Serum and Tissue Biomarkers Associated With Composite of Relevant Endpoints for Sjögren Syndrome (CRESS) and Sjögren Tool for Assessing Response (STAR) to B Cell-Targeted Therapy in the Trial of Anti-B Cell Therapy in Patients With Primary Sjögren Syndrome (TRACTISS).

OBJECTIVE: This study aimed to identify peripheral and salivary gland (SG) biomarkers of response/resistance to B cell depletion based on the novel concise Composite of Relevant Endpoints for Sjögren Syndrome (cCRESS) and candidate Sjögren Tool for Assessing Response (STAR) composite endpoints. METHODS: Longitudinal analysis of peripheral blood and SG biopsies was performed pre- and post-treatment from the Trial of Anti-B Cell Therapy in Patients With Primary Sjögren Syndrome (TRACTISS) combining flow cytometry immunophenotyping, serum cytokines, and SG bulk RNA sequencing. RESULTS: Rituximab treatment prevented the worsening of SG inflammation observed in the placebo arm, by inhibiting the accumulation of class-switched memory B cells within the SG. Furthermore, rituximab significantly down-regulated genes involved in immune-cell recruitment, lymphoid organization alongside antigen presentation, and T cell co-stimulatory pathways. In the peripheral compartment, rituximab down-regulated immunoglobulins  and auto-antibodies together with pro-inflammatory cytokines and chemokines. Interestingly, patients classified as responders  according to STAR displayed significantly higher baseline levels of C-X-C motif chemokine ligand-13 (CXCL13), interleukin (IL)-22, IL-17A, IL-17F, and tumor necrosis factor-α (TNF-α), whereas a longitudinal analysis of serum T cell-related cytokines showed a selective reduction in both STAR and cCRESS responder patients. Conversely, cCRESS response was better associated with biomarkers of SG immunopathology, with cCRESS-responders showing a significant decrease in SG B cell infiltration and reduced expression of transcriptional gene modules related to T cell costimulation, complement activation, and Fcγ-receptor engagement. Finally, cCRESS and STAR response were associated with a significant improvement in SG exocrine function linked to transcriptional evidence of SG epithelial and metabolic restoration. CONCLUSION: Rituximab modulates both peripheral and SG inflammation, preventing the deterioration of exocrine function with functional and metabolic restoration of the glandular epithelium. Response assessed by newly developed cCRESS and STAR criteria was associated with differential modulation of peripheral and SG biomarkers, emerging as novel tools for patient stratification.

The glucose transporter 2 regulates CD8+ T cell function via environment sensing.

T cell activation is associated with a profound and rapid metabolic response to meet increased energy demands for cell division, differentiation and development of effector function. Glucose uptake and engagement of the glycolytic pathway are major checkpoints for this event. Here we show that the low-affinity, concentration-dependent glucose transporter 2 (Glut2) regulates the development of CD8+ T cell effector responses in mice by promoting glucose uptake, glycolysis and glucose storage. Expression of Glut2 is modulated by environmental factors including glucose and oxygen availability and extracellular acidification. Glut2 is highly expressed by circulating, recently primed T cells, allowing efficient glucose uptake and storage. In glucose-deprived inflammatory environments, Glut2 becomes downregulated, thus preventing passive loss of intracellular glucose. Mechanistically, Glut2 expression is regulated by a combination of molecular interactions involving hypoxia-inducible factor-1 alpha, galectin-9 and stomatin. Finally, we show that human T cells also rely on this glucose transporter, thus providing a potential target for therapeutic immunomodulation.

Immunofibroblasts regulate LTα3 expression in tertiary lymphoid structures in a pathway dependent on ICOS/ICOSL interaction.

Immunofibroblasts have been described within tertiary lymphoid structures (TLS) that regulate lymphocyte aggregation at sites of chronic inflammation. Here we report, for the first time, an immunoregulatory property of this population, dependent on inducible T-cell co-stimulator ligand and its ligand (ICOS/ICOS-L). During inflammation, immunofibroblasts, alongside other antigen presenting cells, like dendritic cells (DCs), upregulate ICOSL, binding incoming ICOS + T cells and inducing LTα3 production that, in turn, drives the chemokine production required for TLS assembly via TNFRI/II engagement. Pharmacological or genetic blocking of ICOS/ICOS-L interaction results in defective LTα expression, abrogating both lymphoid chemokine production and TLS formation. These data provide evidence of a previously unknown function for ICOSL-ICOS interaction, unveil a novel immunomodulatory function for immunofibroblasts, and reveal a key regulatory function of LTα3, both as biomarker of TLS establishment and as first driver of TLS formation and maintenance in mice and humans.

Clinical and imaging characteristics of patients with COVID-19 predicting hospital readmission after emergency department discharge: a single-centre cohort study in Italy.

OBJECTIVE: We aimed at identifying baseline predictive factors for emergency department (ED) readmission, with hospitalisation/death, in patients with COVID-19 previously discharged from the ED. We also developed a disease progression velocity index. DESIGN AND SETTING: Retrospective cohort study of prospectively collected data. The charts of consecutive patients with COVID-19 discharged from the Reggio Emilia (Italy) ED (2 March 2 to 31 March 2020) were retrospectively examined. Clinical, laboratory and CT findings at first ED admission were tested as predictive factors using multivariable logistic models. We divided CT extension by days from symptom onset to build a synthetic velocity index. PARTICIPANTS: 450 patients discharged from the ED with diagnosis of COVID-19. MAIN OUTCOME MEASURE: ED readmission within 14 days, followed by hospitalisation/death. RESULTS: Of the discharged patients, 84 (18.7%) were readmitted to the ED, 61 (13.6%) were hospitalised and 10 (2.2%) died. Age (OR=1.05; 95% CI 1.03 to 1.08), Charlson Comorbidity Index 3 versus 0 (OR=11.61; 95% CI 1.76 to 76.58), days from symptom onset (OR for 1-day increase=0.81; 95% CI 0.73 to 0.90) and CT extension (OR for 1% increase=1.03; 95% CI 1.01 to 1.06) were associated in a multivariable model for readmission with hospitalisation/death. A 2-day lag velocity index was a strong predictor (OR for unit increase=1.21, 95% CI 1.08 to 1.36); the model including this index resulted in less information loss. CONCLUSIONS: A velocity index combining CT extension and days from symptom onset predicts disease progression in patients with COVID-19. For example, a 20% CT extension 3 days after symptom onset has the same risk as does 50% after 10 days.

NKp30 Receptor Upregulation in Salivary Glands of Sjögren's Syndrome Characterizes Ectopic Lymphoid Structures and Is Restricted by Rituximab Treatment.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disease resulting from the inflammatory infiltration of exocrine glands, mainly salivary and lacrimal glands, leading to secretory dysfunction and serious complications including debilitating fatigue, systemic autoimmunity, and lymphoma. Like other autoimmune disorders, a strong interferon (IFN) signature is present among subsets of pSS patients, suggesting the involvement of innate immunity in pSS pathogenesis. NCR3/NKp30 is a natural killer (NK) cell-specific activating receptor regulating the cross talk between NK and dendritic cells including type II IFN secretion upon NK-cell activation. A genetic association between single-nucleotide polymorphisms (SNPs) in the NCR3/NKp30 promoter gene and a higher susceptibility for pSS has been previously described, with pSS patients most frequently carrying the major allele variant associated with a higher NKp30 transcript and IFN-γ release as a consequence of the receptor engagement. In the present study, we combined RNA-sequencing and histology from pSS salivary gland biopsies to better characterize NKp30 (NCR3) and its ligand B7/H6 (NCR3LG1) in pSS salivary gland tissues. Levels of NCR3/NKp30 were significantly increased both in salivary glands and in circulating NK cells of pSS patients compared with sicca controls, especially in salivary glands with organized ectopic lymphoid structures. In line with this observation, a strong correlation between NCR3/NKp30 levels and salivary gland infiltrating immune cells (CD3, CD20) was found. Furthermore, NCR3/NKp30 levels also correlated with higher IFN-γ, Perforin, and Granzyme-B expression in pSS SGs with organized ectopic lymphoid structures, suggesting an activation state of NK cells infiltrating SG tissue. Of note, NKp30+ NK cells accumulated at the border of the inflammatory foci, while the NKp30 ligand, B7/H6, is shown to be expressed mainly by ductal epithelial cells in pSS salivary glands. Finally, immunomodulatory treatment, such as the B-cell depleting agent rituximab, known to reduce the infiltration of immune cells in pSS SGs, prevented the upregulation of NCR3/NKp30 within the glands.

Stepwise changes in the murine salivary gland immune response during virally-induced ectopic lymphoid structure formation.

OBJECTIVES: Sjögren's syndrome (SS) is a chronic autoimmune disease characterised by lymphocytic infiltration into the salivary glands (SG) and, in a subset of patients, formation of ectopic lymphoid structures (ELS) in the glands. However, the mechanisms of how ELS form ectopically are not fully elucidated. Here we used a viral inducible murine model of ELS formation in the SG to elucidate the key immunological steps regulating the formation of ELS in the SG. METHODS: We have utilised an inducible murine model of sialadenitis whereby retrograde cannulation of the submandibular SG with a replication-deficient adenovirus 5 leads to the formation of ELS. Flow cytometry, immunofluorescence and gene expression was performed on the SGs at regular time points after cannulation to follow the organisation of ELS. RESULTS: Innate immune cells (neutrophils, eosinophils and monocytes) rapidly infiltrated the SG by 3 days post cannulation (dpc) whereby monocytes started to differentiate into resident macrophages. Myeloid dendritic cells accumulated inside leukocytic aggregates whereas macrophages were excluded from the developing ELS. Meanwhile, CD11b+ cells upregulated Il18, Cxcl13, Ltb, April and other lymphoid genes stimulating the influx of T cells by 12 days and B cells shortly after. Infiltration of T-follicular helper (Tfh) cells correlated with an increase in GL7+ germinal centre B cells, which peaked at 19 dpc. CONCLUSIONS: Immune cell infiltration in virally-infected murine SG follows a highly reproducible step-wise process whereby early innate immune cells reshape the SG myeloid compartment leading to upregulation of genes involved in the ectopic lymphoid neogenesis process. This in turns leads to T and B cell recruitment, differentiation and activation, culminating in the organization of ELS and localised germinal centres responses.

Advanced imaging for quantification of abnormalities in the salivary glands of patients with primary Sjögren's syndrome.

OBJECTIVES: To assess non-invasive imaging for detection and quantification of gland structure, inflammation and function in patients with primary Sjogren's syndrome (pSS) using PET-CT with 11C-Methionine (11C-MET; radiolabelled amino acid), and 18F-fluorodeoxyglucose (18F-FDG; glucose uptake marker), to assess protein synthesis and inflammation, respectively; multiparametric MRI evaluated salivary gland structural and physiological changes. METHODS: In this imaging/clinical/histology comparative study (GSK study 203818; NCT02899377) patients with pSS and age- and sex-matched healthy volunteers underwent MRI of the salivary glands and 11C-MET PET-CT. Patients also underwent 18F-FDG PET-CT and labial salivary gland biopsies. Clinical and biomarker assessments were performed. Primary endpoints were semi-quantitative parameters of 11C-MET and 18F-FDG uptake in submandibular and parotid salivary glands and quantitative MRI measures of structure and inflammation. Clinical and minor salivary gland histological parameter correlations were explored. RESULTS: Twelve patients with pSS and 13 healthy volunteers were included. Lower 11C-MET uptake in parotid, submandibular and lacrimal glands, lower submandibular gland volume, higher MRI fat fraction, and lower pure diffusion in parotid and submandibular glands were observed in patients vs healthy volunteer, consistent with reduced synthetic function. Disease duration correlated positively with fat fraction and negatively with 11C-MET and 18F-FDG uptake, consistent with impaired function, inflammation and fatty replacement over time. Lacrimal gland 11C-MET uptake positively correlated with tear flow in patients, and parotid gland 18F-FDG uptake positively correlated with salivary gland CD20+ B-cell infiltration. CONCLUSION: Molecular imaging and MRI may be useful tools to non-invasively assess loss of glandular function, increased glandular inflammation and fat accumulation in pSS.

One year in review 2020: pathogenesis of primary Sjögren's syndrome.

The pathogenesis of primary Sjögren's syndrome (pSS) remains poorly understood. However, important efforts have been made during the last few months. In this review, following the others of this series we will summarise the most recent literature on pSS pathogenesis focusing in particular on new insights into pSS animal models, genetics and epigenetics, innate and adaptive immune system abnormalities and tertiary lymphoid structures. Hopefully, novel insights into pSS pathogenesis will pave the way to new therapeutic approaches to the disease improving patients' management and prognosis.

The use of digital image analysis in the histological assessment of Sjögren's syndrome salivary glands improves inter-rater agreement and facilitates multicentre data harmonisation.

OBJECTIVES: To assess whether the use of digital image analysis (DIA) in primary Sjögren's syndrome (pSS) for the calculation of the total area of the salivary gland (SG), focus score (FS) and SG area occupied by the inflammatory infiltrate (area fraction, AF), was able to generate reproducible readings among different raters, reducing disagreement. METHODS: Haematoxylin and Eosin digital slides from pSS and non-specific chronic sialadenitis (NSCS) patients were analysed blindly by 4 independent raters among 3 centres. Using an open-source software (QuPath) raters were asked to provide the total area of the gland i) using a grid-based method and ii) a software-based area-calculation tool, iii) the number of inflammatory foci and iv) the total area of the inflammatory infiltrate. Collected data was used to calculate the inter-rater agreement. RESULTS: For the calculation of the total SG area, DIA generated higher agreement among raters than grid-based calculation (inter-class correlation coefficient ICC=0.85 vs 0.98). Agreement for calculated total area of the inflammatory infiltrate (ICC=0.94) and for AF (ICC=0.94) was higher than infiltrates count number (ICC=0.54) and FS (ICC=0.56). AF achieved a 30% improvement over the FS at generating consensus among raters when used as a diagnostic cut-off. CONCLUSIONS: A digital approach achieved a far superior inter-rater agreement when calculating the total area compared to a grid-based approach. The calculation of AF proved superior to FS in correctly classifying pSS vs NSCS biopsies. We suggest that digitally calculated AF should be used alongside FS for large multi-centre studies to improve data harmonisation.

Unique expansion of IL-21+ Tfh and Tph cells under control of ICOS identifies Sjögren's syndrome with ectopic germinal centres and MALT lymphoma.

OBJECTIVES: To explore the relevance of T-follicular-helper (Tfh) and pathogenic peripheral-helper T-cells (Tph) in promoting ectopic lymphoid structures (ELS) and B-cell mucosa-associated lymphoid tissue (MALT) lymphomas (MALT-L) in Sjögren's syndrome (SS) patients. METHODS: Salivary gland (SG) biopsies with matched peripheral blood were collected from four centres across the European Union. Transcriptomic (microarray and quantitative PCR) analysis, FACS T-cell immunophenotyping with intracellular cytokine detection, multicolor immune-fluorescence microscopy and in situ hybridisation were performed to characterise lesional and circulating Tfh and Tph-cells. SG-organ cultures were used to investigate functionally the blockade of T-cell costimulatory pathways on key proinflammatory cytokine production. RESULTS: Transcriptomic analysis in SG identified Tfh-signature, interleukin-21 (IL-21) and the inducible T-cell co-stimulator (ICOS) costimulatory pathway as the most upregulated genes in ELS+SS patients, with parotid MALT-L displaying a 400-folds increase in IL-21 mRNA. Peripheral CD4+CXC-motif chemokine receptor 5 (CXCR5)+programmed cell death protein 1 (PD1)+ICOS+ Tfh-like cells were significantly expanded in ELS+SS patients, were the main producers of IL-21, and closely correlated with circulating IgG and reduced complement C4. In the SG, lesional CD4+CD45RO+ICOS+PD1+ cells selectively infiltrated ELS+ tissues and were aberrantly expanded in parotid MALT-L. In ELS+SG and MALT-L parotids, conventional CXCR5+CD4+PD1+ICOS+Foxp3- Tfh-cells and a uniquely expanded population of CXCR5-CD4+PD1hiICOS+Foxp3- Tph-cells displayed frequent IL-21/interferon-γ double-production but poor IL-17 expression. Finally, ICOS blockade in ex vivo SG-organ cultures significantly reduced the production of IL-21 and inflammatory cytokines IL-6, IL-8 and tumour necrosis factor-α (TNF-α). CONCLUSIONS: Overall, these findings highlight Tfh and Tph-cells, IL-21 and the ICOS costimulatory pathway as key pathogenic players in SS immunopathology and exploitable therapeutic targets in SS.

Impaired Interleukin-27-Mediated Control of CD4+ T Cell Function Impact on Ectopic Lymphoid Structure Formation in Patients With Sjögren's Syndrome.

OBJECTIVE: Ectopic lymphoid structures (ELS) develop at sites of infection, autoimmunity, and cancer. In patients with Sjögren's syndrome (SS), ELS support autoreactive B cell activation and lymphomagenesis. Interleukin-27 (IL-27) is a key regulator of adaptive immunity and limits Th17 cell-driven pathology. We undertook this study to elucidate the role of IL-27 in ELS formation and function in autoimmunity using a murine model of sialadenitis and in patients with SS. METHODS: ELS formation was induced in wild-type and Il27ra-/- mice via salivary gland (SG) cannulation of a replication-deficient adenovirus in the presence or absence of IL-17A neutralization. In SG biopsy samples, IL-27-producing cells were identified by multicolor immunofluorescence microscopy. Lesional and circulating IL-27 levels were determined by gene expression and enzyme-linked immunosorbent assay. The in vitro effect of IL-27 on T cells was assessed using fluorescence-activated cell sorting and cytokine release. RESULTS: In experimental sialadenitis, Il27ra-/- mice had larger and more hyperactive ELS (focus score; P < 0.001), increased autoimmunity, and an expanded Th17 response (P < 0.001), compared to wild-type mice. IL-17 blockade in Il27ra-/- mice suppressed the aberrant ELS response (B and T cell reduction against control; P < 0.01). SS patients displayed increased circulating IL-27 levels (P < 0.01), and in SG biopsy samples, IL-27 was expressed by DC-LAMP+ dendritic cells in association with CD3+ T cells. Remarkably, in SS T cells (but not in T cells from patients with rheumatoid arthritis or healthy controls), IL-27-mediated suppression of IL-17 secretion was severely impaired and associated with an aberrant interferon-γ release upon IL-27 stimulation. CONCLUSION: Our data indicate that the physiologic ability of IL-27 to limit the magnitude and function of ELS through control of Th17 cell expansion is severely impaired in SS patients, highlighting a defective immunoregulatory checkpoint in this condition.

Serum Copeptin levels in the emergency department predict major clinical outcomes in adult trauma patients.

BACKGROUND: Early prognostication in trauma patients is challenging, but particularly important. We wanted to explore the ability of copeptin, the C-terminal fragment of arginine vasopressin, to identify major trauma, defined as Injury Severity Score (ISS) > 15, in a heterogeneous cohort of trauma patients and to compare its performances with lactate. We also evaluated copeptin performance in predicting other clinical outcomes: mortality, hospital admission, blood transfusion, emergency surgery, and Intensive Care Unit (ICU) admission. METHODS: This single center, pragmatic, prospective observational study was conducted at Arcispedale Santa Maria Nuova, a level II trauma center in Reggio Emilia, Italy. Copeptin determination was obtained on Emergency Department (ED) arrival, together with venous lactate. Different outcomes were measured including ISS, Revised Trauma Score (RTS), hospital and ICU admission, blood transfusion, emergency surgery, and mortality. RESULTS: One hundred and twenty five adult trauma patients admitted to the ED between June 2017 and March 2018. Copeptin showed a good ability to identify patients with ISS > 15 (AUC 0.819). Similar good performances were recorded also in predicting other outcomes. Copeptin was significantly superior to lactate in identifying patients with ISS > 15 (P 0.0015), and in predicting hospital admission (P 0.0002) and blood transfusion (P 0.016). Comparable results were observed in a subgroup of patients with RTS 7.84. CONCLUSIONS: In a heterogeneous group of trauma patients, a single copeptin determination at the time of ED admission proved to be an accurate biomarker, statistically superior to lactate for the identification of major trauma, hospital admission, and blood transfusion, while no statistical difference was observed for ICU admission and emergency surgery. These results, if confirmed, may support a role for copeptin during early management of trauma patients.

Interleukin-36 family dysregulation drives joint inflammation and therapy response in psoriatic arthritis.

OBJECTIVES: IL-36 agonists are pro-inflammatory cytokines involved in the pathogenesis of psoriasis. However, their role in the pathogenesis of arthritis and treatment response to DMARDs in PsA remains uncertain. Therefore, we investigated the IL-36 axis in the synovium of early, treatment-naïve PsA, and for comparison RA patients, pre- and post-DMARDs therapy. METHODS: Synovial tissues were collected by US-guided biopsy from patients with early, treatment-naïve PsA and RA at baseline and 6 months after DMARDs therapy. IL-36 family members were investigated in synovium by RNA sequencing and immunohistochemistry, and expression levels correlated with DMARDs treatment response ex vivo. Additionally, DMARDs effects on IL-36 were investigated in vitro in fibroblast-like synoviocytes. RESULTS: PsA synovium displayed a reduced expression of IL-36 antagonists, while IL-36 agonists were comparable between PsA and RA. Additionally, neutrophil-related molecules, which drive a higher activation of the IL-36 pathway, were upregulated in PsA compared with RA. At baseline, the synovial expression of IL-36α was significantly higher in PsA non-responders to DMARDs treatment, with the differential expression being sustained at 6 months post-treatment. In vitro, primary PsA-derived fibroblasts were more responsive to IL-36 stimulation compared with RA and, importantly, DMARDs treatment increased IL-36 expression in PsA fibroblasts. CONCLUSION: The impaired balance between IL-36 agonists-antagonists described herein for the first time in PsA synovium and the decreased sensitivity to DMARDs in vitro may explain the apparent lower efficacy of DMARDs in PsA compared with RA. Exogenous replacement of IL-36 antagonists may be a novel promising therapeutic target for PsA patients.

CXCL13 as biomarker for histological involvement in Sjögren's syndrome.

OBJECTIVES: SS is an autoimmune condition characterized by systemic B-cell activation, autoantibody production and ectopic germinal centres' formation within the salivary gland (SG). The extent of SG infiltrate has been proposed as a biomarker of disease severity. Plasma levels of CXCL13 correlate with germinal centres' activity in animal models and disease severity in SS, suggesting its potential use as a surrogate serum marker to monitor local B-cell activation. The aim of this study was to evaluate the potential role of CXCL13 as a biomarker of SG pathology in two independent SS cohorts. METHODS: 109 patients with SS were recruited at Sapienza University of Rome (Italy) (n = 60), or at Queen Elizabeth Hospital in Birmingham and Barts Health NHS Trust in London (n = 49). Both sera and matched minor SG biopsy were available. Sicca (n = 57) and healthy subjects' (n = 19) sera were used as control. RESULTS: CXCL13 serum level was higher in SS patients compared with controls. Correlations between its serum levels and a series of histomorphological parameters, including size of the aggregates and the presence germinal centres', were observed. CONCLUSION: Our data foster the use of CXCL13 to monitor the extent of local pathology in SS and its validation in longitudinal clinical studies.

NK cell recruitment in salivary glands provides early viral control but is dispensable for tertiary lymphoid structure formation.

Salivary glands (SGs) represent a permissive site for several sialotropic viruses whose persistence is linked to the development of autoimmunity. Natural Killer (NK) cells play a key role in viral clearance but their involvement in viral infection control and in tertiary lymphoid structures (TLS) development within SGs is unknown. By using an inducible model of TLS in the SGs of wild-type C57BL/6 mice, induced by the local delivery of a replication-defective adenovirus (AdV), we demonstrated that circulating NK cells are rapidly recruited to SGs and highly enrich the early inflammatory infiltrate prior to TLS development. NK cells migrating to SGs in response to AdV infection up-regulate NKp46, undergo proliferation, acquire cytotoxic potential, produce Granzyme-B and IFN-γ, and reduce viral load in the acute phase of the infection. Nonetheless, the selective depletion of both circulating and infiltrating NK cells in AdV-infected mice neither affect the development and frequency of TLS nor the onset of autoimmunity. These data demonstrate that, upon local viral delivery of AdV, peripheral NK cells homing to SGs can exert an early control of the viral infection but are dispensable for the formation of TLS and breach of immunologic tolerance.

A clinical and histopathological analysis of the anti-centromere antibody positive subset of primary Sjögren's syndrome.

OBJECTIVES: ACA-positive/primary Sjögren's syndrome (pSS) represents a distinct overlapping entity with intermediate features in between limited systemic sclerosis (lSSc) and pSS. Few data are available on their general risk for lymphoproliferative complications, specifically regarding adverse predictors at the level of minor salivary gland (MSG) histology. The objectives of this work are: a) to characterise, through a detailed immunohistochemistry study, the organisation of the lymphomonocitic infiltrates in ACA-positive/pSS patient vs. ACA-negative/pSS patients focusing on the presence of GC-like structures in minor salivary gland biopsies; b) to compare the frequency of traditional clinical and serological risk factors for lymphoma between the two subgroups. METHODS: We analysed 28 MSG samples from ACA-positive/pSS patients and 43 consecutive MSGs from ACA-negative/pSS, using sequential IHC staining for CD3, CD20 and CD21 in order to define the T/B cell segregation within the periductal infiltrates and presence of ectopic GC-like on the detection of GC-like structures. Clinical and serological data of all the patients were retrieved and analysed. RESULTS: Ectopic lymphoid structures (ELS) with GC-like structures were observed in 7 out of 28 ACA-positive/pSS patients (25%) and in 13 out of 43 ACA-negative/pSS patients (30.2%). Similarly, no statistical significant difference was found between the two groups as far as the classical pSS risk factors for lymphoproliferative complications was concerned (i.e. salivary gland enlargement, purpura, low C4, leukocytopenia, clonal gammopathy). Finally, the 3 cases of non-Hodgkin's lymphoma observed were equally distributed between the two subsets. CONCLUSIONS: Overall, this study indicates that ACA-positive/and ACA-negative pSS patients apparently present a similar risk for lymphoproliferative complications as suggested indirectly by the analogies between the two groups observed at the histopathology level.

Current views on the pathogenesis of Sjögren's syndrome.

PURPOSE OF REVIEW: The purpose of this review is to provide an insight into the pathophysiological mechanisms involved in the pathogenesis of primary Sjögren's 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.

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

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.

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

Sjögren's 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.

Transcriptomic profiling of the development of the inflammatory response in human monocytes in vitro.

Monocytes/macrophages are key players in all phases of physiological and pathological inflammation. To understanding the regulation of macrophage functional differentiation during inflammation, we designed an in vitro model that recapitulates the different phases of the reaction (recruitment, initiation, development, and resolution), based on human primary blood monocytes exposed to sequential changes in microenvironmental conditions. All reaction phases were profiled by transcriptomic microarray analysis. Distinct clusters of genes were identified that are differentially regulated through the different phases of inflammation. The gene sets defined by GSEA analysis revealed that the inflammatory phase was enriched in inflammatory pathways, while the resolution phase comprised pathways related to metabolism and gene rearrangement. By comparing gene clusters differentially expressed in monocytes vs. M1 and vs. M2 macrophages extracted from an in-house created meta-database, it was shown that cells in the model resemble M1 during the inflammatory phase and M2 during resolution. The validation of inflammatory and transcriptional factors by qPCR and ELISA confirmed the transcriptomic profiles in the different phases of inflammation. The accurate description of the development of the human inflammatory reaction provided by this in vitro kinetic model can help in identifying regulatory mechanisms in physiological conditions and during pathological derangements.