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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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BAFF expression and secretion are up-regulated in Poly (I:C)- and IFN-γ-stimulated rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and systemic sclerosis (SSc) human dermal fibroblast (HDF).A, C. BAFF mRNA expression was determined by RT-qPCR in NFLS(n = 4) and RAFLS(n = 4) (A) or NHDF(n = 3) and SScHDF (n = 4) (C) stimulated (depending of the cell types) with BLP (1 µg/ml), LPS (1 µg/ml), Poly (I:C) (10 µg/mL) or IFN-γ (0.1, 1 or 5 ng/mL) for 72 h. Results were normalized to Gapdh and expressed as fold change compared with samples from cells incubated in medium. B, D. BAFF release was quantified by ELISA in culture supernatants of NFLS (n = 4) and RAFLS(n = 4) (B) or NHDF(n = 3) and SScHDF (n = 4) (D) in the same conditions as panels A and C. Data are expressed as the mean of triplicate samples ± SEM. *p<0.05, **p<0.01, ***p<0.001.
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pone-0111266-g001: BAFF expression and secretion are up-regulated in Poly (I:C)- and IFN-γ-stimulated rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and systemic sclerosis (SSc) human dermal fibroblast (HDF).A, C. BAFF mRNA expression was determined by RT-qPCR in NFLS(n = 4) and RAFLS(n = 4) (A) or NHDF(n = 3) and SScHDF (n = 4) (C) stimulated (depending of the cell types) with BLP (1 µg/ml), LPS (1 µg/ml), Poly (I:C) (10 µg/mL) or IFN-γ (0.1, 1 or 5 ng/mL) for 72 h. Results were normalized to Gapdh and expressed as fold change compared with samples from cells incubated in medium. B, D. BAFF release was quantified by ELISA in culture supernatants of NFLS (n = 4) and RAFLS(n = 4) (B) or NHDF(n = 3) and SScHDF (n = 4) (D) in the same conditions as panels A and C. Data are expressed as the mean of triplicate samples ± SEM. *p<0.05, **p<0.01, ***p<0.001.

Mentions: Upregulation of BAFF expression by Poly (I:C)- and IFN-γ-activated RAFLS, but not upon TLR2 or TLR4 activation, has been previously reported [13], [22]. To gain more insights into the physiopathological consequences of this observation, we first compared BAFF expression in FLS isolated from healthy donors or RA patients. As shown in figure 1A and B, we observed that IFN-γ-dependent BAFF expression reaches maximum levels in RAFLS, whereas FLS isolated from healthy donors (NFLS) exhibit reduced cytokine expression at both mRNA and protein levels. Of note, Poly (I:C) stimulation induced very limited BAFF expression and secretion by NFLS, while RAFLS appeared extremely responsive to this stimulus. Next, we tested whether this difference between a healthy and a pathological (inflammatory) state could also be observed in another fibroblastic cell type and for this, we chose Human Dermal Fibroblasts (HDF) isolated from skin biopsies harvested from healthy donors (NHDF) or from patients suffering from systemic sclerosis (SScHDF). IFN-γ stimulation (1 and 5 ng/mL) resulted in a comparable increased expression of BAFF transcripts (figure 1C) and cytokine secretion (figure 1D) by NHDF or SScHDF. Interestingly, up-regulation of BAFF transcripts and protein release in response to TLR3 triggering by Poly (I:C) was only detectable in SScHDF and not from healthy individuals. We also investigated here the ability of Bacterial LipoProteins (BLP, Pam3CSK4) or LipoPolysaccharide (LPS), which are respectively ligands for TLR2 and 4, to stimulate BAFF synthesis by NHDF and SScHDF. As seen in figure 1C and D, these PAMPs (Pathogen Associated Molecular Pattern) are not activators of BAFF transcription.


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)

BAFF expression and secretion are up-regulated in Poly (I:C)- and IFN-γ-stimulated rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and systemic sclerosis (SSc) human dermal fibroblast (HDF).A, C. BAFF mRNA expression was determined by RT-qPCR in NFLS(n = 4) and RAFLS(n = 4) (A) or NHDF(n = 3) and SScHDF (n = 4) (C) stimulated (depending of the cell types) with BLP (1 µg/ml), LPS (1 µg/ml), Poly (I:C) (10 µg/mL) or IFN-γ (0.1, 1 or 5 ng/mL) for 72 h. Results were normalized to Gapdh and expressed as fold change compared with samples from cells incubated in medium. B, D. BAFF release was quantified by ELISA in culture supernatants of NFLS (n = 4) and RAFLS(n = 4) (B) or NHDF(n = 3) and SScHDF (n = 4) (D) in the same conditions as panels A and C. Data are expressed as the mean of triplicate samples ± SEM. *p<0.05, **p<0.01, ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111266-g001: BAFF expression and secretion are up-regulated in Poly (I:C)- and IFN-γ-stimulated rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and systemic sclerosis (SSc) human dermal fibroblast (HDF).A, C. BAFF mRNA expression was determined by RT-qPCR in NFLS(n = 4) and RAFLS(n = 4) (A) or NHDF(n = 3) and SScHDF (n = 4) (C) stimulated (depending of the cell types) with BLP (1 µg/ml), LPS (1 µg/ml), Poly (I:C) (10 µg/mL) or IFN-γ (0.1, 1 or 5 ng/mL) for 72 h. Results were normalized to Gapdh and expressed as fold change compared with samples from cells incubated in medium. B, D. BAFF release was quantified by ELISA in culture supernatants of NFLS (n = 4) and RAFLS(n = 4) (B) or NHDF(n = 3) and SScHDF (n = 4) (D) in the same conditions as panels A and C. Data are expressed as the mean of triplicate samples ± SEM. *p<0.05, **p<0.01, ***p<0.001.
Mentions: Upregulation of BAFF expression by Poly (I:C)- and IFN-γ-activated RAFLS, but not upon TLR2 or TLR4 activation, has been previously reported [13], [22]. To gain more insights into the physiopathological consequences of this observation, we first compared BAFF expression in FLS isolated from healthy donors or RA patients. As shown in figure 1A and B, we observed that IFN-γ-dependent BAFF expression reaches maximum levels in RAFLS, whereas FLS isolated from healthy donors (NFLS) exhibit reduced cytokine expression at both mRNA and protein levels. Of note, Poly (I:C) stimulation induced very limited BAFF expression and secretion by NFLS, while RAFLS appeared extremely responsive to this stimulus. Next, we tested whether this difference between a healthy and a pathological (inflammatory) state could also be observed in another fibroblastic cell type and for this, we chose Human Dermal Fibroblasts (HDF) isolated from skin biopsies harvested from healthy donors (NHDF) or from patients suffering from systemic sclerosis (SScHDF). IFN-γ stimulation (1 and 5 ng/mL) resulted in a comparable increased expression of BAFF transcripts (figure 1C) and cytokine secretion (figure 1D) by NHDF or SScHDF. Interestingly, up-regulation of BAFF transcripts and protein release in response to TLR3 triggering by Poly (I:C) was only detectable in SScHDF and not from healthy individuals. We also investigated here the ability of Bacterial LipoProteins (BLP, Pam3CSK4) or LipoPolysaccharide (LPS), which are respectively ligands for TLR2 and 4, to stimulate BAFF synthesis by NHDF and SScHDF. As seen in figure 1C and D, these PAMPs (Pathogen Associated Molecular Pattern) are not activators of BAFF transcription.

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