Extracellular CIRP Induces an Inflammatory Phenotype in Pulmonary Fibroblasts via TLR4

Abstract

Extracellular cold-inducible RNA-binding protein (eCIRP), a new damage-associated molecular pattern (DAMP), has been recently shown to play a critical role in promoting the development of bleomycin-induced pulmonary fibrosis. Although fibroblast activation is a critical component of the fibrotic process, the direct effects of eCIRP on fibroblasts have never been examined. We studied eCIRP's role in the induction of inflammatory phenotype in pulmonary fibroblasts and its connection to bleomycin-induced pulmonary fibrosis in mice. We found that eCIRP causes the induction of proinflammatory cytokines and differentially expression-related pathways in a TLR4-dependent manner in pulmonary fibroblasts. Our analysis further showed that the accessory pathways MD2 and Myd88 are involved in the induction of inflammatory phenotype. In order to study the connection of the enrichment of these pathways in priming the microenvironment for pulmonary fibrosis, we investigated the gene expression profile of lung tissues from mice subjected to bleomycin-induced pulmonary fibrosis collected at various time points. We found that at day 14, which corresponds to the inflammatory-to-fibrotic transition phase after bleomycin injection, TLR4, MD2, and Myd88 were induced, and the transcriptome was differentially enriched for genes in those pathways. Furthermore, we also found that inflammatory cytokines gene expressions were induced, and the cellular responses to these inflammatory cytokines were differentially enriched on day 14. Overall, our results show that eCIRP induces inflammatory phenotype in pulmonary fibroblasts in a TLR4 dependent manner. This study sheds light on the mechanism by which eCIRP induced inflammatory fibroblasts, contributing to pulmonary fibrosis.

Keywords: bleomicyn; eCIRP; fibroblast; fibrosis; inflammation.

Figure 1

eCIRP induces proinflammatory cytokines in pulmonary fibroblasts in a TLR4-dependent manner. Differential mRNA expression normalized counts for TNF-α (A), IL-1β (D), and IL-6 (G) in pulmonary fibroblasts isolated from wild type (WT) and TLR4^-/- treated with PBS and 1 μg/ml CIRP. Real-time reverse transcription-polymerase chain reaction (RT-PCR) for TNF-α (B), IL-6 (E), and (G) normalized against Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. Western blot (WB) analyses of TNF-α (C) and IL-1β (F), and enzyme-linked immunosorbent assay (ELISA) analysis of IL-6 (I) in WT and TLR4^-/- pulmonary fibroblasts treated with PBS and 1 μg/ml CIRP. Analyses for (A, D, G) are performed by log2 fold change of maximum likelihood estimates (MLE) using the DESeq2 workflow and the p-values were adjusted by Benjamini-Hochberg (BH) with a false discovery cut-off set to 0.1. Analyses for (E, F, H, I) were done using two-way ANOVA with one factor being genetics (WT vs TLR4^-/-) and one factor being treatment (PBS vs. CIRP). *p<0.05 vs. WT PBS. ^#p<0.05 vs. WT CIRP. The groups were checked for Shapiro-Wilk test for normality prior to the application of two-way ANOVA. The mRNA sequencing (3 samples per group, 3 repeats per sample) and RT-PCR (4 samples per group, 2 repeats per sample) were done after the cells were incubated for 24 hours. The WBs (4-5 samples per group) and ELISA (6-12 samples per group, triplicate) were performed when cells were incubated for 48 hours. Representative samples for each lineage (WT vs. TLR4^-/-) are from a single blot shown for western blot analyses.

Figure 2

eCIRP differentially enriches TLR4 and dependent pathways in pulmonary fibroblasts irrespective of TGF-β1. MA plots for differential mRNA expression profile of 1 μg/ml CIRP treatment vs. PBS treatment of pooled pulmonary fibroblasts isolated from wild type (WT) (A) and TLR4^-/- mice (B). Differential enrichment profile transcriptome of WT pulmonary fibroblasts isolated from mice and treated with PBS (orange), 2 ng/ml TGF-β1 (yellow), 1 μg/ml CIRP (purple), and the combination of the two (green) in gene ontologies 0034145 (toll-like receptor 4 signaling pathway) (C), 0002755 (MyD88-dependent toll-like receptor signaling pathway) (D) shown in heatmaps. The dendrogram clustering was made based on the hierarchical clustering of samples across all genes for each ontology profile. The heatmap color spectra were normalized across each gene. Z-score color key and histogram of counts presented in the left upper corner. Pulmonary fibroblasts were collected 24 hours after treatments described and prepared for RNA sequencing.

Figure 3

eCIRP induces MD2 expression in pulmonary fibroblasts in a TLR4 dependent manner. Differential mRNA expression profile (A), real-time reverse transcription-polymerase chain reaction (RT-PCR) (B), and western blot (WB) analyses for MD2 in WT and TLR4^-/- pulmonary fibroblasts treated with PBS and 1 μg/ml CIRP. Analysis for A was performed by log2 fold change of maximum likelihood estimates (MLE) using the DESeq2 workflow and the p-values were adjusted by Benjamini-Hochberg (BH) with a false discovery cut-off set to 0.1. Analyses for (B, C) were done using two-way ANOVA with one factor being genetics (WT vs TLR4^-/-) and one factor being treatment (PBS vs. CIRP). *p<0.05 vs. WT PBS. ^#p<0.05 vs. WT CIRP. The groups were checked for Shapiro-Wilk test for normality prior to the application of two-way ANOVA. The mRNA sequencing (3 samples per group, 3 repeats per instance) and RT-PCR (4 samples per group, 2 repeats per instance) were done after the cells were incubated for 24 hours. The WBs (4-5 samples per group) were performed when cells were incubated for 48 hours. Representative samples for each lineage (WT vs. TLR4^-/-) are from a single blot shown for western blot analyses.

Figure 4

eCIRP induction of proinflammatory phenotype is not dependent on TGF-β1. Principal component analysis (PCA) of the mRNA expression profile of WT pulmonary fibroblasts treated with PBS, 1 μg/ml CIRP, 2 ng/ml TGF-β1, and combination of the two shown in clustering of samples (A) and direction of selected proinflammatory/profibrotic genes (B). Pulmonary fibroblasts were collected 24 hours after treatments and prepared for RNA sequencing. PC1 and PC2 explained 84 and 11% of the variance, respectively. PC, Principal component.

Figure 5

eCIRP differentially enriches proinflammatory transcriptional profiles irrespective of TGF-β1. Differential enrichment profile of WT pulmonary fibroblasts isolated from mice and treated with PBS (orange), 2 ng/ml TGF-β1 (yellow), 1 μg/ml CIRP (purple), and the combination of the two (green) in gene ontologies 0033209 (TNF-α signaling pathway) (A) and 0032755 (positive regulation of IL-6 production) (B) shown in heatmaps. The dendrogram clustering was made based on the hierarchical clustering of samples across all genes for each ontology profile. The heatmap of the color spectra was normalized across each gene. Z-score color key and histogram of counts presented in the left upper corner. Pulmonary fibroblasts were collected 24 hours after treatments described and prepared for RNA sequencing.

Figure 6

Toll-like receptor 4 (TLR4) and related genes are induced, and pathways are differentially enriched on day 14 of bleomycin injection in lung tissue. Examinations of a publicly available gene expression omnibus (GEO) profile: GSE132869. Expression profiles of TLR4 (A), MD-2 (C), and Myd88 (E) mRNA as base 2 logarithmic counts per million over time (days) in lung tissue of mice with the means and 0.95 confidence intervals. The samples are presented as bleomycin injected group (in green), and PBS injected mice (in purple). The plots are smoothed using a generalized linear model. Differential enrichment profile of lung tissues of PBS injected (purple) and bleomycin injected mice on day 14 from the start of injections in gene ontologies 0002224 (toll-like receptor signaling pathway) (B), 0035666 (TRIF-dependent toll-like receptor signaling pathway) (D), and 0002755 (MyD88-dependent toll-like receptor signaling pathway) (F) shown in the heatmap. The dendrogram clustering was made based on the hierarchical clustering of samples across all genes for each ontology profile. The heatmap of the color spectra was normalized across each gene. Z-score color key and histogram of counts presented in the left upper corner. Female mice (~12 weeks old) subcutaneously injected daily either with bleomycin (10 mg/kg/day) or PBS. Injections began on Day 0 and were done five times per week for two weeks. Mice were sacrificed on days 7, 14, 21, 28, and 42, and the lung tissues were collected.

Figure 7

Proinflammatory cytokines are induced, and their cellular responses are enriched on day 14 of bleomycin injection in lung tissue. Examinations of a publicly available gene expression omnibus (GEO) profile: GSE132869. Expression profiles of TNF-α (A) and IL-6 (C) mRNA as base 2 logarithmic counts per million over time (days) in lung tissue of mice with the means and 0.95 confidence intervals. The samples are presented as bleomycin injected group (in green), and PBS injected mice (in purple). The plots are smoothed using a generalized linear model. Differential enrichment profile of lung tissues of PBS injected (purple), and bleomycin injected mice on day 14 from the start of injections in gene ontologies 0071356 (cellular response to TNF-α, B) and 0071354 (cellular response to IL-6, D) shown in the heatmap. The dendrogram clustering was made based on the hierarchical clustering of samples across all genes for each ontology profile. The heatmap of the color spectra was normalized across each gene. Z-score color key and histogram of counts presented in the left upper corner. Female mice (~12 weeks old) subcutaneously injected daily either with bleomycin (10 mg/kg/day) or PBS. Injections began on Day 0 and were done five times per week for two weeks. Mice were sacrificed on days 7, 14, 21, 28, and 42, and the lung tissues were collected.