkappaB.

Expression of p53, cyclin D1 and Ki-67 in pre-malignant and malignant oral lesions: association with clinicopathological parameters.

In this study, a possible association was examined between the immunoexpressions of p53, cyclin D1, Ki-67 and tobacco exposure and the risk of oral cancer (OC) in premalignant and malignant formalin-fixed, paraffin-embedded oral mucosal tissue specimens from patients from Yemen (n=24, all were pre-malignant) and India (n=16, 11 were OCs). Overexpressions of p53, cyclin D1 and Ki-67 were found in 100%, 45.5% and 80% of the OCs, compared to 65.5%, 82.8% and 85.1% of the pre-malignant lesions, respectively. In the pre-malignant lesions, a statistically significant correlation was found between histopathological grading and expressions of cyclin D1 (p = 0.001) and Ki-67 (p = 0.03), and between anatomical site and expression of Ki-67 (p = 0.01). Coexpressions of the three proteins in the cases examined was found to correlate significantly to each other (cyclin D1: p53, r = 0.48, p = 0.002; p53: Ki-67, r = 0.41, p = 0.008) except for cyclin D1: Ki-67. These findings suggest that the expressions of p53, cyclin D1 and Ki-67 might contribute to OC susceptibility in oral mucosal lesions examined from Yemen and India. The importance of the three proteins examined as biomarkers in OC and pre-malignant lesions deserves particular attention because it might offer further understanding of the development of these lesions, particularly in populations heavily exposed to tobacco habits. Abnormalities of both cyclin D1 and Ki-67 might play an important role in the development of oral pre-malignant lesions and warrant further studies. Larger studies are, therefore, necessary in the two countries to examine the role of these biomarkers in OCs and premalignant oral mucosal lesions.