Reprogramming of synovial macrophage metabolism by synovial fibroblasts under inflammatory conditions

Abstract

Background: Macrophages adapt to microenvironments, and change metabolic status and functions to regulate inflammation and/or maintain homeostasis. In joint cavities, synovial macrophages (SM) and synovial fibroblasts (SF) maintain homeostasis. However, under inflammatory conditions such as rheumatoid arthritis (RA), crosstalk between SM and SF remains largely unclear.

Methods: Immunofluorescent staining was performed to identify localization of SM and SF in synovium of collagen antibody induced arthritis (CAIA) model mice and normal mice. Murine arthritis tissue-derived SM (ADSM), arthritis tissue-derived SF (ADSF) and normal tissue-derived SF (NDSF) were isolated and the purity of isolated cells was examined by RT-qPCR and flow cytometry analysis. RNA-seq was conducted to reveal gene expression profile in ADSM, NDSF and ADSF. Cellular metabolic status and expression levels of metabolic genes and inflammatory genes were analyzed in ADSM treated with ADSM-conditioned medium (ADSM-CM), NDSF-CM and ADSF-CM.

Results: SM and SF were dispersed in murine hyperplastic synovium. Isolations of ADSM, NDSF and ADSF to analyze the crosstalk were successful with high purity. From gene expression profiles by RNA-seq, we focused on secretory factors in ADSF-CM, which can affect metabolism and inflammatory activity of ADSM. ADSM exposed to ADSF-CM showed significantly upregulated glycolysis and mitochondrial respiration as well as glucose and glutamine uptake relative to ADSM exposed to ADSM-CM and NDSF-CM. Furthermore, mRNA expression levels of metabolic genes, such as Slc2a1, Slc1a5, CD36, Pfkfb1, Pfkfb3 and Irg1, were significantly upregulated in ADSM treated with ADSF-CM. Inflammation marker genes, including Nos2, Tnf, Il-1b and CD86, and the anti-inflammatory marker gene, Il-10, were also substantially upregulated by ADSF-CM. On the other hand, NDSF-CM did not affect metabolism and gene expression in ADSM.

Conclusions: These findings suggest that crosstalk between SM and SF under inflammatory conditions can induce metabolic reprogramming and extend SM viability that together can contribute to chronic inflammation in RA. Video Abstract.

Keywords: Chronic inflammation; Metabolic reprogramming; Rheumatoid arthritis; Synovial fibroblast; Synovial macrophage.

Fig. 1

Histological analysis of knee joint tissue from CAIA mice. a Appearance of wild type mice with or without experimental arthritis (CAIA). b Sections of normal (left) and CAIA (right) knee joints stained with Hematoxylin and Eosin showing hyperplastic synovium (HS) in the joint (arrow head). Scale bar represents 500 μm. In immunofluorescent staining c F4/80-positive (green) cells were observed in the HS and articular cavity (AC). The inflammatory macrophage markers Nos2 and Tnf (red) co-localized in F4/80^high-positive cells (arrow, yellow). Scale bar represents 20 μm. d Synovial cells were localized randomly in the HS. Panels on the right are high magnification images of boxed areas in the left panels. Podoplanin (Pdpn)-positive (red) cells were also observed in the HS. Scale bar represents 50 μm (left panel) and 20 μm (right panel). Histological data were technically replicated more than 2 times

Fig. 2

Gene expression profile in arthritis tissue-derived synovial macrophages (ADSM), normal tissue-derived synovial fibroblasts (NDSF) and arthritis tissue-derived synovial fibroblasts (ADSF). a ADSM, NDSF and ADSF were isolated from 4 independent ankles with or without CAIA. Representative phase-contrast images of ADSM, NDSF and ADSF. Scale bar represents 100 μm. b Gene expression of pan-macrophage and SF markers in ADSM, NDSF and ADSF were analyzed by RT-qPCR (n = 4). ** indicates P < 0.01 by ANOVA followed by Tukey’s test. Data are presented as average ± SD. c Pan-leukocyte marker (CD45), macrophage markers (F4/80, CD11b), B cell marker (Ly6G) and T cell marker (CD3e) were analyzed in ADSM by flow cytometry. Representative dot plots were shown. Percentage of FITC⁺ and PE⁺ were showed lower left graph (n = 4). ** indicates P < 0.01 versus Ly6G⁺ CD45⁺ and CD3e⁺ CD45⁺ by ANOVA followed by Tukey’s test. Data are presented as average ± SD. d Heatmap of selected genes in ADSM, NDSF and ADSF (n = 3). Log10 transformed read counts are scaled to 0.0 to 3.0. Rows and columns were ordered based on hierarchical clustering by MeV. e Principal-component analysis (PCA) displaying clusters of ADSM, NDSF and ADSF (n = 3). f Venn diagram for the number of specifically expressed genes in NDSF and ADSF. g Gene Ontology (GO) analyses were performed using DAVID Bioinformatics Resources. The top of enriched annotation cluster among 212 genes are illustrated by P-value. All data were obtained from 3–4 independent experiments using ADSM, NDSF and ADSF derived from 3 to 4 independent ankles with or without CAIA

Fig. 3

Cellular metabolism analysis in ADSM. a Representative phase-contrast images of ADSM cultured for 24 h by CM. Scale bar represents 100 μm. b Cellular metabolic status in ADSM was measured by MTT assay (n = 4). ** indicates P < 0.01 versus 0 h by ANOVA followed by Tukey’s test. c The number of nuclei were calculated to assess cell proliferation for 0–48 h (n = 4). N.S.; not significant versus 0 h by ANOVA followed by Tukey’s test. d Oxygen consumption rate (OCR) was assessed after the addition of oligomycin (Oligo), Carbonyl cyanide 4- (trifluoromethoxy) phenylhydrazone (FCCP) and antimycin A/rotenone (AA/ROT) at the indicated times (Left). Basal respiration, ATP production and maximal respiration were measured (Right) (n = 3). ** indicates P < 0.01 by unpaired t‐test. e Extracellular acidification rate (ECAR) was assessed after the addition of glucose and oligomycin (oligo) at the indicated times (Left). Glycolysis and glycolysis capacity were measured (Right) (n = 3). ** indicates P < 0.01 by unpaired t‐test. Data are presented as average ± SD. All data were technically replicated and repeated more than 2 times using ADSM derived from other CAIA ankle

Fig. 4

Metabolic reprogramming and immunological activation in ADSM. a Metabolic gene expression in ADSM treated with CM was analyzed by RT-qPCR (n = 4). b Glucose and c Glutamine uptake rate per 24 h were measured in ADSM treated with CM (n = 4). d Inflammatory and anti-inflammatory gene expression in ADSM treated with CM was analyzed by RT-qPCR (n = 4). e After culturing in CM for 0–24 h, the Tnf concentration in the medium was measured by ELISA (0 h; n = 1, 24 h; n = 4). f Immunocytochemical staining of Nos2 (red) and DAPI (blue) in ADSM treated with CM. Scale bar represents 50 μm. g Summarized schema in this study. * and ** indicate P < 0.05 and P < 0.01, respectively, by ANOVA followed by Tukey’s test. Data are presented as average ± SD. All data were obtained from 4 independent experiments using ADSM derived from 4 independent CAIA ankles