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A multidimensional blood stimulation assay reveals immune alterations underlying systemic juvenile idiopathic arthritis [RNA-Seq]

ABSTRACT: Despite great scientific and technological advances, immune alterations predisposing to sporadic human inflammatory diseases remain mostly unknown. To fill this gap, we developed a strategy to evaluate blood leukocyte responses to innate stimuli, simultaneously at the transcriptional, cellular and secreted protein levels. The data were integrated using weighted gene co-expression network analysis. When applied to systemic juvenile idiopathic arthritis (sJIA), an autoinflammatory disease of unknown etiology, this approach identified gene sets associated with specific cytokine environments and activated leukocyte subsets. During the remission phase of disease and off treatment, sJIA patients displayed dysregulated responses to TLR4, TLR8 and TLR7 stimulation in blood. Isolated sJIA monocytes accumulated higher levels of intracellular IL-1β after stimulation, and underexpressed the IL-1 inhibitor AHR at baseline. In line with the recent demonstration that AHR downregulation in monocytes is linked to macrophage differentiation, we show that the differentiation of sJIA monocytes in vitro was skewed towards macrophages, away from dendritic cell phenotype. This might contribute to the increased incidence of macrophage activation syndrome in these patients. The integrated analysis of these high-dimensional data can thus help unravel underlying immune alterations predisposing to complex inflammatory diseases.

ORGANISM(S): Homo sapiens

PROVIDER: GSE103501 | GEO | 2017/09/19

SECONDARY ACCESSION(S): PRJNA401766

REPOSITORIES: GEO

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Project description: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 number 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 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 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.

Project description:Selective stimulation of IL-4 receptor on smooth muscle induces airway hyper-responsiveness in mice. Abstract: Production of the cytokines IL-4 and IL-13 is increased in both human asthma and mouse asthma models and Stat6 activation by the common IL-4/IL-13R drives most mouse model pathophysiology, including airway hyperresponsiveness (AHR). However, the precise cellular mechanisms through which IL-4Rα induces AHR remain unclear. Overzealous bronchial smooth muscle constriction is thought to underlie AHR in human asthma, but the smooth muscle contribution to AHR has never been directly assessed. Furthermore, differences in mouse vs. human airway anatomy and observations that selective IL-13 stimulation of Stat6 in airway epithelium induces murine AHR raise questions about the importance of direct IL-4R effects on smooth muscle in murine asthma models and relevance of these models to human asthma. Using transgenic mice in which smooth muscle is the only cell type that expresses or fails to express IL-4Rα, we demonstrate that direct smooth muscle activation by IL-4, IL-13, or allergen is sufficient, but not necessary, to induce AHR and show that 5 genes known to promote smooth muscle migration, proliferation and contractility are activated by IL-13 in smooth muscle in vivo. These observations demonstrate that IL-4Rα promotes AHR through multiple mechanisms and provide a model for testing smooth muscle-directed asthma therapeutics. For the microarray aspect of of the study, there were three groups of mice: 1. IL4R gene knockout (KO) mice 2. WT mice 3. IL4R KO mice that were also transgenic for a gene construct that expressed IL4R under the control of the smooth muscle-specific promoter from the SMP8 gene All mice were subjected to intratracheal IL13 exposure for 7 days, and whole lung RNA was prepared for microarray analysis 24 hours after the last instillation. Per treatment and genotype: Two RNA pools were made from four mice each. These were labeled and hybridized to make a total of 6 microarrays. RNA was labeled with the standard Affymetrix 3' labeling protocol to make cDNA that was hybridized to Mouse MOE 430 plus 2.0 GeneChips. Gene transcripts were identified that differed in their relative expression as a function of IL4R expression on the smooth muscle cells.

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A multidimensional blood stimulation assay reveals immune alterations underlying systemic juvenile idiopathic arthritis [RNA-Seq]

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