Davide Lucchesi

IL-22 regulates lymphoid chemokine production and assembly of tertiary lymphoid organs

Significance Ectopic clusters of immune cells that mimic the structure and function of secondary lymphoid organs are defined as tertiary lymphoid organs (TLOs). They have been observed at sites of chronic inflammation for decades, but their formation and function have remained enigmatic. TLOs are thought to contribute to disease pathogenesis by promoting autoreactive lymphocyte survival and autoantibody production. In this study we identify a novel role for the cytokine IL-22 in TLO development and biology. We provide evidence that IL-22 expression within TLOs is instrumental for the production of the lymphoid chemokines, chemokine (C-X-C motif) ligand 13 and chemokine (C-X-C motif) ligand 12, which in turn orchestrate B-cell clustering, lymphoid aggregation, and autoantibody production. Our data provide a strong rationale for targeting IL-22 in TLO-associated autoimmune diseases. The series of events leading to tertiary lymphoid organ (TLO) formation in mucosal organs following tissue damage remain unclear. Using a virus-induced model of autoantibody formation in the salivary glands of adult mice, we demonstrate that IL-22 provides a mechanistic link between mucosal infection, B-cell recruitment, and humoral autoimmunity. IL-22 receptor engagement is necessary and sufficient to promote differential expression of chemokine (C-X-C motif) ligand 12 and chemokine (C-X-C motif) ligand 13 in epithelial and fibroblastic stromal cells that, in turn, is pivotal for B-cell recruitment and organization of the TLOs. Accordingly, genetic and therapeutic blockade of IL-22 impairs and reverses TLO formation and autoantibody production. Our work highlights a critical role for IL-22 in TLO-induced pathology and provides a rationale for the use of IL-22–blocking agents in B-cell–mediated autoimmune conditions.

Inducible tertiary lymphoid structures, autoimmunity, and exocrine dysfunction in a novel model of salivary gland inflammation in C57BL/6 mice.

Salivary glands in patients with Sjögren’s syndrome (SS) develop ectopic lymphoid structures (ELS) characterized by B/T cell compartmentalization, the formation of high endothelial venules, follicular dendritic cell networks, functional B cell activation with expression of activation-induced cytidine deaminase, as well as local differentiation of autoreactive plasma cells. The mechanisms that trigger ELS formation, autoimmunity, and exocrine dysfunction in SS are largely unknown. In this article, we present a novel model of inducible ectopic lymphoid tissue formation, breach of humoral self-tolerance, and salivary hypofunction after delivery of a replication-deficient adenovirus-5 in submandibular glands of C57BL/6 mice through retrograde excretory duct cannulation. In this model, inflammation rapidly and consistently evolves from diffuse infiltration toward the development of SS-like periductal lymphoid aggregates within 2 wk from AdV delivery. These infiltrates progressively acquire ELS features and support functional GL7+/activation-induced cytidine deaminase+ germinal centers. Formation of ELS is preceded by ectopic expression of lymphoid chemokines CXCL13, CCL19, and lymphotoxin-β, and is associated with development of anti-nuclear Abs in up to 75% of mice. Finally, reduction in salivary flow was observed over 3 wk post-AdV infection, consistent with exocrine gland dysfunction as a consequence of the inflammatory response. This novel model has the potential to unravel the cellular and molecular mechanisms that regulate ELS formation and their role in exocrine dysfunction and autoimmunity in SS.

EBV and other viruses as triggers of tertiary lymphoid structures in primary Sjögren’s syndrome

Sjögrens syndrome (SS) is an autoimmune disease that targets salivary (SG) and lachrymal glands, leading to exocrine dysfunction. Several viruses have been associated with SS, although the role of persistent viral infections in triggering and/or perpetuating the disease is still a matter of controversy. Together with exocrine dysfunction, SS is characterised by the production of autoantibodies and the presence of lymphomonocytic periductal aggregates in the SG, which in 30/40% of the patients display features of tertiary lymphoid structures (TLS) supporting an ectopic germinal centre response. Here we first review i) the relevance of TLS in SS and ii) the evidence in support of a role for viruses in SS insurgence and/or persistence; next, iii) we review recent data which links viral infection with TLS formation in the SG and suggests that viral-host interactions within TLS favour breach of tolerance and development of autoimmunity in SS.

The role of viruses in autoreactive B cell activation within tertiary lymphoid structures in autoimmune diseases

TLS, characterized by the formation of ectopic B/T cell follicles with FDCs supporting an ectopic GC response, have been described in the target organs of several autoimmune diseases, including MS, RA, SS, and autoimmune thyroiditis. These structures represent functional niches, whereby autoreactive B cells undergo in situ affinity maturation and differentiation to autoantibody‐producing cells, thus contributing to the progression and persistence of autoimmunity. Increasing evidence demonstrates that TLS can also develop in the context of cancer, as well as chronic infections. In this review, we collect recent evidences that highlights the relationship between persistent viral infection and the development of ectopic lymphoid structures in animal models and patients. Furthermore, we shall discuss the concept that whereas in physiological conditions, inducible TLS are critical for viral clearance and the establishment of protective immunity, but in the context of susceptible individuals, persistent viral infections may contribute, directly or indirectly, to the development of breach of tolerance against self‐antigens and the development of autoimmunity through the formation of TLS.

Current views on the pathogenesis of Sjgrenʼs syndrome

Purpose of review The purpose of this review is to provide an insight into the pathophysiological mechanisms involved in the pathogenesis of primary Sjögrens Syndrome (pSS), highlighting recent findings with potential therapeutic repercussions. Recent findings In the last 2 years, epigenetic analyses provided new insights into pSS pathogenesis. Characterization of DNA methylation patterns, chromatin structures and microRNA confirmed the importance of aberrant interferon and B-cell responses in the development of the disease. The formation of ectopic B-cell follicles with germinal centers is now a well recognized pathogenic mechanism within salivary glands of pSS. In the context of ectopic germinal centers reaction, T/B-cell interactions, that is regarding T-helper 17 and T-follicular helper cells, and their respective counterparts, T-regulatory and T-follicular regulatory cells, appear particularly relevant in pSS pathogenesis as their imbalance is associated with a dysregulation of B-cell dynamics and the production of autoantibodies. Summary Advances in the understanding of pSS pathogenesis have paved the way for clinical trials with novel biologic agents targeting immune pathways regulating T/B-cell interactions and downstream B-cell activation. Reverse translation from these studies provides invaluable novel information of the mechanisms sustaining autoimmunity and chronic inflammation in pSS.

01.09 Myeloid cells drive early inflammation and orchestrate salivary glands tertiary lymphoid structures formation in a model of inducible sialadenitis

Background Tertiary lymphoid structures (TLS) are leukocytes aggregates forming in non-lymphoid tissues in response to chronic inflammation. Salivary glands (SG) of patients with Sjögren’s syndrome (SS) develop TLS characterised by B/T cell compartmentalization, specialised vasculature (HEV), functional B cell activation and local differentiation of autoreactive plasma cells. Although TLS presence in SS SG associates with an aggressive disease progression, the mechanisms triggering their formation in SS are largely unknown. We recently developed a model of inducible TLS formation, breach of self-tolerance and salivary hypofunction upon delivery of a replicationdeficient adenovirus5 (AdV5) into C57BL/6 mice SG. In this model, 3 weeks after infection, we observed fully formed and functional TLS, breach of self-tolerance and loss of salivary function. Here we characterised the presence, function and relevance in TLS formation of the myeloid compartment in the first month post-infection. Materials and methods A luciferase-encoding AdV5 was delivered to the SG, glands were collected between 1 and 4 weeks post-cannulation (WPC), embedded for immunofluorescence staining and/or digested for FACS analysis, and stained for various markers. Sorted myeloid cells were analysed my Q-PCR. Infiltration and differentiation of monocytes was followed via adoptive transfer. Results During the first WPC, innate cells infiltrated the gland in sequential waves, as shown by FACS. Neutrophils and eosinophils were rapidly recruited to the SG and likely contributed to the histological damage observed. Inflammatory monocytes were recruited from the periphery and progressively differentiated into macrophages in-situ. NK and pDC also increased in number in response to viral infection. Gene expression analysis of sorted myeloid cells showed early upregulation of inflammatory mediators (eg, CCL5, IFNg) and a later production of lymphoneogenetic signals (eg, Lt-b, CXCL13) mRNA. Conclusions In our model of inducible ialadenitis, innate immune cells not only mediate early inflammation in the SG, but also take part in driving the lymphocytic infiltration, thus playing a pivotal role in tertiary lymphoid formation.

AB0169 Recruitment and Activation of NK Cells in the Salivary Glands Regulates Early Viral Control but is Dependable for Autoimmunity and Focal Lymphocytic Sialoadenitis in an Inducible Murine Model of Sjogren-Like Disease

Background Dysregulation of Natural Killer (NK) cells has been shown to influence the development of autoimmune diseases1, but their role in Sjögrens syndrome (SS) remain unclear. Objectives The main goal of our study was to investigate the recruitment and functional relevance of NK cells in the early phases of salivary gland (SG) inflammation in an inducible murine model of SS. Methods Sialoadenitis was induced in WT-C57BL/6 mice via SG retro-cannulation of 108 pfu/gland of luciferase-encoding adenovirus-5 (AdV). FACS analysis of NK cells (CD45pos/NK1.1pos/CD3neg) was performed in digested SG, while snap-frozen SG tissues were stained for either cytotoxic NK cells (NK1.1pos/CD3neg/Granzyme Bpos) or B (B220pos) and T cells (CD3pos). Negatively selected CFSEpos NK cells from WT-C57BL/6 mice spleens were adoptively transferred in AdV-infected mice to evaluate NK cell recruitment and proliferation in the SG. Virus infected cell clearance by NK cells was assessed with luciferase activity quantification in SG. To achieve NK cell depletion, 200μg of anti-NK1.1 antibody were administered via intraperitoneal injection every 48h. Anti-nuclear antibodies were assessed on Hep-2-coated microscope slides, and anti-AdV antibodies with ELISA assay. Results AdV infection in SG induced an early NK cell recruitment peaking at 4 days post-cannulation (dpc). This is due to an active NK cell migration to the SG at 1dpc followed by their intra-SG proliferation at 3-5dpc, as shown by cell transfer experiments. NK cells preferentially accumulate before B/T cell aggregates formation. Activating markers NKp46 and CD69 were up regulated from 5dpc onwards when an increased degranulation potential was observed. Accordingly, NK cells, rather then cytotoxic T cells, mainly mediated cytotoxic activity, as measured by granzyme B expression, at 4/5dpc. NK depletion impaired viral control at 3dpc but not at later time-points. Conversely, NK depletion did not affect the formation of SS-like inflammatory foci or lymphoid chemokine expression. Moreover, NK cells did not influence the production of ANA or anti-AdV antibodies. Conclusions Here we show that NK cells are actively recruited to the inflammatory site of the SG and are critically involved in the early immune control of AdV infection in this organ but are dispensable for the development of SS-like inflammatory foci and autoimmunity. References Schleinitz N1, Vély F, Harlé JR, Vivier E. Natural killer cells in human autoimmune diseases. Immunology. 2010 Dec;131(4):451-8. Disclosure of Interest None declared

A6.2 Inducible Tertiary Lymphoid Structures and Autoimmunity in a Novel model of Sialoadenitis in Wild-Type Mice

Introduction Salivary glands of patients with Sjögren’s syndrome (SS) develop ectopic lymphoid structures (ELS) characterised by B/T cell compartmentalisation, the formation of high endothelial venules (HEV), follicular dendritic cell networks (FDCs), functional B cell activation with expression of activation-induced cytidine deaminase (AID) as well as local differentiation of autoreactive plasma cells. The mechanisms triggering ELS formation, autoimmunity and exocrine dysfunction in SS are largely unknown. Here we present a novel model of inducible ectopic lymphoid tissue formation, breach of humoral self-tolerance and salivary hypofunction following delivery of a replication-deficient adenovirus-5 (AdV5) in submandibular glands of C57BL/6 mice through retrograde excretory duct cannulation. Materials and Methods Luciferase- or LacZ-encoding Ad5 were delivered in C57BL/6 mice salivary glands (SG) through retrograde cannulation. SGs were collected at various time-points 1, 2 and 3 weeks post-cannulation (pc) and frozen sections were graded for infiltration and stained for T/B cell segregation, FDCs and HEV markers. Submandibular salivary flow was induced by pilocarpine stimulation and the amount of saliva measured. Expression of TLS-related genes was investigated by TaqMan-PCR. Anti-viral antibodies and autoantibodies were detected by IF and western blot. Results In this model, inflammation rapidly and consistently evolves from diffuse infiltration towards the development of SS-like periductal lymphoid aggregates within 2 weeks from AdV delivery. These infiltrates progressively acquire ELS features and support functional GL7+/AID+ germinal centres. Formation of ELS is preceded by ectopic expression of lymphoid chemokines CXCL13, CCL21 and CCL19 as well as lymphotoxin-β and is associated with development of anti-nuclear antibodies in 75% of the mice. Finally, reduction in salivary flow was observed over 3 weeks post AdV infection consistent with exocrine gland dysfunction as a consequence of the inflammatory response. Conclusions This novel model has the potential to unravel the cellular and molecular mechanisms regulating ELS formation and their role in exocrine dysfunction and autoimmunity in SS.