[Proinflammatory cytokines and cardio-vascular risk: From myocardial infarction tocytokine storm of COVID-19]. / Cytokines proinflammatoires et risque cardio-vasculaire : de l'infarctus du myocarde à la tempête cytokinique du COVID-19.

The mechanisms of inflammatory diseases involve the key inflammatory cytokines IL-1, TNF, IL-6, and IL-17 which are now therapeutic targets with biotherapies. They contribute to the local manifestations of clinically different diseases. In addition to these local aspects, these cytokines have systemic effects from their action on the liver, muscle, adipose tissue and the cardiovascular system. All these diseases have in common an increase in cardiovascular risk. In the general population, the same concepts are applicable, as shown by the link between an even modest rise in CRP and cardiovascular risk. More recently, the cytokine storm of severe forms of COVID-19 has shown that synergistic interactions between cytokines first described in vitro are further amplified in the clinical picture with multiple and severe impairment of key organs. In these chronic and acute contexts, control of inflammation by targeting cytokines is a new vascular treatment option, with already important results for IL-1.

Interleukin-17 and lupus: enough to be a target? For which patients?

The involvement of the interleukin (IL)-17 axis in many inflammatory and autoimmune diseases is now well established, and this has led to the development of successful targeted therapies. Its role in systemic lupus erythematosus (SLE) is less described, since SLE is characterized by the impairment of many other immune actors. However, results from animal models and patients strongly suggest that IL-17 and its producing cells are involved in SLE pathogenesis. Circulating levels of IL-17 are increased in lupus, and tissue staining shows the presence of IL-17-producing cells in organ lesions. Through different mechanisms, the IL-17 axis promotes autoantibody production, immune complex deposition, complement activation and then tissue damage. There are also many interactions with other immune and non-immune actors, which account for the broad spectrum of clinical manifestations and disease heterogeneity. SLE treatment faces challenges with many disappointing trials and persistent unmet needs. The identification of subsets of SLE patients with an IL-17-driven disease now constitutes the key priority before starting trials. More preclinical studies are needed to improve the selection of the right patients able to respond and tolerate the many inhibitors that are already available.

IL-17 and TNF-α co-operation contributes to the proinflammatory response of hepatic stellate cells.

Hepatic stellate cells (HSCs) have a central role in liver inflammation and fibrosis by producing inflammatory and fibrotic mediators. Their activation is regulated through direct cell-cell interactions, but also through systemic and local effects of soluble factors such as cytokines. The effects of the proinflammatory cytokines interleukin (IL)-17 and tumor necrosis factor (TNF)-α and cell interactions with hepatocytes on HSC activation were assessed. Human HSC and HepaRG cells were exposed to IL-17 and/or TNF-α. IL-17 and TNF-α contribution from immune cells was determined in a co-culture model with phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMC), HSC and/or hepatocytes. IL-17 enhanced TNF-α effects on the induction of IL-6, IL-1ß, and the chemokine IL-8, chemokine (C-C motif) ligand 20 (CCL20) and monocyte chemoattractant protein-1 (MCP-1) expression/secretion in isolated HSC cultures. HSC-hepatocyte interactions did not enhance IL-6, IL-8 and CCL20 production compared to hepatocyte alone. However, HSC-hepatocyte interactions increased C-reactive protein expression. IL-17 and/or TNF-α had no direct profibrotic effects on collagen 1 α1, tissue inhibitor of matrix metalloproteinase (TIMP) and matrix metalloproteinase (MMP) 2 gene expression, whereas mRNA levels of MMP3, an enzyme involved in matrix destruction, were up-regulated in HSCs. The use of specific inhibitors of IL-17 and TNF-α indicated their contribution to the strong increase of IL-6 and IL-8 production induced by PBMC, HSC and/or hepatocyte interactions. As chronic liver inflammation leads to liver fibrosis, IL-17 and/or TNF-α neutralization can be of interest to control liver inflammation and therefore its effects on fibrosis.

Synergistic effect of interleukin-17 and tumour necrosis factor-α on inflammatory response in hepatocytes through interleukin-6-dependent and independent pathways.

The proinflammatory cytokines interleukin (IL)-17 and tumour necrosis factor (TNF)-α are targets for treatment in many chronic inflammatory diseases. Here, we examined their role in liver inflammatory response compared to that of IL-6. Human hepatoma cells (HepaRG, Huh7.5 and HepG2 cells) and primary human hepatocytes (PHH) were cultured with IL-6, IL-17 and/or TNF-α. To determine the contribution of the IL-6 pathway in the IL-17/TNF-α-mediated effect, an anti-IL-6 receptor antibody was used. IL-17 and TNF-α increased in synergy IL-6 secretion by HepaRG cells and PHH but not by Huh7.5 and HepG2 cells. This IL-17/TNF-α synergistic cooperation enhanced the levels of C-reactive protein (CRP) and aspartate aminotransferase (ASAT) in HepaRG cell and PHH cultures through the induction of IL-6. IL-17/TNF-α also up-regulated IL-8, monocyte chemoattractant protein (MCP)-1 and chemokine (C-C motif) ligand 20 (CCL20) chemokines in synergy through an IL-6-independent pathway. Interestingly, first exposure to IL-17, but not to TNF-α, was crucial for the initiation of the IL-17/TNF-α synergistic effect on IL-6 and IL-8 production. In HepaRG cells, IL-17 enhanced IL-6 mRNA stability resulting in increased IL-6 protein levels. The IL-17A/TNF-α synergistic effect on IL-6 and IL-8 induction was mediated through the activation of extracellular signal-regulated kinase (ERK)-mitogen-activated protein kinase, nuclear factor-κB and/or protein kinase B (Akt)-phosphatidylinositol 3-kinase signalling pathways. Therefore, the IL-17/TNF-α synergistic interaction mediates systemic inflammation and cell damage in hepatocytes mainly through IL-6 for CRP and ASAT induction. Independently of IL-6, the IL-17A/TNF-α combination may also induce immune cell recruitment by chemokine up-regulation. IL-17 and/or TNF-α neutralization can be a promising therapeutic strategy to control both systemic inflammation and liver cell attraction.

Role of podoplanin in the high interleukin-17A secretion resulting from interactions between activated lymphocytes and psoriatic skin-derived mesenchymal cells.

In the context of psoriasis, T helper type 17 (Th17) cells infiltrate the inflammatory site and interact with local mesenchymal cells, including skin fibroblasts. The aim of this work was to study the interactions of skin-derived fibroblasts with peripheral blood mononuclear cells (PBMC) with a focus on the Th17 pathway and to identify a mechanism which leads to a high interleukin (IL)-17 secretion. A co-culture system between PBMC and skin fibroblasts was developed. Healthy and patient PBMC were added to non-lesional or lesional skin fibroblasts at a 5:1 ratio for 48 h in the presence or not of activation with phytohaemagglutinin (PHA). Monocytes were removed or not by adherence before the co-culture. An anti-podoplanin antibody was also used during the co-culture. Cytokine production (IL-8, IL-6, IL-1ß and IL-17) was measured by enzyme-linked immunosorbent assay (ELISA) and cell staining (CD3, CD4, IL-17 and podoplanin) by flow cytometry. Without T cell receptor (TCR) activation, IL-8, IL-6 and IL-1ß production increased in PBMC-fibroblast co-culture compared to PBMC alone. No additional effect was observed with TCR activation, with no difference in the Th17 cell percentage in activated-PBMC alone or co-cultured. Conversely, IL-17 production was increased highly only in co-cultures between control and patient activated-PBMC and skin fibroblasts. Removal of monocytes decreased cytokine production, notably that of IL-17. Addition of an anti-podoplanin antibody decreased IL-17 secretion by 60%. Interactions between resting PBMC and fibroblasts induce the IL-8, IL-6 and IL-1ß production. PBMC activation and cell interactions are critical for a high IL-17 secretion. Podoplanin contributes largely to this massive IL-17 secretion.

Biomarkers and personalised medicine in rheumatoid arthritis: a proposal for interactions between academia, industry and regulatory bodies.

Rheumatoid arthritis (RA) is one of the most appropriate conditions for the application of personalised medicine as a high degree of heterogeneity has been recognised, which remains to be explained. Such heterogeneity is also reflected in the large number of treatment targets and options. A growing number of biologics as well as small molecules are already in use and there are promising new drugs in development. In order to make the best use of treatment options, both targeted and non-targeted biomarkers have to be identified and validated. To this aim, new rules are needed for the interaction between academia and industry under regulatory control. Setting up multi-centre biosample collections with clear definition of access, organising early, possibly non-committing discussions with regulatory authorities, and defining a clear route for the validation, qualification and registration of the biomarker-drug combination are some of the more critical areas where effective collaboration between the drug industry, academia and regulators is needed.

Esophageal dysmotility associated with systemic sclerosis: a high-resolution manometry study.

Esophageal involvement occurs in about 80% of patients with systemic sclerosis, with a marked diminution of peristaltic pressures in the distal two-thirds of the esophagus. Our aims were to more fully characterize esophageal motility disorders in systemic sclerosis using high-resolution manometry (HRM) and to determine predictive factors of esophageal involvement. Fifty-one patients (46 females) with systemic sclerosis were included in this retrospective study. Esophageal motility was characterized with HRM. The demographic data, esophageal symptoms, presence of other organ involvement, and autoantibody profile (anti-Scl70 antibodies [Scl70], anticentromere antibodies [ACA]) were recorded for all patients. Esophageal body dysmotility was present in 33 patients (67.3%) and was associated with hypotensive esophagogastric junction in 27 patients (55.1%). The velocity of proximal contractions was higher in patients with esophageal body dysmotility compared to patients with normal peristalsis (median 10.8 cm/s vs. 5.5, P = 0.04). The amplitude of middle esophageal contraction but not of distal esophageal contraction was reduced in patients with hypoperistalsis. Diffuse esophageal skin involvement, presence of Scl70 and absence of ACA were associated with esophageal involvement. Esophageal symptoms encountered in 87.5% of patients were not predictive of esophageal dysmotility. This HRM series confirms the high prevalence of esophageal body dysmotility in systemic sclerosis. Diffuse skin involvement, positive Scl70 and negative ACA, but not esophageal symptoms, may predict esophageal body dysmotility.

Expression of Toll-like receptor 3 and Toll-like receptor 7 in muscle is characteristic of inflammatory myopathy and is differentially regulated by Th1 and Th17 cytokines.

OBJECTIVE: To assess the expression of Toll-like receptor 3 (TLR-3) and TLR-7 in muscle tissue from patients with polymyositis (PM) and dermatomyositis (DM) and to investigate the function and regulation of TLR-3 in cultured muscle cells. METHODS: The expression of TLR-3, TLR-7, HLA class I, and CD56, a marker of immature myoblast precursors, was analyzed using immunohistochemistry. TLR-3 regulation and signaling were assessed in myoblasts and in differentiated myotubes with the TLR-3 agonist poly(I-C), necrotic myoblasts, and Th1 and Th17 cytokines, in the presence or absence of neutralizing anti-TLR-3 antibody. Levels of TLR-3 messenger RNA (mRNA) were quantified by reverse transcription-polymerase chain reaction. Levels of interleukin-6 (IL-6), CCL20, and IL-8 were determined by enzyme-linked immunosorbent assay. RESULTS: TLR-3 and TLR-7 were expressed in PM/DM tissues, but not in noninflammatory muscle tissues, and were primarily detected in inflammatory infiltrates, although a few muscle cells were also positive. These TLR-3- and TLR-7-positive fibers expressed high levels of CD56 and HLA class I antigens. A synergy between poly(I-C) and IL-17 was observed for the production of IL-6 and CCL20. Similarly, stimulation with necrotic myoblasts increased IL-6 production, and stimulation with necrotic myoblasts in combination with IL-17 further increased the induction of IL-6. TLR-3 blockade decreased the inducing effect of necrotic myoblasts and IL-17 on IL-6 production. Stimulation with interferon-gamma (IFNgamma) increased TLR-3 mRNA levels, but IL-17 down-regulated the inducing effect of IFNgamma. CONCLUSION: Our findings indicate that TLR-3 and TLR-7 are expressed in inflammatory myopathic tissues, particularly in immature myoblast precursors. Necrotic muscle cells activate cytokine production, in part, through the TLR-3 pathway, with a differential regulatory effect of Th1 and Th17 cytokines.

Rheumatoid arthritis and ankylosing spondylitis - pathology of acute inflammation.

Histomorphological analysis of inflammatory lesions in rheumatoid arthritis (RA) and ankylosing spondylitis (AS) display similarities but also major differences. Ankylosing spondylitis is characterised by two key pathological findings: sacroiliac joint and spinal inflammation and new bone formation with the possible consequence of bone fusion, usually in the axial skeleton. In AS the primary site of inflammation is located at the enthesis or subchondral bone marrow with bone marrow oedema, lymphocytic infiltrates, increased osteoclast density and increased microvessel density are typical findings in acute inflammation. In RA joint inflammation has its origin in the synovial membrane of peripheral joints. Osteitis in the subchondral bone marrow reveals similar findings compared to AS and it is suggested to occur secondary to inflammation in the synovial membrane. Structural damage defines the outcome in both diseases. However, in AS it is defined by new bone formation and in RA by the destruction of cortical bone.

Increase in human endogenous retrovirus HERV-K (HML-2) viral load in active rheumatoid arthritis.

To study the viral loads of human endogenous retrovirus HERV-K (HML-2) type 1 and type 2 in rheumatoid arthritis (RA), we measured the viral loads of HERV-K (HML-2) type 1 and type 2 using nucleic acid sequence-based amplification (NASBA) technology. We analyzed plasma samples from RA patients (n = 79) and healthy volunteers (HV, n = 46) and synovial fluid samples from RA (n = 10) and osteoarthritis (OA, n = 10) patients. HERV-K type 1 and type 2 viruses were detected and quantified for the majority of plasma and synovial fluid samples from RA patients. HERV-K type 1 and type 2 viral loads were significantly elevated in RA patients compared with HV in plasma (P < 0.0001) and from RA patients compared with OA patients in synovial fluid (type 1: P = 0.0007; type 2: P = 0.023). Moreover, an association was observed between the HERV-K type 1 viral load in plasma and the disease activity in RA patients (RA patients with low activity versus high activity P = 0.0129; RA patients with intermediate activity versus high activity P = 0.037). Our findings showed that HERV-K (HML-2) viral load can be detected in plasma samples from RA patients, with higher levels observed for those with active disease. There was an association of HERV-K type 1 levels with the disease activity.

Nitrated type III collagen as a biological marker of nitric oxide-mediated synovial tissue metabolism in osteoarthritis.

OBJECTIVES: Nitric oxide (NO) is a major mediator of joint tissue inflammation and damage in osteoarthritis (OA) and mediates the nitration of tyrosine (Y*) residues in proteins. We investigated the nitration of type III collagen, a major constituent of synovial membrane, in knee OA. METHODS: A polyclonal antibody directed against the nitrated QY*DSY*DVKSG sequence from type III collagen N-telopeptide was generated. Synovial tissues from patients with knee OA (n=4) and rheumatoid arthritis (RA, n=4) were analyzed by immunohistochemistry for IIINys. Serum IIINys levels were measured by enzyme-linked immunosorbent assay in 87 patients with painful knee OA (mean age: 63.0+/-8.0 years, Kellgren-Lawrence score II-III) and in 40 sex and age-matched healthy controls. RESULTS: Competition experiments using various nitrated and un-nitrated type III collagen and derived sequences, showed that the antibody was highly specific for the nitrated IIINys sequence. High IIINys immunoreactivity was detected in the synovial tissues from all patients with OA and RA with a preferential localization in the intimal layer. Serum IIINys levels were on average 1.5-fold higher (P<0.0001) in patients with knee OA than in healthy controls and significantly correlated with C-reactive protein values (r=0.40, P<0.005). CONCLUSIONS: Nitration of tyrosine residues of type III collagen N-telopeptide is increased in the synovial tissue of patients with knee OA. Measurements of serum IIINys level may be useful for the clinical investigation of oxidative-related alterations of synovial tissue metabolism in OA.

Chemokines and dendritic cells in inflammatory myopathies.

This review focuses on the contribution of the local production of chemokines and cytokines and of dendritic cells (DC) to the pathogenesis of inflammatory myopathies. DC are the most efficient professional antigen-presenting cells (APC), which are critical for the development of innate and adaptive immune responses. Chemokines are important mediators of the immune response as they regulate leucocyte recruitment to tissue and play a key role in inflammatory diseases by acting on T-cell and DC migration. Recent advances indicate that the muscle cell itself could participate in the inflammatory process. Furthermore, the T-helper (Th) type 1 and Th17 proinflammatory cytokines, present in myositis samples, are associated with the migration, differentiation and maturation of inflammatory cells and allow a network of interactions between all the components of the immune response. An understanding of such interactions is essential because it can lead to therapeutic applications.

Effects of infliximab therapy on biological markers of synovium activity and cartilage breakdown in patients with rheumatoid arthritis.

BACKGROUND: Defining the remission criteria of rheumatoid arthritis (RA) remains a critical issue. Markers of synovium activity, urinary glucosyl-galactosyl-pyridinoline (Glc-Gal-PYD) and of cartilage destruction, urinary C-terminal crosslinking telopeptide of type II collagen (CTX-II) have been shown to reflect disease activity and joint damage progression in RA. METHODS: The prospective study cohort comprised 66 RA patients treated with infliximab and methotrexate and 76 healthy controls. Measurements of urinary Glc-Gal-PYD and CTX-II were performed at baseline and at 1 year of infliximab therapy. RESULTS: At baseline, urinary Glc-Gal-PYD and CTX-II levels were increased in patients with RA and correlated with modified Sharp scores and progression of joint damage. Patients with more progressive joint destruction had higher Glc-Gly-PYD and CTX-II baseline levels. CONCLUSION: These markers reflected bone erosion evolution and might be useful for treatment monitoring and evaluation of RA. Markers remained high even in clinical responders after infliximab, suggesting persistence of synovitis.

Apolipoprotein A-I and platelet factor 4 are biomarkers for infliximab response in rheumatoid arthritis.

OBJECTIVES: The use of biologicals such as infliximab has dramatically improved the treatment of rheumatoid arthritis (RA). However, factors predictive of therapeutic response need to be identified. A proteomic study was performed prior to infliximab therapy to identify a panel of candidate protein biomarkers of RA predictive of treatment response. METHODS: Plasma profiles of 60 patients with RA (28 non-responders (as defined by the American College of Rheumatology 20% improvement criteria (ACR20)) negative and 32 responders (ACR70 positive) to infliximab) were studied by surface enhanced laser desorption/ionisation time-of-flight mass spectrometry (SELDI-TOF MS) technology on two types of arrays, an anion exchange array (SAX2) and a nickel affinity array (IMAC3-Ni). Biomarker characterisation was carried out using classical biochemical methods (purification by ammonium sulfate precipitation or metal affinity chromatography) and identification by matrix assisted laser desorption/ionisation time-of-flight (MALDI-TOF) MS analysis. RESULTS: Two distinct protein profiles were observed on both arrays and several proteins were differentially expressed in both patient populations. Five proteins at 3.86, 7.77, 7.97, 8.14 and 74.07 kDa were overexpressed in the non-responder group, whereas one at 28 kDa was increased in the responder population (sensitivity>56%, specificity>77.5%). Moreover, combination of several biomarkers improved the sensitivity and specificity of the detection of patient response to over 97%. The 28 kDa protein was characterised as apolipoprotein A-I and the 7.77 kDa biomarker was identified as platelet factor 4. CONCLUSIONS: Six plasma biomarkers are characterised, enabling the detection of patient response to infliximab with high sensitivity and specificity. Apolipoprotein A-1 was predictive of a good response to infliximab, whereas platelet factor 4 was associated with non-responders.

Early occupational therapy programme increases hand grip strength at 3 months: results from a randomised, blind, controlled study in early rheumatoid arthritis.

AIM: The goal of occupational therapy (OT) is to facilitate adjustments to lifestyle and to prevent function loss. This study evaluated the effects of an early OT programme in early rheumatoid arthritis (RA). METHODS: We conducted a randomised, blind, controlled trial enrolling 60 patients with early RA, divided into 2 groups. At baseline, group 1 received the full information programme and group 2 received no information. In an extension phase, patients in group 2 received the full information programme at 3 months and were assessed at 6 months. The main outcomes were grip strength of hands (as objective assessment) and Health Assessment Questionnaire (HAQ) score (as subjective assessment). RESULTS: At 3 months, grip strength of the dominant and non-dominant hands increased more in group 1 than in group 2 (p = 0.021 and 0.047 respectively). HAQ score decreased more in group 1 than in group 2 (p<0.001). In the extension phase, changes in grip strength and HAQ score in group 2 were similar to those seen in group 1 between baseline and 3 months. CONCLUSIONS: This study comparing two schedules of OT programme showed that an early extended information programme improved hand function in patients with early RA.

Physiopathology of haemophilic arthropathy.

Haemophilic arthropathy, which shares some clinical and biological injury characteristics with rheumatoid arthritis, is characterized by two main features: chronic proliferative synovitis and cartilage destruction. It is the consequence of repeated extravasation of blood into joint cavities, but its exact pathogenesis, particularly with regard to early changes in the joint, is still incompletely understood. This review presents recent findings obtained in experiments performed in vitro and using animal models, which have improved our knowledge of the pathogenesis of haemophilic arthropathy. These experimental studies show that haemophilic arthropathy is a multifactorial event in which the deposit of iron in the joints appears to exert a central role. First, iron may promote the apoptosis of chondrocytes by catalysing the formation of oxygen metabolites; this may explain the fact that intra-articular blood exerts a directly harmful effect on cartilage before, and independent of synovial changes. Secondly, iron may also act on the synovial membrane by favouring its proliferation through the induction of proto-oncogenes involved in cellular proliferation and stimulation of inflammatory cytokines as well as abrogation of apoptosis. These two processes, one degenerative and cartilage-mediated, the other inflammatory and synovium-mediated could occur in parallel or sequentially. Overall, it may be expected that these experimental results will yield new therapeutic strategies capable of effectively preventing the occurrence of this still serious and common complication in patients with severe haemophilia.

Etanercept treatment for three months is safe in patients with rheumatological manifestations associated with hepatitis C virus.

OBJECTIVE: The treatment of the rheumatological manifestations associated with hepatitis C virus (HCV) remains difficult. To examine the safety of anti-tumour necrosis factor-alpha treatment, nine patients having rheumatological manifestations associated with HCV were treated with etanercept 25 mg twice a week for 3 months. METHODS: Five patients had a positive viral load at study entry (Group I), four were negative (Group II). Clinical data recorded were: disease duration, painful and swollen joint count, patient global and physician global assessment, the number of 18 specified fibromyalgia tender points and the Health Assessment Questionnaire score. Laboratory studies included checking for the presence of cryoglobulinaemia and transaminase levels. Quantitative HCV viral RNA was performed by real-time polymerase chain reaction (PCR). RESULTS: At 3 months, no patient was found to have evidence of increased hepatic inflammation based on serial serum transaminase levels. In the five patients from Group I with detectable HCV RNA, no significant viral load increase was observed. No reactivation was observed in the four patients from Group II with undetectable HCV RNA. The effect on the clinical rheumatological manifestations was more heterogeneous but appears to be lower than that observed in rheumatoid arthritis. CONCLUSION: In this phase II open short-term study, etanercept appeared to be safe in patients with articular manifestations associated with HCV.

Extensive multiallelic analysis of the relationship between HLA-DRB1 and rheumatoid arthritis using a Bayesian partition model.

To analyse the association between individual HLA-DRB1 locus genotypes and rheumatoid arthritis (RA) susceptibility, taking in account the multiallelic nature of the shared epitope (SE). In total, 538 patients and 536 controls were genotyped for 12 alleles of the HLA-DRB1 locus. A Bayesian partition model and multivariate logistic models were used to assess the role of the SE and of its individual components. The SE was associated with RA susceptibility (odds ratio (OR) 2 versus 0 SE copy=9.99 (95 CI 4.69-15.30) and OR 1 versus 0 SE copy=3.16 (95% CI 2.42-4.12)). The Bayesian partition model supplied a permutation of the HLA-DRBA locus alleles ordered by increasing disease risk. Alleles associated with highest risks are those that code for the SE. The individual OR estimations for the HLA-DRB1 locus genotypes went from OR=1.00 (95% CI 1.00-1.25) for the less associated genotype to OR=21.40 (95% CI 8.02-65.79) for the most associated one. In conclusion, the allele order risk and the OR estimations for individual genotypes of the HLA-DRB1 locus were consistent with the SE theory. Using an exploratory statistical method without a priori hypothesis, our study allowed a detailed analysis of the multiallelic nature of the SE.