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Gene expression profiling of lip salivary gland (LSG) has shown that C-X-C motif chemokine 10 (CXCL10) and matrix metalloproteinase 9 (MMP9) expression is up-regulated in primary Sjögren's syndrome (pSS) patients. Although CXCL10 and MMP-9 are both associated with pSS pathogenesis, the potential relationship between these two factors has not been investigated. In this study, we used LSG sections from pSS patients and human salivary gland cell lines to investigate the relationship between CXCL10 and MMP-9. Immunofluorescence analyses revealed that CXCL10 and MMP-9 were co-expressed in the LSG of pSS patients, particularly in expanded ductal cells. Furthermore, RT-qPCR analyses on human salivary gland ductal NS-SV-DC cells confirmed that CXCL10 expression was induced by interferon (IFN)-γ, whereas that of MMP9 was stimulated by IFN-α, tumor necrosis factor-α, and interleukin 1β. Remarkably, MMP-9 inhibition in IFN-γ-stimulated NS-SV-DC cells significantly decreased CXCL10 mRNA and secreted protein levels. Further analyses established that MMP-9 inhibition in IFN-γ-stimulated NS-SV-DC cells decreased STAT1 phosphorylation and hence suppressed IFN-γ signaling. Collectively, these results suggest that in addition to its reported role in the destruction of acinar structures, MMP-9 is involved in the IFN-γ-induced production of CXCL10 in pSS lesions. We believe that our findings open the door to the development of novel treatments for pSS, based on the modulation of MMP-9 activity.

Sjogren’s syndrome (SS) is a chronic autoimmune disease targeting salivary and lacrimal glands. C-X-C motif chemokine ligand 10 (CXCL10) expression is upregulated in lip salivary glands (LSGs) of primary SS (pSS) patients, and CXCL10 involved in SS pathogenesis via immune-cell accumulation. Moreover, interferon (IFN)-γ enhances CXCL10 production via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. We investigated the effects of baricitinib, a selective JAK1/2 inhibitor, on both IFN-γ-induced CXCL10 production and immune-cell chemotaxis. We used immunohistochemical staining to determine the expression levels and localization of JAK1 and JAK2 in LSGs of SS patients (n = 12) and healthy controls (n = 3). We then evaluated the effect of baricitinib in an immortalized normal human salivary gland ductal (NS-SV-DC) cell line. Immunohistochemical analysis of LSGs from pSS patients revealed strong JAK1 and JAK2 expression in ductal and acinar cells, respectively. Baricitinib significantly inhibited IFN-γ-induced CXCL10 expression as well as the protein levels in an immortalized human salivary gland ductal-cell clone in a dose-dependent manner. Additionally, western blot analysis showed that baricitinib suppressed the IFN-γ-induced phosphorylation of STAT1 and STAT3, with a stronger effect observed in the case of STAT1. It also inhibited IFN-γ-mediated chemotaxis of Jurkat T cells. These results suggested that baricitinib suppressed IFN-γ-induced CXCL10 expression and attenuated immune-cell chemotaxis by inhibiting JAK/STAT signaling, suggesting its potential as a therapeutic strategy for pSS.

Background: Mechanisms underlying immune cells' recruitment and activation into the inflammatory lesions of lip salivary glands (LSGs) from primary Sjogren's syndrome (pSS) patients are incompletely understood. Chemokines play pivotal roles in these processes, so we investigated the clinical significance of chemokine receptor CXCR3 and its ligands in the autoimmune lesions of pSS. Methods: We histologically determined the grade of LSG samples from 22 pSS patients and subjected the samples to immunofluorescence analysis to determine the expressions of CXCR3 and its ligands: CXCL9, CXCL10, and CXCL11. To identify the immune cells expressing CXCR3 in the LSGs, we performed double immunofluorescence analysis using antibodies against CD3 (pan-T cells), CD80 (M1 macrophages), CD163 (M2 macrophage), and CD123 (plasmacytoid dendritic cells: pDCs). The relationship between the grade of lymphocytic infiltration and the number of positively stained cells was analyzed by Spearman's rank correlation test. Results: The expressions of CXCL9 and CXCL10 showed particularly intense staining in the LSG samples' ductal cells. The CXCR3 expression was detected mainly in CD80+ and CD163+ macrophages. The number of CXCR3+CD163+ macrophages inversely correlated with the LSG inflammatory lesions' severity (rs= −0.777, p

CXCL10, a CXC chemokine induced by interferon-gamma [IFN-γ], has been observed in a wide variety of chronic inflammatory disorders and autoimmune conditions. Although CXCL10 is known to be overexpressed in the salivary glands of individuals with primary Sjögren's syndrome (pSS), it is unclear which cells produce CXCL10 under what types of stimulations. Here, we investigated the precise molecular mechanisms by which CXCL10 was produced in human salivary gland ductal (NS-SV-DC) and acinar (NS-SV-AC) cell lines. Our results demonstrated that NS-SV-DC cells produced higher levels of CXCL10 compared to NS-SV-AC cells. In addition, our findings demonstrated that the regulator of the enhancement of CXCL10 was different between NS-SV-DC and NS-SV-AC cells, i.e., interferon-gamma (IFN-γ) had more potential than interferon-alpha (IFN-α), tumor necrosis factor (TNF)-α, and interleukin (IL)1-β in the induction of CXCL10 production in NS-SV-DC cells, whereas TNF-α had potential to induce CXCL10 production in NS-SV-AC cells. A Western blot analysis demonstrated that IFN-γ enhanced the production of CXCL10 via both the JAK/STAT1 pathway and the NF-κB pathway in NS-SV-DC cells, whereas TNF-α enhanced the production of CXCL10 via the NF-κB pathway in NS-SV-AC cells. The results of study suggest that the CXCL10 overexpression in the salivary glands is caused mainly by IFN-γ-stimulated salivary gland ductal cells. The enhanced production of CXCL10 by IFN-γ from ductal cells may result in the inflammation of pSS lesions.

Cepharanthine, a biscolaurine alkaloid isolated from the plant Stephania cephalantha Hayata, has been reported to have potent anti-inflammatory properties. Here, we investigated the effects of cepharanthine on the expression of CXCL10 (a CXC chemokine induced by interferon-gamma [IFN-γ] that has been observed in a wide variety of chronic inflammatory disorders and autoimmune conditions) in IFN-γ-treated human salivary gland cell lines. We observed that IFN-γ-induced CXCL10 production in NS-SV-DC cells (a human salivary gland ductal cell line), but not in NS-SV-AC cells (a human salivary gland acinar cell line). Cepharanthine inhibited the IFN-γ-induced CXCL10 production in NS-SV-DC cells. A Western blot analysis showed that cepharanthine prevented the phosphorylation of JAK2 and STAT1, but did not interfere with the NF-κB pathway. Moreover, cepharanthine inhibited the IFN-γ-mediated chemotaxis of Jurkat T cells. These results suggest that cepharanthine suppresses IFN-γ-induced CXCL10 production via the inhibition of the JAK2/STAT1 signaling pathway in human salivary gland ductal cells. Our findings also indicate that cepharanthine could inhibit the chemotaxis of Jurkat T cells by reducing CXCL10 production.