CD4 T lymphocyte autophagy is upregulated in the salivary glands of primary Sjögren’s syndrome patients and correlates with focus score and disease activity

Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disease characterized by infiltration of exocrine glands by T and B lymphocytes that, by producing chemokines and cytokines, play a fundamental role in coordinating the chronic inflammatory process [1, 2]. Different subsets of effector T cells, including Th1, Th2, Th17, regulatory T cells, and follicular helper T cells, have been demonstrated to participate in the pathogenesis of pSS [3‐5]. In particular, follicular T-helper cells seem to be relevant biomarkers of pSS [6] and cytokines such as interleukin (IL)-21 and IL-23 play a major role in their modulation [6, 7]. Although the aberrant activation of the T cell response observed in pSS has partially been explained by the interaction of T lymphocytes both with the ductal epithelium and the infiltrating B cells via costimulatory molecules (i.e., CD40/CD40L) and the BAFF axis, other immunological mechanisms could play a crucial role.

Autophagy is a lysosome-mediated catabolic process that allows cells to degrade damaged organelles and misfolded proteins, and to recycle nutrients [8]. During autophagy, portions of cytoplasm are sequestered by double-membrane vesicles, the autophagosomes, and degraded after fusion with lysosomes for subsequent recycling. Autophagy is a genetically regulated process that requires the activity of autophagy-related gene (Atg) proteins [9]. Atg5 plays a central role in the autophagic process by binding to Atg12, acting as a dimeric complex together with Atg16, inducing the curvature of the phagophore. Atg7 acts like an E1 ubiquitin-activating enzyme [10]. The Atg5-Atg12 complex contributes to the conjugation of microtubule-associated protein 1 light-chain 3 (MAP1LC3, also known as LC3, the homolog of Atg8 in yeast) to the lipid phosphatidylethanolamine. The unlipidated cytosolic form of LC3 is called LC3-I, whereas the lipidated form, referred to as LC3-II, is localized into the autophagosomal membranes throughout the maturation process of the autophagosome, therefore being considered an autophagic marker [11]. Other different genes and proteins finely regulate the autophagy process, among them: ATG16L1, the main component of a large protein complex essential for autophagy; the immunity-associated GTPase family M (IRGM), which regulates autophagy formation in response to intracellular pathogens; ATG5, a ubiquitin ligase essential for autophagosomal elongation; HSPA8/HSC70, a protein that binds to nascent polypeptides to facilitate correct protein folding; HSP90AA1, involved in the proper folding of specific target proteins; p62/SQSTM1, a protein that links ubiquitinated proteins to the autophagic machinery to enable their degradation [12].

Double-membrane autophagosomes have been identified in both human and murine T lymphocytes [13, 14], and a crucial role for autophagy in T lymphocyte development, survival, and proliferation has been observed [15, 16]. Moreover, aberrant regulation of autophagy has been implicated in an increasing number of autoimmune disorders such as systemic lupus erythematous (SLE), rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, Crohn disease, and vitiligo [17‐19]. To date, only a few studies have been able to evaluate the potential role of autophagy in pSS induction and development. In animal models, it has been shown how activation of the autophagosomal pathway is involved in preventing or alleviating salivary and lacrimal gland dysfunctions [20‐22]. More recently, Katsiougiannis et al. [23] demonstrated that, in human salivary glands (HSG) cells, endoplasmic reticulum (ER) stress induced autophagy and apoptosis in both patients with SS and controls. However, all the studies focused on the possible role of the autophagy pathway in salivary gland epithelial cells and there is no evidence of its effect on T cells.

In this study, we investigated the autophagy process in human T lymphocytes from salivary gland tissues and peripheral blood of patients with pSS, hypothesizing a possible role of autophagy in the increasing survival of autoreactive T cells. We also assessed the possible relationship between the level of T lymphocyte autophagy and the clinical features of pSS. Evidence provided in this study strongly supports a prominent role for CD4⁺ T lymphocyte autophagy in the local immune processes in salivary glands of patients affected by pSS.

In this study, we provided the first demonstration of autophagy dysregulation in both salivary and peripheral T lymphocytes from pSS patients. A positive correlation was found between autophagy level and CD4⁺ T lymphocyte infiltration as well as tissue expression of the proinflammatory cytokines IL-21 and IL-23 in salivary glands of pSS patients. Additionally, we found that the level of autophagy in peripheral blood T lymphocytes positively correlated with pSS disease activity indexes.

pSS is a chronic systemic autoimmune disease characterized by the infiltration of T and B lymphocytes into exocrine glands [1, 2]. Evidence involves both innate and adaptive immune responses in pSS pathogenesis. The chronic inflammation provokes ductal epithelial cell death with exocrine gland dysfunction, contributing to causing xerostomia and keratoconjunctivitis sicca, typical of pSS. The upregulation of autophagy-related markers (at both the mRNA and protein levels) observed in this study in the salivary glands of pSS, mainly in CD4⁺ T cells, is intriguing considering previous studies showing that T cells, mostly CD4⁺ T cells, predominate in the salivary gland lesions and play a crucial role in the induction and/or maintenance of pSS (e.g., providing a stimulus for B lymphocytes and promoting the destruction of the gland through cytotoxicity) [33]. Growing evidence suggests that autophagy has a positive, cytoprotective, and homeostatic role in activated T cells [16] and dysregulation of this process, as herein observed, could favor the persistence of pathogenetic CD4⁺ T cells in salivary glands. For instance, autophagy supports metabolic functions of T cells at various stages of their maturation and effector function and is crucial for T cell development at the precursor stage, also regulating peptide presentation in stromal cells and professional antigen-presenting cells. Indeed, in other autoimmune diseases, and prevalently in SLE, the role of autophagy in T lymphocytes has been extensively studied: atg5 ⁻/⁻ CD8⁺ T lymphocytes displayed increased cell death, and atg5⁻/⁻ CD4⁺ and CD8⁺ T cells demonstrated deficient proliferation after T cell receptor stimulation [15]; atg7-deficient T lymphocytes had increased cell death and also showed expanded mitochondria, expanded ER content, and altered calcium flux [33]. Similarly, atg3-deficent mature T cells showed defective ER homeostasis and mitochondrial clearance and elevated levels of reactive oxygen species, which might induce autophagy and consequently the apoptotic pathway [34]. Although the precise mechanisms leading to autophagic dysregulation in SLE are still not understood, this pathway has been implicated in promoting survival of both human and murine T cells [18, 35].

The role of autophagy in salivary gland homeostasis and pathology has been studied exclusively in the epithelial cells, demonstrating its prosurvival and homeostatic role [20, 21]. More recently, Katsiougiannis et al. [23] investigated the relation between ER stress and autophagy induction in salivary epithelial cells. In this study, thapsigargin was used as an autophagy inducer, but the authors speculate that, in real life, other inducers of ER stress can be responsible for this phenomenon, such as an epithelial cell viral infection or sympathetic hormone insults leading to autoimmune initiation, as reflected by autoantibody redistribution on the cell surface [23]. Therefore, further studies are needed to elucidate the role and the possible pathogenetic effect of autophagy in pSS. Our study innovatively investigates the possible pathogenetic role of autophagy in a different cell population, T cells, demonstrating that autophagy is increased in salivary glands T lymphocytes and speculating that this could promote the survival of a subset of cells that can initiate autoimmunity. In our study, the autophagy level in salivary glands was also significantly correlated with FS and damage indexes. These results are expected because patients with a higher FS or GCs in their salivary glands are more likely to have worse disease and prognosis [36, 37]. Moreover, according to the prognostic role of T cell autophagy in pSS, it would be interesting to evaluate the role of autophagy in lymphoproliferative complications in pSS. In fact, the identification of biomarkers that could be clinically valuable for making diagnoses and determining disease progression in lymphoid malignancies are currently lacking.

Previous studies have demonstrated a role for autophagy in modulating the expression of IL-23, a cytokine strongly implicated in the pathogenesis of pSS and correlated with clinical manifestations [31]. Interestingly, we found a significant correlation between the level of Atg genes and that of IL-23p19 and IL-21 in salivary glands from pSS patients. Modulation of IL-23 production by autophagy has been recently demonstrated, suggesting a role of autophagy in bridging innate and adaptive immune responses [38]. The correlation between autophagy gene expression and IL-21 in the inflamed salivary glands of pSS represents, in our opinion, an important and novel finding. Although no direct correlation between autophagy and IL-21 production has been demonstrated, the differentiation of IL-21-producing Th17 cells seems to be dependent on MHC II expression by CD11c⁺ cells [39]. In this regard, it has been recently demonstrated that pathogens and pattern-recognition receptors can directly activate autophagy to upregulate MHC II presentation [40]. In light of this evidence, the autophagy activation in pSS salivary glands might be the result of a viral or bacterial stimulation and might be involved in driving the differentiation of IL-21-producing cells. Nevertheless, in our study no difference in serum levels of IL-23 and IL-21 between patients with pSS and HC was observed. We speculate that the discrepant results in blood compared to tissue might reflect local rather than systemic cytokine modulation.

Finally, as expected, circulating T cells in the pSS population were demonstrated to have a general state of activation, given the imbalance of naïve and memory T cell distribution and the increased expression of T cell activation markers. Interestingly, despite the absence of significant differences in terms of autophagy levels between pSS and controls, a significant correlation between peripheral T lymphocyte autophagy and disease activity and damage indexes in pSS was found.

This is the first study that investigates autophagy in T lymphocytes in patients with pSS. According to our results, an increase in the autophagy levels in T lymphocytes from salivary glands in pSS patients could represent the local origin of the pathogenic process in which an immune stimulus could activate the autophagic pathway and therefore promote the survival of a subset of lymphocytes. Our findings may be disease-specific or simply reflect the general increase in inflammation seen in this condition and further studies over larger cohorts of patients are mandatory to validate the possible use of T lymphocyte autophagy-related molecules as prognostic peripheral biomarkers and to evaluate the implications of autophagy as a therapeutic target in pSS.