Differential effects of interleukin-17A and 17F on cell interactions between immune cells and stromal cells from synovium or skin.

We compared the contribution of IL-17A and IL-17F in co-culture systems mimicking cell interactions as found in inflamed synovium and skin. Synoviocytes or skin fibroblasts were co-cultured with activated PBMC, with IL-17A, IL-17 A/F, IL-17F, IL-23, anti-IL-17A, anti-IL-17A/F or anti-IL-17F antibodies. IL-17A, IL-17F, IL-6 and IL-10 production was measured at 48 h. mRNA expression of receptor subunits for IL-23, IL-12 and IL-17 was assessed at 24 h. Both cell activation and interactions were needed for a high IL-17A secretion while IL-17F was stimulated by PHA activation alone and further increased in co-cultures. IL-17F levels were higher than IL-17A in both co-cultures (p < 0.05). IL-17F addition decreased IL-17A secretion (p < 0.05) but IL-17A addition had no effect on IL-17F secretion. Interestingly, IL-17A and IL-17F upregulated IL-17RA and IL-17RC mRNA expression in PBMC/skin fibroblast co-cultures (p < 0.05) while only IL-17F exerted this effect in synoviocytes (p < 0.05). Monocyte exclusion in both co-cultures increased IL-17A and IL-17F (twofold, p < 0.05) while decreasing IL-10 and IL-6 secretion (twofold, p < 0.05). IL-17A and F had differential effects on their receptor expression with a higher sensitivity for skin fibroblasts highlighting the differential contribution of IL-17A and F in joint vs. skin diseases.

Heterogeneous effects of S100 proteins during cell interactions between immune cells and stromal cells from synovium or skin.

Cell interactions represent an important mechanism involved in the pathogenesis of chronic inflammation. The key S100 proteins A8 and A9 have been studied in several models of chronic inflammatory diseases with highly heterogeneous conclusions. In this context, the aim of this study was to determine the role of cell interactions on S100 protein production and their effect on cytokine production during cell interactions, between immune and stromal cells from synovium or skin. Peripheral blood mononuclear cells (PBMC) were cultured alone or with synoviocytes or skin fibroblasts, with or without phytohemagglutinin, exogenous A8, A9, A8/A9 proteins or anti-A8/A9 antibody. Production of IL-6, IL-1ß, IL-17, TNF, A8, A9, and A8/A9 was measured by ELISA. Cell interactions with synoviocytes had no effect on A8, A9, or A8/A9 secretion, while cell interactions with skin fibroblasts decreased A8 production. This highlights the importance of stromal cell origin. The addition of S100 proteins in co-cultures with synoviocytes did not increase the production of IL-6, IL-17, or IL-1ß, except for an increase of IL-6 secretion with A8. The presence of anti-S100A8/A9 antibody did not show obvious effects. Low concentration or absence of serum in the culture medium decreased the production of IL-17, IL-6, and IL-1ß but despite these conditions, the addition of S100 proteins did not increase cytokine secretion. In conclusion, the role of A8/A9 in cell interactions during chronic inflammation appears complex and heterogeneous, depending on multiple factors, notably the origin of stromal cells that can affect their secretion.

Evaluation of the Performance of an Indirect Immunofluorescence Assay for the Detection of Anti-MDA5 Antibodies.

Anti-melanoma differentiation-associated protein 5 (MDA5) antibody (Ab) positive dermatomyositis (anti-MDA5 DM) is a rare systemic autoimmune disease; further, its prognosis can be rapidly fatal due to pulmonary involvement. The identification and quantification of anti-MDA5 Abs, which serve as a highly specific biomarker of the disease, is a critical step for the establishing of both the diagnosis and monitoring of the disease's activity. The development of a simple, fast, low-cost, and specific detection system of anti-MDA5 Ab is therefore highly desirable for the purposes of routine laboratory diagnosis. Here, we developed a human cell line that stably expresses MDA5 and evaluated its analytical performance in order to detect anti-MDA5 Abs by the utilization of indirect immunofluorescence (IIF). Serum samples from 23 anti-MDA5 DM patients and 22 anti-MDA5 Abs negative myositis readings, which were obtained at time of diagnosis, were analyzed by IIF on MDA5-transfected cells. The results were compared with those obtained with specific semi-quantitative (immunodot) and quantitative (ELISA) assays. A specific cytoplasmic pattern was found solely with the sera of anti-MDA5 DM patients. The sensitivity and specificity of IIF on MDA5-transfected cells were 96% and 100%, respectively, compared with ELISA. The anti-MDA5 Abs titers that were determined by this approach were consistent with the quantitative results obtained by ELISA. Baseline concentrations of anti-MDA5 Abs, either by ELISA or IIF, were not significantly different between surviving and deceased patients; further, they did not differ significantly according to clinical phenotypes. Overall, an IIF cell-based assay constitutes a simple, fast, and low-cost approach to identify and quantify anti-MDA5 Abs; moreover, it is as efficient as ELISA.

Synoviocytes and skin fibroblasts show opposite effects on IL-23 production and IL-23 receptor expression during cell interactions with immune cells.

BACKGROUND: The IL-23/IL-17 axis is involved in inflammatory diseases including arthritis and psoriasis. However, the response to IL-23 or IL-17 inhibitors is different depending on the disease. The aim was to compare the effects of interactions between immune and stromal cells on the IL-23 axis to understand these differences. METHODS: Peripheral blood mononuclear cells were co-cultured with RA synoviocytes or Pso skin fibroblasts, with or without phytohemagglutinin, IL-23, or anti-IL-23 antibody. Production of IL-6, IL-1ß, IL-23, IL-17, IL-12, and IFNγ was measured by ELISA. IL-23 and cytokine receptor gene expression (IL-17RA, IL-17RC, IL-12Rß1, IL-12Rß2, and IL-23R) was analyzed by RT-qPCR. IL-12Rß1 and IL-23R subunits were analyzed by flow cytometry. RESULTS: The production of IL-6, IL-1ß, IL-17, IL-12, and IFNγ with synoviocytes or skin fibroblasts was rather similar, and cell interactions with immune cells increased their production, specifically that of IL-17. A major difference was observed for IL-23. Interactions with synoviocytes but not with skin fibroblasts decreased IL-23 secretion while mRNA level was increased, mainly with synoviocytes, reflecting a major consumption difference. IL-23 addition had only one effect, the increase of IL-17 secretion. Cell activation induced similar effects on cytokine receptor gene expression in co-cultures with synoviocytes or skin fibroblasts. The key difference was the cell interaction effects depending on the stromal cell origin. Interactions with synoviocytes increased the expression of both IL-23 receptor subunits at mRNA levels and IL-23R at the surface expression level while interactions with skin fibroblasts decreased their expression at the mRNA level and had no effect at the surface expression level. CONCLUSION: Interactions between immune and stromal cells are crucial in cytokine production and their receptor expression. The origin of stromal cells had a major influence on the production of IL-23 and its receptor expression. Such differences may explain part of the heterogeneity in treatment response.

Monoclonal antibodies from B cells of patients with anti-MDA5 antibody-positive dermatomyositis directly stimulate interferon gamma production.

Anti-melanoma differentiation-associated gene 5 (MDA5) antibody (Ab) positive dermatomyositis (anti-MDA5 DM) is a rare entity associated with poor prognosis and multiple immunologic abnormalities. These include the presence of autoAbs and deleterious interferon (IFN)-gamma production in the severe form of the disease. Here, we show that the autoAbs profile differs between patients, depending on disease severity, and that autoAbs from B cells of patients directly stimulate IFN-gamma production by peripheral blood cells. Serum of 29 anti-MDA5 DM patients were analyzed by indirect immunofluorescence (IIF) on Hep-2 cells, to identify patterns associated with poor outcome. Seventeen (59%) serum gave a specific cytoplasmic MDA5 pattern on Hep-2 cells, while 12 (41%) gave an unspecific pattern. Specific MDA5 pattern was associated with a higher risk to develop interstitial lung disease (p = 0.003). Monoclonal autoAbs were generated from B cell clones of two patients with extreme clinical presentation, one who developed a lethal form of the disease, and the other with a favorable outcome. Supernatants of the autoreactive B cell clones that gave an IIF cytoplasmic pattern were tested for their abilities to stimulate IFN-gamma production by peripheral blood cells. Out of 120,000 B cell clones analyzed, 12 produced monoclonal Abs that triggered direct IFN-gamma secretion by peripheral blood cells, by a monocyte-dependent mechanism. None of them was directed against the MDA5 antigen. Altogether, these findings demonstrated that autoAbs other than the highly specific anti-MDA5 Ab are direct contributors of the IFN-gamma upregulation that is linked to the severity of the disease.

Synoviocytes from pigmented villonodular synovitis are less sensitive to cadmium-induced cell death than synoviocytes from rheumatoid arthritis.

Pigmented villonodular synovitis (PVNS) is a rare inflammatory articular disease sharing common characteristics with rheumatoid arthritis (RA), notably hyperplasia of the synovium due to a hyperproliferation of synoviocytes, and with cancer owing to mutations of the CSF1/M-CCSF gene. Targeting synovium hyperplasia by the local delivery of Cadmium (Cd) has been already tested in vitro and in vivo models of RA and could be applied to PVNS. PVNS and RA synoviocytes were exposed to low doses of Cd. After different culture time points, a qualitative analysis was done by microscopy and quantitative measurements of apoptosis, cell viability and IL-6 production were carried. IL-6 production by PVNS synovial tissue was also quantified after Cd treatment with or without the presence of pro-inflammatory cytokines (IL-17 + TNF). Addition of Cd induced cell death in both PVNS (1 ppm) and RA (0.1 ppm) synoviocytes, which increased with time and Cd concentrations. Cd increased the percentage of apoptotic cells and decreased cell viability and IL-6 production. In all these experiments, PVNS synoviocytes were tenfold less sensitive to Cd than RA synoviocytes. Cd decreased IL-6 production by PVNS synovial tissue and its effect was enhanced with pro-inflammatory cytokines. In summary, PVNS synoviocytes show resistance to Cd-induced cell death and decreased inflammation. Intra-articular use of Cd could represent a potential therapeutic tool in PVNS.

Update on Tenosynovial Giant Cell Tumor, an Inflammatory Arthritis With Neoplastic Features.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that leads to joint destruction and bone erosion. Even if many treatments were developed with success in the last decades, some patients fail to respond, and disease chronicity is still a burden. Mechanisms involved in such resistance may include molecular changes in stromal cells. Other explanations can come from observations of tenosynovial giant cell tumor (TGCT), first considered as an inflammatory arthritis, but with unusual neoplastic features. TGCT leads to synovium hypertrophy and hyperplasia with hemosiderin deposition. It affects young adults, resulting in secondary osteoarthritis and increased morbidity. TGCT shows clinical, histological and genetic similarities with RA but affecting a single joint. However, the monoclonality of some synoviocytes, the presence of translocations and rare metastases also suggest a neoplastic disease, with some features common with sarcoma. TGCT is more probably in an intermediate situation between an inflammatory and a neoplastic process, with a main involvement of the proinflammatory cytokine CSF-1/CSF1R signaling axis. The key treatment option is surgery. New treatments, derived from the RA and sarcoma fields, are emerging. The tyrosine kinase inhibitor pexidartinib was recently FDA-approved as the first drug for severe TGCT where surgery is not an option. Options directly targeting the excessive proliferation of synoviocytes are at a preclinical stage.

Addition of Fibroblast-Stromal Cell Markers to Immune Synovium Pathotypes Better Predicts Radiographic Progression at 1 Year in Active Rheumatoid Arthritis.

Objectives: This study aims to investigate if addition of fibroblast-stromal cell markers to a classification of synovial pathotypes improves their predictive value on clinical outcomes in rheumatoid arthritis (RA). Methods: Active RA patients with a knee needle synovial biopsy at baseline and finished 1-year follow-up were recruited from a real-world prospective cohort. Positive staining for CD20, CD38, CD3, CD68, CD31, and CD90 were scored semiquantitatively (0-4). The primary outcome was radiographic progression defined as a minimum increase of 0.5 units of the modified total Sharp score from baseline to 1 year. Results: Among 150 recruited RA patients, 123 (82%) had qualified synovial tissue. Higher scores of CD20+ B cells, sublining CD68+ macrophages, CD31+ endothelial cells, and CD90+ fibroblasts were associated with less decrease in disease activity and greater increase in radiographic progression. A new fibroblast-based classification of synovial pathotypes giving more priority to myeloid and stromal cells classified samples as myeloid-stromal (57.7%, 71/123), lymphoid (31.7%, 39/123), and paucicellular pathotypes (10.6%, 13/123). RA patients with myeloid-stromal pathotype showed the highest rate of radiographic progression (43.7% vs. 23.1% vs. 7.7%, p = 0.011), together with the lowest rate of Boolean remission at 3, 6, and 12 months. Baseline synovial myeloid-stromal pathotype independently predicted radiographic progression at 1 year (adjusted OR: 3.199, 95% confidence interval (95% CI): 1.278, 8.010). Similar results were obtained in a subgroup analysis of treatment-naive RA. Conclusions: This novel fibroblast-based myeloid-stromal pathotype could predict radiographic progression at 1 year in active RA patients which may contribute to the shift of therapeutic decision in RA.

Extensive Phenotype of Human Inflammatory Monocyte-Derived Dendritic Cells.

Inflammatory monocyte-derived dendritic cells (Mo-DCs) have been described in several chronic inflammatory disorders, such as rheumatoid arthritis (RA), and are suspected to play a detrimental role by fueling inflammation and skewing adaptive immune responses. However, the characterization of their phenotype is still limited, as well as the comprehension of the factors that govern their differentiation. Here, we show that inflammatory Mo-DCs generated in vitro expressed a large and atypical panel of C-type lectin receptors, including isoforms of CD209 and CD206, CD303 and CD207, as well as intracellular proteins at their surfaces such as the lysosomal protein CD208. Combination of these markers allowed us to identify cells in the synovial fluid of RA patients with a close phenotype of inflammatory Mo-DCs generated in vitro. Finally, we found in coculture experiments that RA synoviocytes critically affected the phenotypic differentiation of monocytes into Mo-DCs, suggesting that the crosstalk between infiltrating monocytes and local mesenchymal cells is decisive for Mo-DCs generation.

Reactivation of latent tuberculosis with TNF inhibitors: critical role of the beta 2 chain of the IL-12 receptor.

Tumor necrosis factor (TNF) inhibitors have improved a lot the treatment of numerous diseases, with the well-known example of rheumatoid arthritis (RA). In the early 2000s, postmarketing data quickly revealed an alarming number of severe tuberculosis (TB) under such treatment. These findings were consistent with previous results in mice where TNF is essential for lymph node formation and granuloma organization. The effects of TNF inhibition on RA synovium structure are very similar to those on granuloma, with changes in cellular interactions, cytokine, and chemokine production. In addition to the role of TNF in granuloma, the interleukin (IL)-12/interferon (IFN)-γ pathway is required for an efficient host defense against TB. Primary and secondary immunodeficiencies affecting this pathway lead to severe bacillus Calmette-Guérin (BCG) reaction or full TB. Any chronic inflammation as in RA induces a systemic Th1 defect that predisposes to TB through specific downregulation of the IL-12Rß2 chain. When TNF inhibitors are initiated, this transiently increases this risk of TB, through effects on cellular interactions in a latent TB granuloma. At a later stage, when a better control disease activity is obtained, the risk of TB is reduced but not abrogated. Given the clear benefit from TNF inhibition, latent TB infection screening at baseline is essential for an optimal safety.

Additive or Synergistic Interactions Between IL-17A or IL-17F and TNF or IL-1ß Depend on the Cell Type.

Background: IL-17A has effects on several cell types and is a therapeutic target in several inflammatory diseases. IL-17F shares 50% homology and biological activities with IL-17A. It is now of interest to target both cytokines. The objective was to compare the IL-17A and IL-17F effect on cytokine production by RA synoviocytes, and to extend to other cells. Methods: Cells (RA synoviocytes, psoriasis skin fibroblasts, endothelial cells, myoblasts, and hepatocytes) were cultured in the presence or not of: IL-17A, IL-17F, TNF, IL-1ß alone or their combinations, IL-17A/TNF, IL-17A/IL-1ß, IL-17A/TNF/IL-1ß, IL-17F/TNF, IL-17F/IL-1ß, and IL-17F/TNF/IL-1ß. All experiments were performed in parallel to reduce variability. After 48 h, supernatants were recovered and IL-6 and IL-8 levels were measured by ELISA. Results: IL-17A and IL-17F alone increased significantly IL-6 and IL-8 productions by synoviocytes, with a stronger effect for IL-17A. For IL-6 production, TNF or IL-1ß alone had the largest effect on myoblasts (5-fold increase), while for IL-8 production, it was on skin fibroblasts (5-fold increase). The IL-17A/TNF synergistic increase was observed on all cells for IL-6; and for IL-8, except for endothelial cells. For IL-17F/TNF, except with endothelial cells, a synergistic effect was also observed, but less powerful than with IL-17A/TNF. IL-17A/IL-1ß or IL-17F/IL-1ß effect was cell-type dependent, with an additive effect for synoviocytes (1.6 and 2-fold increase, respectively for IL-6, and 1.8 and 2-fold increase, respectively for IL-8) and a synergistic effect for hepatocytes (3.8 and 4.2-fold increase, respectively for IL-6, and 6 and 2-fold increase, respectively for IL-8). The three-cytokine combination induced an additive effect for synoviocytes and a synergistic effect for skin fibroblasts. Conclusion: IL-17A and IL-17F acted similarly by inducing pro-inflammatory cytokine secretion, with a stronger response intensity with IL-17A. Their activities were potentiated by the combination with TNF and IL-1ß, with an effect dependent on the cell type.

Two phase kinetics of the inflammatory response from hepatocyte-peripheral blood mononuclear cell interactions.

Active liver diseases are characterized by an infiltration of inflammatory immune cells, which interact locally with hepatocytes. Co-cultures between non- and -activated human peripheral blood mononuclear cells (PBMCs) and human hepatoma HepaRG cells were used to determine the role of these cell interactions in the inflammatory response. At the early stage, PBMC-HepaRG cell interactions increased mRNA expression and/or secretion of IL-6, IL-8, CCL-20 and MCP-1, in part through direct cell contact and the induction was higher in PHA-activated conditions. The pro-inflammatory cytokines IL-17 and/or TNFα contributed to the increase of IL-6 and IL-8 secretion. HepaRG cells modulated T cell polarization by increasing Th1 cell transcription factor expression and by reducing CD3+ CD4+ IL-17+ cell frequency when PBMCs were activated with PHA. At a later stage, the presence of HepaRG cells inhibited PHA-induced HLA-DR expression on PBMCs, and PBMC proliferation. In contrast, the presence of skin fibroblasts had no effect of PBMC proliferation induced by PHA. After a first pro-inflammatory phase, PBMC-HepaRG cell interactions may down-regulate the immune response. The PBMC-hepatocyte interactions can thus participate first to the initiation of hepatitis and later to the maintenance of immune tolerance in liver, possibly contributing to chronicity.

Cryoglobulins Today: Detection and Immunologic Characteristics of 1,675 Positive Samples From 13,439 Patients Obtained Over Six Years.

OBJECTIVE: Cryoglobulins are cold-precipitating immunoglobulins. Through progress in techniques, we undertook this study to update information on the biologic characteristics of cryoglobulins in a very large population. METHODS: A cohort of 13,439 patients was tested for cryoglobulins from January 2010 to December 2016. The analysis included cryoglobulin isotype, clonality, concentration, and IgM rheumatoid factor (IgM-RF) in cryoprecipitate, as well as serum complement and RF. Markers of gammopathy, viral infection, and autoimmunity were also investigated. RESULTS: Of the 13,439 patients, 1,675 (12.5%) tested positive for cryoglobulins: 155 patients (9.3%) with type I, 788 (47%) with type II, and 732 (43.7%) with type III cryoglobulins. Nine percent of patients who were retested after initially testing negative for cryoglobulins showed a positive result on a follow-up test (196 of the 2,213 retested patients). In type I cryoglobulins, IgM was more frequent but occurred at lower concentrations than IgG. Mixed cryoglobulins were found in 34.8% of the tested patients who were positive for hepatitis C virus and <5% of those who were positive for hepatitis B virus or HIV. Of the patients with anti-double-stranded DNA, anti-SSA, or anti-cyclic citrullinated peptide autoantibodies, 25.4% tested positive for mixed cryoglobulins, with type III occurring more frequently than type II. Both cryoprecipitate and serum were RF-positive in 21.6% of type II and 10.1% of type III cryoglobulins. A decrease of C4, with or without accompanying decreases of C3 and CH50, was found in 23.6% of cryoglobulin samples. CONCLUSION: Obtained with the use of modern assays, our findings from this very large collection of cryoglobulins provide an update on cryoglobulin distribution and characteristics, with minimal selection bias. Despite strict preanalytical conditions, a negative finding for the presence of cryoglobulin must be confirmed in a second sample. RF activity and complement decreases were rarely detected.

Activation of the Peroxisome Proliferator-Activated Receptor γ Coactivator 1ß/NFATc1 Pathway in Circulating Osteoclast Precursors Associated With Bone Destruction in Rheumatoid Arthritis.

OBJECTIVE: Activation of osteoclastogenesis at the bone site in rheumatoid arthritis (RA) is well established. The mechanisms by which circulating osteoclast precursors contribute are still unclear. Peroxisome proliferator-activated receptor γ coactivator 1ß (PGC-1ß) is implicated in transcriptional regulation of osteoclastogenesis in mouse models. This study was undertaken to investigate the contribution of PGC-1ß to circulating osteoclast precursors and its link to bone destruction in RA. METHODS: PGC-1ß expression in RA peripheral blood CD14+ monocytes was increased and showed correlation with joint destruction shown on radiographs. Cells from RA patients or healthy controls were transfected with a lentivirus vector for PGC-1ß gene silencing or overexpression and cultured with macrophage colony-stimulating factor and RANKL. Bone resorption activity, bone-degrading enzymes, and signaling molecules were measured in these mature osteoclasts. RESULTS: Increased nuclear accumulation of PGC-1ß was observed in RA peripheral blood CD14+ monocytes, and these cells had stronger osteoclastogenesis than in healthy controls. PGC-1ß protein expression was positively correlated with radiographic joint destruction (r = 0.396-0.413; all P < 0.05). PGC-1ß knockdown suppressed (51-82% reduction) the expression of cathepsin K, tartrate-resistant acid phosphatase (TRAP), and matrix metalloproteinase 9 (MMP-9), as well as osteoclast differentiation and bone resorption activity. Conversely, PGC-1ß overexpression increased these markers (by 1.5-1.8-fold) and osteoclastogenesis. VIVIT, an inhibitor of NFATc1 activation, inhibited the effect of overexpressed PGC-1ß by reducing cathepsin K, TRAP, and MMP-9 expression. Chromatin immunoprecipitation assay and dual-luciferase reporter gene assay showed PGC-1ß bound to NFATc1 promoter, leading to transcriptional activation. CONCLUSION: Activation of the PGC-1ß/NFATc1 pathway in circulating osteoclast precursors was associated with bone destruction in RA. This may represent a new treatment target.

Prealbumin, platelet factor 4 and S100A12 combination at baseline predicts good response to TNF alpha inhibitors in rheumatoid arthritis.

OBJECTIVES: Tumour necrosis factor-alpha inhibitors (TNFi) are effective treatments for Rheumatoid Arthritis (RA). Responses to treatment are barely predictable. As these treatments are costly and may induce a number of side effects, we aimed at identifying a panel of protein biomarkers that could be used to predict clinical response to TNFi for RA patients. METHODS: Baseline blood levels of C-reactive protein, platelet factor 4, apolipoprotein A1, prealbumin, α1-antitrypsin, haptoglobin, S100A8/A9 and S100A12 proteins in bDMARD naive patients at the time of TNFi treatment initiation were assessed in a multicentric prospective French cohort. Patients fulfilling good EULAR response at 6 months were considered as responders. Logistic regression was used to determine best biomarker set that could predict good clinical response to TNFi. RESULTS: A combination of biomarkers (prealbumin, platelet factor 4 and S100A12) was identified and could predict response to TNFi in RA with sensitivity of 78%, specificity of 77%, positive predictive values (PPV) of 72%, negative predictive values (NPV) of 82%, positive likelihood ratio (LR+) of 3.35 and negative likelihood ratio (LR-) of 0.28. Lower levels of prealbumin and S100A12 and higher level of platelet factor 4 than the determined cutoff at baseline in RA patients are good predictors for response to TNFi treatment globally as well as to Infliximab, Etanercept and Adalimumab individually. CONCLUSION: A multivariate model combining 3 biomarkers (prealbumin, platelet factor 4 and S100A12) accurately predicted response of RA patients to TNFi and has potential in a daily practice personalized treatment.

Live-stream characterization of cadmium-induced cell death using visible CdTe-QDs.

Characterization of cell death currently requires the use of indirect markers, which has largely limited the ability to monitor cell death processes inside the cell. Here, we introduce a new method for the characterization of cell death mechanisms using cadmium telluride quantum dots (CdTe-QDs). Using visible CdTe-QDs with mesenchymal cells (e.g. synoviocytes), live-stream imaging allowed for visualization of cadmium-induced cell death, combining characteristics of apoptosis and autophagy. Initially, similar anti-proliferative effect was observed between 10 µg/ml Cd2+ and CdTe-QDs at 24 h (cell index/cell density ratio decreased from 0.6 to -16.6, p < 0.05) using techniques that do not require the capacity of CdTe-QDs. Apoptosis was confirmed by the quantification of morphological parameters (reduced surface area, increased cell thickness) and positive labeling with annexin V. Autophagy was confirmed by monodansylcadaverine staining, identifying similar autophagic vacuoles with both Cd2+ and CdTe-QD. However, QD imaging allowed for visualization of cadmium elements inside cell structures and their kinetic changes leading to cell death. Cell death characteristics were similar in inflammatory and non-inflammatory environment but were induced up to 4 h earlier in the former. Therefore, live-stream imaging of a visible cytotoxic agent has useful applications not currently possible with indirect methods, including chronological monitoring of cell death.

Differential effects of TNF-α and IL-1ß on the control of metal metabolism and cadmium-induced cell death in chronic inflammation.

OBJECTIVE: Interleukin-1-beta (IL-1ß) and tumour necrosis factor-alpha (TNF-α) are both monocyte-derived cytokines. Both cytokines have been previously described to exert a role in rheumatoid arthritis (RA) pathogenesis synergizing with other pro-inflammatory mediators, such as interleukin-17 (IL-17) on target cells, for the perpetuation of the inflammatory response (e.g. IL-6 production). In the context of experimental RA, Cd addition has an anti-proliferative and anti-inflammatory effect when associated to IL-17/TNF-α stimulation, due to its accumulation in synoviocytes. The aim of this work was to evaluate if IL-1ß interaction with IL-17 also contributes to metal-import mechanisms and its effects on cell viability and inflammation. METHODS: IL-17 and IL-1ß were added to synoviocyte cultures with or without exogenous Cd addition (0.1 ppm, 0.89 µM). IL-6 production, Cd import kinetics, gene expression of ZIP-8 importer and metallothioneins (MTs) and cell viability were evaluated by ELISA, inductively-coupled mass spectrometry (ICP-MS), q-RT-PCR and viability assays (neutral red and annexin V) respectively. RESULTS: IL-17 and IL-1ß acted in synergy on synoviocytes to induce IL-6 production similarly to the IL-17/TNF-α combination. Metal import was lower with IL17/ IL-1ß in comparison to IL-17/TNF-α exposed-synoviocytes, as the expression of ZIP-8 and MT-1F was less induced. Monocyte and PBMCs exposure to Cd resulted in a reduced production of IL-1ß and an increased production of TNF-α and this result was confirmed in co-cultures of synoviocytes and PBMCs. The IL-17/IL-1ß combination with Cd slightly reduced cell viability in comparison to the IL-17/TNF-α combination and resulted in a strong induction of IL-6 production. CONCLUSION: IL-17/TNF-α combination but not IL-17/IL-1ß combination mainly drives the accumulation of Cd in synoviocytes and its effects on cell viability and inflammation.

[Reactivation of tuberculosis during treatment with inhibitors of TNF]. / Réactivation de la tuberculose au cours des traitements par inhibiteurs du TNF.

Reactivation of tuberculosis during treatment with inhibitors of TNF. The inhibition of the tumor necrosis factor (TNF) has been a major progress in the treatment of chronic inflammatory diseases by acting on their local and systemic manifestations. However, this treatment can be responsible for severe infections, specifically reactivation of tuberculosis. The underlying mechanisms of these infections in this context are now better understood. First, chronic inflammatory diseases are associated with a cellular mediated immune defect, specifically affecting the Th1 pathway. On the other hand, TNF has a central role in granuloma formation. TNF inhibition allows the escape of tuberculosis bacillus from residual sites and its rapid diffusion. This understanding has allowed the implementation of prevention measures with screening and treatment of latent tuberculosis.

Réactivation de la tuberculose au cours des traitements par inhibiteurs du TNF. L'inhibition du tumor necrosis factor (TNF) a été un progrès majeur dans le traitement des maladies inflammatoires chroniques en agissant sur leur expression locale et systémique. Cependant, ce traitement peut être responsable d'infections graves, en particulier de réactivation de la tuberculose. Les mécanismes de ces infections sont maintenant mieux compris. D'une part, les maladies inflammatoires chroniques s'accompagnent d'un déficit de l'immunité à médiation cellulaire, touchant particulièrement la voie Th1. D'autre part, le TNF a un rôle central dans la formation des granulomes. Son inhibition permet la libération du bacille de la tuberculose à partir des sites résiduels et sa propagation rapide. Cette compréhension a permis des actions de prévention avec le dépistage et le traitement des tuberculoses latentes.

Dual IL-17A and IL-17F neutralisation by bimekizumab in psoriatic arthritis: evidence from preclinical experiments and a randomised placebo-controlled clinical trial that IL-17F contributes to human chronic tissue inflammation.

OBJECTIVE: Interleukin (IL)-17A has emerged as pivotal in driving tissue pathology in immune-mediated inflammatory diseases. The role of IL-17F, sharing 50% sequence homology and overlapping biological function, remains less clear. We hypothesised that IL-17F, together with IL-17A, contributes to chronic tissue inflammation, and that dual neutralisation may lead to more profound suppression of inflammation than inhibition of IL-17A alone. METHODS: Preclinical experiments assessed the role of IL-17A and IL-17F in tissue inflammation using disease-relevant human cells. A placebo-controlled proof-of-concept (PoC) clinical trial randomised patients with psoriatic arthritis (PsA) to bimekizumab (n=39) or placebo (n=14). Safety, pharmacokinetics and clinical efficacy of multiple doses (weeks 0, 3, 6 (240 mg/160 mg/160 mg; 80 mg/40 mg/40 mg; 160 mg/80 mg/80 mg and 560 mg/320 mg/320 mg)) of bimekizumab, a humanised monoclonal IgG1 antibody neutralising both IL-17A and IL-17F, were investigated. RESULTS: IL-17F induced qualitatively similar inflammatory responses to IL-17A in skin and joint cells. Neutralisation of IL-17A and IL-17F with bimekizumab more effectively suppressed in vitro cytokine responses and neutrophil chemotaxis than inhibition of IL-17A or IL-17F alone. The PoC trial met both prespecified efficacy success criteria and showed rapid, profound responses in both joint and skin (pooled top three doses vs placebo at week 8: American College of Rheumatology 20% response criteria 80.0% vs 16.7% (posterior probability >99%); Psoriasis Area and Severity Index 100% response criteria 86.7% vs 0%), sustained to week 20, without unexpected safety signals. CONCLUSIONS: These data support IL-17F as a key driver of human chronic tissue inflammation and the rationale for dual neutralisation of IL-17A and IL-17F in PsA and related conditions. TRIAL REGISTRATION NUMBER: NCT02141763; Results.