Background
Autophagy is a cell-protective catabolic process aimed at eliminating damaged organelles, misfolded proteins, and intracellular pathogens [
1]. Thanks to the ability to shape the adaptive immune response, deregulation of autophagy mechanisms seems implicated in the development of autoimmunity [
2]. During stressful conditions, autophagy seems able to shape the adaptive immune response, orchestrating the regulation of lymphocyte survival, differentiation, and activation [
3]. Several autophagy (Atg)-related proteins are implicated in this pathway, leading to the downstream conjugation of the microtubule-associated protein 1A/1B-light chain 3-I (LC3-I) with phosphatidylethanolamine and subsequent formation of LC3-II (LC3-phosphatidylethanolamine conjugate) [
4]. LC3-II plays a crucial role in cargo selection and autophagosome biogenesis and is thereby widely utilized as a marker of autophagy activation [
5]. LC3, which is identified by three different forms named LC3A, LC3B, and LC3C [
6], is a structural protein of the autophagosomal membranes used as a biomarker of autophagy. Specifically, the cytoplasmic presence of LC3B, which is measured by immunofluorescence as an increase in punctate LC3 named “LC3 puncta” stain, is widely used to monitor autophagy [
7,
8].
We previously investigated the engagement of autophagy in the pathogenesis of several autoimmune conditions, demonstrating a role in both promoting the exposure of immunogenic peptides and supporting immune cell survival in patients with rheumatoid arthritis [
9]. Additionally, we demonstrated a natural resistance of T lymphocytes to autophagy in systemic lupus erythematous [
10]. More recently, we provided the first evidence of autophagy mechanisms in CD4+ T lymphocytes infiltrating Sjögren’s syndrome (SS) minor salivary glands (MSG) [
11].
SS is a chronic systemic autoimmune disease mainly affecting exocrine glands and characterized by autoantibody production and systemic manifestations. Whilst, B cell hyperactivity is central to SS pathogenesis [
12], a concomitant role of T cells in disease development and progression has been recognized [
13]. The formation of lymphocyte aggregates in MSG is a histological hallmark of SS, and their organization in T and B cell areas reflects lesion severity [
14]. Within those aggregates, the development of ectopic germinal centers (GC), characterized by full segregation in T and B cell zones, formation of CD21+ follicular dendritic cell networks has also been described, in association with local B cell proliferation and affinity maturation [
15]. This phenomenon is associated with disease severity, autoantibody production, and lymphoma development [
16].
Evidence on the role of autophagy in SS is limited. Previous studies pointed at a dysregulation of autophagy in salivary glands of SS mice models [
17,
18], and a protective role of this pathway specifically in salivary glands’ epithelial cells following induction of endoplasmic reticulum stress [
19]. Nonetheless, the role of autophagy in supporting lymphocyte survival, proliferation, and GC formation in SS is still unknown. Critical unanswered questions pertain to the role of this pathway and its association with the severity of salivary gland infiltrates. This study aims to further explore the role of autophagy in infiltrating and circulating lymphocytes of patients with SS and to investigate its role in disease histopathological progression.
Discussion
The present study suggests that autophagy is a feature of T and B cells infiltrating MSG of patients with SS. In comparison to lymphocytes inhabiting secondary lymphoid organs, lymphocytes infiltrating SS MSG display a clear expression of the autophagy marker LC3B, as confirmed by the “LC3 puncta” stain [
8] in both CD3+ and CD20+ cells. The presence of upregulated autophagy genes in lymphocytes invading SS MSG could be differently interpreted. Being autophagy a mechanism of cell survival, it is reasonable to hypothesize that during MSG inflammation, this pathway is used by lymphocytes to prevent death and sustain proliferation. Accordingly, upregulation of autophagy genes possibly reflects the presence of lymphocytes with increased lifespan and/or slower turnover due to the concomitant inflammatory activation. In line with this finding, our group recently demonstrated overexpression of molecules favoring lymphocyte survival and proliferation, such as PI3Kδ, especially in large and organized SS MSG infiltrates [
22]. Interestingly, in this study, we demonstrated a higher expression of the autophagy genes Atg5 and MAP1LC3A in large and organized infiltrates compared to small infiltrates, thus suggesting that autophagy might be implicated in the organization of salivary gland infiltrates and, possibly, in their severity. Especially for MAP1LC3A, the different expression of autophagy genes according to the size of infiltrates was confirmed also in individual cases. Although this subanalysis was available only for two patients, this finding likely supports the hypothesis that the different expression of autophagy genes between small and large foci is related more to the type of infiltrate than to a specific patient “autophagic state.” This finding is further supported by the variability in autophagy gene expression levels observed among the same type of infiltrate belonging to a singular patient.
Since autophagy is essential for lymphocyte survival, differentiation, and activation [
2], as well as for their secretion of inflammatory proteins [
23], it is not unexpected that deregulation of this mechanism might be identified in patients with autoimmune conditions, such as SS. In SS, inflammatory infiltrates may differ among patients and their aspect is typically correlated with patients’ serological and clinical features [
16]. Large and organized infiltrates are usually associated with the presence of autoantibodies and higher disease activity [
16,
24,
25]. Conversely, small and non-organized infiltrates are more commonly observed in seronegative patients with milder disease activity [
16]. The evidence of higher autophagy in large and organized infiltrates strongly suggests the involvement of this pathway in their progression.
Our data not only demonstrate that lymphocytes’ autophagy is associated with SS histological severity but also highlight an aberrant regulation of this pathway in SS GC-like structures. Specifically, we identified an overexpression of autophagy compared to the classic GC inhabiting human tonsils, which, as we know, share structural and molecular similarities including the presence of a CD21+ network. Although the definition of GC in SS is still subject of debate, following identification by H&E, the expression of CD21 is largely accepted by the community as a validation of GC-like formation [
26]. To this regard, we evaluated autophagy specifically in aggregates displaying both histological appearance of GC and confirmed to express CD21 and we found a significant overexpression in GC-like structures compared to the normal GC. This finding highlights the presence of functional differences between the two structures, highlighting the role of GC-like structures in SS as a pathogenic hub for autoreactive B cell survival, autoantibody production [
15], increased systemic disease activity, and more severe disease progression [
24,
27]. Thus, the evidence of increased lymphocyte autophagy in these peculiar structures raises the possibility of a role for autophagy in the development of autoimmunity rather than a simple function in supporting lymphoid organization. Nonetheless, the pathogenic role of autophagy in GC formation and maintenance is still unknown. Early studies failed to unveil a role for autophagy in GC of secondary lymphoid organs; however, more recently, a non-canonical autophagy mechanism regulating B cell survival during the GC’ reaction has been described [
28]. The presence of GC-like structures in SS MSG is undoubtedly associated with more active and systemic disease [
16] and, possibly, with the development of lymphoma [
26]. Thus, a relationship between the upregulation of autophagy in SS GC and their pathogenic function is expected.
Of the several effector proteins implicated in autophagy pathways [
1], LC3 is commonly utilized as an autophagy marker [
5]. In this study, we observed the expression of increased amount of LC3B on both T and B cells infiltrating MSG by the evidence of “LC3 puncta” stain [
8]. Interestingly, this finding was not confirmed in lymphocytes inhabiting secondary lymphoid organs.
In T cells, autophagy is involved both in survival homeostatic mechanisms and in their activation [
29]. Here, we highlight the presence of autophagy in CD3+ cells infiltrating MSG. In our previous work, we investigated the expression of this pathway specifically on this cell subset identifying a co-localization of Atg5/LC3II markers with CD4+ and CD8+ lymphocytes [
11]. However, along SS course, there is also a progressive increase in the foci B cell component [
14]. Autophagy is a crucial survival mechanism across differentiation stages of the B cell cycle [
30]. In the present study, we demonstrate, for the first time, the presence of autophagic B cells in infiltrating SS MSG.
However, recent studies have suggested that increased expression of cytoplasmic LC3-positive structures may also indicate impaired autophagosome degradation [
7], and LC3 puncta stain can be also detected when autophagosome formation is abrogated due to LC3-I sequestration to p62 aggregates which accumulate into the cytoplasm [
31]. Thus, the presence of LC3 puncta in lymphocytes infiltrating SS salivary glands also raises the possibility of impaired autophagy. However, taking together data from gene expression analysis and our previous findings [
11,
22], we are inclined to consider the high expression of LC3B puncta as result of autophagy activation. Either way, a deregulation of autophagy mechanisms in SS infiltrating lymphocytes is observed and further studies are highly recommended to clarify this aspect.
Although lymphocytes’ autophagy was associated with escalating histological severity, when determining its activation in peripheral circulating lymphocytes, we did not identify any difference compared to HC. The absence of increased autophagy in SS circulating lymphocytes further supports a predominant role of this process at tissue level. Moreover, as pathogenic mechanisms identified with histologic evaluations might be diluted out and not identifiable in the periphery, the lack of differences in peripheral autophagy levels between SS and HC is not unexpected.
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