Contributions
Abstract: EP1475
Type: ePoster
Abstract Category: Pathology and pathogenesis of MS - 14 Genetics/Epigenetics
Background: Fingolimod (FTY) is a second-line drug approved for Relapsing Remitting Multiple Sclerosis (RRMS). It is known to prevent lymphocyte egress outside lymph nodes, thus reducing peripheral lymphocytes counts.
Aims: To investigate transcriptional changes induced by FTY in immune cell subtypes using RNA-seq technology, in order to better elucidate FTY mechanism of action at the molecular and pathway level.
Methods: 24 RRMS patients were sampled at baseline and after 6 months of FTY treatment. CD3+
T cells, CD20+ B cells and CD 14+ monocytes were sorted through the MACS MicroBeads system. RNA sequencing was performed on the three cell types using the Illumina TruSeq-Stranded mRNA preparation kit, on the NextSeq500 platform. Differentially expressed genes (DEGs) were identified for each cell type using DESeq2 R package. Genes strongly modulated by FTY (fold change [FC]>2 or
FC< 0.5 and false discovery rate [FDR]< 5%) were considered for a pathway enrichment analysis performed using WebGestalt tool, based on KEGG and Reactome databases.
Results: A marked up-regulation was observed in both T and B lymphocytes (695 up- and 492 down-regulated genes in T cells; 857 up- and 261 down-regulated genes in B cells), with evidence of significant overlap between the 2 cell-types; a less pronounced transcriptional induction was observed in monocytes, mainly regarding down-regulated genes (n=189). Most of FTY-responsive genes had immune-related function; among the top 10 DEGs, CCL4 was up-regulated in T cells (adjusted p-value [adjp]=5.4x10-55, FC=4.5), FCGR3A in B cells (adjp=1.4x10-31, FC=5), whereas CCR7 was down-regulated in monocytes (adjp=3.1x10-30, FC=0.2). Up-regulated DEGs from T and B cells were enriched of genes involved in shared immune-related pathways (e.g. cytokine-cytokine receptor interaction, chemokine signaling pathways); similarly, also down-regulated genes in monocytes seemed to be involved in immune modulation (e.g. Th1 and Th2 cell differentiation pathways).
Conclusions: FTY induces major transcriptional changes at the immune level, that appear to be shared between T and B lymphocytes, whereas the induced gene expression modulation in monocytes is quite different. Our data suggest that at least part of the immunomodulatory action of FTY seems to be regulated at the transcriptional level.
Disclosure:
L. Ferrè, F. Clarelli, P. Provero, E. Mascia, G. Sferruzza and M. Sorosina report no disclosures.
L. Moiola received honoraria for speaking at meetings or for attending to advisory board from Sanofi-Genzyme, Biogen-Idec, Novartis and TEVA.
V. Martinelli has received honoraria for consulting and speaking activities from Biogen-Idec, Merck, Bayer, TEVA, Novartis and Genzyme.
G. Comi has received compensation for consulting services with the following companies: Novartis, Teva, Sanofi, Genzyme, Merck, Biogen, Excemed, Roche, Almirall, Chugai, Receptos, Forward Pharma and compensation for speaking activities from Novartis, Teva, Sanofi, Genzyme, Merk, Biogen, Excemed, Roche.
F. Martinelli Boneschi has received compensation for activities with Teva Neuroscience, Biogen Idec, Merck Serono as speaker and/or advisor.
F. Esposito received honoraria from TEVA, Almirall and Genzyme.
Abstract: EP1475
Type: ePoster
Abstract Category: Pathology and pathogenesis of MS - 14 Genetics/Epigenetics
Background: Fingolimod (FTY) is a second-line drug approved for Relapsing Remitting Multiple Sclerosis (RRMS). It is known to prevent lymphocyte egress outside lymph nodes, thus reducing peripheral lymphocytes counts.
Aims: To investigate transcriptional changes induced by FTY in immune cell subtypes using RNA-seq technology, in order to better elucidate FTY mechanism of action at the molecular and pathway level.
Methods: 24 RRMS patients were sampled at baseline and after 6 months of FTY treatment. CD3+
T cells, CD20+ B cells and CD 14+ monocytes were sorted through the MACS MicroBeads system. RNA sequencing was performed on the three cell types using the Illumina TruSeq-Stranded mRNA preparation kit, on the NextSeq500 platform. Differentially expressed genes (DEGs) were identified for each cell type using DESeq2 R package. Genes strongly modulated by FTY (fold change [FC]>2 or
FC< 0.5 and false discovery rate [FDR]< 5%) were considered for a pathway enrichment analysis performed using WebGestalt tool, based on KEGG and Reactome databases.
Results: A marked up-regulation was observed in both T and B lymphocytes (695 up- and 492 down-regulated genes in T cells; 857 up- and 261 down-regulated genes in B cells), with evidence of significant overlap between the 2 cell-types; a less pronounced transcriptional induction was observed in monocytes, mainly regarding down-regulated genes (n=189). Most of FTY-responsive genes had immune-related function; among the top 10 DEGs, CCL4 was up-regulated in T cells (adjusted p-value [adjp]=5.4x10-55, FC=4.5), FCGR3A in B cells (adjp=1.4x10-31, FC=5), whereas CCR7 was down-regulated in monocytes (adjp=3.1x10-30, FC=0.2). Up-regulated DEGs from T and B cells were enriched of genes involved in shared immune-related pathways (e.g. cytokine-cytokine receptor interaction, chemokine signaling pathways); similarly, also down-regulated genes in monocytes seemed to be involved in immune modulation (e.g. Th1 and Th2 cell differentiation pathways).
Conclusions: FTY induces major transcriptional changes at the immune level, that appear to be shared between T and B lymphocytes, whereas the induced gene expression modulation in monocytes is quite different. Our data suggest that at least part of the immunomodulatory action of FTY seems to be regulated at the transcriptional level.
Disclosure:
L. Ferrè, F. Clarelli, P. Provero, E. Mascia, G. Sferruzza and M. Sorosina report no disclosures.
L. Moiola received honoraria for speaking at meetings or for attending to advisory board from Sanofi-Genzyme, Biogen-Idec, Novartis and TEVA.
V. Martinelli has received honoraria for consulting and speaking activities from Biogen-Idec, Merck, Bayer, TEVA, Novartis and Genzyme.
G. Comi has received compensation for consulting services with the following companies: Novartis, Teva, Sanofi, Genzyme, Merck, Biogen, Excemed, Roche, Almirall, Chugai, Receptos, Forward Pharma and compensation for speaking activities from Novartis, Teva, Sanofi, Genzyme, Merk, Biogen, Excemed, Roche.
F. Martinelli Boneschi has received compensation for activities with Teva Neuroscience, Biogen Idec, Merck Serono as speaker and/or advisor.
F. Esposito received honoraria from TEVA, Almirall and Genzyme.