Transcriptional profiling of murine macrophages stimulated with cartilage fragments revealed a strategy for treatment of progressive osteoarthritis

Abstract

Accumulating evidence suggests that synovitis is associated with osteoarthritic process. Macrophages play principal role in development of synovitis. Our earlier study suggests that interaction between cartilage fragments and macrophages exacerbates osteoarthritic process. However, molecular mechanisms by which cartilage fragments trigger cellular responses remain to be investigated. Therefore, the current study aims at analyzing molecular response of macrophages to cartilage fragments. To this end, we analyzed the transcriptional profiling of murine macrophages exposed to cartilage fragments by RNA sequencing. A total 153 genes were differentially upregulated, and 105 genes were down-regulated in response to cartilage fragments. Bioinformatic analysis revealed that the most significantly enriched terms of the upregulated genes included scavenger receptor activity, integrin binding activity, TNF signaling, and toll-like receptor signaling. To further confirm our results, immunohistochemical staining was performed to detected regulated molecules in synovial tissues of OA patients. In consistence with RNA-seq results, MARCO, TLR2 and ITGα5 were mainly detected in the intima lining layer of synovial tissues. Moreover, blockade of TLR2 or ITGα5 but not Marco using specific antibody significantly reduced production of TNF-α in stimulated macrophages by cartilage fragments. Our data suggested that blocking TLR2 or ITGα5 might be promising therapeutic strategy for treating progressive osteoarthritis.

Figure 1

Gene expression profile of macrophages stimulated by cartilage fragments. (A) Scatter plot analysis for transcript expression levels of significantly up-or down-regulated genes in macrophages response to cartilage fragments (p < 0.05) (n = 3). (B) Hierarchical clustering for the expression of significantly up-and-down-regulated genes in macrophages (p < 0.05). A scale bar for intense color change from −2 or below indicated by blue color or 2 or above indicated by red. (C) Venn diagram analysis for the numbers of genes which are significantly upregulated in response to cartilage fragments, LPS and TNF-α.

Figure 2

Gene enrichment analysis of differentially expressed genes in macrophages stimulated by cartilage fragments. (A) GO terms for cellular component. (B) GO terms for molecular function. (C) Heat map for clustered gene family. (D) GO terms for biological process. (E) GO terms for mouse response phenotypes. (F) Heat map for the transcript expression levels of enriched genes in inflammation. A scale bar for intense color change from −1.5 or below indicated by blue color or 1.5 or above indicated by red. (G) GO terms for human diseases.

Figure 3

KEGG pathways and transcriptional factors of the differentially expressed genes in macrophages response to cartilage fragments. (A) Top enriched pathways. (B) TNF signaling pathway generated from KEGG pathway database for the regulated genes in response to cartilage fragments. Red indicates the upregulated genes identified in our study. (C) Heat map for the most enriched transcription factors. A scale bar for intense color change from −1.5 or below indicated by blue color or 1.5 or above indicated by red.

Figure 4

Identification of macrophage receptors to cartilage fragments and functional blocking assay. (A) A predicated model of macrophage activation by cartilage fragments based on bioinformatic analysis. (B) Detection of MARCO, TLR2 and ITGα5 in synovial tissues of OA patients. Representative images form sections of synovial tissues of 8 OA patients. Lower panels show section stained by rabbit IgG and mouse IgG2 as negative controls. Scale bars are indicated on each image. (C) Effects of functional blocking antibodies to MARCO, TLR2 and ITGα5 on the secretion of TNF-α. Macrophages were pretreated with 10 µg/ml for 30 min and then cultured with cartilage fragments. Results are presented as means ± standard errors of the means from 4 wells values for each treatment. Significant difference was determined by one-way ANOVA followed by Tukey’s multiple-comparison procedure. The results are representative of two independent experiments. Experiments were repeated twice for reproducibility of data.