The classical NLRP3 inflammasome controls FADD unconventional secretion through microvesicle shedding.

Fas-associated death domain (FADD) is a key adaptor molecule involved in numerous physiological processes including cell death, proliferation, innate immunity and inflammation. Therefore, changes in FADD expression have dramatic cellular consequences. In mice and humans, FADD regulation can occur through protein secretion. However, the molecular mechanisms accounting for human FADD secretion were still unknown. Here we report that canonical, non-canonical, but not alternative, NLRP3 inflammasome activation in human monocytes/macrophages induced FADD secretion. NLRP3 inflammasome activation by the bacterial toxin nigericin led to the proinflammatory interleukin-1ß (IL-1ß) release and to the induction of cell death by pyroptosis. However, we showed that FADD secretion could occur in absence of increased IL-1ß release and pyroptosis and, reciprocally, that IL-1ß release and pyroptosis could occur in absence of FADD secretion. Especially, FADD, but not IL-1ß, secretion following NLRP3 inflammasome activation required extracellular glucose. Thus, FADD secretion was an active process distinct from unspecific release of proteins during pyroptosis. This FADD secretion process required K+ efflux, NLRP3 sensor, ASC adaptor and CASPASE-1 molecule. Moreover, we identified FADD as a leaderless protein unconventionally secreted through microvesicle shedding, but not exosome release. Finally, we established human soluble FADD as a new marker of joint inflammation in gout and rheumatoid arthritis, two rheumatic diseases involving the NLRP3 inflammasome. Whether soluble FADD could be an actor in these diseases remains to be determined. Nevertheless, our results advance our understanding of the mechanisms contributing to the regulation of the FADD protein expression in human cells.

Modulatory role of the anti-apoptotic protein kinase CK2 in the sub-cellular localization of Fas associated death domain protein (FADD).

The Fas associated death domain protein (FADD) is the key adaptor molecule of the apoptotic signal triggered by death receptors of the TNF-R1 superfamily. Besides its crucial role in the apoptotic machinery, FADD has proved to be important in many biological processes like tumorigenesis, embryonic development or cell cycle progression. In a process to decipher the regulatory mechanisms underlying FADD regulation, we identified the anti-apoptotic kinase, CK2, as a new partner and regulator of FADD sub-cellular localization. The blockade of CK2 activity induced FADD re-localization within the cell. Moreover, cytoplasmic FADD was increased when CK2ß was knocked down. In vitro kinase and pull down assays confirmed that FADD could be phosphorylated by the CK2 holoenzyme. We found that phosphorylation is weak with CK2α alone and optimal in the presence of stoichiometric amounts of CK2α catalytic and CK2ß regulatory subunit, showing that FADD phosphorylation is undertaken by the CK2 holoenzyme in a CK2ß-driven fashion. We found that CK2 can phosphorylate FADD on the serine 200 and that this phosphorylation is important for nuclear localization of FADD. Altogether, our results show for the first time that multifaceted kinase, CK2, phosphorylates FADD and is involved in its sub-cellular localization. This work uncovered an important role of CK2 in stable FADD nuclear localization.

Implication of clusterin in TNF-α response of rheumatoid synovitis: lesson from in vitro knock-down of clusterin in human synovial fibroblast cells.

Recently clusterin (CLU) was reported to be an inhibitor of NF-κB pathway and involved in rheumatoid arthritis (RA) synovitis. This study was designed to decipher the molecular network linked to CLU expression in FLS (fibroblast-like synoviocytes) and evaluate the consequences of its low expression in conditions of TNF-α stimulation. FLS were transfected with siRNA for CLU or not and cultured for 24 and 48 h with TNF-α or not. Pan-genomic gene expression was assayed by DNA microarray. The gene network around CLU and gene interactions were analyzed with the Ingenuity Pathway Analysis software. Downregulation of CLU resulted in modification of the expression of genes known to be directly linked to CLU and for almost 5% of the tested genes (857 out of 17,225); the upregulation of a small group of gene (e.g., TIAM1) emphasizes the hypothetical role of CLU in the pseudotumoral characteristic of FLS. The comparison of gene expression with or without TNF stimulation allowed the classification of sampled with good concordance. Moreover, differential comparison showed that CLU downregulation in RA led to a profound modification of the TNF-α response as three sets of genes emerged: 497 genes modulated by siCLU transfection with TNF stimulation, 356 genes modified because of TNF stimulation only, and 484 genes modulated during TNF stimulation with CLU expression (e.g., IL-8 and Wnt signaling genes). Using a global two-way ANOVA we could identify a set of genes defining a molecular signature of TNF response directly influenced by CLU. These results (based on differential gene expression patterns) argue that CLU downregulation in FLS alters their aggressiveness in RA synovitis.

Treatment of collagen-induced arthritis by Natura-alpha via regulation of Th-1/Th-17 responses.

Cytokines and CD4(+) Th cells play a crucial role in the pathogenesis of rheumatoid arthritis. Among the Th populations, Th-1 and Th-17 have been described as pathogenic in collagen-induced arthritis (CIA) whereas Th-2 and Treg were found to have protective effects. The objective of this study was to examine the affect of Natura-alpha, a newly developed cytokine regulator, on CIA and on Th cell development. Natura-alpha treatment was administered before or during arthritis induction. Anti-type II collagen antibodies and cytokine expression were evaluated by ELISA. Emergence of CD4(+)CD25(+)Foxp3(+) T cells was assessed by flow cytometry. Th-17 differentiation of naive CD4 T cells was assessed in cultures with anti-CD3 and anti-CD28. We showed that Natura-alpha both prevented and treated CIA. We further demonstrated that in vivo treatment with Natura-alpha inhibited IL-17 production and anti-type II collagen IgG development. We showed in vitro, using an APC-free system, that Natura-alpha acted directly on differentiating T cells and inhibiting the formation of Th-1 and Th-17 cells but did not affect Th-2 cells. Since Natura-alpha inhibits a large spectrum of important pathogenic factors in CIA, it may provide a new and powerful approach to the treatment of rheumatoid arthritis and other inflammatory diseases.

Adenosine receptors control a new pathway of Fas-associated death domain protein expression regulation by secretion.

FADD is the key adaptor transmitting the apoptotic signal mediated by death receptors. We have previously shown that FADD protein expression could be lost in vivo in cancerous cells, in mice and humans, and be used as prognostic factor. Furthermore, loss of FADD could contribute to tumor progression and aggressiveness. However, the mechanism accounting for the loss of FADD was unknown. Using in vitro-cultured mouse organ models, we demonstrated that loss of FADD occurred through a new regulatory pathway of FADD expression by secretion. The secretion of FADD is an active release following shedding of microvesicles derived from the plasma membrane. In our experimental settings, this phenomenon was restricted to 6 of 12 FADD-expressing organs. This process is calcium- and adenosine-dependent. Moreover, we identified the two receptors with low affinity to adenosine, namely A(2B) and A(3) adenosine receptors, as regulators of the FADD secretion process. Furthermore, we showed that modulating A(3) adenosine receptor can convert a nonsecreting organ into a FADD-secreting one. Finally, we reported that mouse FADD release occurred in vivo during tumor disease. These results demonstrate the existence of a new localization site (in microvesicles) and regulatory mechanism (by secretion) of the FADD protein, and the implication of adenosine receptors in this process. These data open a new field of investigation consisting of the possibility to regulate FADD expression via the modulation of adenosine receptors, which constitutes a therapeutic target in diseases in which FADD-mediated signaling is impaired.

Insights into spatial configuration of a galactosylated epitope required to trigger arthritogenic T-cell receptors specific for the sugar moiety.

The immunodominant epitope of bovine type II collagen (CII256-270) in Aq mice carries a hydroxylysine-264 linked galactose (Gal-Hyl264), the recognition of which is central to the development of collagen-induced arthritis. This study explores the molecular interactions involved in the engagement of T-cell receptors (TCRs) with such epitopes. Responses of three anti-CII T-cell hybridomas and clone A9.2 (all sharing close TCR sequences) to a panel of CII256-270 analogues incorporating Gal-Hyl264 with a modified side chain were determined. Recognition of naturally occurring CII256-270 peptides by either group of T cells depended strictly upon the presence of the carbohydrate and, more precisely, its intact HO-4 group. Modifications of primary amino group on the hydroxylysine side chain eliminated T-cell reactivity, notwithstanding the presence of the galactosyl moiety. Moderate stereochemical changes, such as altered sugar orientation and methylation at the galactose anchor position, were still permissive. Conversely, robust transformations affecting the relative positions of the key elements were detrimental to TCR recognition. To conclude, these data provide strong new experimental evidence that integrity of both galactose HO-4 and hydroxylysine side chain primary amino groups are mandatory for activation of anti-Gal-Hyl264 TCRs. They also indicate that there is a certain degree of TCR plasticity in peptide-TCR interactions.

Characterization and functional consequences of underexpression of clusterin in rheumatoid arthritis.

We previously compared by microarray analysis gene expression in rheumatoid arthritis (RA) and osteoarthritis (OA) tissues. Among the set of genes identified as a molecular signature of RA, clusterin (clu) was one of the most differentially expressed. In the present study we sought to assess the expression and the role of CLU (mRNA and protein) in the affected joints and in cultured fibroblast-like synoviocytes (FLS) and to determine its functional role. Quantitative RT-PCR, Northern blot, in situ hybridization, immunohistochemistry, and Western blot were used to specify and quantify the expression of CLU in ex vivo synovial tissue. In synovial tissue, the protein was predominantly expressed by synoviocytes and it was detected in synovial fluids. Both full-length and spliced isoform CLU mRNA levels of expression were lower in RA tissues compared with OA and healthy synovium. In synovium and in cultured FLS, the overexpression of CLU concerned all protein isoforms in OA whereas in RA, the intracellular forms of the protein were barely detectable. Transgenic overexpression of CLU in RA FLS promoted apoptosis within 24 h. We observed that CLU knockdown with small interfering RNA promoted IL-6 and IL-8 production. CLU interacted with phosphorylated IkappaBalpha. Differential expression of CLU by OA and RA FLS appeared to be an intrinsic property of the cells. Expression of intracellular isoforms of CLU is differentially regulated between OA and RA. We propose that in RA joints, high levels of extracellular CLU and low expression of intracellular CLU may enhance NF-kappaB activation and survival of the synoviocytes.

Activation of IFN pathways and plasmacytoid dendritic cell recruitment in target organs of primary Sjögren's syndrome.

Gene expression analysis of target organs might help provide new insights into the pathogenesis of autoimmune diseases. We used global gene expression profiling of minor salivary glands to identify patterns of gene expression in patients with primary Sjögren's syndrome (pSS), a common and prototypic systemic autoimmune disease. Gene expression analysis allowed for differentiating most patients with pSS from controls. The expression of 23 genes in the IFN pathways, including two Toll-like receptors (TLR8 and TLR9), was significantly different between patients and controls. Furthermore, the increased expression of IFN-inducible genes, BAFF and IFN-induced transmembrane protein 1, was also demonstrated in ocular epithelial cells by quantitative RT-PCR. In vitro activation showed that these genes were effectively modulated by IFNs in salivary gland epithelial cells, the target cells of autoimmunity in pSS. The activation of IFN pathways led us to investigate whether plasmacytoid dendritic cells were recruited in salivary glands. These IFN-producing cells were detected by immunohistochemistry in all patients with pSS, whereas none was observed in controls. In conclusion, our results support the pathogenic interaction between the innate and adaptive immune system in pSS. The persistence of the IFN signature might be related to a vicious circle, in which the environment interacts with genetic factors to drive the stimulation of salivary TLRs.

Interleukin-32, CCL2, PF4F1 and GFD10 are the only cytokine/chemokine genes differentially expressed by in vitro cultured rheumatoid and osteoarthritis fibroblast-like synoviocytes.

Since cytokines and chemokines are important actors in rheumatoid arthritis (RA), the aim of this study was to compare the gene expression profiles in cultured fibroblast-like synoviocytes (FLS) obtained from patients with either RA, or osteoarthritis (OA), focusing our analysis on genes for cytokines and chemokines, and their respective receptors. Gene expression in cultured FLS (third passage) from eight patients with RA (RA-FLS) were compared with gene expression in cultured FLS from nine patients with OA (OA-FLS) using Affymetrix Human Genome U133 Plus 2.0 Array microarray, allowing analysis of over 54,000 transcripts. Among the 171 genes studied (241 probes), limiting the selection of differentially expressed genes to a significant value (p < 0.05), and a differential ratio of expression > 1.6, only four genes, namely IL-32, CCL2, PF4F1 and GDF10 were found to be differentially expressed. Out of these four genes, only higher expression of CCL2 has been reported previously in RA. The newly described cytokine IL-32 was the most prominently differentially expressed gene in the present study, with higher expression in RA-FLS than in OA-FLS (p < 0.0073). IL-32 might have a previously unidentified pivotal role in RA.

Loss of FADD protein expression results in a biased Fas-signaling pathway and correlates with the development of tumoral status in thyroid follicular cells.

Downregulation of proapoptotic molecules like Fas or caspase 8, or upregulation of antiapoptotic molecules like FLICE inhibitory protein has been suggested to be a regulatory mechanism set up by tumor cells to block the death signal received via death receptors. In an in-depth study of the Fas/FasL-signaling pathway in thyroid tumor development, we have demonstrated that tumor cells specifically downregulate the multideath receptor adapter Fas-associated death domain (FADD). The regulation of FADD expression occurred only at the protein level. Furthermore, in the absence of FADD, Fas-signaling resulted in accelerated growth of thyrocytes. Since thyrocytes also acquired FasL expression during tumor development, the absence of FADD protein could lead to greater resistance to numerous death receptor-mediated apoptosis, stimulation of their own proliferation through Fas/FasL interaction, and the capacity to counter-attack the infiltrating lymphocytes.

IL-10 is necessary for FasL-induced protection from experimental autoimmune thyroiditis but not for FasL-induced immune deviation.

Ectopic expression of FasL on thyrocytes confers immune privilege status to the thyroid by inducing apoptosis of Fas-expressing autoimmune effector T cells and anti-thyroglobulin (Tg) immune deviation away from the T1 type. Fas-mediated apoptosis of lymphoid cells leads to rapid production of anti-inflammatory cytokines such as IL-10. On the other hand, cytokines play a crucial role in the immunoregulation and pathology of experimental autoimmune thyroiditis (EAT), and systemic and local administration of IL-10 has a curative effect on EAT. To test the effect of endogenous IL-10 production in EAT, and to find out whether IL-10 production could be involved in FasL-induced protection, EAT was induced in IL-10(-/-) and in IL-10(-/-)xFasL-transgenic CBA/J mice. The results demonstrated that wild-type and IL-10 knockout (KO) animals developed similar EAT. In contrast, lack of endogenous IL-10 abolished the protective effect of FasL. Polymorphonuclear cells were observed significantly more frequently in the inflammatory cell infiltrates from IL-10(-/-)xFasL animals compared to IL-10(-/-) animals, but they were never detected in wild-type or IL-10(+/+)/FasL-transgenic mice. A shift away from T1 response was observed in FasL-transgenic mice irrespective of their IL-10 status, demonstrating that in our model, endogenous IL-10 plays no part in the T1-towards-T2 anti-Tg immune balance induced by FasL. In summary, endogenous IL-10 is not essential in EAT, or for the immune deviation induced by thyroid FasL expression, whereas it is necessary for the immune privilege status of the thyroid conferred by FasL expression on thyrocytes.