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. 2021 Apr 9:12:663736.
doi: 10.3389/fimmu.2021.663736. eCollection 2021.

MicroRNA Expression Differences in Blood-Derived CD19+ B Cells of Methotrexate Treated Rheumatoid Arthritis Patients

Fatima Heinicke  1 , Xiangfu Zhong  1 , Siri T Flåm  1 , Johannes Breidenbach  2 , Magnus Leithaug  1 , Marthe T Mæhlen  3 , Siri Lillegraven  3 , Anna-Birgitte Aga  3 , Ellen S Norli  4 , Maria D Mjaavatten  3 , Espen A Haavardsholm  3 , Manuela Zucknick  5 , Simon Rayner  1 , Benedicte A Lie  1
Affiliations

MicroRNA Expression Differences in Blood-Derived CD19+ B Cells of Methotrexate Treated Rheumatoid Arthritis Patients

Fatima Heinicke et al. Front Immunol. .

Abstract

Rheumatoid arthritis (RA) is a complex disease with a wide range of underlying susceptibility factors. Recently, dysregulation of microRNAs (miRNAs) in RA have been reported in several immune cell types from blood. However, B cells have not been studied in detail yet. Given the autoimmune nature of RA with the presence of autoantibodies, CD19+ B cells are a key cell type in RA pathogenesis and alterations in CD19+ B cell subpopulations have been observed in patient blood. Therefore, we aimed to reveal the global miRNA repertoire and to analyze miRNA expression profile differences in homogenous RA patient phenotypes in blood-derived CD19+ B cells. Small RNA sequencing was performed on CD19+ B cells of newly diagnosed untreated RA patients (n=10), successfully methotrexate (MTX) treated RA patients in remission (MTX treated RA patients, n=18) and healthy controls (n=9). The majority of miRNAs was detected across all phenotypes. However, significant expression differences between MTX treated RA patients and controls were observed for 27 miRNAs, while no significant differences were seen between the newly diagnosed patients and controls. Several of the differentially expressed miRNAs were previously found to be dysregulated in RA including miR-223-3p, miR-486-3p and miR-23a-3p. MiRNA target enrichment analysis, using the differentially expressed miRNAs and miRNA-target interactions from miRTarBase as input, revealed enriched target genes known to play important roles in B cell activation, differentiation and B cell receptor signaling, such as STAT3, PRDM1 and PTEN. Interestingly, many of those genes showed a high degree of correlated expression in CD19+ B cells in contrast to other immune cell types. Our results suggest important regulatory functions of miRNAs in blood-derived CD19+ B cells of MTX treated RA patients and motivate for future studies investigating the interactive mechanisms between miRNA and gene targets, as well as the possible predictive power of miRNAs for RA treatment response.

Keywords: CD19+ B cells; NGS; methotrexate; miRNA; microRNA; rheumatoid arthritis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Workflow of the study representing (A) the three study phenotypes and major steps during wet-lab and computational analysis, (B) detailed representation of the kit, technology and tools used during miRNA library preparation, sequencing and bioinformatics analysis, and (C) detailed presentation of the steps included in the data analysis. DE, differential expression analysis; MTX, Methotrexate; HC, healthy controls. Images from Servier Medical Art (Servier. www.servier.com, licensed under a Creative Commons Attribution 3.0 Unported License) were used in the figure.
Figure 2
Figure 2
The miRNA repertoire observed in blood-derived CD19+ B cells (A) across the different study phenotypes and (B) comparing the miRNA repertoire of healthy controls in the present study with those of a reference dataset (29). Interactions are indicated by the blue dots and lines. The number of miRNAs in the intersection is represented by the black bars.
Figure 3
Figure 3
PCA plots for the first two principal components on low read count filtered and rlog transformed miRNA read counts of the three study phenotypes. (A) The study phenotypes are indicated by different colors. The percentage of variance explained by the principal component is indicated. (B) MTX dosage is indicated by different colors while different shapes were used to differentiate the study phenotypes.
Figure 4
Figure 4
Differential expression analysis. (A) Differentially expressed miRNAs are represented with their specific log2 fold changes. (B) Boxplots representing the abundance of miR-155-5p in CPM based on study phenotype.
Figure 5
Figure 5
Properties of the miRNA-target enrichment analysis based on the differentially expressed miRNAs identified from comparing MTX treated RA patients to healthy controls: (A) Enriched miRNA-target interactions. The x-axis shows the specific enriched target gene and the y-axis the specific targeting miRNA. (B) Gene expression correlation network in mature B cells generated using the Immuno-Navigator database. Target genes with correlated expressions are connected by an edge. The thicker and more saturated the line, the stronger the correlation.
See this image and copyright information in PMC

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