Molecular Regulation of Bone Turnover in Juvenile Idiopathic Arthritis: Animal Models, Cellular Features and TNFα.

We review the abnormal bone turnover that is the basis of idiopathic inflammatory or rheumatoid arthritis and bone loss, with emphasis on Tumor Necrosis Factor-alpha (TNFα)-related mechanisms. We review selected data on idiopathic arthritis in juvenile human disease, and discuss mouse models focusing on induction of bone resorbing cells by TNFα and Receptor Activator of Nuclear Factor kappa B Ligand (RANKL). In both humans and animal models, macrophage-derived cells in the joint, particularly in the synovium and periosteum, degrade bone and cartilage. Mouse models of rheumatoid arthritis share with human disease bone resorbing cells and strong relation to TNFα expression. In humans, differences in therapy and prognosis of arthritis vary with age, and results from early intervention for inflammatory cytokines in juvenile patients are particularly interesting. Mechanisms that contribute to inflammatory arthritis reflect, in large part, inflammatory cytokines that play minor roles in normal bone turnover. Changes in inflammatory cytokines, particularly TNFα, are many times larger, and presented in different locations, than cytokines that regulate normal bone turnover. Recent data from in vitro and mouse models include novel mechanisms described in differentiation of bone resorbing cells in inflammatory arthritis dependent on the Transient Receptor Potential Channel (TRPC) family of calcium channels. Low-molecular weight (MW) inhibitors of TRPC channels add to their potential importance. Associations with inflammatory arthritis unrelated to TNFα are briefly summarized as pointing to alternative mechanisms. We suggest that early detection and monoclonal antibodies targeting cytokines mediating disease progression deserves emphasis.

Lower hair cortisol concentration in adolescent and young adult patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome and Q-Fever Fatigue Syndrome compared to controls.

BACKGROUND: In patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS), momentary cortisol concentrations in blood, urine, and saliva are lower compared to healthy controls. Long-term cortisol concentration can be assessed through hair, but it is unclear whether these concentrations are also lower. Additionally, it is unknown if lower cortisol extends to other patients suffering from persistent fatigue and how hair cortisol concentration (HCC) relates to fatigue levels. Therefore, this study examines HCC in fatigued patients with ME/CFS, Q fever Fatigue Syndrome (QFS), Post-COVID-19 condition (PCC), and Juvenile Idiopathic Arthritis (JIA). METHODS: Adolescent and young adult patients with ME/CFS (n=12), QFS (n=20), PCC (n=8), JIA (n=19), and controls (n=57) were included. Patients participated in a randomized cross-over trial (RCT) targeting fatigue through lifestyle and dietary self-management strategies. HCC was measured pre-post RCT in patients and once in controls, quantified using a LC-MS/MS-based method. Fatigue severity was measured with the Checklist Individual Strength-8. HCC was compared between groups with ANOVAs. Relations between HCC, fatigue severity, and other variables were investigated using linear regression analyses. RESULTS: The ME/CFS (p=.009) and QFS (p=.047) groups had lower HCC compared to controls. Overall, HCC was negatively associated with the presence of symptoms related to chronic fatigue syndromes (e.g., sleeping issues, often feeling tired, trouble thinking clearly; ß=-0.018, p=.035), except in the QFS group (ß=.063, p<.001). Baseline HCC did not predict fatigue improvement during the RCT (p=.449), and HCC increased during the trial (Mdif=.076, p=.021) regardless of clinically relevant fatigue improvement (p=.658). CONCLUSION: Lower cortisol concentration can also be observed in the long-term. Lower HCC is not limited to ME/CFS, as it was also observed in QFS. The role of cortisol may differ between these diagnoses and appears to be unrelated to fatigue levels.

Blood brain barrier permeability and astrocyte-derived extracellular vesicles in children with juvenile idiopathic arthritis: a cross-sectional study.

BACKGROUND: Juvenile idiopathic arthritis (JIA) is the most prevalent rheumatic disease in children, and the inflammatory process is widely studied, primarily characterized by its impact on joint health. Emerging evidence suggests that JIA may also affect the central nervous system (CNS). This study investigates the potential CNS involvement in JIA by analyzing the presence of astrocyte-derived extracellular vesicles (EVs) and the S100B protein in plasma, both of which are indicative of astrocyte activity and blood-brain barrier (BBB) integrity. METHODS: EDTA plasma from 90 children diagnosed with JIA and 10 healthy controls, matched by age and gender, was analyzed for extracellular vesicles by flow cytometric measurement. Astrocyte-derived EVs were identified using flow cytometry with markers for aquaporin 4 (AQP-4) and glial fibrillary acidic protein (GFAP). Levels of the S100B protein were measured using a commercial ELISA. Disease activity was assessed using the Juvenile Arthritis Disease Activity Score (JADAS27, 0-57), and pain levels were measured using a visual analogue scale (VAS, 0-10 cm). RESULTS: Our analyses revealed a significantly higher concentration of astrocyte-derived EVs in the plasma of children with JIA compared with healthy controls. Furthermore, children with JADAS27 scores of 1 or higher exhibited notably higher levels of these EVs. The S100B protein was detectable exclusively in the JIA group. CONCLUSION: The elevated levels of astrocyte-derived EVs and the presence of S100B in children with JIA provide evidence of BBB disruption and CNS involvement, particularly in those with higher disease activity. These findings underscore the importance of considering CNS health in the comprehensive management of JIA. Further research is required to elucidate the mechanisms behind CNS engagement in JIA and to develop treatments that address both joint and CNS manifestations of the disease.

Th17/1 and ex-Th17 cells are detected in patients with polyarticular juvenile arthritis and increase following treatment.

BACKGROUND: A better understanding of the pathogenesis of polyarticular juvenile idiopathic arthritis (polyJIA) is needed to aide in the development of data-driven approaches to guide selection between therapeutic options. One inflammatory pathway of interest is JAK-STAT signaling. STAT3 is a transcription factor critical to the differentiation of inflammatory T helper 17 cells (Th17s). Previous studies have demonstrated increased STAT3 activation in adult patients with rheumatoid arthritis, but less is known about STAT3 activation in polyJIA. We hypothesized that Th17 cells and STAT3 activation would be increased in treatment-naïve polyJIA patients compared to pediatric controls. METHODS: Blood from 17 patients with polyJIA was collected at initial diagnosis and again if remission was achieved (post-treatment). Pediatric healthy controls were also collected. Peripheral blood mononuclear cells were isolated and CD4 + T cell subsets and STAT activation (phosphorylation) were evaluated using flow cytometry. Data were analyzed using Mann-Whitney U and Wilcoxon matched-pairs signed rank tests. RESULTS: Treatment-naïve polyJIA patients had increased Th17 cells (CD3 + CD4 + interleukin(IL)-17 +) compared to controls (0.15% v 0.44%, p < 0.05), but Tregs (CD3 + CD4 + CD25 + FOXP3 +) from patients did not differ from controls. Changes in STAT3 phosphorylation in CD4 + T cells following ex vivo stimulation were not significantly different in patients compared to controls. We identified dual IL-17 + and interferon (IFN)γ + expressing CD4 + T cells in patients, but not controls. Further, both Th17/1 s (CCR6 + CD161 + IFNγ + IL-17 +) and ex-Th17s (CCR6 + CD161 + IFNγ + IL-17neg) were increased in patients' post-treatment (Th17/1: 0.3% v 0.07%, p < 0.05 and ex-Th17s: 2.3% v 1.4%, p < 0.05). The patients with the highest IL-17 expressing cells post-treatment remained therapy-bound. CONCLUSIONS: Patients with polyJIA have increased baseline Th17 cells, potentially reflecting higher tonic STAT3 activation in vivo. These quantifiable immune markers may identify patients that would benefit upfront from pathway-focused biologic therapies. Our data also suggest that inflammatory CD4 + T cell subsets not detected in controls but increased in post-treatment samples should be further evaluated as a tool to stratify patients in remission on medication. Future work will explore these proposed diagnostic and prognostic biomarkers.

Methotrexate inhibits BMP4 and abrogates the hypertrophic chondrocyte phenotype of synovial fibroblasts in juvenile idiopathic arthritis.

BACKGROUND: Juvenile Idiopathic Arthritis (JIA) induces growth disturbances in affected joints. Fibroblast-like synoviocytes (FLS) play a crucial role in JIA pathogenesis. FLS overexpress bone morphogenetic protein 4 (BMP4) and have a chondrocyte-like phenotype. FLS contribute directly to joint growth disturbances through endochondral bone formation. We investigated the ability of methotrexate to inhibit BMP4 expression and alter the hypertrophic chondrocyte-like phenotype of JIA FLS. METHODS: We selected primary cells from three subjects with persistent oligoarticular JIA, three subjects who eventually extended to a polyarticular disease course, which we termed extended-to-be (ETB), and three subjects who had polyarticular arthritis at time of diagnosis. We treated cells with methotrexate and two BMP4 inhibitors: noggin and chordin. We measured protein concentration from three chondrocyte cell markers: collagen II, aggrecan, and collagen X as well as BMP4. RESULTS: ColX, marker of chondrocyte hypertrophy, was significantly increased in polyarticular FLS when compared to both persistent FLS and ETB FLS, making polyarticular FLS the most like hypertrophic chondrocytes. Methotrexate caused significant decreases in BMP4 and ColX expression in persistent, ETB, and polyarticular FLS when compared to respective untreated cells. Ligand-binding BMP4 antagonists, noggin and chordin, caused significant decreases in ColX expression in FLS from all three disease courses and significant increases in collagen II protein, an early chondrocyte marker, when compared to respective untreated cells. CONCLUSIONS: Methotrexate, the first-line therapy in the treatment of JIA, mimics BMP4 antagonists by effectively lowering BMP4 and ColX expression in FLS. Inhibiting FLS from undergoing hypertrophy could prevent these cells from contributing to joint growth disturbances via endochondral bone formation.

Macrophage IL-1ß-positive microvesicles exhibit thrombo-inflammatory properties and are detectable in patients with active juvenile idiopathic arthritis.

Objective: IL-1ß is a leaderless cytokine with poorly known secretory mechanisms that is barely detectable in serum of patients, including those with an IL-1ß-mediated disease such as systemic juvenile idiopathic arthritis (sJIA). Leukocyte microvesicles (MVs) may be a mechanism of IL-1ß secretion. The first objective of our study was to characterize IL-1ß-positive MVs obtained from macrophage cell culture supernatants and to investigate their biological functions in vitro and in vivo. The second objective was to detect circulating IL-1ß-positive MVs in JIA patients. Methods: MVs were purified by serial centrifugations from PBMCs, or THP-1 differentiated into macrophages, then stimulated with LPS ± ATP. MV content was analyzed for the presence of IL-1ß, NLRP3 inflammasome, caspase-1, P2X7 receptor, and tissue factor (TF) using ELISA, Western blot, or flow cytometry. MV biological properties were studied in vitro by measuring VCAM-1, ICAM-1, and E-selectin expression after HUVEC co-culture and factor-Xa generation test was realized. In vivo, MVs' ability to recruit leukocytes in a murine model of peritonitis was evaluated. Plasmatic IL-1ß-positive MVs were studied ex vivo in 10 active JIA patients using flow cytometry. Results: THP-1-derived macrophages stimulated with LPS and ATP released MVs, which contained NLRP3, caspase-1, and the 33-kDa precursor and 17-kDa mature forms of IL-1ß and bioactive TF. IL-1ß-positive MVs expressed P2X7 receptor and released soluble IL-1ß in response to ATP stimulation in vitro. In mice, MVs induced a leukocyte peritoneal infiltrate, which was reduced by treatment with the IL-1 receptor antagonist. Finally, IL-1ß-positive MVs were detectable in plasma from 10 active JIA patients. Conclusion: MVs shed from activated macrophages contain IL-1ß, NLRP3 inflammasome components, and TF, and constitute thrombo-inflammatory vectors that can be detected in the plasma from active JIA patients.

MicroRNA-27a-3p enhances the inflammatory phenotype of Juvenile Idiopathic Arthritis fibroblast-like synoviocytes.

BACKGROUND: Juvenile Idiopathic Arthritis (JIA) is the most prevalent chronic pediatric rheumatic disorder. In joints of JIA patients, aggressive phenotypic changes in fibroblast-like synoviocytes (FLS) of the synovial lining play a key role in inflammation. MicroRNAs are dysregulated in rheumatoid arthritis and JIA, including miR-27a-3p. However, it is not understood if miR-27a-3p, enriched in JIA synovial fluid (SF) and leukocytes, alters FLS function. METHODS: Primary JIA FLS cells were transfected with a miR-27a-3p mimic or a negative control microRNA (miR-NC) and stimulated with pooled JIA SF or inflammatory cytokines. Viability and apoptosis were analyzed by flow cytometry. Proliferation was evaluated using a 3H-thymidine incorporation assay. Cytokine production was assessed by qPCR and ELISA. Expression of TGF-ß pathway genes was determined using a qPCR array. RESULTS: MiR-27a-3p was constitutively expressed in FLS. Overexpression of miR-27a-3p caused increased interleukin-8 secretion in resting FLS, and interleukin-6 was elevated in SF-activated FLS compared to miR-NC. Furthermore, stimulation with pro-inflammatory cytokines augmented FLS proliferation in miR-27a-3p-transfected FLS relative to miR-NC. Expression of multiple TGF-ß pathway genes was modulated by overexpression of miR-27a-3p. CONCLUSIONS: MiR-27a-3p significantly contributes to FLS proliferation and cytokine production, making it a potential candidate for epigenetic therapy that targets FLS in arthritis.

Assessment of Cardiovascular Risk Factors in Patients with Juvenile Idiopathic Arthritis.

INTRODUCTION: The aim of this study was to assess the exposure to cardiovascular disease (CVD) risk factors in patients with juvenile idiopathic arthritis (JIA). Intima-media complex thickness (IMT), selected metabolic parameters and health behaviors were assessed in the course of the study. METHODS: The study included study group, which consisted of 45 patients with JIA and 37 healthy age- and sex-matched children in the control group. Analyses in both groups included anthropometric parameters, laboratory tests, IMT and a questionnaire on exposure to modifiable CVD risk factors. RESULTS: The study confirmed that CVD risk factors were present in both groups of patients. Significantly more children with JIA had abnormal BMI (p = 0.006) compared to the control group. Children in the study group were more likely to consume fruit regularly (p = 0.021) and less likely to consume fast food (p = 0.011) and sweetened beverages (p = 0.042) than children in the control group. Only 1 patient with JIA met criteria for ideal cardiovascular health. Dietary habits were not associated with IMT values, BMI, presence of joint pain or biochemical parameters in the study group. CONCLUSIONS: Patients with JIA are exposed to cardiovascular risk factors equally to their healthy peers. Ideal cardiovascular health should be pursued in the pediatric population with particular attention paid to patients with chronic diseases (i.e., JIA). The application of carotid artery IMT measurement in the assessment of CVD risk requires studies on a larger group of patients.

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OBJECTIVES: To study the expression of V-domain Ig suppressor of T cell activation (VISTA) in peripheral blood of children with juvenile idiopathic arthritis (JIA) and its role in the pathogenesis of JIA. METHODS: In this prospective study, peripheral blood was collected from 47 children with different subtypes of JIA and 10 healthy children. Flow cytometry was used to measure the expression levels of VISTA, interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) on CD14+ mononuclear cells, CD4+ T lymphocytes, and CD8+ T lymphocytes. RESULTS: The children with JIA had a significantly lower expression level of VISTA than the healthy children (P<0.05). There was a significant difference in the expression of VISTA between the children with different subtypes of JIA, with the lowest expression level in those with systemic JIA (P<0.05). There was also a significant difference in the expression of VISTA between different immune cells, with a significantly higher expression level on the surface of monocytes (P<0.05). Correlation analysis showed that VISTA was negatively correlated with the expression of IFN-γ and TNF-α on CD4+ T cells (r=-0.436 and -0.382 respectively, P<0.05), CD8+ T cells (r=-0.348 and -0.487 respectively, P<0.05), and CD14+ mononuclear cells (r=-0.582 and -0.603 respectively, P<0.05). CONCLUSIONS: The insufficient expression of VISTA may be associated with the pathogenesis of JIA, and enhancing the immunomodulatory effect of VISTA might be one option for the treatment of JIA in the future.

Biomarker significance of interleukins, IL-37 and IL-38 in patients with juvenile idiopathic arthritis.

INTRODUCTION: Juvenile idiopathic arthritis (JIA) is the most common rheumatic condition that develops during child age and adolescence. Unbalanced production of proinflammatory cytokines is suggested to participate in the etio-pathogenesis of JIA, so the objective of this study is to evaluate the role of interleukins (IL), IL-37 and IL-38, in patients with JIA. MATERIALS AND METHODS: Sixty patients with JIA (19 males, 41 females) and 90 healthy controls (35 males, 55 females) were included in this study. Participants were assessed using the juvenile arthritis disease activity score-27 and underwent laboratory tests, including measurements for C-reactive protein, rheumatoid factor, and IL-37 and IL-38. RESULTS: Mean ages of JIA patients and controls were 10.37±4.21 years and 11.13±3.84 years, respectively. Compared to controls, serum IL-37 levels were increased in patients with JIA (117.98±209.282ng/ml vs. 37.87±24.496ng/ml; p<0.01), whereas serum IL-38 titres were diminished in individuals with JIA (106.2±95.781ng/ml vs. 182.24±108.428 ng/ml; p<0.01). CONCLUSION: This study provides a further layer of evidence for the role played by IL-37 in JIA and creates new questions about the potential role of IL-38 in this condition.

Mitigated suppressive function of regulatory T cells (Treg) upon Th17-inducing cytokines in oligo- and polyarticular Juvenile Idiopathic Arthritis (JIA) patients.

BACKGROUND: The plasticity of T helper-17 (Th17) and regulatory T (Treg) cells may be a clue to pathogenesis of Juvenile Idiopathic Arthritis (JIA). It is still unclear, whether targeted suppression of Interleukin (IL)-17 is able to influence regulatory function of Treg to control pro-inflammatory effectors in JIA. This study aimed to assess the effect of a Th17-stimulating cytokine environment and of IL-17A-inhibition on phenotype plasticity and suppressive function of Treg derived from JIA patients. METHODS: Th17 and Treg characteristics of CD4+ helper T cells were investigated in blood samples of JIA patients with oligo- and polyarticular pattern and healthy controls (HC). Isolated CD4+CD25+CD127- cells defined as Treg were cultivated with Th17-inducing cytokine environment as well as with IL-17A-inhibitors and analyzed for plasticity of phenotype by flow cytometry. Furthermore, inhibitory function of Treg on autologous effectors after cultivation with these stimuli was determined by suppression assays. RESULTS: Our findings demonstrated significantly elevated proportions of Th17 and Th17-like Treg in JIA compared to HC. After incubation with Th17-inducing stimuli, increased FoxP3 expression in separated Treg in JIA and an impaired suppressive capacity in JIA and HC were found. Blockade of IL-17A resulted in adjustment of FoxP3-expression in JIA to proportions found in controls and in regular suppressive function. CONCLUSIONS: Our results demonstrate an induction of FoxP3 expressing Treg by Th17-inducing cytokines with concomitant mitigated suppressive function. In contrast, specific IL-17A blockade maintains suppressive Treg function and adjusted FoxP3-expression in JIA to levels found in controls. These findings may help to provide experimental evidence for the successful clinical use of IL-17A inhibition in JIA patients.

Serine, N-acetylaspartate differentiate adolescents with juvenile idiopathic arthritis compared with healthy controls: a metabolomics cross-sectional study.

BACKGROUND: In comparison with the general population, adolescents with juvenile idiopathic arthritis (JIA) are at higher risk for morbidity and mortality. However, limited evidence is available about this condition's underlying metabolic profile in adolescents with JIA relative to healthy controls. In this untargeted, cross-sectional metabolomics study, we explore the plasma metabolites in this population. METHODS: A sample of 20 adolescents with JIA and 20 controls aged 13-17 years were recruited to complete surveys, provide medical histories and biospecimens, and undergo assessments. Fasting morning plasma samples were processed with liquid chromatography-mass spectrometry. Data were centered, scaled, and analyzed using generalized linear models accounting for age, sex, and medications (p-values adjusted for multiple comparisons using the Holm method). Spearman's correlations were used to evaluate relationships among metabolites, time since diagnosis, and disease severity. RESULTS: Of 72 metabolites identified in the samples, 55 were common to both groups. After adjustments, 6 metabolites remained significantly different between groups. Alpha-glucose, alpha-ketoglutarate, serine, and N-acetylaspartate were significantly lower in the JIA group than in controls; glycine and cystine were higher. Seven additional metabolites were detected only in the JIA group; 10 additional metabolites were detected only in the control group. Metabolites were unrelated to disease severity or time since diagnosis. CONCLUSIONS: The metabolic signature of adolescents with JIA relative to controls reflects a disruption in oxidative stress; neurological health; and amino acid, caffeine, and energy metabolism pathways. Serine and N-acetylaspartate were promising potential biomarkers, and their metabolic pathways are linked to both JIA and cardiovascular disease risk. The pathways may be a source of new diagnostic, treatment, or prevention options. This study's findings contribute new knowledge for systems biology and precision health approaches to JIA research. Further research is warranted to confirm these findings in a larger sample.

EXPRESSION OF TOLL-LIKE RECEPTORS 4 ON CD14 + MONOCYTES IN JUVENILE IDIOPATHIC ARTHRITIS.

OBJECTIVE: The aim: The work is aimed at determining the relationship between TLR4 expression on CD14+monocytes in whole heparinized blood and B1a lymphocyte synthesis in various subtypes of JIA. PATIENTS AND METHODS: Materials and methods: 64children aged3to17years were examined, including42children with different subtypes of JIA and22healthy children. The intensity of TLR4 expression onCD14+monocytes was determined in whole heparinized blood incubated with a CD14-FITC/TLR4-PE monoclonal antibody cocktail(Biolegend, USA)using flow cytometry. Monoclonal antibodies (BD Bioscience) were used to determine the main subpopulations of lymphocytes. RESULTS: Results: A statistically significant increase in TLR4 expression has been determined in JIA compared to the control group. The most prominent TLR4 expression was detected in children with oligoarthritis, while in systemic arthritis, there was no statistical difference compared to healthy children. High TLR4 expression on peripheral CD14+monocytes inversely depends on the activity of the autoimmune process, which may have a protective effect against the aseptic inflammation.Increased TLR4 expression involves a statis¬tically significant increase in the percentage and quantity of В1а lymphocytes(p≤0.05). CONCLUSION: Conclusions: A statistically significant increase in TLR4 expression on CD14+monocytes in whole heparinized blood was detected in patients with JIA compared to healthy children. Children with oligoarthritis had the highest rates, which indicates possible differences in the development of pathogenetic processes in different subtypes of arthritis. Determining the degree of TLR4 activation on CD14+monocytes is reasonable for predicting JIA activity.

Studying Juvenile Idiopathic Arthritis through a Network Medicine Approach / Estudando a Artrite Idiopática Juvenil mediante uma abordagem de medicina de redes

Juvenile Idiopathic Arthritis (JIA) is a group of inflammatory conditions of unknown etiology whose underlying molecular pathophysiology is still not well characterized. Several studies have attempted to fill this gap by characterizing the gene expression profiles of JIA patients. However, there is a lack of systematic assessment of the reliability of these transcriptome results on the disease classification, prescription, and monitoring. In addition, despite this disease is more common in females, none of these studies have tried to assess the impact of sex on disease pathophysiology. In this project, we performed a comprehensive systematic review and a gene expression meta-analysis to reveal the core molecular JIA pathophysiology taking into consideration the patient sex. We gathered and cataloged more than 60,000 entries of genomic features reported as JIA-related in the functional genomics literature, and found a dramatic disparity among the JIA transcriptome studies. Near 15,000 genes have been reported as perturbed in JIA leukocytes. Less than one percent of these genes were reported in at least a quarter of the reviewed studies. We then removed the study-specific analytical bias by re-analyzing more than 700 unique pediatric transcriptome profiles from nine JIA studies using a common analytical framework. The differential expression results from different studies were combined using a random effect model meta-analysis approach. We implemented this differential gene expression meta-analysis methodology in the MetaVolcanoR R package that we made available in Bioconductor. Using this package, we confirmed several gene expression signatures previously associated with JIA and uncover new genes whose expression was perturbed in JIA patients. The effect sizes of the topmost reported perturbed genes coincide with our meta-analysis results. Through a meta-coexpression approach, we characterized the cell type signatures of circulating leukocytes in the JIA affected children. Additionally, we characterized the JIA sexual dimorphism. We found that systemic JIA female patients over-activate a gene expression signature which comprises early myelocytes and band neutrophil expression markers. This signature is correlated with the disease status and response to IL-1 receptor blockade. This suggests that sJIA pathophysiology is characterized by a sexually dimorphic neutrophilia that impacts disease progression and the response to anti-IL-1 treatments. We further assessed this immature neutrophil and female-biased signature in other contexts. We found that this signature presents a sex-dependent expression over human lifetime, in other inflammatory diseases, and its expression increases during pregnancy

A Artrite Idiopática Juvenil (AIJ) é um grupo de condições inflamatórias de etiologia desconhecida, cuja patofisiologia molecular subjacente ainda não está bem caracterizada. Vários estudos tentaram preencher essa lacuna, caracterizando os perfis de expressão gênica de pacientes com AIJ. No entanto, há uma falta de avaliação sistemática desses resultados transcriptômicos na classificação, prescrição e monitoramento da doença. Além disso, apesar de esta doença ser mais comum em mulheres, nenhum desses estudos tentou avaliar o impacto do sexo na fisiopatologia da doença. Neste projeto, realizamos uma revisão sistemática abrangente e uma metanálise de expressão gênica para revelar a fisiopatologia molecular da AIJ levando em consideração o sexo do paciente. Reunimos e catalogamos mais de 60.000 entradas de características genômicas reportadas como relacionadas à AIJ na literatura. Entre os estudos de transcriptoma, encontramos uma disparidade dramática. Cerca de 15.000 genes foram reportados como perturbados nos leucócitos da AIJ, sendo que menos de um por cento desses genes foram relatados em pelo menos um quarto dos estudos revisados. Em seguida, re-analisamos mais de 700 transcriptomas pediátricos de nove estudos usando uma abordagem analítica comum. Os resultados de expressão diferencial foram combinados usando meta-análise de modelo de efeitos aleatórios. Implementamos esta abordagem de meta-análise de expressão gênica diferencial no pacote MetaVolcanoR R que disponibilizamos no Bioconductor. Usando este pacote, confirmamos várias assinaturas de expressão gênica previamente associadas à AIJ e descobrimos novos genes cuja expressão está perturbada em pacientes com AIJ. Os tamanhos dos efeitos dos genes mais reportados como perturbados coincidem com os resultados da nossa meta-análise. Por meio de uma análise de meta-co-expressão, caracterizamos as assinaturas dos tipos de leucócitos circulantes. Além disso, caracterizamos o dimorfismo sexual da AIJ. Descobrimos que pacientes do sexo feminino com AIJ sistêmica super-ativam genes característicos de mielócitos precoces e neutrófilos bastonetes. Esta assinatura está correlacionada com o estado clínico da doença e à resposta ao tratamento por bloqueio do receptor de IL-1. Isto sugere que a fisiopatologia da AIJs é caracterizada por uma neutrofilia sexualmente dimórfica que afeta a progressão da doença e a resposta aos tratamentos anti-IL-1. Avaliamos ainda esta assinatura neutrofílica em outros contextos. Descobrimos que essa assinatura apresenta uma expressão dependente do sexo ao longo da vida humana, em outras doenças inflamatórias, e sua expressão aumenta durante a gravidez

Infection with SARS-CoV-2 causes flares in patients with juvenile idiopathic arthritis in remission or inactive disease on medication.

BACKGROUND: Flares of juvenile idiopathic arthritis (JIA) have been described in the context of various infections. Flares of rheumatic diseases in adults have been described following infection with SARS-CoV-2 in several cohorts. So far, the effect of infection with SARS-CoV-2 on the course of JIA is unknown. METHODS: The database of the German Center for Pediatric and Adolescent Rheumatology was searched for patients with confirmed infection with SARS-CoV-2 and subsequent disease flare, admitted from July 2020 until June 2021. cJADAS-27, ESR and C-reactive protein, as well as uveitis activity, medication at the time of flare and treatment of flare was extracted. Patient cases were described individually. RESULTS: Out of 988 patients admitted, five patients with remission off medication (n = 2) or inactive disease on medication (n = 3) were identified, with flare symptoms up to four weeks after infection with SARS-CoV-2. CONCLUSIONS: Flares can occur after infection with SARS-CoV-2 in patients with JIA in remission or inactive disease on medication. Treating physicians need to be aware of this fact, especially when counseling patients with rheumatic diseases about the respective dangers of COVID-19 and vaccination against SARS-CoV-2.

Immunometabolic factors in adolescent chronic disease are associated with Th1 skewing of invariant Natural Killer T cells.

Invariant Natural Killer T (iNKT) cells respond to the ligation of lipid antigen-CD1d complexes via their T-cell receptor and are implicated in various immunometabolic diseases. We considered that immunometabolic factors might affect iNKT cell function. To this end, we investigated iNKT cell phenotype and function in a cohort of adolescents with chronic disease and immunometabolic abnormalities. We analyzed peripheral blood iNKT cells of adolescents with cystic fibrosis (CF, n = 24), corrected coarctation of the aorta (CoA, n = 25), juvenile idiopathic arthritis (JIA, n = 20), obesity (OB, n = 20), and corrected atrial septal defect (ASD, n = 25) as controls. To study transcriptional differences, we performed RNA sequencing on a subset of obese patients and controls. Finally, we performed standardized co-culture experiments using patient plasma, to investigate the effect of plasma factors on iNKT cell function. We found comparable iNKT cell numbers across patient groups, except for reduced iNKT cell numbers in JIA patients. Upon ex-vivo activation, we observed enhanced IFN-γ/IL-4 cytokine ratios in iNKT cells of obese adolescents versus controls. The Th1-skewed iNKT cell cytokine profile of obese adolescents was not explained by a distinct transcriptional profile of the iNKT cells. Co-culture experiments with patient plasma revealed that across all patient groups, obesity-associated plasma factors including LDL-cholesterol, leptin, and fatty-acid binding protein 4 (FABP4) coincided with higher IFN-γ production, whereas high HDL-cholesterol and insulin sensitivity (QUICKI) coincided with higher IL-4 production. LDL and HDL supplementation in co-culture studies confirmed the effects of lipoproteins on iNKT cell cytokine production. These results suggest that circulating immunometabolic factors such as lipoproteins may be involved in Th1 skewing of the iNKT cell cytokine response in immunometabolic disease.

Application of Weighted Gene Coexpression Network Analysis to Identify Key Modules and Hub Genes in Systemic Juvenile Idiopathic Arthritis.

Systemic juvenile idiopathic arthritis (sJIA) is a severe autoinflammatory disorder with a still not clearly defined molecular mechanism. To better understand the disease, we used scattered datasets from public domains and performed a weighted gene coexpression network analysis (WGCNA) to identify key modules and hub genes underlying sJIA pathogenesis. Two gene expression datasets, GSE7753 and GSE13501, were used to construct the WGCNA. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were applied to the genes and hub genes in the sJIA modules. Cytoscape was used to screen and visualize the hub genes. We further compared the hub genes with the genome-wide association study (GWAS) genes and used a consensus WGCNA to verify that our conclusions were conservative and reproducible across multiple independent datasets. A total of 5,414 genes were obtained for WGCNA, from which highly correlated genes were divided into 17 modules. The red module demonstrated the highest correlation with the sJIA module (r = 0.8, p = 3e -29), whereas the green-yellow module was found to be closely related to the non-sJIA module (r = 0.62, p = 1e -14). Functional enrichment analysis demonstrated that the red module was mostly enriched in the activation of immune responses, infection, nucleosomes, and erythrocytes, and the green-yellow module was mostly enriched in immune responses and inflammation. Additionally, the hub genes in the red module were highly enriched in erythrocyte differentiation, including ALAS2, AHSP, TRIM10, TRIM58, and KLF1. The hub genes from the green-yellow module were mainly associated with immune responses, as exemplified by the genes KLRB1, KLRF1, CD160, and KIRs. We identified sJIA-related modules and several hub genes that might be associated with the development of sJIA. Particularly, the modules may help understand the mechanisms of sJIA, and the hub genes may become biomarkers and therapeutic targets of sJIA in the future.

Lipopolysaccharide stimulation test on cultured PBMCs assists the discrimination of cryopyrin-associated periodic syndrome from systemic juvenile idiopathic arthritis.

Systemic juvenile idiopathic arthritis (sJIA) and cryopyrin-associated periodic syndrome (CAPS) share many common manifestations. We aim to identify an applicable method to assist disease discrimination. Inflammatory cytokines were measured in the plasma of patients with CAPS, sJIA with persistent disease course and healthy controls. Supernatants collected from non-stimulated peripheral blood mononuclear cells (PBMCs) and those undergone inflammasome stimulation tests utilizing lipopolysaccharide (LPS) with and without adenosine triphosphate (ATP) were investigated. Inflammatory cytokines in patient plasma fail to differentiate sJIA from CAPS. PBMCs from sJIA secrets higher amount of IL-1ß and IL-18 while CAPS PBMCs produces more caspase-1 without stimulation. IL-1ß, IL-18, and caspase-1 were significantly elevated among CAPS PBMCs (all p < 0.05) upon LPS stimulation, but not when additional ATPs were provided. Levels of cytokines and PBMC responses to the stimulation assays were similar among all sJIA patients regardless of their history of macrophage activation syndrome. Unstimulated PBMC activities and the LPS inflammasome stimulation assay without exogenic ATPs can assist the differentiation of CAPS from sJIA with persistent disease course.

The culture microenvironment of juvenile idiopathic arthritis synovial fibroblasts is favorable for endochondral bone formation through BMP4 and repressed by chondrocytes.

BACKGROUND: We examined influences of conditioned media from chondrocytes (Ch) on juvenile idiopathic arthritis synovial fibroblasts (JFLS) and potential for JFLS to undergo endochondral bone formation (EBF). METHODS: Primary cells from three control fibroblast-like synoviocytes (CFLS) and three JFLS were cultured in Ch-conditioned media and compared with untreated fibroblast-like synoviocytes (FLS). RNA was analyzed by ClariomS microarray. FLS cells cultured in conditioned media were exposed to either TGFBR1 inhibitor LY3200882 or exogenous BMP4 and compared with FLS cultured in conditioned media from Ch (JFLS-Ch). Media supernatants were analyzed by ELISA. RESULTS: In culture, JFLS downregulate BMP2 and its receptor BMPR1a while upregulating BMP antagonists (NOG and CHRD) and express genes (MMP9, PCNA, MMP12) and proteins (COL2, COLX, COMP) associated with chondrocytes. Important TGFß superfamily member gene expression (TGFBI, MMP9, COL1A1, SOX6, and MMP2) is downregulated when JFLS are cultured in Ch-conditioned media. COL2, COLX and COMP protein expression decreases in JFLS-Ch. BMP antagonist protein (NOG, CHRD, GREM, and FST) secretion is significantly increased in JFLS-Ch. Protein phosphorylation increases in JFLS-Ch exposed to exogenous BMP4, and chondrocyte-like phenotype is restored in BMP4 presence, evidenced by increased secretion of COL2 and COLX. Inhibition of TGFBR1 in JFLS-Ch results in overexpression of COL2. CONCLUSIONS: JFLS are chondrocyte-like, and Ch-conditioned media can abrogate this phenotype. The addition of exogenous BMP4 causes JFLS-Ch to restore this chondrocyte-like phenotype, suggesting that JFLS create a microenvironment favorable for endochondral bone formation, thereby contributing to joint growth disturbances in juvenile idiopathic arthritis.