Genes differentially expressed by methylprednisolone in vivo in CD4 T lymphocytes from multiple sclerosis patients: potential biomarkers

Abstract

Intravenous methylprednisolone (IVMP) is the gold standard treatment in acute relapses of multiple sclerosis. Knowing the response to IVMP in advance could facilitate earlier selection of patients for subsequent courses of therapy. However, molecular mechanisms and changes in gene expression induced by methylprednisolone remain unknown. The aim of the study was to identify in vivo differentially expressed genes in relapsing-remitting multiple sclerosis patients after 3-6 days of treatment with IVMP. For this purpose, whole-genome transcription profiling of CD4+ T lymphocytes was performed before and after treatment with IVMP in 8 relapsing-remitting multiple sclerosis patients during relapse using Human GE 4x44K v2 microarrays. Differentially expressed genes were identified using a paired t test on GeneSpring v13.0 software. A P-value <0.001 and a twofold change were considered significant. Microarray data were confirmed using real-time PCR. Microarray revealed changes in gene expression: four genes were downregulated (B3GNT3, ZNF683, IFNG and TNF) and seven upregulated (DEFA4, CTSG, DEFA8P, AZU1, MPO, ELANE and PRTN3). Pathway analysis revealed the transforming growth factor-β signaling pathway to be affected. Comparison with previously published data on in vitro methylprednisolone-regulated genes showed that SMAD7, TNF and CHI3L1 were also downregulated in vivo in relapsing-remitting multiple sclerosis patients. In summary, we performed the first in vivo transcriptome analysis in CD4+ T lymphocytes before and after the treatment with IVMP in patients with multiple sclerosis. Identification of differentially expressed genes in patients receiving IVMP could improve our understanding of the molecular mechanisms underlying the therapeutic effects of IVMP and highlight potential biomarkers of the response to IVMP.

Differential Expression of SMAD Genes and S1PR1 on Circulating CD4+ T Cells in Multiple Sclerosis and Crohn’s Disease

“…SMAD7 was the only SMAD gene downregulated in MS. This finding is in agreement with those of a previous study, in which SMAD2, SMAD3, SMAD4, and SMAD7 were measured in methylprednisolone-treated RRMS patients [44]. However, in CD, we found downregulation of SMAD2, SMAD3, and SMAD4, in addition to SMAD7, probably owing to more pronounced downregulation of SMAD transcription in CD than in MS, although this hypothesis requires further investigation.…”

“…RNA was isolated from CD4+ T cells, measured and integrity verified as described [44]. When necessary, RNA was concentrated using Concentrator 5301 (Eppendorf, Hamburg, Germany).…”

“…Good quality samples (RNA integrity number > 8 were selected. cDNA was generated from 1000 ng (for StellARrays) or 500 ng (for qRT-PCR) of total RNA as described [44].…”

“…Changes in the selected genes were quantified by qRT-PCR in 20 RRMS patients in remission, 19 healthy donors (HD), 42 RRMS patients during a relapse, and in 16 Crohn's disease patients during a relapse. Real-time PCR was performed in triplicate using 2 µL/well of a dilution performed for 1/10 of each cDNA (0.04 µM) for SMAD7, TNF, S1PR1, SMAD2, SMAD3, SMAD4, GAPDH, and HPRT1 (SMAD7 forward, 5 -ACC CGA TGG ATT TTC TCA A-3 ; SMAD7 reverse, 5 -AGG GGC CAG ATA ATT CGT TC'; TNF forward, 5 -CAG CCT CTT CTC CTT CCT GAT-3 ; TNF reverse, 5 -GCC AGA GGG CTG ATT AGA GA-3 ; S1PR1 forward, 5 -AAC TTC GCC CTG CTT GAG-3; S1PR1 reverse, 5 -TCC AGG CTT TTT GTG TAG CTT-3 ; SMAD2 forward, AAA GGG TGG GGA GCA GAA TA; SMAD2 reverse, GAA GTT CAA TCC AGC AAG GAG T; SMAD3 forward, CCA TCC CCG AAA ACA CTA AC; SMAD3 reverse, TCC ATC TTC ACT CAG GTA GCC; SMAD4 forward, CCT GTT CAC AAT GAG CTT GC; SMAD4 reverse, GCA ATG GAA CAC CAA TAC TCA G; GAPDH forward, 5 -AGC CAC ATC GCT CAG ACA C-3 , GAPDH reverse, 5 -GCC CAA TAC GAC CAA ATC C-3 , HPRT1 forward, 5 -GAC CAG TCA ACA GGG GAC AT-3 , HPRT1 reverse, 5 -GTG TCA ATT ATA TCT TCC ACA ATC AAG-3 ), 1× SYBR Green PCR Master Mix (Roche Applied Science, Penzberg, Germany) as described [44]. GAPDH and HPRT1 and were used as normalization genes [49].…”

Activity-based Protein Profiling Approaches for Transplantation

“…49 Interestingly, PRTN3 transcription may be regulated in part by steroidresponsive elements as it becomes highly expressed in CD4 T cells following treatment with methylprednisolone. 50 These data raise the possibility of neutrophil, monocyte, CD4 T cell involvement, or an alternate cellular source of this enzyme during rejection. Clearly, this requires more comprehensive analysis to determine if there is an association between PRTN3 activity and graft status and to characterize the source and relevance of its activity.…”