Follistatin-like protein 1 modulates IL-17 signaling via IL-17RC regulation in stromal cells

Abstract

Follistatin-like protein 1 (FSTL-1) possesses several newly identified roles in mammalian biology, including interleukin (IL)-17-driven inflammation, though the mechanism underlying FSTL-1 influence on IL-17-mediated cytokine production is unknown. Using parallel in vitro bone marrow stromal cell models of FSTL-1 suppression, we employed unbiased microarray analysis to identify FSTL-1-regulated genes and pathways that could influence IL-17-dependent production of IL-6 and granulocyte colony-stimulating factor. We discovered that FSTL-1 modulates Il17rc gene expression. Specifically, FSTL-1 was necessary for Il17rc gene transcription, IL-17RC surface protein expression and IL-17-dependent cytokine production. This work identifies a mechanism by which FSTL-1 influences IL-17-driven inflammatory signaling in vitro and reveals a novel function for FSTL-1, as a modulator of gene expression. Thus enhanced understanding of the interplay between FSTL-1 and IL-17-mediated inflammation may provide insight into potential therapeutic targets of IL-17-mediated diseases and warrants ongoing study of in vivo models and clinical scenarios of FSTL-1-influenced diseases.

Figure 1. Follistatin-like protein 1 suppression by shRNA reduces IL-17/TNFα mediated cytokine production

In the ST2 cell line (A) Fstl1 transcript abundance and (B) FSTL-1 protein production increased following IL-17/TNFα stimulation, but was reduced in cells transfected with shRNA targeting Fstl1 (gray bars) compared to control shRNA (black bars) IL-17/TNFα dependent cytokine production is reduced by Fstl1 inhibition as determined by (C, E) transcript induction and protein secretion (D, F). ** p<0.01, ***p<0.001, ****p<0.0001

Figure 2. Follistatin-like protein 1 suppression by gene deletion reduces IL-17/TNFα mediated cytokine production

In primary bone marrow stromal cells (A) Fstl1 transcript abundance and (B) FSTL-1 protein production increased following IL-17A and TNFα stimulation, but was reduced in cells from FSTL-1 KO mice (gray bars) compared to wild-type littermate control mice (black bars) IL-17/TNFα dependent cytokine production is reduced by Fstl1 inhibition as determined by (C, E) transcript induction and protein secreton (D, F) *p<0.05. ** p<0.01, ***p<0.001, ****p<0.0001

Figure 3. Microarray and pathway analysis of the role of FSTL-1 in IL-17 signaling

cDNA from ST2 shControl and shFSTL-1 cells, as well as primary WT and FSTL-1 KO BMSCs, with and without IL-17A/TNFa stimulation underwent Microarray analysis. Differential expression (≥1.1 fold-change) for FSTL-1 influenced targets were identified for each condition by t-test with Bonferroni correction (p<0.05) of non-transformed data. (A) Forty-six symmetrically upregulated and (B) twenty-three downregulated genes in both cell systems with and without IL-17A/TNFa stimulation are displayed schematically in a Venn diagram corresponding to Table 1.

Figure 4. Follistatin-like protein 1 attenuation is associated with reduced IL-17RC mRNA and protein expression

Il17rc transcript abundance is reduced in (A) ST2 cells with shRNA FSTL-1 suppression as well as (B) primary BMSCs from FSTL-1 KO mice when compared to controls. Surface expression of IL-17RC is reduced as measured by FACS determined by (C) %IL17RC+ and (D) mean fluorescence intensity as noted by (E) representative histograms of isotype (dark grey), FSTL-1 KO (light grey) and WT BMSCs (dashed line). *p<0.05, **p<0.01.

Figure 5. Ectopic Il17rc expression rescues cytokine production in FSTL-1 KO cells

FSTL-1 KO BMSC transfection with pCMV-il17rc rescues Il17rc transcript abundance (A). pCMV-Il17rc complementation in FSTL-1 KO BMSCs rescues IL-17A stimulated transcript levels of (B) Il6 and (C) Csf3. * p<0.05, ** p<0.01, ** p<0.001

Figure 6. Ectopic Fstl1 expression rescues Il17rc expression and cytokine production in FSTL-1 KO cells

FSTL-1 KO BMSC transfection with pCMV-fstl1 rescues (A) Fstl1 and (B) Il17rc transcript abundance. pCMV-Fstl1 complementation in FSTL-1 KO BMSCs rescues IL-17A/TNF stimulated transcript levels of (C) Il6 and (D) Csf3. *p<0.05, ** p<0.01

Figure 7. FSTL-1 influences Il17rc transcript abundance via transcriptional regulation

Following ActinomycinD treatment, FSTL-1 KO cells had reduced (A) Fstl1 and (8) Il17rc transcript abundance before ActinomycinD treatment and at 1, 2, 4 and 6 hours post-treatment compared with WT BMSCs. Linear regression of Il17rc transcript abundance (C) at various timepoints following ActinomycinD treatment showed similar slopes for WT and FSTL-1 KO BMSCs (−3.254±1.200 and −2.457±1.429, p=0.6748). Newly synthesized mRNA (5-EU labeled) was similar for (D) Hprt, but reduced for (E) Fstl1 and (F) Il17rc, transcripts in FSTL-1 KO cells compared to WT * p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001

Figure 8. Fstl1 expression correlates with Il17rc expression in the CD45 negative bone marrow population

CD45 negative cells from wild-type and FSTL-1 Hypomorphic mouse bone marrow had slightly reduced (A) Fstl1, (B) Il17rc and (C) Il17ra expression. Correlation analysis revealed highly significant association between (D) Fstl1 and Il17rc (r=0.9512, R2=0.9048, p<0.0001) but not (E) Fstl1 and Il17ra (r=−0.1982, R2=0.0393, p=0.5162).