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. 2022 Jun 4;24(1):134.
doi: 10.1186/s13075-022-02817-7.

Semaphorin 3G exacerbates joint inflammation through the accumulation and proliferation of macrophages in the synovium

Jumpei Shoda #  1   2 , Shigeru Tanaka #  1 , Keishi Etori  1 , Koto Hattori  1 , Tadamichi Kasuya  1 , Kei Ikeda  1 , Yuko Maezawa  1 , Akira Suto  1 , Kotaro Suzuki  1 , Junichi Nakamura  2 , Yoshiro Maezawa  3 , Minoru Takemoto  3   4 , Christer Betsholtz  5 , Koutaro Yokote  3 , Seiji Ohtori  2 , Hiroshi Nakajima  6
Affiliations

Semaphorin 3G exacerbates joint inflammation through the accumulation and proliferation of macrophages in the synovium

Jumpei Shoda et al. Arthritis Res Ther. .

Abstract

Objectives: Methotrexate (MTX) is an anchor drug for the treatment of rheumatoid arthritis (RA). However, the precise mechanisms by which MTX stalls RA progression and alleviates the ensuing disease effects remain unknown. The aim of the present study was to identify novel therapeutic target molecules, the expression patterns of which are affected by MTX in patients with RA.

Methods: CD4+ T cells from 28 treatment-naïve patients with RA before and 3 months after the initiation of MTX treatment were subjected to DNA microarray analyses. The expression levels of semaphorin 3G, a differentially expressed gene, and its receptor, neuropilin-2, were evaluated in the RA synovium and collagen-induced arthritis synovium. Collagen-induced arthritis and collagen antibody-induced arthritis were induced in semaphorin3G-deficient mice and control mice, and the clinical score, histological score, and serum cytokines were assessed. The migration and proliferation of semaphorin 3G-stimulated bone marrow-derived macrophages were analyzed in vitro. The effect of local semaphorin 3G administration on the clinical score and number of infiltrating macrophages during collagen antibody-induced arthritis was evaluated.

Results: Semaphorin 3G expression in CD4+ T cells was downregulated by MTX treatment in RA patients. It was determined that semaphorin 3G is expressed in RA but not in the osteoarthritis synovium; its receptor neuropilin-2 is primarily expressed on activated macrophages. Semaphorin3G deficiency ameliorated collagen-induced arthritis and collagen antibody-induced arthritis. Semaphorin 3G stimulation enhanced the migration and proliferation of bone marrow-derived macrophages. Local administration of semaphorin 3G deteriorated collagen antibody-induced arthritis and increased the number of infiltrating macrophages.

Conclusions: Upregulation of semaphorin 3G in the RA synovium is a novel mechanism that exacerbates joint inflammation, leading to further deterioration, through macrophage accumulation.

Keywords: Macrophage; Methotrexate; Neuropilin-2; Rheumatoid arthritis; Semaphorin.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
The enhanced expression of Sema3G in the inflamed synovium in humans and mice. A Genes differentially expressed before and after MTX treatment. Genes differentially expressed are highlighted in red (upregulated after MTX) or blue (downregulated after MTX). B Sema3G expression in the synovium of OA or RA patients. The synovium specimens were stained with anti-Sema3G antibody and visualized with DAB. Bars indicate 200 μm (Low magnification) or 20 μm (high magnification). C The cumulative data of the Sema3G-positive area. Data are expressed as the means ± SEM. The statistical analyses were performed using an unpaired t-test. D Sema3G expression in the synovium of CIA. The hind paws of the control and CIA-induced mice were stained with anti-Sema3G antibody. The representative data are shown. Similar results were obtained in three independent experiments. Bars indicate 200 μm
Fig. 2
Fig. 2
Nrp2 expression in activated macrophages during joint inflammation. A Nrp2 expression in immune cells in the CIA synovium. The hind paws of CIA-induced mice were digested and subjected to flow-cytometric analysis. The representative histograms of Nrp2 staining and the cumulative data are shown. Data are expressed as the means ± SD. N = 3 from three independent experiments. B The characteristics of Nrp2-high macrophages. The representative histograms of CD86 and MHC class II expression on joint-infiltrating macrophages (left) and their cumulative data (right) are shown. N = 6 from two independent experiments. C Nrp2 expression on BMMs. BMMs were cultured under the indicated conditions, and Nrp2 expression was determined by flow cytometry. The representative histograms and the cumulative data are shown. N = 3 from three independent experiments. D NRP2 expression in synovium-infiltrating cells in RA. The deposited single-cell RNA-seq data were reanalyzed, and several cell types were defined by UMAP. NRP2 expression is denoted by purple dots. The statistical analyses were performed using an unpaired t-test
Fig. 3
Fig. 3
Attenuated joint inflammation in Sema3G-deficient mice. A The clinical score of Sema3G-deficient (Sema3G−/−) mice and their littermate controls (Sema3G+/−) in CIA. Data were obtained from four independent experiments (N = 17 in each group). B The pathological scores of CIA. The front paws were subjected to H&E staining, and the inflammation, the cartilage damage, and the erosion scores were separately assessed. C The inflammatory cytokines in sera. The sera were collected on day 42 and subjected to ELISA to measure IL-6 and TNFα. D The clinical score of Sema3G−/− mice and Sema3G+/− in CAIA. Data were obtained from four independent experiments (N = 13 in each group). E The pathological scores of CAIA. The front paws were subjected to H&E staining, and the inflammation, the cartilage damage, and the erosion scores were separately assessed. F The inflammatory cytokines in sera. The sera were collected on day 9 and subjected to ELISA to measure IL-6 and TNFα. Data are expressed as the means ± SEM. For the analyses of the clinical score, 2-way ANOVA was used. For the pathological scores, an unpaired t-test was used
Fig. 4
Fig. 4
Enhanced macrophage proliferation by Sema3G. A Chemotaxis assay. LPS-stimulated BMMs were subjected to a transwell migration assay. PBS (negative control), MCP1 (positive control), or various concentrations of Sema3G were added to the lower chambers. Cell migration towered to the lower chamber was determined by Hoechst staining. The pictures were taken for five different areas, and Hoechst-positive round cell numbers were counted. N = 7 from three independent experiments. One-way ANOVA test, followed by Dunnet test, was used for the statical analysis. B The volcano plot of Sema3G-stimulated BMMs. Genes differentially expressed are plotted as red dots (upregulated by Sema3G) or blue dots (downregulated by Sema3G). Some immune-related gene names are labeled. C EdU uptake in Sema3G-stimulated BMMs. Sema3G-stimulated or control (PBS) BMMs were pulsed with 10 μM EdU for 2 h, and EdU and Hoechst were detected by immunofluorescent analysis. The pictures were taken for ten different areas, and the percentage of EdU-positive cells over Hoechst-positive cells was calculated. Data were obtained from three independent experiments. N = 6. D The clinical score of Sema3G- or PBS-injected paws during CAIA. WT mice were subjected to CAIA. Sema3G (100 ng) was injected into the right footpad, and PBS was injected into the left footpad daily from day 3 to day 9. N = 11 from two independent experiments. E Infiltrating cell subsets to each paw on day 9 of CAIA. The hind paws were digested, and cells recovered were analyzed by flow cytometry. The percentages of T cells, B cells, and macrophages are shown. A paired t-test was used for the statistical analyses
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