Fig 1: Tumor necrosis factor-α (TNF-α), interleukin 12 (IL12p40), C-C motif chemokine ligand (CCL) 17, and 22 concentration in the plasma of wildtype (wt) and Nod2 knockout (ko) mice at day 5 after ileocecal resection. Mice received intraperitoneal injections of muramyl dipeptide (MDP, 100 µg/mouse) or NaCl (vehicle, veh 100 µL) 2 h prior to surgery at Day 2 and Day 5. Values are given as mean ± SD. Wildtype treated with vehicle (wt veh) n = 9–10; wildtype treated with muramyl dipeptide (wt MDP) n = 9–10; Nod2 knockout treated with vehicle (ko veh) n = 5–8; Nod2 knockout treated with muramyl dipeptide (ko MDP) n = 6–10. p values as indicated in the figure. Mann–Whitney U test.
Fig 2: Tissue-resident macrophages show distinct phagocytic speeds and inflammatory gene expression upon stimulation in vitroMG-LCs are shown in yellow, KC-LCs in green, PM-LCs in pink, and AM-LCs in blue. One of three to eight independent experiments is shown. Cells were gated as depicted in Figure S7.(A) Representative histograms of PE-pHrodo labeling after 5, 10, 15, and 30 min of TRM-LCs phagocytosis of PE-pHrodo-coupled zymosan beads in vitro.(B) Quantification of percentage of TRM-LCs labeled with PE-pHrodo after 5, 10, 15, and 30 min. Mean ± SEM is shown; n = 3–8; *p < 0.05, **p < 0.01, and ***p < 0.001.(C) Quantification of percentage of differently cultured BMDMs labeled with PE-pHrodo after 5, 10, 15, and 30 min. BMDMs in normoxia + GM-CSF are shown in white, BMDMs in normoxia + M-CSF are shown in gray, BMDMs in hypoxia + M-CSF are shown in dark gray. Mean ± SEM is shown; n = 3–6; *p < 0.05, **p < 0.01, and ***p < 0.001.(D–F) Levels of secreted cytokines are measured in mean fluorescence intensity (MFI). Control is shown in blue, LPS in red, poly(I:C) in green, and zymosan in violet. Released cytokine levels of IL-1ß are shown in (D), IL-6 in (E), and TNF-a in (F). Mean ± SEM is shown; n = 3/group; *p < 0.05, **p < 0.01, and ***p < 0.001.See also Figures S6 and S7.
Fig 3: Tendon-derived mEPs can induce macrophage infiltration and cytokine release.a Chemotaxis assay for RAW 264.7 cells toward tendon-derived mEPs and (b) corresponding quantitative colorimetric measurements are presented as three biological replicates from three independent experiments. RAW 264.7 cells that migrated to the bottom of the insert microporous membrane in the Boyden chamber were stained. The mEPs from tendon constructs receiving 0%, 3%, 6%, or 9% strain were used as chemoattractants in the lower chamber. Scale bar, 50 μm. c xCell analysis of bulk RNA sequencing data from healthy tendons and tendons with tendinopathy showing macrophage infiltration in diseased human tendons. The dots in orange indicate that P < 0.2. d Gating strategy for identification of different cytokines by a bead-based immunoassay. A total of 13 bead populations were distinguished by size and allophycocyanin (APC) fluorescence. e Bead-based immunoassay showed that the mEPs from tendon constructs that received 0%, 3%, 6%, or 9% strain induced macrophages to secrete different cytokines (n = 3 per group). Lipopolysaccharide (LPS) (500 ng/mL) was used as the positive control to induce cytokine release from RAW 264.7 cells. Phosphate-buffered saline (PBS) as the carrier was used as another control. f The bead-based immunoassay showed that mEPs mediated RAW 264.7 cells to release IL-6, CXCL1 and IL-18 in a dose-dependent manner (n = 3 each group). Two-way ANOVA with Tukey’s multiple comparisons test was used for statistical analysis of the main concentration effect at different doses. Two-way ANOVA with Tukey’s multiple comparisons test was used to compare the effects of different doses within mEP9% for simple effect analysis. The data are presented as the means ± SEMs. One-way ANOVA with Tukey’s multiple comparisons test was used for statistical analysis. ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant.
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