Objective Systemic juvenile idiopathic arthritis–associated lung disease (SJIA‐LD) is a life‐threatening disease complication. Key questions remain regarding clinical course and optimal treatment approaches. The objectives of the study were to detail management strategies after SJIA‐LD detection, characterize overall disease courses, and measure long‐term outcomes. Methods This was a prospective cohort study. Clinical data were abstracted from the electronic medical record, including current clinical status and changes since diagnosis. Serum biomarkers were determined and correlated with presence of LD. Results We enrolled 41 patients with SJIA‐LD, 85% with at least one episode of macrophage activation syndrome and 41% with adverse reactions to a biologic. Although 93% of patients were alive at last follow‐up (median 2.9 years), 37% progressed to requiring chronic oxygen or other ventilator support, and 65% of patients had abnormal overnight oximetry studies, which changed over time. Eighty‐four percent of patients carried the HLA‐DRB1*15 haplotype, significantly more than patients without LD. Patients with SJIA‐LD also showed markedly elevated serum interleukin‐18 (IL‐18), variable C‐X‐C motif chemokine ligand 9 (CXCL9), and significantly elevated matrix metalloproteinase 7. Treatment strategies showed variable use of anti–IL‐1/6 biologics and addition of other immunomodulatory treatments and lung‐directed therapies. We found a broad range of current clinical status independent of time from diagnosis or continued biologic treatment. Multidomain measures of change showed imaging features were the least likely to improve with time. Conclusion Patients with SJIA‐LD had highly varied courses, with lower mortality than previously reported but frequent hypoxia and requirement for respiratory support. Treatment strategies were highly varied, highlighting an urgent need for focused clinical trials.

Background: Multisystem inflammatory syndrome in children (MIS-C) is a potentially life-threatening hyperinflammatory syndrome that occurs after primary SARS-CoV-2 infection. The pathogenesis of MIS-C remains undefined, and whether specific inflammatory biomarker patterns can distinguish MIS-C from other hyperinflammatory syndromes including Kawasaki disease (KD) and macrophage activation syndrome (MAS) is unknown. Methods: We studied a prospective cohort of nineteen MIS-C and nine KD patients and an established cohort of eleven new onset SJIA and nine MAS associated SJIA patients. Clinical and laboratory features as well as S100A8/A9, S100A12, IL-18, CXCL9 and IL-6 levels were compared between disease groups. Findings: KD and MIS-C patients have similar S100 proteins and IL-18 profiles but are distinguished by significantly higher levels of the IFN-γ-induced chemokine CXCL9 in MIS-C. Stratifying MIS-C patients by CXCL9 levels revealed differential severity of clinical and laboratory presentation. MIS-C with high CXCL9 levels was associated with acute kidney injury, altered mental status, a higher frequency of shock (40 vs 90%), myocardial dysfunction (20 vs 50%), and more severe systemic inflammatory markers, cytopenia, and coagulopathy. The low CXCL9 MIS-C group in contrast resembled KD patients including the frequency of coronary involvement. We also found that elevated S100A8/A9, S100A12 and IL-18 were useful in distinguishing SJIA from KD with high sensitivity and specificity. Interpretation: Our findings show MIS-C is distinguished from KD primarily by elevated CXCL9. The stratification of CXCL9 levels of MIS-C patients provides support for MAS pathophysiology in patients with severe MIS-C, suggesting new approaches for diagnosis and management. Funding: This work was supported by an Academic Research Clinical (ARC) award to AG and GS from the Cincinnati Children’s Research Foundation. GS was supported by NIAMS/NIH K08-AR072075, AG by P30-AR070549, and JRS and GC by T32-AR069512. EV was supported by the Deutsche Forschungsgemeinschaft (German Research Foundation, DFG/448863690). Declaration of Interests: AG has served as a consultant and received research support from Novartis, Sobi, NovImmune and AB2Bio. GS consulting fees from Novartis and SOBI. All other authors report no disclosures. Ethics Approval Statement: The study was approved by the Institutional Review Board (CCHMC IRB2018-2408).

Objective Macrophage activation syndrome (MAS) is a life-threatening complication of systemic juvenile idiopathic arthritis (sJIA) characterised by a vicious cycle of immune amplification that can culminate in overwhelming inflammation and multiorgan failure. The clinical features of MAS overlap with those of active sJIA, complicating early diagnosis and treatment. We evaluated adenosine deaminase 2 (ADA2), a protein of unknown function released principally by monocytes and macrophages, as a novel biomarker of MAS. Methods We established age-based normal ranges of peripheral blood ADA2 activity in 324 healthy children and adults. We compared these ranges with 173 children with inflammatory and immune-mediated diseases, including systemic and non-systemic JIA, Kawasaki disease, paediatric systemic lupus erythematosus and juvenile dermatomyositis. Results ADA2 elevation beyond the upper limit of normal in children was largely restricted to sJIA with concomitant MAS, a finding confirmed in a validation cohort of sJIA patients with inactive disease, active sJIA without MAS or sJIA with MAS. ADA2 activity strongly correlated with MAS biomarkers including ferritin, interleukin (IL)-18 and the interferon (IFN)-γ-inducible chemokine CXCL9 but displayed minimal association with the inflammatory markers C reactive protein and erythrocyte sedimentation rate. Correspondingly, ADA2 paralleled disease activity based on serial measurements in patients with recurrent MAS episodes. IL-18 and IFN-γ elicited ADA2 production by peripheral blood mononuclear cells, and ADA2 was abundant in MAS haemophagocytes. Conclusions These findings collectively identify the utility of plasma ADA2 activity as a biomarker of MAS and lend further support to a pivotal role of macrophage activation in this condition.

Background: MicroRNAs (miRNAs) are small noncoding RNAs which post-transcriptionally regulate gene expression. The miR-17-92 cluster is well studied in cancer biology and cellular differentiation; its overexpression has been found to serve a major oncogenic role in the targeting and downregulation of tumor-suppressive pathways, such as PTEN or TGFß. Our previous work identified several members of the cluster – miR-18, miR-19a/b, miR-20a, and miR-92a – with significantly higher levels in monocytes from patients with active Systemic Juvenile Idiopathic Arthritis (SJIA). SJIA is a chronic inflammatory disease of childhood with features of autoinflammation, and innate immune cells including monocytes have important roles in disease pathogenesis. Children with SJIA are at risk for life-threatening complications including Macrophage Activation Syndrome (MAS), an episode of overwhelming inflammation characterized by macrophage proliferation and driven by IFNγ. Objectives: Characterize the regulation of the miR-17-92 cluster, define key targets, and determine the cluster’s role in inflammation and SJIA. Methods: MiRNA levels were examined in THP-1 cells, as well as primary human monocytes isolated from healthy donors over the course of the monocyte to monocyte-derived macrophage (MDM) transition. MiR-17, miR-19a, and miR-20a were overexpressed via transfection in CD14+ monocytes for 2 days. Transcriptional profiles were performed using Ampliseq Transcriptome and the Ion Torrent S5 system and analyzed using AltAnalyze. Potential targets of the miR-17-92 cluster determined from sequencing analysis were then validated via dual-luciferase reporter assay. Results: Neither blood monocytes nor fully differentiated THP-1 cells showed significant changes in miR-17-92 levels under standard polarization conditions, including M1, M2a, and M2b conditions, or IL-6 and IL-10 stimulation. The most sizable changes in miR-17-92 levels were found during monocyte to macrophage transition. Interestingly, primary monocytes showed increases in miR-17-92 levels within the first 48 hours of differentiation towards MDM, variable by miRNA and experiment, similar to that seen in SJIA monocytes. In contrast, both PMA-differentiated THP1 cells and fully differentiated MDMs showed decreased miR-17-92 compared to undifferentiated monocytic cells. MiR-17-92 was overexpressed in vitro in primary monocytes to model these early transition changes. Genome-wide transcriptional profiling showed an upregulation of genes involved in Type I and II Interferon pathways, including response to interferon-alpha (adjusted p=2.71x10-12) and interferon-gamma (adjusted p=7.81x10-9). Analysis of genes significantly downregulated by miR-17, miR-19a, or miR-20a identified several putative and previously validated miR-17-92 cluster targets, including ATG5, IFRD2, JAK1, PPARG, and PTPN2 which have interferon-regulatory functions. Dual-luciferase reporter assay experiments support that these genes are direct targets of miR-17, miR-19a, and/or miR-20a. Conclusion: MiR-17-92 cluster members demonstrate initial increase followed by subsequent decrease in expression during 2-week human monocyte to macrophage differentiation. Overexpression of miR-17-92 miRNAs upregulates Type I and II interferon pathway genes, and these miRNAs target multiple genes involved in regulating interferon signaling and/or inflammatory response. Taken together, miR-17-92 cluster overexpression in SJIA monocytes may suggest a more differentiated phenotype, and contribute to IFNγ sensitivity and risk for MAS. Disclosure of Interests: : None declared

ObjectiveSystemic juvenile idiopathic arthritis (JIA) is associated with a recently recognized, albeit poorly defined and characterized, lung disease (LD). The objective of this study was to describe the clinical characteristics, risk factors, and histopathologic and immunologic features of this novel inflammatory LD associated with systemic JIA (designated SJIA‐LD).MethodsClinical data collected since 2010 were abstracted from the medical records of patients with systemic JIA from the Cincinnati Children's Hospital Medical Center. Epidemiologic, cellular, biochemical, genomic, and transcriptional profiling analyses were performed.ResultsEighteen patients with SJIA‐LD were identified. Radiographic findings included diffuse ground‐glass opacities, subpleural reticulation, interlobular septal thickening, and lymphadenopathy. Pathologic findings included patchy, but extensive, lymphoplasmacytic infiltrates and mixed features of pulmonary alveolar proteinosis (PAP) and endogenous lipoid pneumonia. Compared to systemic JIA patients without LD, those with SJIA‐LD were younger at the diagnosis of systemic JIA (odds ratio [OR] 6.5, P = 0.007), more often had prior episodes of macrophage activation syndrome (MAS) (OR 14.5, P < 0.001), had a greater frequency of adverse reactions to biologic therapy (OR 13.6, P < 0.001), and had higher serum levels of interleukin‐18 (IL‐18) (median 27,612 pg/ml versus 5,413 pg/ml; P = 0.047). Patients with SJIA‐LD lacked genetic, serologic, or functional evidence of granulocyte–macrophage colony‐stimulating factor pathway dysfunction, a feature that is typical of familial or autoimmune PAP. Moreover, bronchoalveolar lavage (BAL) fluid from patients with SJIA‐LD rarely demonstrated proteinaceous material and had less lipid‐laden macrophages than that seen in patients with primary PAP (mean 10.5% in patients with SJIA‐LD versus 66.1% in patients with primary PAP; P < 0.001). BAL fluid from patients with SJIA‐LD contained elevated levels of IL‐18 and the interferon‐γ–induced chemokines CXCL9 and CXCL10. Transcriptional profiling of the lung tissue from patients with SJIA‐LD identified up‐regulated type II interferon and T cell activation networks. This signature was also present in SJIA‐LD human lung tissue sections that lacked substantial histopathologic findings, suggesting that this activation signature may precede and drive the lung pathology in SJIA‐LD.ConclusionPulmonary disease is increasingly detected in children with systemic JIA, particularly in association with MAS. This entity has distinct clinical and immunologic features and represents an uncharacterized inflammatory LD.

OBJECTIVES:Systemic juvenile idiopathic arthritis (SJIA) confers high risk for macrophage activation syndrome (MAS), a life-threatening cytokine storm driven by interferon (IFN)-γ. SJIA monocytes display IFN-γ hyper-responsiveness, but the molecular basis of this remains unclear. The objective of this study is to identify circulating monocyte and bone marrow macrophage (BMM) polarisation phenotypes in SJIA including molecular features contributing to IFN response. METHODS:Bulk RNA-seq was performed on peripheral blood monocytes (n=26 SJIA patients) and single cell (sc) RNA-seq was performed on BMM (n=1). Cultured macrophages were used to define consequences of tripartite motif containing 8 (TRIM8) knockdown on IFN-γ signalling. RESULTS:Bulk RNA-seq of SJIA monocytes revealed marked transcriptional changes in patients with elevated ferritin levels. We identified substantial overlap with multiple polarisation states but little evidence of IFN-induced signature. Interestingly, among the most highly upregulated genes was TRIM8, a positive regulator of IFN-γ signalling. In contrast to PBMC from SJIA patients without MAS, scRNA-seq of BMM from a patient with SJIA and MAS identified distinct subpopulations of BMM with altered transcriptomes, including upregulated IFN-γ response pathways. These BMM also showed significantly increased expression of TRIM8. In vitro knockdown of TRIM8 in macrophages significantly reduced IFN-γ responsiveness. CONCLUSIONS:Macrophages with an 'IFN-γ response' phenotype and TRIM8 overexpression were expanded in the bone marrow from an MAS patient. TRIM8 is also upregulated in SJIA monocytes, and augments macrophage IFN-γ response in vitro, providing both a candidate molecular mechanism and potential therapeutic target for monocyte hyper-responsiveness to IFNγ in cytokine storms including MAS.

CD163 facilitates regulation and resolution of inflammation and removal of free hemoglobin and is highly expressed in myeloid cells from patients with inflammatory disorders, such as systemic juvenile idiopathic arthritis (SJIA) and macrophage activation syndrome (MAS). Our recent studies indicate that regulation of CD163 mRNA expression is a key functional property of polarized monocytes and macrophages and is mediated at the transcriptional and posttranscriptional level, including via microRNAs. The goal of the current study is to develop a multiparameter flow cytometry panel incorporating detection of CD163 mRNA for polarized monocyte and macrophage populations in disorders such as SJIA and MAS. THP-1 cells and CD14+ human monocytes were stained using fluorochrome-conjugated Abs to myeloid surface markers, along with CD163 mRNA. Staining for mRNA could reliably detect CD163 expression while simultaneously detecting different macrophage populations using Abs targeting CD14, CD64, CD80, CD163, and CD209. This approach was found to be highly sensitive for increased mRNA expression when macrophages were polarized with IL-10 [M(IL-10)], with a strong signal over a broad range of IL-10 concentrations, and showed distinct kinetics of CD163 mRNA and protein induction upon IL-10 stimulation. Finally, this panel demonstrated clear changes in polarization markers in unstimulated monocytes from patients with SJIA and MAS, including upregulated CD163 mRNA and increased CD64 expression. This approach represents a robust and sensitive system for RNA flow cytometry, useful for studying CD163 expression as part of a multimarker panel for human monocytes and macrophages, with broad applicability to the pathogenesis of hyperinflammatory diseases.

Systemic juvenile idiopathic arthritis (SJIA) is a severe childhood arthropathy with features of autoinflammation. Monocytes and macrophages in SJIA have a complex phenotype with both pro- and anti-inflammatory properties that combine features of several well characterized in vitro conditions used to activate macrophages. An important anti-inflammatory phenotype is expression of CD163, a scavenger receptor that sequesters toxic pro-inflammatory complexes that is highly expressed in both active SJIA and macrophage activation syndrome (MAS). CD163 is most strongly up-regulated by IL-10 (M(IL-10)), and not by other conditions that reflect features seen in SJIA monocytes such as M(LPS+IC). MicroRNA plays key roles in integrating cellular signals such as those in macrophage polarization, and as such we hypothesize microRNAs regulate macrophage functional responses in SJIA including CD163 expression. We find that 2 microRNAs previously found to be elevated in active SJIA, miR-125a-5p and miR-181c, significantly reduced macrophage CD163 expression through 2 distinct mechanisms. Neither microRNA was elevated in M(IL-10) with robust CD163 expression, but were instead induced in M(LPS+IC) where they restricted CD163 mRNA expression. Mir-181 species directly targeted CD163 mRNA for degradation. In contrast, miR-125a-5p functions indirectly, as transcriptome analysis of miR-125a-5p overexpression identified cytokine-cytokine receptor interactions as the most significantly repressed gene pathway, including decreased IL10RA, required for IL-10-mediated CD163 expression. Finally, overexpression of miR-181c inhibited CD163 anti-inflammatory responses to hemoglobin or high mobility group box 1 (HMGB1) complexes. Together, these data show that microRNA utilizes multiple mechanisms to integrate well-characterized polarization phenotypes and regulate macrophage functional properties seen in SJIA.

Background: Systemic juvenile idiopathic arthritis (SJIA) is a chronic childhood arthropathy with features of autoinflammation. Early inflammatory SJIA is associated with expansion and activation of neutrophils with a sepsis-like phenotype, but neutrophil phenotypes present in longstanding and clinically inactive disease (CID) are unknown. The objective of this study was to examine activated neutrophil subsets, S100 alarmin release, and gene expression signatures in children with a spectrum of SJIA disease activity. Methods: Highly-purified neutrophils were isolated using a two-step procedure of density-gradient centrifugation followed by magnetic-bead based negative selection prior to flow cytometry or cell culture to quantify S100 protein release. Whole transcriptome gene expression profiles were compared in neutrophils from children with both active SJIA and CID. Results: Patients with SJIA and active systemic features demonstrated a higher proportion of CD16+CD62Llo neutrophil population compared to controls. This neutrophil subset was not seen in patients with CID or patients with active arthritis not exhibiting systemic features. Using imaging flow cytometry, CD16+CD62Llo neutrophils from patients with active SJIA and features of macrophage activation syndrome (MAS) had increased nuclear hypersegmentation compared to CD16+CD62L+ neutrophils. Serum levels of S100A8/A9 and S100A12 were strongly correlated with peripheral blood neutrophil counts. Neutrophils from active SJIA patients did not show enhanced resting S100 protein release; however, regardless of disease activity, neutrophils from SJIA patients did show enhanced S100A8/A9 release upon PMA stimulation compared to control neutrophils. Furthermore, whole transcriptome analysis of highly purified neutrophils from children with active SJIA identified 214 differentially expressed genes (DEG) compared to neutrophils from healthy controls. The most significantly upregulated gene pathway was Immune System Process, including AIM2, IL18RAP, and NLRC4. Interestingly, this gene set showed intermediate levels of expression in neutrophils from patients with long-standing CID yet persistent serum IL-18 elevation. Indeed, all patient samples regardless of disease activity demonstrated elevated inflammatory gene expression, including inflammasome components and S100A8. Conclusion: We identify features of neutrophil activation in SJIA patients with both active disease and CID, including a proinflammatory gene expression signature, reflecting persistent innate immune activation. Taken together, these studies expand understanding of neutrophil function in chronic autoinflammatory disorders such as SJIA.

BACKGROUND:Autoinflammatory disorders are distinguished by seemingly random episodes of systemic hyperinflammation, driven in particular by IL-1. Recent pre-clinical work has shown a key role for IL-1 in epilepsy in animal models, and therapies for autoinflammation including IL-1 blockade are proposed for refractory epilepsy.CASE PRESENTATION:Here, we report an adolescent female with signs of persistent systemic inflammation and epilepsy unresponsive to multiple anti-epileptic drugs (AED). She was diagnosed with generalized epilepsy with a normal brain MRI and an electroencephalogram (EEG) showing occasional generalized spike and slow wave discharges. Her diagnostic evaluation showed no signs of autoimmunity or genetic causes of epilepsy or periodic fever syndromes but persistently elevated serum inflammatory markers including S100 alarmin proteins. She experienced prompt clinical response to IL-1 blockade with first anakinra and then canakinumab, with near complete resolution of clinical seizures. Additionally, she displayed marked improvements in quality of life and social/academic functioning. Baseline gene expression studies on peripheral blood mononuclear cells (PBMC) from this patient showed significantly activated gene pathways suggesting systemic immune activation, including focal adhesion, platelet activation, and Rap1 signaling, which is an upstream regulator of IL-1β production by the NLRP3 inflammasome. It also showed activation of genes that characterize inflammasome-mediated autoinflammatory disorders and no signs of interferon activation. This gene expression signature was largely extinguished after anakinra treatment.CONCLUSIONS:Together, these findings suggest that patients with epilepsy responsive to immune modulation may have distinct autoinflammatory features supporting IL-1 blockade. As such, IL-1 blockade may be highly efficacious adjunctive medication for certain refractory epilepsy syndromes.

Background CD163 is a hemoglobin scavenger receptor and serves as a marker of activated monocytes and macrophages. It is also expressed on monocytes from children with active systemic juvenile idiopathic arthritis, and highly enriched on hemophagocytic macrophages that characterize the emergence of macrophage activation syndrome. However, the regulation of CD163 expression during monocyte and macrophage polarization is poorly understood. MicroRNA are small non-coding RNA that post-transcriptionally regulate gene expression, often serving as feedback loops to “fine tune” gene expression programs. Objectives Determine microRNA regulation of CD163 at the protein and mRNA levels using novel flow cytometry techniques. Methods Human THP-1 monocytic cells and primary monocytes and monocyte-derived macrophages (MDMs) from healthy donors were polarized with activating conditions including M1 (LPS and IFNg), M2a (IL-4), M2b (LPS and immune complexes) and M2c (TGFb or IL-10). CD163 expression was determined by quantitative RT-PCR or on a single-cell level using RNA flow cytometry. Transcripts were detected using the PrimeFlow kit, with probes specific for CD163 mRNA and other cell surface markers. Cells were also transfected with specific microRNA mimics. Results Significantly increased CD163 mRNA levels were detected only in monocytes and macrophages polarized towards M2c by IL-10 treatment. Similarly, single-cell RNA analysis showed markedly increased CD163 mRNA upon IL-10 treatment, with more modest increases in protein expression. Several candidate microRNA may regulate CD163. As such, our studies indicate that overexpression of miR-125a and miR-181c significantly reduced CD163 mRNA expression in IL-10 polarized macrophages. Notably, both of these microRNAs were significantly increased in monocytes from children with active systemic JIA. In vitro, miR-125a and miR-181c were both elevated in polarizing conditions that restrict CD163 expression. Interestingly, these microRNAs regulated CD163 expression through distinct mechanisms. Computationally, miR-181 is predicted to bind directly to CD163 mRNA. Indeed, using RNA immunoprecipitation (RIP) we found that overexpression of miR-181c significantly increased delivery of CD163 mRNA to the RNA-induced silencing complex. In contrast, CD163 does not have a predicted miR-125a binding site. Using whole transcriptome analysis of macrophages overexpressing miR-125a-5p, significantly reduced levels of mRNA encoding several macrophage receptors, including IL10RA and TGFbR2, was observed. This suggests that miR-125a-5p exerts more global effects to restrict M2c polarization. Conclusions These data show that microRNAs regulate the IL-10-induced expression of CD163 in human macrophages through distinct mechanisms, directly targeting of CD163 mRNA and indirectly inhibiting cytokine receptor expression. These findings illustrate how microRNA “fine-tune” innate immune activation during inflammatory responses, and expand the understanding of the phenotype of activated monocytes and macrophages in systemic JIA. Disclosure of Interest S. Thornton: None declared, T. Do: None declared, R. Tan: None declared, A. Sproles: None declared, M. Bennett: None declared, M. Medvedovic: None declared, M. DeLay: None declared, N. Shen: None declared, A. Grom Grant/research support from: Novartis, Novimmune, Consultant for: Novartis, Novimmune, G. Schulert: None declared