Interplay between inflammation, autoimmunity and regeneration in the NOD mouse model of type 1 diabetes and Sjogren’s Syndrome.

PhDA continuous process of tissue remodelling and regeneration is a fundamental feature of the homeostatic response of the target organ of several autoimmune diseases. In type 1 diabetes (T1D) the β cell mass is in a constant process of death and renewal in order to regenerate the islets damaged by the autoimmune process. The relationship linking inflammation and regeneration during autoimmunity remains elusive. Reg genes, a multigene family discovered using cDNA libraries derived from rat regenerating islets, have been suggested to play an important role in epithelial regeneration not only in the pancreas but also in the salivary glands (SG) of Sjogren’s Syndrome (SS) during autoimmune sialoadenitis. Both in patients and animal models of T1D and SS, the chronic inflammatory/autoimmune process is heterogeneous and display high immunological variability. In particular, in a sizeable subset of cases, inflammatory lesions display ectopic lymphoid structures (ELS) characterised by T/B cell segregation, follicular dendritic cells networks and differentiation of germinal center B cells. However, there is limited evidence on the cellular and molecular mechanisms underlying ELS formation and their contribution to autoimmunity in the pancreas during autoimmune insulitis and in SG during autoimmune sialoadenitis. In this PhD project, I used the NOD mouse model of T1D and SS in order to investigate i) the cellular and molecular mechanisms regulating ELS formation, ii) the functionality of ELS in supporting in situ autoreactive B cell differentiation and iii) the relationship between formation of ELS and the expression of REG genes. In this work I showed that ELS formation was preceded by local up-regulation of lymphotoxins (LTαβ) and lymphoid chemokines CXCL13 and CCL19 and that, once formed, ELS were fully functional in promoting autoreactive B cell activation. Importantly, inhibition of the LT-β pathway prevented the formation of ELS and B cell autoimmunity. Finally, I showed that the expression pattern of Reg genes was strictly related to the development of inflammatory infiltrates in NOD 7 mice and that Reg proteins were target of the autoimmune process itself, as shown by the development of anti-Reg1 antibodies in patients with T1D. Overall, these results suggest that the processes of destruction and regeneration occurring in chronic autoimmune/inflammatory diseases are strongly interdependent whereby autoimmunity may be further enhanced by the attempt to regenerate

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

Background: Primary Sjögren’s syndrome (pSS) is a common chronic autoimmune disease characterized by lymphocytic infiltration of exocrine glands and peripheral lymphocyte perturbation. In the current study, we aimed to investigate the possible pathogenic implication of autophagy in T lymphocytes in patients with pSS. Methods: Thirty consecutive pSS patients were recruited together with 20 patients affected by sicca syndrome a nd/or chronic sialoadenitis and 30 healthy controls. Disease activity and damage were evaluated according to SS disease activity index, EULAR SS disease activity index, and SS disease damage index. T lymphocytes were analyzed for the expression of autophagy-specific markers by biochemical, molecular, and histological assays in peripheral blood and labial gland biopsies. Serum interleukin (IL)-23 and IL-21 levels were quantified by enzyme-linked immunosorbent assay. Results: Our study provides evidence for the first time that autophagy is upregulated in CD4+ T lymphocyte salivary glands from pSS patients. Furthermore, a statistically significant correlation was detected between lymphocyte autophagy levels, disease activity, and damage indexes. We also found a positive correlation between autophagy enhancement and the increased salivary gland expression of IL-21 and IL-23, providing a further link between innate and adaptive immune responses in pSS. Conclusions: These findings suggest that CD4+ T lymphocyte autophagy could play a key role in pSS pathogenesis. Additionally, our data highlight the potential exploitation of T cell autophagy as a biomarker of disease activity and provide new ground to verify the therapeutic implications of autophagy as an innovative drug target in pSS

Cytokines and inflammatory mediators: 25. Certolizumab Pegol has a Different Profile from the other Anti-TNFS, Including Golimumab, in a Variety of in Vitro Assays

Background: Activities of the anti-TNFs, certolizumab pegol (CZP), etanercept (ETA), infliximab (IFX) and adalimumab (ADA), have been compared in a range of in vitro assays. CZP is the only licensed PEGylated Fab' anti-TNF; ETA is a fusion protein with an IgG1 Fc, and IFX and ADA are both antibodies with an IgG1 Fc. Golimumab (GLM) is a monoclonal IgG1 TNF inhibitor recently approved for a number of indications; it is thus of interest to assess the in vitro activity of GLM. In vitro assays previously used were neutralisation of TNF in the L929 bioassay, inhibition of LPS-driven cytokine production by monocytes, induction of apoptosis in activated lymphocytes and monocytes, and induction of neutrophil necrosis. Methods: Neutralisation of human TNF was assessed in the L929 bioassay using a range of concentrations of the anti-TNFs and a fixed concentration of TNF (100 pg/mL). Activity of the anti-TNFs at inhibiting LPS-driven IL-1β secretion by monocytes was assessed by incubating peripheral blood monocytes with various concentrations of the anti-TNF for 1 hour (hr) and then washing the cells. LPS was added for 4 hrs, the supernatants collected and the IL-1β level measured by ELISA. To assess induction of apoptosis, peripheral blood lymphocytes were activated for 2 days with 2 μg/mL CD3/CD28 and monocytes with 300 U/mL IL-4 and GMCSF for 3 days. The effect of the anti-TNFs on apoptosis was assessed by Annexin V staining using flow cytometry 24 hrs later. The effect of the anti-TNFs on neutrophil necrosis was determined by measuring myeloperoxidase release after 12 hrs. An isotype-matched control was used in all assays except the L929 bioassay. Results: IC90 neutralisation activity of the anti-TNFs in the L929 bioassay was 0.3 ng/mL for ETA, 4 ng/mL for GLM, 15 ng/mL for ADA, and 20 ng/mL for IFX, compared with 2.5 ng/mL for CZP. CZP was the most potent inhibitor of LPS-driven IL-1β secretion (IC50 ∼0.1 ng/mL), followed by GLM (20 ng/mL) and IFX (50 ng/mL). GLM, ADA, IFX and ETA induced apoptosis of monocytes and lymphocytes to a similar degree reaching a level of 23% and ∼40% at 100 μg/mL, respectively. CZP caused no increase in apoptosis above the levels seen with the isotype-matched control. In the neutrophil necrosis assay, ADA,IFX and GLM caused ∼70% necrosis at 100 μg/mL, and ETA 48%. CZP did not increase the level of necrosis above the level of the control. Conclusions: Bioactivity of the IgG1 molecules GLM, IFX and ADA in neutralising human TNF was inferior to that of CZP and ETA. CZP, the only PEGylated anti-TNF, had a different profile to the other anti-TNFs as it was the most potent at inhibiting LPS-driven IL-1β production by monocytes, did not induce apoptosis of activated monocytes and lymphocytes, and did not cause neutrophil necrosis. The clinical relevance of these in vitro effects is unknown. Nevertheless, these assays show interesting in vitro differences between the anti-TNFs. Disclosure statement: G.F. and A.N. are employees of UC

CX3CL1 and CX3CR1 expression in tertiary lymphoid structures in salivary gland infiltrates: fractalkine contribution to lymphoid neogenesis in Sjogren's syndrome

Methods. We assessed the presence of CX3CL1 protein in sera by ELISA in 21 patients with primary SS, 11 patients with Sicca syndrome (Sicca), 20 RA patients and 10 blood donors. Histological evaluation was performed on sequential sections of salivary gland tissue. Using TaqMan RT-PCR we studied CX3CL1 and CX3CR1 mRNA expression in salivary gland tissues from a molecular point of view. Results. Increased serum levels of CX3CL1 protein were observed in SS patients compared with controls (P < 0.0001) and in RA patients compared with controls (P < 0.0001), but no difference was found between Sicca patients and controls (P = 0.22). We identified histologically the cells expressing CX3CL1 and CX3CR1 in salivary glands of SS patients and we localized the molecule within tertiary lymphoid structures. Finally, the mRNA levels of the CK and its receptor were up-regulated in SS salivary glands. Conclusion. We believe that our findings point to the need for future studies on CX3CL1 and CX3CR1 proteins as contributors to the formation of ectopic GCs and possibly as a new tool in the evaluation and diagnosis of SS

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 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 CD4CXC-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 CD4CD45ROICOSPD1 cells selectively infiltrated ELS+ tissues and were aberrantly expanded in parotid MALT-L. In ELS+SG and MALT-L parotids, conventional CXCR5CD4PD1ICOSFoxp3 Tfh-cells and a uniquely expanded population of CXCR5CD4PD1ICOSFoxp3 Tph-cells displayed frequent IL-21/interferon-γ double-production but poor IL-17 expression. Finally, ICOS blockade in 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

Additional file 1: Figure S1. of CD4 T lymphocyte autophagy is upregulated in the salivary glands of primary Sjögren’s syndrome patients and correlates with focus score and disease activity

IL-23p19 and IL-21 mRNA level correlations with mRNA levels of autophagy genes. IL-23p19 and IL-21 mRNA levels were directly and significantly correlated with the mRNA levels of ATG16L1, ATG5, and IRGM autophagy genes. (TIF 50 kb