pSS is a systemic autoimmune disease that targets the exocrine glands, such as the salivary and lacrimal glands, resulting in sicca symptoms in affected patients. Histopathologically, pSS is characterized by the lymphocytic infiltrate surrounding the ductal epithelium and the progressive destruction of the acinar structure [1, 2]. During the last few decades, clarification of the underlying pathogenetic pathway in SS has fueled international efforts toward the discovery of novel therapeutic modalities targeting both innate and adaptive immune pathways [21]. IFNs are involved in innate immunity, and show potential as SS treatment targets. Microarray and gene expression studies confirmed the upregulation of type I and II IFN-regulated genes in both peripheral blood and affected salivary gland tissues of patients with pSS [22–25]. IFNs bind to their receptor, leading to phosphorylation of JAKs, activation of the STAT pathway and the upregulation of IFN-inducible genes [26]. In recent years, there has been growing interest in modulating the JAK/STAT pathway for the treatment of rheumatoid arthritis (RA) and other inflammatory autoimmune diseases [27].
JAK inhibitors undergo competitive ATP binding, thus blocking the phosphorylation of cytokine receptors and inhibiting gene transcription, leading to reduced production of multiple cytokines and impaired differentiation of Th1, Th2, and Th17 cells [28]. Although JAK inhibitors are expected to act on immune cells, our study revealed that salivary gland duct cells produce CXCL10 via the JAK/STAT pathway following stimulation with type-II IFNs [10]. This suggests that JAK inhibitors have an effect on not only immune cells but also target organs. A randomized study with JAK inhibitors has been conducted in patients with pSS; however, the expression pattern of JAKs in the salivary glands of patients with pSS is not well known. In this study, we investigated the localization of JAK1, JAK2, phosphorylated JAK1, and phosphorylated JAK2 in LSGs of patients with pSS and evaluated their expression levels using a semi-quantitative score. Moreover, we examined the association between the expression levels of JAKs and the degree of lymphocytic infiltration.
The salivary gland structure consists of clusters of acinar cells, which secrete protein/fluid, and ductal cells, which modify the composition of saliva and its transport into the oral cavity. Myoepithelial cells surround the acinar units and other supportive tissues, including blood vessels, and provide an exchange of nutrients and waste, while nerves stimulate the glands to secrete saliva [29]. JAK1 and JAK2 are ubiquitously expressed in mammals [17]. Immunohistochemical staining of LSGs revealed the expression of JAK1 and JAK2 in both the acinar and ductal epithelium in human salivary glands, but their expression patterns differed between healthy controls and patients with pSS. Our study showed that the expression of JAK1, JAK2, and phosphorylated JAK1 in the acinar epithelium of patients with pSS was significantly lower than that in healthy controls. In contrast, we found that the expression of phosphorylated JAK1 and JAK2 in the ductal epithelium of patients with pSS patients was significantly higher than that in healthy controls.
JAKs are involved in the signaling pathway of a plethora of cytokines [30]. JAK1 is essential for the activation and secretion of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. These cytokines also depend on JAK3 expression. JAK1 is also essential for the activation of a family of cytokines including IL-6, IL-11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNF) as well as IFNs and granulocyte colony-stimulating factor (G-CSF). JAK2 is important in the signaling pathway of some IFNs [17], hormone-like cytokines such as growth hormone, prolactin, erythropoietin, thrombopoietin, and a family of cytokines that signal through the IL-3 receptor (IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor). The immunohistochemical staining revealed that JAK1 and JAK2 are expressed in both the acinar epithelium and ductal epithelium of human salivary glands, which implies that stimulation of these cytokines, including IFNs, activates the JAK/STAT pathway in salivary gland epithelial cells.
In pSS, a dysregulated cytokine network impairs glandular function and induces chronic systemic inflammation [31]. Several cytokine families are involved in the etiopathogenesis of pSS, including the IFN, TNF, IL-6, IL-2, IL-10, and IL-17 families [32]. We have reported that IFN-γ enhances the production of CXCL10 via the JAK/STAT pathway in an immortalized normal human salivary gland ductal cell line, resulting in the accumulation of CXCR3+ immune cells [10]. Based on these previous studies, the high expression of phosphorylated JAK1 and JAK2 in the ductal epithelium of patients with pSS is considered to be the effect of cytokines such as IFN-γ. Interestingly, we found that the expression levels of JAK1 and JAK2 in the ductal epithelium of patients with pSS did not decrease, even if the phosphorylation of JAK1 and JAK2 was increased. Moreover, the expression levels of JAK1, JAK2, phosphorylated JAK1, and phosphorylated JAK2 in the ductal epithelium was not associated with the degree of inflammation. These results suggest that JAK1 and JAK2 may be therapeutic targets for the treatment of pSS at any disease stage.
Immunofluorescence analysis of LSGs from patients with pSS demonstrated increased JAK2 phosphorylation in the infiltrated CD3+ T cells, but not increased JAK1 phosphorylation. A randomized controlled trial using filgotinib (a JAK1 inhibitor) has been conducted on 152 adult subjects with SS (ClinicalTrials.gov NCT03100942). While there are no data on glandular lesions, the European League Against Rheumatism Sjögren's syndrome Disease Activity Index (ESSDAI) and European League Against Rheumatism Sjögren's syndrome Patient Index (ESSPRI) were reportedly not significantly different from the placebo group. Our findings suggest that JAK2 inhibitors may suppress CD3+ T cell activation and control inflammatory lesions in pSS.
In summary, we detected high levels of phosphorylated JAK1 and JAK2 in the ductal epithelium of LSGs from patients with pSS by immunohistochemical analysis. In addition, infiltrated CD3+ T cells strongly expressed phosphorylated JAK2. Our results suggest that JAK inhibitors may be effective therapeutic agents for pSS.