MiR-30a-3p Negatively Regulates BAFF Synthesis in Systemic Sclerosis and Rheumatoid Arthritis Fibroblasts

<div><p>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.</p></div

miR-30a-3p expression is down-regulated in Poly (I:C)- and IFN-γ-stimulated RAFLS and SScHDF.

<p>A. miR-30a-3p is predicted to target BAFF 3′ UTR mRNA. B, C. miR-30a-3p expression was determined by RT-qPCR in RAFLS (n = 4) (B) and SScHDF (n = 4) (C) stimulated with Poly (I:C) (10 µg/mL) or IFN-γ (0.1 or 5 ng/mL) for 6 h, 48 h and 72 h. Results were normalized to U6snRNA and expressed as fold change compared with samples from cells incubated in medium. Data are expressed as the mean of triplicate samples ± SEM. *p<0.05, ***p<0.001.</p

miR-30a-3p transfection affects BAFF mRNA expression and BAFF secretion in RAFLS and SScHDF.

<p>A, C. RAFLS (n = 5) (A) and SScHDF (n = 4) (C) were transfected with miR-30a-3p mimic (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, depending on cell types) or medium for 72 h. BAFF mRNA expression was determined by RT-qPCR. Results were normalized to Gapdh and expressed as fold change compared with samples from cells incubated with medium. B, D. BAFF release was determined by ELISA in culture supernatants in the same conditions as panel A and C. E, F. IL-6 release was determined by ELISA in culture supernatants in the same conditions as panel A and C. Data are expressed as the mean of triplicate samples ± SEM. *p<0.05; **p<0.01; ***p<0.001.</p

miR-30a-3p expression in RAFLS and SScHDF regulates BAFF-dependent B cells survival in vitro.

<p>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.</p

miR-30a-3p directly targets the 3′UTR of BAFF mRNA.

<p>A. Luciferase reporter constructs with wild-type or mutated (for miR-30-3p binding sites) BAFF 3′UTR were generated. B. HEK293 cells were transiently co-transfected with reporter constructs and with miR-30a-3p mimic (20 pM). Firefly Luciferase activities were measured 48 h after transfection and normalized to Renilla Luciferase expressed by the control psi-CHECK-2 vector devoid of 3′UTR sequences. Data are expressed as the mean of triplicate samples ± SEM and are representative of three independent experiments. **p<0.01.</p

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).

<p>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.</p

MiR-30a-3p represses BAFF secretion by healthy FLS and HDF.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111266#pone-0111266-g005" target="_blank">Figure 5</a>. *p<0.05; **p<0.01.</p