Background/Purpose: We have previously demonstrated a role for neutrophils in polyarticulartar juvenile idiopathic arthritis (JIA) disease pathogenesis. For example, JIA neutrophils show specific abnormalities in gene expression that do not correct when children reach remission. The studies here were intended to gain insights into mechanisms of therapeutic response in JIA by examining molecular events in neutrophils early in therapy  

Methods: We examined 35 children with JIA who had active, untreated disease (ADU) and 26 children with active disease (ADM) who had been on therapy with methotrexate (MTX) for periods ranging from 2 weeks to 2 months.  We also studied 15 healthy control children (HC) for comparison.  We performed transcriptome analysis using Affymetrix exon and miRNA hybridization-based microarrays. Exon gene expression data were processed using RMA software in the Affy package of Bioconductor and analyzed using t-tests to detect differentially expressed genes (DEGs) between JIA and HC as well as between ADM individuals and healthy controls. Enrichment analyses to determine the associated functions for DEGs were performedd using by DAVID Functional Annotation Tools (http://david.abcc.ncifcrf.gov/).

Results: 216 genes were detected showing differential expression between JIA and HC.  More than half of these genes (51%) are involved in regulating gene/exon splicing. Treatment with MTX was found to have substantial impact on JIA transcriptome, even while children still maintained active disease status. Of the 216 genes that were differentially expressed between HC and ADU, 112 of them (52%) reverted to normal expression levels.  However, early treatment with MTX was associated with extensive re-ordering of the neutrophil transcriptome, as shown by the large number of DEGs.  A total of 1271 DEGs were detected between ADM and HC, of which 1167 (92%) are unique to ADM when compared to ADU.  These DEGs are associated with a broad range of functions, including more than 20 biological processes and 40 gene groups. Similar results were obtained from both isoform-level and miRNA  expression data analysis. While 193 and 73 differentially expressed miRNAs were detected in ADM and ADU, respectively, 53 of them are common to both groups (p = 1.3E-22).  At  the isoform level, we found 2808 isoforms from ADM and 446 isoforms from ADU displaying differential exon splicing, 275 of them are common to both groups (p < 6.5E-322).  Functional analysis indicated that the 446 isoforms were associated with pathways of spliceosome, apoptosis, endocytosis, B and T cell receptor signaling pathways.  On the other hand, the 275 common isoforms display enrichment in the pathways of spliceosome and endocytosis.

Conclusion: Upon initiation of therapy with methotrexate, substantial re-ordering of the neutrophil transcriptome occurs.  How these changes relate to eventual therapeutic response, and whether and how they relate to or lead to changes in adaptive immune function are ongoing areas of investigation in our research groups.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/early-treatment-with-methotrexate-is-associated-with-extensive-re-ordering-of-the-neutrophil-transcriptome-in-juvenile-idiopathic-arthritis/