Fig 1: TREM2 deficiency accelerates the clearance of mycobacterial infection.a, b WT or Trem2−/− BMDMs were infected in vitro with M. bovis BCG (MOI 10) for 4 h. After washing, the cells were further cultured for 24 h, and nitrite concentration in the culture supernatant was measured by Griess assay (a). The cells were collected, lysed, and CFUs of M. bovis BCG were counted (b). The statistical significance was calculated by two-tailed unpaired t test. c–f WT or Trem2 −/− mice (day 3, n = 5; day 14, n = 7) were infected intratracheally with 7.5 × 106 CFU of M. bovis BCG. The lung tissues were collected at days 3 and 14 after infection, homogenized, and CFU were determined (c). The expressions of Ccl2, Adgre1, and Nos2 in the lungs from uninfected control (n = 5) and infected mice were analyzed by qRT-PCR (d–f). Data are presented as the mean ± SEM and are representative of two independent experiments. The statistical significance was calculated by two-tailed unpaired t test. *p < 0.05, **p < 0.01. Source data are provided as a Source data file.
Fig 2: Ablation of autophagy in myeloid cells depletes M2 macrophages and reduces M2:M1 ratio. Skin cells from myeloid autophagy-deficient (atg7 cKO) mice were harvested for flow cytometric analysis of PTPRC (myeloid), ADGRE1 (macrophage), LY6C (M1), and MRC1 (M2). Representative plots illustrate the distribution of PTPRC+ ADGRE1+ cells into LY6C+ MRC1− (M1) and LY6C− MRC1+ (M2) macrophage populations in atg7 cKO and littermates in groups (a) control, (b) UV, and (c) UV+VD. Arrows indicate absence of MRC1+ M2 macs in VD-treated atg7 cKO compared to M2 macs restored in VD-treated littermate mice. (d) Quantitation of M2 to M1 ratio in atg7 cKO and littermates. For atg7 cKO n = 14 for control, n = 11 for UV, n = 12 for UV+VD, littermates n = 13 for control and UV, and n = 16 for UV+VD. Number on each plot is its M2:M1 ratio ± sem, p˂0.03. (e) Quantitation of percent M1 and M2 macs comparing atg7 cKO and littermate mice treated with UV+VD, (p = 0.04).
Fig 3: Vitamin D enhances macrophage-specific autophagy and diminishes inflammation in an autophagy dependent manner. Skin sections were subject to (a-f) confocal microscopy at 72 h and (g-i) real time PCR at 48 h post UV. (a-f) Representative images of skin tissue sections stained for immunofluorescent detection of ADGRE1 (green), LC3 (red), (yellow, indicative of co-localization, is marked with arrows), and DAPI (blue). Tissue gene expression of anti-inflammatory mediator (g) Pparg, and pro-inflammatory mediators (h) Tnf, and (i) Mmp9 were quantified following blockade of autophagy by pharmacological inhibitor 3-MA, (p ≤ 0.03). For Tnf and Mmp9, n = 6 for all groups except UV+3-MA n = 4, for Pparg n = 6 for all groups except control n = 5 and UV+VD+3-MA n = 10. Scale bar: 20 µm.
Fig 4: Efferocytosis is significantly reduced in SjSS mice. (A,B) Bone marrow-derived macrophages (BMDM) were cultured from mice and incubated with pHrodo green (ThermoFisher) labeled apoptotic thymocytes. (A) B6 (n = 3) and SjSS (n = 3) BMDMs were stained with CellTracker CMTPX Red (ThermoFisher) before 90-min incubation with pHrodo green-labeled apoptotic thymocytes; following washing, efferocytosis was observed using a Nikon Ti-E fluorescent microscope at 200X magnification. (B) B6 (n = 3), MerKO (n = 3), and SjSS (n = 3) BMDMs were incubated with pHrodo-labeled apoptotic thymocytes for 90 min, and were washed, detached, and stained for macrophage markers F4/80 and CD11b. Efferocytosis was determined by flow cytometry. (C) Resident peritoneal macs (pmacs) were collected from B6 (n = 3), MerKO (n = 3), and SjSS (n = 3) mice and were incubated with pHrodo-labeled apoptotic thymocytes for 90 min, and were washed, detached, and stained for macrophage markers F4/80 and CD11b. Efferocytosis was determined by flow cytometry. (D) B6 (n = 3) and SjSS mice (n = 3) were injected with pHrodo-stained apoptotic cells i.p. and pmacs were collected 45 min later, stained, and analyzed for efferocytosis, as above. The statistical significance was calculated by two-tailed unpaired t-tests or one-way ANOVA where error bars indicate SEM * p < 0.05, ** p < 0.01, and **** p < 0.0001.
Fig 5: An ultralow dose of DPI inhibits the infiltration and migration of macrophages. Immunohistochemical staining of F4/80, CD68, CD19, and CD3ε in the paraffin-embedded sections (Bar = 100 µm) and (B) quantification of the positive cells by Image-Pro Plus 5.0. (C) Serum levels of MCP-1 were measured by ELISA on day 15 after the DSS treatment (n = 5 per group). (D) MCP-1 levels in serum of mice in the peritonitis models (n = 6 per group). (E) The number of peritoneal macrophages isolated from the control and DPI groups in the thioglycolate-elicited peritonitis models (n = 9 per group). (F) The expression of the phenotypic markers of macrophages was determined by flow cytometry. Scatter gram of cells expressing F4/80 and CD11b in the control and DPI groups. Peritoneal cells were collected on day 4 after the thioglycolate injection. (G) The proportion of macrophages in CD45+ cells (n = 6 per group). (H) Images showing the migration of the THP-1 and RAW264.7 cells. Cells were incubated with supernatant containing DPI for 12 h at 37 °C. The experiment was repeated three times. (I) The cell number in each group in the migration assay. The results are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Supplier Page from BioLegend for PE anti-mouse F4/80