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Although lymphatic neovascularization may be a key feature of chronic inflammation, it is almost unexplored in primary Sjögren's syndrome (pSS). A recent study revealed a pro-lymphangiogenic function of interleukin (IL)-17, a leading player in pSS pathogenesis. The aims of the study were to investigate lymphangiogenic mediators and lymphatic vasculature in pSS, as well as their possible association with IL-17. Circulating lymphatic endothelial precursor cells (LEPCs) and Th17 cells were enumerated in pSS patients and healthy donors. VEGF-C and IL-17 levels were assessed in paired serum samples. Lymphatic vasculature, VEGF-C/VEGF receptor (VEGFR)-3 and IL-17 were evaluated in pSS minor salivary glands (MSGs) and compared with normal and non-specific chronic sialadenitis (NSCS) MSGs. Circulating LEPCs were expanded in pSS and correlated with circulating Th17 cells, IL-17 and VEGF-C. In pSS MSGs, a newly formed lymphatic capillary network was found within periductal inflammatory infiltrates and the number of interlobular lymphatic vessels was significantly increased compared with normal and NSCS MSGs. Strong VEGF-C expression was detected in pSS ductal epithelial cells and periductal inflammatory cells. Numerous VEGFR-3(+) infiltrating mononuclear cells were exclusively observed in pSS MSGs. VEGFR-3 expression was strongly increased in lymphatic capillaries of pSS MSGs. IL-17(+) inflammatory cells were preferentially observed around lymphatic vessels in pSS MSGs. This study supports the notion that lymphvasculogenesis and lymphangiogenesis are active in pSS, thereby unmasking a novel aspect of disease pathogenesis. In addition, our results suggest another possible pathogenic role of IL-17 in pSS, further supporting its therapeutic targeting in this disease., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Introduction: There is evidence that interferon is involved in the pathogenesis of primary Sjögren's syndrome (pSS). The interferon-inducible IFI16 protein, normally expressed in cell nuclei, may be overexpressed, mislocalized in the cytoplasm and secreted in the extracellular milieu in several autoimmune disorders. This leads to tolerance breaking to this self-protein with consequent development of anti-IFI16 antibodies. The aim of this study was to identify the pathogenic and clinical significance of IFI16 and anti-IFI16 in pSS., Methods: IFI16 and anti-IFI16 were assessed in the serum of 67 pSS patients and over 100 healthy donors by enzyme-linked immunosorbent assay. IFI16 was also evaluated by immunohistochemistry in minor salivary glands of 15 pSS patients and 10 subjects with sicca symptoms but without any clinical, serological or histological features of pSS., Results: pSS patients display higher serum levels of both IFI16 and anti-IFI16 compared to healthy donors. IFI16 concentration was directly correlated with disease duration and focus score and inversely correlated with age at diagnosis. Moreover, IFI16 positivity was associated with concurrent positivity for rheumatoid factor. Interestingly, the direct correlation between IFI16 positivity and focus score was independent of disease duration and age at diagnosis. pSS minor salivary glands display marked expression and cytoplasmic mislocalization of IFI16 by acinar and ductal epithelial cells as well as infiltrating lymphocytes and peri/intralesional endothelium compared to minor salivary glands with normal architecture or nonspecific chronic sialadenitis. Within the mononuclear cell infiltrate, IFI16 expression appears to parallel the distribution of T lymphocytes., Conclusion: Our data suggest that the IFI16 protein may be involved in the pathogenesis of glandular inflammation occurring in pSS.

It has been recently reported that telocytes, a stromal (interstitial) cell subset involved in the control of local tissue homeostasis, are hampered in the target organs of inflammatory/autoimmune disorders. Since no data concerning telocytes in minor salivary glands (MSGs) are currently available, aim of the study was to evaluate telocyte distribution in MSGs with normal architecture, non-specific chronic sialadenitis (NSCS) and primary Sjögren's syndrome (pSS)-focal lymphocytic sialadenitis. Twelve patients with pSS and 16 sicca non-pSS subjects were enrolled in the study. MSGs were evaluated by haematoxylin and eosin staining and immunofluorescence for CD3/CD20 and CD21 to assess focus score, Tarpley biopsy score, T/B cell segregation and germinal center (GC)-like structures. Telocytes were identified by immunoperoxidase-based immunohistochemistry for CD34 and CD34/platelet-derived growth factor receptor α double immunofluorescence. Telocytes were numerous in the stromal compartment of normal MSGs, where their long cytoplasmic processes surrounded vessels and encircled both the excretory ducts and the secretory units. In NSCS, despite the presence of a certain degree of inflammation, telocytes were normally represented. Conversely, telocytes were markedly reduced in MSGs from pSS patients compared to normal and NSCS MSGs. Such a decrease was associated with both worsening of glandular inflammation and progression of ectopic lymphoid neogenesis, periductal telocytes being reduced in the presence of smaller inflammatory foci and completely absent in the presence of GC-like structures. Our findings suggest that a loss of MSG telocytes might have important pathophysiological implications in pSS. The specific pro-inflammatory cytokine milieu of pSS MSGs might be one of the causes of telocyte loss., (© 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Objective: Chronic autoimmune diseases are associated with increased risk of cardiovascular death. Endothelial dysfunction represents the first stage of subclinical atherosclerosis and multiple factors contribute to endothelial injury. Among these, an altered balance between endothelial microparticle (EMP) release and endothelial progenitor cell (EPC) generation promotes endothelial dysfunction. The role of EMPs and EPCs in promoting endothelial damage in primary SS (pSS) has never been investigated. Our aim was to evaluate the role of EMPs and EPCs as markers of endothelial damage in pSS and their correlation with disease clinical and immunological features., Methods: Circulating EMPs (CD31(+)/CD42(-)), true EPCs (CD34(+)/KDR(+)/CD133(+)) and mature EPCs (CD34(+)/KDR(+)/CD133(-)) were quantified by FACS analysis in 34 pSS patients and 18 age- and sex-matched controls. Correlation between EMP and EPC levels and parameters of disease activity and damage, clinical features and markers of immunological dysfunction was performed., Results: Patients displayed higher EMP numbers with respect to healthy controls [HCs; mean 450 n/μl (S.D. 155) vs 231 (110), P < 0.0001]. EPC and mature EPC levels were higher in patients compared with HCs [mean 226 n/ml (S.D. 181) vs 69 (53), P < 0.001 and 166 (161) vs 36 (32), P < 0.0001, respectively). EMP levels directly correlated with disease duration from symptoms and diagnosis (ρ = 0.5, P < 0.01). Early EPCs inversely correlated with disease duration from symptoms (ρ = -0.5, P < 0.01) and diagnosis (ρ = -0.4, P < 0.05)., Conclusion: This is the first demonstration of chronic endothelial fragmentation characterizing pSS. The reparative potentiality of the endothelial layer appears to be preserved in the earliest stages of disease. During the course of the disease, progressive exhaustion of the precursor endothelial pool may be hypothesized, leading to defective vascular layer restoration and endothelial dysfunction., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Historically, primary Sjögren's syndrome (pSS) was thought to be a T helper (h) 1 driven disease due to the predominance of CD4(+)T lymphocytes and their products in target organs and peripheral blood of patients. In the last decades, the identification of a number of T cell subsets, including Th17, T regulatory (Treg), and follicular helper T cells, challenged this long-standing paradigm and prompted to identify their role in pSS pathogenesis. In addition the impact of abnormal proinflammatory cytokine production, such as IL-6, IL-17, IL-22, and IL-23, has also attracted considerable attention. However, although several studies have been carried out in experimental models and patients with pSS, many aspects concerning the role of Treg cells and IL-17/Th17 cell system in pSS pathogenesis are not fully elucidated. In particular, the role played by different IL-17-producing T cell subsets as well as the effects of pharmacological therapies on Treg/Th17 cell balance represents an intriguing issue. The aim of this review article is to provide an overview of current knowledge on Treg cells and IL-17-producing T cells in pSS pathogenesis. We believe that these insights into pSS pathogenesis may provide the basis for successful therapeutic intervention in this disease.

Objective: Human umbilical cord Wharton jelly-derived mesenchymal stem cells (hUCMS) are easy to retrieve in bulk. They may interact with immune cells by either cell contact or soluble factors. Little evidence is currently available on potential therapeutic application of hUCMS to systemic autoimmune disorders such as primary SS (pSS). We have recently developed an endotoxin-free alginate gel that can be used to microencapsulate different cell types for graft into non-immunosuppressed hosts. We aimed to assess the in vitro effects of IFN-γ-pretreated microencapsulated (CpS)-hUCMS on T cells of pSS., Methods: Ten pSS patients and 10 healthy donors were selected. Peripheral blood mononuclear cells (PBMCs) were obtained from venous blood to establish co-cultures with CpS-hUCMS. Lymphocyte proliferation and phenotypic analysis was performed by flow cytometry and real-time PCR on IFN-γ-pretreated hUCMS was performed before PBMCs co-culture., Results: We found that CpS-hUCMS suppress pSS T cell proliferation and restore the Treg/Th17 ratio, thereby possibly positively impacting the pSS disease process., Conclusion: We have developed a new biohybrid drug delivery system that now waits for clinical application in autoimmune diseases, including pSS., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Compelling evidence suggests that the IL-17 axis plays a pivotal role in the pathogenesis of several autoimmune disorders including primary Sjögren's syndrome (pSS). However, although several studies have been carried out in experimental models and patients with pSS, many aspects of this field are not fully elucidated. In particular, the role played by different Th17 cell subsets as well as the effects of pharmacological therapies on IL-17 balance represent an intriguing issue. Furthermore, the understanding of IL-17 axis pathogenic role in pSS may be of interest for therapeutic purposes as a variety of compounds targeting IL-17, IL-17 receptor and other related cytokines and transcription factors involved in Th17 cell commitment are under intense investigation. The aim of this review article is to provide an overview of current knowledge in IL-17/Th17 cells in pSS and discuss their potential therapeutic targeting in this disease., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objectives: Several histological scoring systems, including the focus score, performed in minor salivary glands (MSGs) by hematoxylin-eosin (H&E) staining, have been employed in clinical practice to assess the inflammatory infiltrate and provide the diagnosis of primary Sjo¨gren׳s syndrome (pSS). Aims of this study were to integrate different scoring systems and identify potential differences in the molecular profile of lymphoid cytokines related to germinal center (GC) formation and clinical subsets in pSS., Methods: Overall, 104 pSS patients and 40 subjects with sicca non-pSS were retrospectively evaluated. MSG biopsies were evaluated by H&E and immunofluorescence to assess histological pattern, Chisholm and Mason grading system, Tarpley score, a grading for the severity of inflammatory infiltrate, T-/B-cell segregation, and the presence of GC. MSGs from 50 pSS patients and 30 sicca non-pSS patients were processed by real-time PCR to assess LTα, LTβ, BAFF, CXCR4, CXCL12, CXCR5, CXCL13, CCR7, CCL19, and CCL21., Results: GCs presence was associated with anti-Ro/SSA and anti-La/SSB antibodies, hypergammaglobulinemia, salivary gland swelling, higher Tarpley score and focus score, and extraglandular involvement but, at multivariate analysis, only extraglandular involvement was independently associated to GC. pSS patients displayed higher level of all cytokines compared to those with sicca symptoms. GC(+) pSS patients displayed higher level of all cytokines compared to those GC(-)., Conclusions: Our study demonstrates that different histopathological patterns, including GC presence, reflect different cytokine expression and different clinical subsets. We believe that the combined immunofluorescence/molecular approach in MSGs would help to tailor diagnostic and therapeutic approach for different subsets of pSS patients., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objectives: Growing evidence suggests that IL-17-producing T cells, lacking both CD4 and CD8 molecules and defined as double negative (DN) cells, play a pivotal role in the pathogenesis of a number of systemic autoimmune disorders. We recently demonstrated that this T-cell subset is expanded in the peripheral blood (PB) of patients with primary Sjögren's syndrome (pSS), produces IL-17 and accumulates in minor salivary glands (MSGs). We aimed to investigate glandular and PB DN T cells in early pSS in order to verify a possible correlation with MSGs histological patterns and clinical parameters., Methods: Paired samples of PB mononuclear cells and MSGs from pSS patients were evaluated at the diagnosis by flow cytometry and immunofluorescence staining respectively. Histological analysis to identify histological scores, B/T cell segregation and the presence of germinal center (GC)-like structures was also performed., Results: In early stages of pSS, circulating DN T cells appear to be not yet expanded and inversely correlated with circulating CD4(+)Th17 cells. The number of infiltrating DN T cells were associated with extent of glandular involvement, presence of GC-like structures and dryness symptoms and were inversely correlated with circulating DN T cells., Conclusions: Our findings suggest that DN T cells are actively involved in the pathogenic mechanisms leading to glandular dysfunction and damage in pSS and may play a role in ectopic lymphoneogenesis development occurring during the disease., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Objective: CD4(+)CD25(low)GITR(+) T lymphocytes expressing FoxP3 and showing regulatory function have been recently described in healthy donors (HD). The objective of the study was to investigate their presence and role in patients with primary SS (pSS)., Methods: CD4(+)CD25(low)GITR(+) cells circulating in peripheral blood (PB) of patients with pSS were isolated by MACS technique, their phenotype was studied by flow cytometry and real-time PCR, and their function was studied by in vitro co-culture. CD4(+)CD25(low)GITR(+) cells infiltrating salivary glands (SGs) were revealed by immunohistochemistry., Results: Results indicated that conventional CD4(+)CD25(high) regulatory T cells (Tregs) are decreased, whereas CD4(+)CD25(low)GITR(+) cells are expanded in the PB of pSS as compared with HD. Phenotypic analysis demonstrated that CD4(+)CD25(low)GITR(+) cells display Treg markers, including FoxP3, TGF-β and IL-10, and functional experiments demonstrated that they exert a strong inhibitory activity against autologous effector cells. CD4(+)CD25(low)GITR(+) cells were detectable in great number in the SG inflammatory infiltrate. Interestingly, PB CD4(+)CD25(low)GITR(+) cell expansion was evident only in patients with inactive disease, while conventional CD4(+)CD25(high) Treg number was not associated with disease activity., Conclusion: The present data demonstrate that circulating CD4(+) cells expressing GITR, but with low levels of CD25 (CD4(+)CD25(low)GITR(+)), are detectable in pSS patients. These cells, displaying Treg phenotype and function, are present in SG inflamed tissues and are expanded in the PB of subjects with inactive disease. Data suggest that the expansion of CD4(+)CD25(low)GITR(+) cells in pSS may represent a counter-regulatory attempt against autoimmune-driven inflammation and may provide a new target for future treatment strategies.

Objectives: It has been recently observed that a T-cell subset, lacking of both CD4 and CD8 molecules and defined as double negative (DN), is expanded in the blood of patients with systemic lupus erythematosus, produces IL-17 and accumulates in the kidney during nephritis. Since IL-17 production is enhanced in salivary gland infiltrates of primary Sjögren's syndrome (SS) patients, we investigated whether DN T cells may be involved in the pathogenesis of salivary gland damage., Methods: Phenotypic characterisation of peripheral blood mononuclear cells from SS patients and controls was performed by flow cytometry in freshly isolated and anti-CD3-stimulated cells. SS minor salivary glands were processed for immunofluorescence staining., Results: CD3(+)CD4(-)CD8(-) DN T cells were major producers of IL-17 in SS and expressed ROR-γt. They were expanded in the peripheral blood, spontaneously produced IL-17 and infiltrated salivary glands. In addition, the expansion of αβ-TCR(+) DN T cells was associated with disease activity. Notably, IL-17-producing DN T cells from SS patients, but not from healthy controls, were strongly resistant to the in vitro effect of dexamethasone., Conclusions: These findings appear to be of great interest since the identification of a peculiar T-cell subset with pro-inflammatory activity, but resistant to corticosteroids, in an autoimmune disorder such as SS may help to design new specific treatments for the disease.

Abstract: Objectives: Growing evidence suggests that IL-17-producing T cells, lacking both CD4 and CD8 molecules and defined as double negative (DN) cells, play a pivotal role in the pathogenesis of a number of systemic autoimmune disorders. We recently demonstrated that this T-cell subset is expanded in the peripheral blood (PB) of patients with primary Sjögren's syndrome (pSS), produces IL-17 and accumulates in minor salivary glands (MSGs). We aimed to investigate glandular and PB DN T cells in early pSS in order to verify a possible correlation with MSGs histological patterns and clinical parameters. Methods: Paired samples of PB mononuclear cells and MSGs from pSS patients were evaluated at the diagnosis by flow cytometry and immunofluorescence staining respectively. Histological analysis to identify histological scores, B/T cell segregation and the presence of germinal center (GC)-like structures was also performed. Results: In early stages of pSS, circulating DN T cells appear to be not yet expanded and inversely correlated with circulating CD4+Th17 cells. The number of infiltrating DN T cells were associated with extent of glandular involvement, presence of GC-like structures and dryness symptoms and were inversely correlated with circulating DN T cells. Conclusions: Our findings suggest that DN T cells are actively involved in the pathogenic mechanisms leading to glandular dysfunction and damage in pSS and may play a role in ectopic lymphoneogenesis development occurring during the disease. [Copyright &y& Elsevier]