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. Author manuscript; available in PMC: 2023 Jun 23.
Published in final edited form as: Nat Metab. 2022 Jun 23;4(6):759–774. doi: 10.1038/s42255-022-00585-x

Fig. 2. Synovial macrophages are resistant to glucose deprivation.

Fig. 2

Synovial Mϕ were isolated from synovial tissue biopsies collected from patients with RA. Tissues were categorized as having low-grade or high-grade synovitis.

a. Tissue glucose concentrations were measured in synovial tissue lysates and correlated with the synovitis inflammatory score (n=40).

b-d. Synovial Mϕ from tissues with low-grade or high-grade inflammation were isolated and kept in glucose depleted medium for one week. Survival capacity was measured by Almar Blue (b, n=11) or LDH release (c, n=11). Live and dead cells were distinguished by dual probe fluorescence (d, FITC calcein and Texas Red BOBO-3 iodide as live and dead cell indicators, respectively, n=4).

e. Tissue sections from synovial biopsies of RA patients were doubled-stained for the macrophage marker CD68 (brown) and the proliferation marker Ki67 (blue). CD68+ Mϕ, Ki67+ proliferating cells and double positive Ki67+ CD68+ Mϕ were enumerated in 9 tissues.

f-h. Low grade human synovial tissue was implanted into NSG mice. Monocyte-derived Mϕ from either RA patients or matched healthy controls were labeled with CFSE and adoptively transferred into the mice. On day 7, tissue grafts were harvested. (f) Glucose concentrations measured in synovial tissue lysates (n=22 for Con Mϕ, n=24 for RA Mϕ). (g) Mϕ were isolated from the synovial explants and absolute numbers of CD3neg CFSE+ CD68+ cells were determined by flow cytometry (n=11). (h) Proliferation indices of Mϕ isolated from the tissue were determined based on CSFE dilution (n=9). Data are in box and whisker plots.

i-j. Monocyte-derived Mϕ were generated ex vivo from RA patients and age-matched controls and survival under nutrient stress was tested (n=4). Mϕ were kept in either nutrient-rich complete medium or in glucose-depleted medium for one week. Survival was measured by LDH release and Almar Blue cell viability assays on day 7.

Data are mean ± SEM. (a) Pearson’s correlation coefficients test. (b-d, f-j) Two-tailed unpaired t test. (i-j) Two-way ANOVA with post hoc Tukey’s multiple comparisons test.