Abnormal distribution of AQP4 in minor salivary glands of primary Sjögren’s syndrome patients

A decreased saliva production occurs in primary Sjögren’s syndrome (pSS), an autoimmune disease characterized by oral and ocular dryness due to dysfunction of the lacrimal and salivary glands (SGs). Since water movement is involved in saliva secretion, the expression, localization, and function of the water channels aquaporins (AQPs) have been extensively studied in SGs. To date, the presence of AQP4 remains controversial and ambiguous in human SGs. We investigated by immunohistochemistry, high-resolution confocal microscopy and quantitative image analysis, Western blot and real-time RT-PCR, the presence of the AQP4 gene, and the distribution of AQP4 protein in healthy controls and pSS SG biopsies. Through the immunohistochemical analysis, we demonstrated that AQP4 presence is confined to the basal region of acini, to the lateral and apical membrane of intercalated and striated ducts in both control and pSS glands. The most striking observation was the discovery of AQP4 localization in myoepithelial cells (MECs) that surround acini lobules and intercalated ducts, and the demonstration of AQP4-downregulated immunoreactivity in pSS MECs. Our studies suggest that the capacity for water flow across the membrane of MECs may be altered in pSS, identifying AQP4 as a promising new therapeutic agent to treat xerostomia.     

Predicting lymphoma development in patients with Sjögren’s syndrome

ABSTRACT

Introduction: The issue of predicting lymphoma in primary Sjögren’s syndrome (pSS) starts from its clinical and biologic essence, i.e., an autoimmune exocrinopathy with sicca syndrome, inflammation and lymphoproliferation of MALT (mucosa-associated lymphoid tissue) in exocrine glands.

Areas covered: The two major predictors to be firstly focused are persistent salivary gland (SG) swelling and cryoglobulinemic vasculitis with related features as purpura and low C4, or the sole serum cryoglobulinemia repeatedly detected. They are pathogenetically linked and reflect a heavier MALT involvement by histopathology, with the expansion of peculiar rheumatoid factor (RF)-positive clones/idiotypes. Other predictors include lymphadenopathy, splenomegaly, neutropenia, lymphopenia, serum beta2-microglobulin, monoclonal immunoglobulins, light chains, and RF. Composite indexes/scores may also predict lymphoma.

Expert opinion: Prediction at baseline needs amelioration, and must be repeated in the follow-up. Careful clinical characterization, with harmonization and stratification of large cohorts, is a relevant preliminary step. Validated and new biomarkers are needed in biologic fluids and tissues. SG echography with automatic scoring could represent a future imaging biomarker, still lacking. Scoring MALT involvement in pSS, as an additional tool to evaluate disease activity and possibly to predict lymphoma, is welcomed. All these efforts are now ongoing within the HarmonicSS project and in other research initiatives in pSS.     

Type I IFN signature in primary Sjögren’s syndrome patients

Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease characterized by lymphocytic infiltrates in salivary and lacrimal glands. Clinical manifestations range from ocular and oral dryness to vasculitis and severe fatigue. pSS is a disease with heterogeneous symptoms and a variable response to the available treatment. Recently, a key role for Interferon (IFN) type I has been implicated in the pathogenesis of pSS. As type I IFN consists of 17 different subtypes, it cannot be easily assessed using a conventional ELISA. Therefore the expression of type I IFN inducible genes – the so-called type I IFN signature – is assessed in salivary gland tissue and blood from patients as a readout for type I IFN activity. In this review we discuss the potential of type I IFN as a novel biomarker for disease activity, subclassification of patients, prediction of therapy response and most importantly as a target for therapeutic intervention.     

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.     

Classification criteria and treatment modalities in primary Sjögren’s syndrome

Primary Sjögren’s syndrome is a systemic autoimmune disease, characterized by a lymphocytic exocrinopathy. Oral and ocular dryness, asthenia and pain represent hallmarks of the disease. Systemic manifestations concern a third of patients, including lymphoma in 5% of the patients. The American European Consensus Group classification criteria have been used in current practice and clinical trials since 2002. New classification criteria were recently proposed by the American Congress of Rheumatology. A group of international experts are currently working to reach a new consensus between the American European Consensus Group classification criteria and the American Congress of Rheumatology proposal for disease classification. In addition, international consensus disease activity scores were recently established. Regarding treatment modalities, symptomatic treatments remain the cornerstone of therapy in pSS, but new biologic treatments are currently evaluated.     

Selective down-regulation of aquaporin-1 in salivary glands in primary Sjögren's syndrome

Salivary and lacrimal gland secretions are reduced in primary Sj?gren's syndrome (pSS). Aquaporins (AQPs) are involved in transmembrane water transport, and different isoforms show specific cellular and subcellular distributions in salivary and lacrimal glands. Changes in expression of AQP molecules have therefore been suggested to contribute to the glandular dysfunction in pSS. AQP-5 is present in the apical membrane of acinar cells, where it mediates fluid outflow; however, we have recently shown that its expression is not altered in pSS. We therefore studied whether expression of other isoforms of AQP would be altered in pSS. Using high-resolution confocal microscopy, we determined the distribution of AQP-1 and AQP-3 in labial salivary gland biopsies from 11 patients with pSS and 9 healthy controls. AQP-1 is present in myoepithelial cells surrounding acini, and its expression in these cells was decreased by 38% in pSS glands. By contrast, expression of AQP-1 in endothelial cells of nonfenestrated capillaries was not altered in pSS. AQP-3 was expressed in the basolateral membrane of acinar epithelial cells, and its expression was not altered in disease. We therefore conclude that AQP-1 expression in myoepithelial cells is selectively down-regulated in pSS and that myoepithelial cell dysfunction may play a crucial role in the pathology of this disease.     

Detection of ABCA7-positive cells in salivary glands from patients with Sjögren's syndrome

ABCA7 is a member of the subfamily A of adenosine triphosphate‐binding cassette (ABC) proteins, and highly homologous to ABCA1, which mediates the release of cellular cholesterol and phospholipid to form high‐density lipoprotein. ABCA1 and ABCA7 contain two large extracellular domains, ECD1 and 2, which are thought to be important for their functions. Interestingly, part of ECD1 of ABCA7 is deposited as an autoantigen of Sjögren's syndrome. To determine the relationship between ABCA7 and Sjögren's syndrome, an immunohistochemical study was conducted with salivary gland biopsy samples from patients with Sjögren's syndrome. ECD1 of human ABCA7 (amino acids 45–549) was expressed in Escherichia coli as a protein fused with glutathione‐S‐transferase and a monoclonal antibody, KM3095, was generated. KM3095‐immunoreactive cells were observed in salivary glands from 10 of 18 patients with Sjögren's syndrome. Immunostaining of serial sections with the plasma cell marker NCL‐PC indicated that most of the plasma cells infiltrating salivary glands of patients with Sjögren's syndrome were KM3095‐immunoreactive. Although the pathological or biological significance is not clear, it will be intriguing to further examine the relationship between ABCA7 and Sjögren's syndrome.     

Activation of Toll-like receptor 7 signaling in labial salivary glands of primary Sjögren's syndrome patients

The aim of this study was to determine the expressions of Toll-like receptors (TLRs) 7–9 and type I interferon (IFN) signal in labial salivary glands (LSGs) and cultured salivary gland epithelial cells (SGECs) from primary Sjögren's syndrome (pSS) patients. We performed an immunohistochemistry analysis of LSGs from 11 patients with pSS as defined by American–European Consensus Group classification criteria and five healthy subjects. The pSS patients' SGECs were analyzed by immunofluorescence and western blotting. IFN-α expression was examined by immunosorbent assay and flow cytometry. Mononuclear cells (MNCs) from pSS patients' LSGs showed TLR-7-dominant expression. B cells, plasma cells and plasmacytoid dendritic cells (pDCs) co-expressed with TLR-7. Myeloid differentiation primary response gene 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF6) and interferon regulatory factor 7 (IRF7) co-expressed with the pDC marker CD303 in LSGs. Ducts from pSS patients dominantly expressed TLR-7, and TLR-7 in the ducts co-expressed with MyD88, TRAF6 and IRF7. Type I IFNs including IFN-α and IFN-β were detected in MNCs and ducts in pSS patients' LSGs. Increased TRAF6 expression and the nuclear translocation of IRF7 in SGECs were detected by immunofluorescence following loxoribine (a TLR-7 ligand) stimulation despite IFN-β pretreatment. Western blotting showed increased TRAF6 expression in SGECs following IFN-β and loxoribine stimulation. Although no increase in IFN-α was detected in supernatant from stimulated SGECs, the IFN-α in supernatant from stimulated peripheral blood pDCs from pSS patients was significantly increased. Our findings suggest that TLR-7 is dominantly expressed in both MNCs and ducts with downstream signals for type I IFNs, indicating that TLR7-dominant innate immunity is related to the development of sialadenitis in pSS.     

Low number of memory B cells in the salivary glands of patients with primary Sjögren's syndrome

We have previously shown that patients with primary Sjögren's Syndrome (pSS) show a significant reduction of autoantigen specific CD27⁺ memory B cells and an abnormally elevated level of autoantibody producing plasma cells in peripheral blood (PB) compared to controls. Because both memory B cells and plasma cells have been detected in salivary glands (SG) of pSS patients, we aimed to study the B cell pattern in SG biopsies. Double immunohistochemical staining of CD20 and CD27 was carried out on paraffin-embedded SG tissue from 10 pSS patients to distinguish CD20⁺/CD27⁺ memory B cells, and identify the CD20⁺ glandular B cell zones (BCZ). Given that plasma blasts and plasma cells are CD27⁺⁺ and CD20^? , additional CD138 single staining of serial sections allowed the distinction of CD27⁺⁺/CD138^? plasma blasts located within the BCZ from CD27⁺⁺/CD138⁺ plasma cells that were found mostly on the periphery of the BCZ and also observed interstitially. Both BCZ and the memory B cell populations were then quantified. Contrary to what has been reported earlier through immunoflourescent staining of memory B cells in SG tissue, we have shown that there is a low number of memory B cells located within the glandular BCZ. Plasma blasts and plasma cells, however, were more abundant in the SG. Together our findings suggest that these low numbers of memory B cells in both PB and SG of pSS patients may be the result of activation of these cells into plasma cells at the site of inflammation.     

Cardamonin inhibits pro-inflammatory cytokine production and suppresses NO pathway in PBMCs from patients with primary Sjögren’s syndrome

Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disorder with a complex pathophysiology primarily affecting exocrine glands, leading to compromised secretory function. Recent studies imply that many inflammatory mediators, such as pro-inflammatory cytokines and nitric oxide, are critical in the development and perpetuation of pSS systemic manifestations. In the current study, we aimed to investigate the ex vivo immunomodulatory effect of cardamonin (C₁₆H₁₄O₄), on pro-inflammatory cytokines, TNF-α, IL-6 and inducible nitric oxide synthase (iNOS) expression during pSS. For this purpose, peripheral blood mononuclear cells isolated from pSS patients and healthy controls were cultured with different concentrations of cardamonin. Cytokine levels were measured by ELISA and NO production was assessed using the Griess method. Inducible nitric oxide synthase expression and NF-κB activity were analyzed by immunofluorescence staining. Our results suggest that cardamonin inhibits TNF-α, IL-6 and NO production and downregulates iNOS expression and NF-κB activation. Collectively, our results highlight the ex vivo immunomodulatory effects of cardamonin on pro-inflammatory cytokine production and NO pathway in pSS patients. Therefore, cardamonin is a potential candidate for controlling inflammation during pSS.     

Abnormal distribution of aquaporin-5 water channel protein in salivary glands from Sjögren's syndrome patients

Patients with Sj?gren's syndrome (SS) suffer from deficient secretion of saliva due to an autoimmune destruction of salivary glands, however, glandular dysfunction also occurs without destruction. Based upon its abnormal distribution in SS salivary glands, a potential role for the water channel protein aquaporin-5 (AQP5) is proposed in the pathogenesis of SS. The immunohistochemical distribution of AQP5 was compared in minor salivary gland biopsies obtained from women after informed consent: primary SS (53.2 +/- 14 years old, n = 10), healthy volunteers (46.2 +/- 17 years old, n = 10), patients with sarcoidosis (37 and 48 years old), and patients with non-specific sialoadenitis (54 and 61 years old). Biopsies from normal subjects revealed AQP5 primarily at the apical membrane of the salivary gland acinus. In contrast, biopsies from SS patients revealed AQP5 primarily at the basal membranes of the acinus. The AQP5 distribution in biopsies from patients with other dry mouth disorders, such as non-specific sialoadenitis or sarcoidosis, was similar to biopsies from control subjects. Computer-assisted microscopy was performed to quantitatively evaluate AQP5 distribution in the immunoreactive acini of both SS and control subjects. Biopsies from SS patients had higher labeling indices (percentage of acinus area immunoreactive for AQP5) at the basal membrane when compared with biopsies from control subjects. In contrast, biopsies of SS patients exhibited lower labeling indices at the apical membrane when compared with biopsies from control subjects. To verify the specificity of the AQP5 antibody, Western blot analysis was performed on membranes from Xenopus oocytes injected with AQP5 cRNA or on membranes from minor salivary glands of control subjects and SS patients. In each case, the immunoblots had a 27 kd band, corresponding to the expected molecular weight of AQP5. Abnormal distribution of AQP5 in salivary gland acini is likely to contribute to the deficiency of fluid secretion, which is a defining feature of Sj?gren's syndrome.     

Involvement of aquaporin 5 in Sjögren’s syndrome

Sjögren’s syndrome (SS) is a chronic autoimmune disease with the pathological hallmark of lymphoplasmacytic infiltration of exocrine glands - more specifically salivary and lacrimal glands - resulting in a diminished production of tears and saliva (sicca syndrome). The pathophysiology underscoring the mechanisms of the sicca symptoms in SS has still yet to be unraveled but recent advances have identified a cardinal role of aquaporin-5 (AQP5) as a key player in saliva secretion as well as salivary gland epithelial cell dysregulation. AQP5 expression and localization are significantly altered in salivary glands from patients and mice models of the disease, shedding light on a putative mechanism accounting for diminished salivary flow.Furthermore, aberrant expression and localization of AQP5 protein partners, such as prolactin-inducible protein and ezrin, may account for altered AQP5 localization in salivary glands from patients suffering from SS and are considered as new players in SS development. This review provides an overview of the role of AQP5 in SS salivary gland epithelial cell dysregulation, focusing on its trafficking and protein-protein interactions.     

Epigenetic alterations in Sjögren’s syndrome patient saliva

Epigenetic mechanisms have been implicated in the pathogenesis of Sjögren’s syndrome (SS). Extensive alterations in DNA methylation have been described in minor salivary gland (MSG) epithelial cells and lymphocytes derived from SS patients compared to sicca controls. In an effort to identify novel potential epigenetic markers that could prove useful in diagnosis and disease monitoring, we explored whether DNA methylation differences can also be detected in saliva from SS patients compared to sicca controls. We performed DNA methylation analysis by methylation-sensitive restriction digestion followed by quantitative real-time polymerase chain reaction of selected genomic loci in saliva samples of 16 SS patients and 10 sicca controls with negative MSG biopsy. We identified reduced DNA methylation of the imprinting control region (ICR) of the H19 locus in SS patient saliva compared to sicca controls. Levels of saliva H19 ICR methylation were negatively correlated with C4 serum complement levels. Consistent with the reduced methylation of the ICR, H19 RNA levels were increased in SS patient peripheral blood mononuclear cells (PBMCs), while no significant change was observed in MSG H19 RNA levels compared to sicca controls. Our findings support that H19 ICR methylation could be a useful molecular epigenetic marker in monitoring patients with SS, highlighting saliva as a valuable biological sample in SS research and clinical practice. The role of H19 in SS pathogenesis remains to be addressed.     

Plummer-Vinson syndrome in primary Sjögren syndrome: a case-based review

This study aimed to describe a patient with Sjögren syndrome who developed Plummer-Vinson syndrome, and to review the literature and describe shared aspects of this rare association. A systematic screening of articles was conducted in PubMed/MEDLINE, LILACS, SciELO, Scopus, Web of Science, and Cochrane, dating 1940 to 2020. All the articles included the association between Sjögren syndrome and Plummer-Vinson syndrome. No language restriction was applied. The following terms were used: “Sjögren syndrome” or “sicca syndrome” and “Plummer-Vinson syndrome” or “Paterson-Kelly syndrome.” We performed our analysis by adding our present case, with a total of 4 cases. Three out of four were female (75%), age varied from 56 to 58 years old. In 2 cases, Sjögren syndrome preceded Plummer-Vinson syndrome diagnosis, and in 1 report, Plummer-Vinson syndrome appeared before Sjögren syndrome. Disease duration varied from 7 to 20 years. In two cases, autoantibodies were available, and antinuclear antibodies and anti-Ro/SS-A were positive in both, and anti-La/SS-B in one of them was associated with anti-dsDNA; however, no data regarding lupus was available in the article. Treatment involved iron supplementation in 3/3. Two out of three received parenteral iron supplementation, and in these two cases, mechanical esophageal dilatation was needless. In the other case, an additional endoscopic esophageal dilatation was necessary to receive the oral iron supplement. All 3 cases had a good outcome. This case illustrates a patient with Sjögren syndrome who developed the rare Plummer-Vinson syndrome. In Sjögren syndrome, the presence of iron-deficiency anemia, dysphagia, and weight loss should alert the physician to search for associated Plummer-Vinson syndrome.

     

Fatty infiltration of the minor salivary glands is a selective feature of aging but not Sjögren’s syndrome

Objective: Determine the presence and assess the extent of fatty infiltration of the minor salivary glands (SG) of primary SS patients (pSS) as compared to those with non-SS sicca (nSS).

Methods: Minor SG biopsy samples from 134 subjects with pSS (n?=?72) or nSS (n?=?62) were imaged. Total area and fatty replacement area for each glandular cross-section (n?=?4–6 cross-sections per subject) were measured using Image J (National Institutes of Health, Bethesda, MD). The observer was blinded to subject classification status. The average area of fatty infiltration calculated per subject was evaluated by logistic regression and general linearized models (GLM) to assess relationships between fatty infiltration and clinical exam results, extent of fibrosis and age.

Results: The average area of fatty infiltration for subjects with pSS (median% (range) 4.97 (0.05–30.2)) was not significantly different from that of those with nSS (3.75 (0.087–41.9). Infiltration severity varied widely, and subjects with fatty replacement greater than 6% were equivalently distributed between pSS and nSS participants (χ² p?=?.50). Age accounted for all apparent relationships between fatty infiltration and fibrosis or reduced saliva flow. The all-inclusive GLM for prediction of pSS versus non-SS classification including fibrosis, age, fatty replacement, and focus score was not significantly different from any desaturated model. In no iteration of the model did fatty replacement exert a significant effect on the capacity to predict pSS classification.

Conclusions: Fatty infiltration is an age-associated phenomenon and not a selective feature of Sjögren’s syndrome. Sicca patients who do not fulfil pSS criteria have similar rates of fatty infiltration of the minor SG.