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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 represses BAFF secretion by healthy FLS and HDF.A, B. miR-30a-3p expression was determined by RT-qPCR in NFLS (n = 4)/RAFLS (n = 4) (A) and NHDF (n = 3)/SScHDF (n = 4) (B) stimulated with Poly(I:C) (10 µg/mL), IFN-γ (0.1 or 5 ng/mL) or medium for 48 h and 72 h. Results were normalized to U6snRNA and expressed as fold change compared with samples from RAFLS (A) or SScHDF (B) incubated with medium. C. NFLS (n = 3) and NHDF (n = 3) were transfected with miR-30-3p antisense oligonucleotides (20 pM/sample) or with an AllStars negative control (CT). After 24 h, cells were activated with Poly(I:C) (10 µg/mL), IFN-γ (0.1 or 5 ng/mL) or medium for 72 h. BAFF release was determined by ELISA in culture supernatants. D. NHDF (n = 3) transfected with miR-30-3p antisense, were stimulated with poly (I:C). The supernatant was then treated with control IgG or with anti-BAFF antibodies and added to purified B cells. B cells survival was next evaluated as in panel Figure 5. *p<0.05; **p<0.01.
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pone-0111266-g006: MiR-30a-3p represses BAFF secretion by healthy FLS and HDF.A, B. miR-30a-3p expression was determined by RT-qPCR in NFLS (n = 4)/RAFLS (n = 4) (A) and NHDF (n = 3)/SScHDF (n = 4) (B) stimulated with Poly(I:C) (10 µg/mL), IFN-γ (0.1 or 5 ng/mL) or medium for 48 h and 72 h. Results were normalized to U6snRNA and expressed as fold change compared with samples from RAFLS (A) or SScHDF (B) incubated with medium. C. NFLS (n = 3) and NHDF (n = 3) were transfected with miR-30-3p antisense oligonucleotides (20 pM/sample) or with an AllStars negative control (CT). After 24 h, cells were activated with Poly(I:C) (10 µg/mL), IFN-γ (0.1 or 5 ng/mL) or medium for 72 h. BAFF release was determined by ELISA in culture supernatants. D. NHDF (n = 3) transfected with miR-30-3p antisense, were stimulated with poly (I:C). The supernatant was then treated with control IgG or with anti-BAFF antibodies and added to purified B cells. B cells survival was next evaluated as in panel Figure 5. *p<0.05; **p<0.01.

Mentions: Finally, we considered the possibility that miR-30a-3p family members could represent an essential mechanism to maintain BAFF expression at a very low level necessary in healthy conditions. Indeed, excessive BAFF secretion leading to increased B cells activation constitutes a major trigger promoting auto-immunity. A first insight into this model is provided by our initial observation of augmented BAFF secretion in the supernatant of RAFLS compared to NFLS in response to Poly(I:C) and IFN-γ (figure 1B). Therefore, we compared the expression of miR-30a-3p between healthy donor and patients cells in response to Poly(I:C) and IFN-γ for 48 h and 72 h. Interestingly, we showed that control NFLS as well as Poly(I:C)- and IFN-γ-stimulated cells expressed higher levels of miR-30a-3p compared to RAFLS under the same conditions (figure 6A). Moreover, NHDF which did not release BAFF in response to Poly(I:C) (figure 1D), also expressed higher levels of miR-30a-3p in the same conditions (figure 6B). These observations strongly suggest that miR-30a-3p could be a basal repressor of BAFF expression in these cells. Similar results were obtained upon transfection of miR-30e-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 represses BAFF secretion by healthy FLS and HDF.A, B. miR-30a-3p expression was determined by RT-qPCR in NFLS (n = 4)/RAFLS (n = 4) (A) and NHDF (n = 3)/SScHDF (n = 4) (B) stimulated with Poly(I:C) (10 µg/mL), IFN-γ (0.1 or 5 ng/mL) or medium for 48 h and 72 h. Results were normalized to U6snRNA and expressed as fold change compared with samples from RAFLS (A) or SScHDF (B) incubated with medium. C. NFLS (n = 3) and NHDF (n = 3) were transfected with miR-30-3p antisense oligonucleotides (20 pM/sample) or with an AllStars negative control (CT). After 24 h, cells were activated with Poly(I:C) (10 µg/mL), IFN-γ (0.1 or 5 ng/mL) or medium for 72 h. BAFF release was determined by ELISA in culture supernatants. D. NHDF (n = 3) transfected with miR-30-3p antisense, were stimulated with poly (I:C). The supernatant was then treated with control IgG or with anti-BAFF antibodies and added to purified B cells. B cells survival was next evaluated as in panel Figure 5. *p<0.05; **p<0.01.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111266-g006: MiR-30a-3p represses BAFF secretion by healthy FLS and HDF.A, B. miR-30a-3p expression was determined by RT-qPCR in NFLS (n = 4)/RAFLS (n = 4) (A) and NHDF (n = 3)/SScHDF (n = 4) (B) stimulated with Poly(I:C) (10 µg/mL), IFN-γ (0.1 or 5 ng/mL) or medium for 48 h and 72 h. Results were normalized to U6snRNA and expressed as fold change compared with samples from RAFLS (A) or SScHDF (B) incubated with medium. C. NFLS (n = 3) and NHDF (n = 3) were transfected with miR-30-3p antisense oligonucleotides (20 pM/sample) or with an AllStars negative control (CT). After 24 h, cells were activated with Poly(I:C) (10 µg/mL), IFN-γ (0.1 or 5 ng/mL) or medium for 72 h. BAFF release was determined by ELISA in culture supernatants. D. NHDF (n = 3) transfected with miR-30-3p antisense, were stimulated with poly (I:C). The supernatant was then treated with control IgG or with anti-BAFF antibodies and added to purified B cells. B cells survival was next evaluated as in panel Figure 5. *p<0.05; **p<0.01.
Mentions: Finally, we considered the possibility that miR-30a-3p family members could represent an essential mechanism to maintain BAFF expression at a very low level necessary in healthy conditions. Indeed, excessive BAFF secretion leading to increased B cells activation constitutes a major trigger promoting auto-immunity. A first insight into this model is provided by our initial observation of augmented BAFF secretion in the supernatant of RAFLS compared to NFLS in response to Poly(I:C) and IFN-γ (figure 1B). Therefore, we compared the expression of miR-30a-3p between healthy donor and patients cells in response to Poly(I:C) and IFN-γ for 48 h and 72 h. Interestingly, we showed that control NFLS as well as Poly(I:C)- and IFN-γ-stimulated cells expressed higher levels of miR-30a-3p compared to RAFLS under the same conditions (figure 6A). Moreover, NHDF which did not release BAFF in response to Poly(I:C) (figure 1D), also expressed higher levels of miR-30a-3p in the same conditions (figure 6B). These observations strongly suggest that miR-30a-3p could be a basal repressor of BAFF expression in these cells. Similar results were obtained upon transfection of miR-30e-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