Fig 2: DEXs regulated macrophage polarization via Treg cells. A Representative flow cytometry analysis images of different groups. NST, non-treated macrophages; LPS, LPS treated macrophages; IL-4/IL-13, IL-4 and IL-13 treated macrophages; DEX, macrophages co-cultured with DEXs; Treg, macrophages co-cultured with Treg cells. B–E Analysis of the proportions of iNOS+F4/80+ cells, CD206+F4/80+ cells, CD206+iNOS− cells, iNOS+CD206− cells in different groups. n = 3–4, *p < 0.05, **p < 0.01, ***p < 0.001

Fig 3: Macrophage Sub1 upregulates M1‐skewing Irf1 expression in a Ck2‐dependent manner. Unless otherwise stated, the following experiments employed Sub1 flox/flox (wild‐type, WT), LysM Cre/−/Sub1 flox/wt (hemizygous, HEMI), and LysM Cre/−/Sub1 flox/flox (knockout, KO) bone marrow‐derived macrophages (BMDMs). A) qPCR of Irf1 mRNA levels. B) Chromatin immunoprecipitation (ChIP)‐based detection of Sub1's interaction with the Irf1 promoter in WT BMDMs (top). Open confirmation of Irf1 promoter detected by H3K4me3 antibody ChIP in BMDMs (bottom). Human THP‐1 cells were transfected with the WT IRF1 promoter or mutant (MUT) IRF1 promoter alone or in combination with C) CK2 shRNA or scrambled control shRNA, or D) Lenti‐GIII‐CMV‐CK2‐HA or control vector. Renilla luciferase vector was cotransfected as normalization control. Luciferase reporter activity was detected 48 h post‐transfection. E–N) BMDMs were transfected with Lenti‐GIII‐CMV‐Irf1‐HA (LV‐Irf1) to enable stable Irf1 overexpression. Where indicated, BMDMs were stimulated with M1‐polarizing LPS (100 ng mL−1, 8 h) or M2‐polarizing IL4 (5 ng mL−1, 8 h). E) Immunoblotting of Irf1. F) qPCR of M1 marker genes under vehicle (Ctrl) or LPS conditions. G) qPCR of M2 marker genes under vehicle (Ctrl) or IL4 conditions. ELISA of H) Tnf‐α, I) Il‐1β, and J) Ccl2 secretion under vehicle (Ctrl) or LPS conditions. K) Immunoblotting of iNOS protein expression and L) nitrite‐based iNOS activity under vehicle (Ctrl) or LPS conditions. M) Immunoblotting of Arg1 and Retnlb protein expression and N) urea‐based Arg1 activity under vehicle (Ctrl) or IL4 conditions. Data reported as means ± SDs. n = 3 biological replicates × 3 technical replicates. *p < 0.05 and **p < 0.01 (A: one‐way ANOVA with Fisher's LSD; B–N) two‐way ANOVA with Fisher's LSD; comparing n = 3 in vitro biological replicates per group).

Fig 4: The response of RAW 264.7 to EV-hybrid TNT under inflammatory environment with LPS stimulation. (A) CLSM images of macrophages response to EV-hybrid TNT. Inflammatory environment was induced by 1000 ng/mL LPS stimulation for 6 h. Cells were washed by PBS and cocultured with different EV-hybrid scaffolds. The actin was stained with phalloidin (red), and nuclei were stained with DAPI (blue). Representative CLSM images are shown in the figure (n = 3). in (a–d) refer to LPS, 3d EV, MSC EV, and 3d EV/MSC EV hybrid, respectively Scale bar =100 μm (low mag); scale bar =20 μm (high mag). (B) Representative SEM images of macrophages response to EV-hybrid TNT under inflammatory environment. In (a–d) refer to LPS, 3d EV, MSC EV, and 3d EV/MSC EV hybrid, respectively. Scale bar =6 μm (low mag); Scale bar =2 μm (high mag). (C) Relative expression of pro-inflammatory and anti-inflammatory genes in macrophages cocultured with EV-hybrid TNT. The values were normalized to GAPDH as a housekeeping gene. *Indicates statistically significant difference (P < 0.05) compared to LPS group. **Indicates statistically significant difference (P < 0.01) compared to LPS group. (D) Expression of IL-6 produced in response to EV-hybrid TNT under inflammatory environment. The level of IL-6 was measured using cytokine-specific ELISA. All data are expressed as mean ± SD. **Indicates statistically significant difference (P < 0.01) compared to LPS group. (E) CLSM images of iNOS expression. All scale bar: 100 μm.

Fig 5: Effect of miR-185 expression on the expression of NOS2. (A) Expression of miR-185 in HMEC-1 cells after transfection with agomiR-NC and agomiR-185. (B) Expression of NOS2 mRNA in HMEC-1 cells after transfection with agomiR-NC and agomiR-185. (C) Expression of NOS2 protein in HMEC-1 cells after transfection with agomiR-NC and agomiR-185. *P<0.05 and **P<0.01 vs. agomiR-NC group. N=3. miR, microRNA; NOS2, nitric oxide synthase 2; NC, negative control.