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MiR-30a-3p negatively regulates BAFF synthesis in systemic sclerosis and rheumatoid arthritis fibroblasts.

Alsaleh G, François A, Philippe L, Gong YZ, Bahram S, Cetin S, Pfeffer S, Gottenberg JE, Wachsmann D, Georgel P, Sibilia J - PLoS ONE (2014)

Bottom Line: Moreover, we demonstrated that transfecting miR-30a-3p mimic in Poly(I:C)- and IFN-γ-activated RAFLS and SScHDF showed a strong decrease on BAFF synthesis and release and thus B cells survival in our model.Transfection of miR-30a-3p antisense in Poly(I:C)- and IFN-γ-activated NFLS and NHDF upregulated BAFF secretion, confirming that this microRNA is a basal repressors of BAFF expression in cells from healthy donors.Our data suggest a critical role of miR-30a-3p in the regulation of BAFF expression, which could have a major impact in the regulation of the autoimmune responses occurring in RA and SSc.

View Article: PubMed Central - PubMed

Affiliation: Immunorhumatologie moléculaire, INSERM UMR S_1109, Centre de Recherche en Immunologie et Hématologie, Faculté de Médecine, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, Strasbourg, France and Service de Rhumatologie, Centre National de Référence pour les Maladies Systémiques Autoimmunes Rares, Hôpitaux Universitaires de Strasbourg, Strasbourg, France.

ABSTRACT
We evaluated micro (mi) RNA-mediated regulation of BAFF expression in fibroblasts using two concomitant models: (i) synovial fibroblasts (FLS) isolated from healthy controls (N) or Rheumatoid Arthritis (RA) patients; (ii) human dermal fibroblasts (HDF) isolated from healthy controls (N) or Systemic Sclerosis (SSc) patients. Using RT-qPCR and ELISA, we first showed that SScHDF synthesized and released BAFF in response to Poly(I:C) or IFN-γ treatment, as previously observed in RAFLS, whereas NHDF released BAFF preferentially in response to IFN-γ. Next, we demonstrated that miR-30a-3p expression was down regulated in RAFLS and SScHDF stimulated with Poly(I:C) or IFN-γ. Moreover, we demonstrated that transfecting miR-30a-3p mimic in Poly(I:C)- and IFN-γ-activated RAFLS and SScHDF showed a strong decrease on BAFF synthesis and release and thus B cells survival in our model. Interestingly, FLS and HDF isolated from healthy subjects express higher levels of miR-30a-3p and lower levels of BAFF than RAFLS and SScHDF. Transfection of miR-30a-3p antisense in Poly(I:C)- and IFN-γ-activated NFLS and NHDF upregulated BAFF secretion, confirming that this microRNA is a basal repressors of BAFF expression in cells from healthy donors. Our data suggest a critical role of miR-30a-3p in the regulation of BAFF expression, which could have a major impact in the regulation of the autoimmune responses occurring in RA and SSc.

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miR-30a-3p expression in RAFLS and SScHDF regulates BAFF-dependent B cells survival in vitro.RAFLS (n = 4) (left) and SScHDF (n = 4) (right) were transfected with miR-30a-3p mimic (20 pM/sample) or with an AllStars negative control (CT). After 24 h, cells were activated with IFN-γ (0.1 or 5 ng/mL depending on the cell type) or medium for 72 h. Then, supernatants were harvested and cultured with purified blood B cells isolated from healthy subjects. B cells viability was determined by FACS analysis; vital B cells were brightly positive when stained with DiOC6 and excluded PI. Data are expressed as the mean of triplicate samples ± SEM. *p<0.05.
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pone-0111266-g005: miR-30a-3p expression in RAFLS and SScHDF regulates BAFF-dependent B cells survival in vitro.RAFLS (n = 4) (left) and SScHDF (n = 4) (right) were transfected with miR-30a-3p mimic (20 pM/sample) or with an AllStars negative control (CT). After 24 h, cells were activated with IFN-γ (0.1 or 5 ng/mL depending on the cell type) or medium for 72 h. Then, supernatants were harvested and cultured with purified blood B cells isolated from healthy subjects. B cells viability was determined by FACS analysis; vital B cells were brightly positive when stained with DiOC6 and excluded PI. Data are expressed as the mean of triplicate samples ± SEM. *p<0.05.

Mentions: Next, we checked the physiological relevance of BAFF regulation by miR-30a-3p. To this end, we measured the faculty of stimulated RAFLS and SScHDF to promote B cells survival following transfection of miR-30a-3p mimic. RAFLS and SScHDF were transfected with miR-30a-3p mimic or with the AllStars negative control siRNA (CT) for 24 h and then activated with IFN-γ for 72 h. The supernatants (conditioned medium) were harvested and added in the culture medium of B cells. After 3 days, survival of CD19-gated B cells was assessed by FACS analysis. As shown in figure 5, addition of supernatant from IFN-γ-stimulated fibroblasts significantly (p<0.05) increases the proportion of viable B cells, which shifts from 10 to 26% in the case of RAFLS supernatant and from 35 to 56% in the case of SScHDF. Importantly, increasing miRNA activity upon transfection of miR-30a-3p mimic in activated fibroblasts lowers B cells viability (15% and 35%, respectively. p<0.05). Additionally, we analyzed the supernatant of Poly (I:C)-activated RAFLS and SScHDF but we could not detect any difference in B cells survival (data not shown). Indeed, Poly (I:C), unlike IFN-γ, is also a potent inducer of IL-6 release by RAFLS and SScHDF (figure 4 E–F) which could regulate B cells survival. Similar results were obtained upon transfection of miR-30e-3p and miR-30d-3p (data not shown).


MiR-30a-3p negatively regulates BAFF synthesis in systemic sclerosis and rheumatoid arthritis fibroblasts.

Alsaleh G, François A, Philippe L, Gong YZ, Bahram S, Cetin S, Pfeffer S, Gottenberg JE, Wachsmann D, Georgel P, Sibilia J - PLoS ONE (2014)

miR-30a-3p expression in RAFLS and SScHDF regulates BAFF-dependent B cells survival in vitro.RAFLS (n = 4) (left) and SScHDF (n = 4) (right) were transfected with miR-30a-3p mimic (20 pM/sample) or with an AllStars negative control (CT). After 24 h, cells were activated with IFN-γ (0.1 or 5 ng/mL depending on the cell type) or medium for 72 h. Then, supernatants were harvested and cultured with purified blood B cells isolated from healthy subjects. B cells viability was determined by FACS analysis; vital B cells were brightly positive when stained with DiOC6 and excluded PI. Data are expressed as the mean of triplicate samples ± SEM. *p<0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4216016&req=5

pone-0111266-g005: miR-30a-3p expression in RAFLS and SScHDF regulates BAFF-dependent B cells survival in vitro.RAFLS (n = 4) (left) and SScHDF (n = 4) (right) were transfected with miR-30a-3p mimic (20 pM/sample) or with an AllStars negative control (CT). After 24 h, cells were activated with IFN-γ (0.1 or 5 ng/mL depending on the cell type) or medium for 72 h. Then, supernatants were harvested and cultured with purified blood B cells isolated from healthy subjects. B cells viability was determined by FACS analysis; vital B cells were brightly positive when stained with DiOC6 and excluded PI. Data are expressed as the mean of triplicate samples ± SEM. *p<0.05.
Mentions: Next, we checked the physiological relevance of BAFF regulation by miR-30a-3p. To this end, we measured the faculty of stimulated RAFLS and SScHDF to promote B cells survival following transfection of miR-30a-3p mimic. RAFLS and SScHDF were transfected with miR-30a-3p mimic or with the AllStars negative control siRNA (CT) for 24 h and then activated with IFN-γ for 72 h. The supernatants (conditioned medium) were harvested and added in the culture medium of B cells. After 3 days, survival of CD19-gated B cells was assessed by FACS analysis. As shown in figure 5, addition of supernatant from IFN-γ-stimulated fibroblasts significantly (p<0.05) increases the proportion of viable B cells, which shifts from 10 to 26% in the case of RAFLS supernatant and from 35 to 56% in the case of SScHDF. Importantly, increasing miRNA activity upon transfection of miR-30a-3p mimic in activated fibroblasts lowers B cells viability (15% and 35%, respectively. p<0.05). Additionally, we analyzed the supernatant of Poly (I:C)-activated RAFLS and SScHDF but we could not detect any difference in B cells survival (data not shown). Indeed, Poly (I:C), unlike IFN-γ, is also a potent inducer of IL-6 release by RAFLS and SScHDF (figure 4 E–F) which could regulate B cells survival. Similar results were obtained upon transfection of miR-30e-3p and miR-30d-3p (data not shown).

Bottom Line: Moreover, we demonstrated that transfecting miR-30a-3p mimic in Poly(I:C)- and IFN-γ-activated RAFLS and SScHDF showed a strong decrease on BAFF synthesis and release and thus B cells survival in our model.Transfection of miR-30a-3p antisense in Poly(I:C)- and IFN-γ-activated NFLS and NHDF upregulated BAFF secretion, confirming that this microRNA is a basal repressors of BAFF expression in cells from healthy donors.Our data suggest a critical role of miR-30a-3p in the regulation of BAFF expression, which could have a major impact in the regulation of the autoimmune responses occurring in RA and SSc.

View Article: PubMed Central - PubMed

Affiliation: Immunorhumatologie moléculaire, INSERM UMR S_1109, Centre de Recherche en Immunologie et Hématologie, Faculté de Médecine, Fédération de Médecine Translationnelle de Strasbourg (FMTS), Université de Strasbourg, Strasbourg, France and Service de Rhumatologie, Centre National de Référence pour les Maladies Systémiques Autoimmunes Rares, Hôpitaux Universitaires de Strasbourg, Strasbourg, France.

ABSTRACT
We evaluated micro (mi) RNA-mediated regulation of BAFF expression in fibroblasts using two concomitant models: (i) synovial fibroblasts (FLS) isolated from healthy controls (N) or Rheumatoid Arthritis (RA) patients; (ii) human dermal fibroblasts (HDF) isolated from healthy controls (N) or Systemic Sclerosis (SSc) patients. Using RT-qPCR and ELISA, we first showed that SScHDF synthesized and released BAFF in response to Poly(I:C) or IFN-γ treatment, as previously observed in RAFLS, whereas NHDF released BAFF preferentially in response to IFN-γ. Next, we demonstrated that miR-30a-3p expression was down regulated in RAFLS and SScHDF stimulated with Poly(I:C) or IFN-γ. Moreover, we demonstrated that transfecting miR-30a-3p mimic in Poly(I:C)- and IFN-γ-activated RAFLS and SScHDF showed a strong decrease on BAFF synthesis and release and thus B cells survival in our model. Interestingly, FLS and HDF isolated from healthy subjects express higher levels of miR-30a-3p and lower levels of BAFF than RAFLS and SScHDF. Transfection of miR-30a-3p antisense in Poly(I:C)- and IFN-γ-activated NFLS and NHDF upregulated BAFF secretion, confirming that this microRNA is a basal repressors of BAFF expression in cells from healthy donors. Our data suggest a critical role of miR-30a-3p in the regulation of BAFF expression, which could have a major impact in the regulation of the autoimmune responses occurring in RA and SSc.

Show MeSH
Related in: MedlinePlus