Journal: MedComm

Article Title: Oxidized mtDNA Contributes to Pulmonary Inflammation and Fibrosis in Bleomycin‐Induced Lung Injury

doi: 10.1002/mco2.70664

Figure Lengend Snippet: BLM‐induced oxidized mtDNA promotes M2 macrophage polarization. (A and B) Mice were intratracheally administered BLM (2 mg/kg), and lung tissues were harvested at the indicated time points for FCM analysis. Representative scatterplots with percentages of the gated M2 macrophages (CD45 + F4/80 + CD206 + CD11c − ) are shown in (A) and quantified in (B) ( n = 3 mice). (C–F) Oxid‐mtDNA promoted M2‐like polarization of BMDMs. BMDMs were derived from C57BL/6 mice and cultured for 4 days. On Day 4, the medium was replaced with fresh culture medium containing 5 µg/mL nDNA, 5 µg/mL DNase‐treated mtDNA, 5 µg/mL DNase‐treated oxid‐mtDNA, mtDNA, or Oxid‐mtDNA (1, 5, or 10 µg/mL as indicated in group labels), and cells were further incubated for 48 h. Cells were then collected for subsequent analyses. Representative histograms of the gated M2 macrophages (CD45 + CD11c − F4/80 + CD206 + ) are shown in the left panel in (C), and the MFI value is shown in the right panel in (D). Relative expression of CD206 (E) or Arg‐1 (F) in BMDMs was quantified by qRT‐PCR ( n = 3 biologically independent samples). (G and H) The levels of IL‐10 (G) and TGF‐β (H) in the supernatants of BMDMs from control, mtDNA, and oxid‐mtDNA were detected by ELISA ( n = 3 biologically independent samples). (I) Mice were intratracheally administered BLM (2 mg/kg), and lung tissues were harvested on Day 21. Immunofluorescence staining of 8‐OHdG (green), F4/80 (red), CD206 (far‐red), and DAPI (blue) in lung tissues of mice. Scale bars represent 50 µm. Data are represented as mean ± SD. Statistical significances in (B) were determined by a two‐sided unpaired t ‐test. Statistical significances in (D–F and H–K) were determined by one‐way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. BLM, bleomycin; BMDMs, bone marrow‐derived macrophages; ELISA, enzyme‐linked immunosorbent assay; FCM, flow cytometry; MFI, mean fluorescence intensity; mtDNA, mitochondrial DNA; oxid‐mtDNA, oxidative mitochondrial DNA.

Article Snippet: Primary antibodies used for immunofluorescence analysis included goat anti‐8‐OHdG (Novus, #NB600‐1508), rabbit anti‐TOMM20 (Abcam, #ab186735), F4/80 (D2S9R) XP Rabbit mAb (CST, #70076), and Alexa Fluor 594 anti‐mouse CD206 (MMR) (Biolegend, #141726).

Techniques: Derivative Assay, Cell Culture, Incubation, Expressing, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Flow Cytometry, Fluorescence

Journal: Journal of Extracellular Vesicles

Article Title: Clostridioides difficile ‐Derived Extracellular Vesicles Induce Proinflammatory Responses in Macrophages

doi: 10.1002/jev2.70251

Figure Lengend Snippet: TLR5‐dependent internalization of CD630‐EVs by macrophages. THP‐1 cells, differentiated with 100 nM PMA for 72 h, were incubated with Dil‐labelled CD630 EVs (7 × 10 7 particles/mL; labelled with 5 µM Dil) for 12 h. Compared to the untreated control, pre‐treatment with the TLR5 inhibitor TH1020 (0.85 µM) significantly attenuates EVs uptake. Images show Dil‐EVs signal (red) inside F4/80‐immunostained macrophages (green). Nuclei are stained with DAPI (blue). Scale bars: 10 µm. All images were acquired at 1000× magnification.

Article Snippet: Anti‐ADGRE1 (F4/80) rabbit monoclonal antibody (1:100 dilution, Servicebio, China) was applied dropwise, and slides were covered and incubated overnight at 4°C to label mature murine macrophages.

Techniques: Incubation, Control, Staining