Phd in IINNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Thesis title: IL-1 PATHWAY IN SJÖGREN’S SYNDROME: THE ROLE IN AUTOIMMUNE EPITHELITIS

1. ABSTRACT Background. Sjögren syndrome (SS) is an autoimmune disorder characterized by chronic inflammation of exocrine glands and histological features of focal lymphocytic sialoadenitis. While the innate immune mechanisms display a major role at early stages of the disease, the adaptive immune system is the main driver of inflammation at later phases. Although the innate immune pathway of IL-1 displays great pro-inflammatory effect in different inflammatory conditions, its role in SS is still poorly defined. The so called ”activation” of salivary gland epithelial cells (SGECs) is key in the pathogenesis of SS and we recently described how this process is intrinsically linked to the activation of autophagy. The mechanisms behind SGECs activation remain unclear as well as their relationship with innate immune pathways and, in particular, with the IL-1 pathway. The few available data suggest that the expression of IL-1β is upregulated in activated epithelial cells from SS lacrimal glands; accordingly, increased assembly of the inflammasome complex NLRP3 has been described in the SG of SS mice models. Little is known about the role of the other isoform of IL-1 named IL-1α, an “alarmin” passively released by cells following damage with interesting effects in different rheumatic conditions. Aim of the study: Aim of this study is to investigate the IL-1 pathway, with particular regard to the expression of the two isoforms of IL-1 (α and β), in SS salivary glands and to assess its role in SGECs activation and homeostasis. Materials and methods. The study was divided in two parts, a descriptive and a functional one. Descriptive study – Paraffin embedded minor SG biopsies (MSG) were collected from patients with SS and sicca syndrome (controls). Immunofluorescence staining for IL-1α and IL-1β detection were performed along with markers to localize their expression; these markers included SGECs markers (panCK, CK7, CK14) and lymphocytes markers (CD45, CD3, CD20). Functional study – SGECs cultures from SS and sicca MSG were produced. Supernatants were analysed by ELISA for the detection of IL-1 family cytokines. On a human SG cell line (HSG), in vitro treatments with activating stimuli [Poly(I:C) and LPS] and then with IL-1 inhibitors (Anakinra, blocking both IL-1α and IL-1β; Canakinumab, selectively binding IL-1β) were performed. Following treatments, changes in the expression of autophagy (LC3IIB and p62), apoptosis (annexin V) and activation (ICAM-1) markers were assessed by Western Blot and flow cytometry. Results. Descriptive study - IL-1α expression was detected both in MSG sections from patients with SS and in MSG from patients with sicca; its expression was mainly localized in luminal CK7 positive ductal epithelial cells. No expression was detected in acinar cells and infiltrates. The expression of IL-1β was not detectable neither in infiltrates nor in SGECs. Functional study - Analyses of supernatants from SGECs cultures revealed slightly higher levels of IL-1α and IL-1β in SS compared to sicca, however the difference was not statistically significant. Following HSG stimulation with Poly(I:C) and LPS, an increase in autophagy (increased LC3IIB, decreased p62) and activation (ICAM-1), and a slight increase in apoptosis (annexin V), were observed. Both treatments with Anakinra and Canakinumab induced a reduction in autophagy and a slight downregulation of activation markers; only anakinra determined a decrease in apoptosis while canakinumab displayed opposite effects. Conclusions. The lack of expression of IL1β at tissue level might suggest that this pathway is mainly involved in the early phases of the disease rather than during the course of an established disease. Despite the absence of specificity for SS, the expression of IL1α at ductal epithelial cell level is quite interesting and worth of further investigation. In addition, the indirect evidence of an alteration in homeostasis and activation of SGECs following inhibition of IL-1 pathways, might suggest a role of this molecule in two of the major pathogenic mechanisms driving the development of an autoimmune epithelitis .