Mir-155 is overexpressed in systemic sclerosis fibroblasts and is required for NLRP3 inflammasome-mediated collagen synthesis during fibrosis

Abstract

Background: Despite the important role that microRNAs (miRNAs) play in immunity and inflammation, their involvement in systemic sclerosis (SSc) remains poorly characterized. miRNA-155 (miR-155) plays a role in pulmonary fibrosis and its expression can be induced with interleukin (IL)-1β. SSc fibroblasts have activated inflammasomes that are integrally involved in mediating the myofibroblast phenotype. In light of this, we investigated whether miR-155 played a role in SSc and if its expression was dependent on inflammasome activation.

Methods: miR-155 expression was confirmed in SSc dermal and lung fibroblasts by quantitative polymerase chain reaction (PCR). Wild-type and NLRP3-deficient murine fibroblasts were utilized to explore the regulation of miR-155 during inflammasome activation. miR-155-deficient fibroblasts and retroviral transductions with a miR-155 expression or control vectors were used to understand the contribution of miR-155 in fibrosis.

Results: miR-155 was significantly increased and the highest expressing miRNA in SSc lung fibroblasts. Its expression was dependent on inflammasome activation as miR-155 expression could be blocked when inflammasome signaling was inhibited. In the absence of miR-155, inflammasome-mediated collagen synthesis could not be induced but was restored when miR-155 was expressed in miR-155-deficient fibroblasts.

Conclusions: miR-155 is upregulated in SSc. These results suggest that the inflammasome promotes the expression of miR-155 and that miR-155 is a critical miRNA that drives fibrosis.

Keywords: Fibrosis; IL-1; NLRP3 inflammasome; Systemic sclerosis; miR-155.

Fig. 1

miR-155 has increased expression in systemic sclerosis (SSc) lung and dermal fibroblasts. a Lung fibroblasts (n = 9 SSc; n = 5 control) and b dermal fibroblasts (n = 5 SSc; n = 6 control) were assayed for miR-155 levels as described in the methods. miR-155 levels were normalized to SNORD44. Statistical analyses was by Mann-Whitney t test

Fig. 2

miR-155 expression requires the NLRP3 inflammasome. a Systemic sclerosis (SSc) lung fibroblasts (n = 7) were treated with 20 μM YVAD for 48 h. miRNA was extracted and the resulting cDNA was assayed for miR-155 levels normalized to SNORD44. b Hydroxyproline was measured in the culture supernatants from a. Statistical analyses for a and b used the Wilcoxon ranked paired t test. c In mouse cells, miR-155 expression was induced with 20 μM bleomycin (Bleo) + 20 μM YVAD for 48 h and analyzed for miR-155 expression normalized to SNORD47. Data are presented as the average from two independent experiments with three replicates (n = 6 for each condition) + SEM. d Hydroxyproline was measured from the culture supernatants from c. Statistical analyses for c and d used the Mann-Whitney t test

Fig. 3

Collagen synthesis mediated by the inflammasome requires miR-155. a Hydroxyproline levels were measured in the culture media from B6 fibroblasts and miR-155-deficient fibroblasts (miR-155KO) + 10 μM bleomycin (Bleo) after 48 h. b Culture media hydroxyproline levels from miR-155KO fibroblasts transduced with the control (Ctl) vector or the miR-155 expressing vector + 10 μM Bleo after 48 h. Data for both a and b are presented as the average from two independent experiments with three replicates (n = 6 for each condition) + SEM using the Mann-Whitney t test. ns Not significant

Fig. 4

miR-155 regulates fibrosis via interleukin-1 (IL-1) and IL-1 induces miR-155. a miR-155KO fibroblasts were transduced with the miR-155 expression vector or the control (Ctl) vector. Some of the dishes received 10 μM bleomycin (Bleo) at 0 h and some of the cells also received 50 ng/ml IL-1 receptor antagonist (IL-1RA) at 0 and 24 h. Media was recovered after 48 h and hydroxyproline was measured (n = 9 replicates). b Dose-dependent induction of miR-155 in fibroblasts (n = 7 replicates). c Graphical representation of transforming growth factor beta (TGF-β) levels in miR-155KO and B6 cells ± bleo. d Representative Western blot (one of three samples independently tested). Data are presented as averages + SEM using the Mann-Whitney t test. ns Not significant

Fig. 5

miR-155 and interleukin-1 (IL-1) provide a feed-forward mechanism during fibrosis. In fibroblasts, activation of the inflammasome drives miR-155 expression via IL-1 autocrine signaling that further enhances IL-1 transcription and leads to fibrosis. Blockade of the IL-1 receptor or the inflammasome abrogates this process