Journal: The American journal of pathology

Article Title: Macrophages are critical inducers of bleomycin-induced fibrosis in a systemic scleroderma model

doi: 10.1016/j.ajpath.2025.09.009

Figure Lengend Snippet: ( A ) Schematic representation of bleomycin-induced scleroderma. Bleomycin (30 μg/mouse/day) or vehicle (NaCl) were injected subcutaneously on indicated days (arrows). ( B ) Body weight was measured on indicated days and is presented as % of starting weight in mice that received bleomycin (BLM, n=20) or vehicle (n=16). Data shown is pooled from four independent experiments and presented as mean ± SEM ****p<0.0001; 2-way ANOVA and Sidak’s multiple comparison test. ( C ) Collagen content in BLM (n=10) and vehicle (n=6) treated mice shown as mean ± SEM. *p<0.05; **p<0.01; Mann-Whitney test. Data shown is from two pooled experiments (skin) or one experiment (lung). ( D-E ) Skin ( D ) and lung ( E ) sections from BLM (n=10) and vehicle (n=10) treated mice were stained with hematoxylin-eosin-saffron (HES), Picrosirius red and Masson’s trichrome (scale bars indicate 100 μm for skin, 250 μm for lungs). Histological analysis of dermis and epidermis thickness and cell infiltration (per 1.5 mm 2 of skin or per 1500 mm 2 of lung), based on HES staining, and fibrosis based on Picrosirius red staining are shown. Data shown is pooled from two independent experiments and presented as mean ± SEM. **p<0.01; ***p<0.001; ****p<0.0001; Student’s t-test. F . CD45 + cells were quantified in the skin and lung biopsies from BLM (n=26) and vehicle (n=22) treated mice by flow cytometry. Data shown is pooled from 5 independent experiments and presented per 1 cm 2 of skin or per 1 lung. **p<0.01; ***p<0.001; Mann-Whitney test. ( G-H ) Macrophage (CD45 + CD11b + F4/80 + ) percentage ( G ) and absolute number ( H ) in the skin and lung. ( I ) Correlation between macrophage number and collagen level in skin and lung biopsies from two experiments with 17 mice per group total. Simple linear regression. **p<0.01; ****p<0.0001; Pearson r test. ( J ) Macrophage gating strategy for panels K-L of indicated markers in the skin and lungs of BLM- and vehicle- treated mice. ( K ) Ly6c low CD11b + F4/80 + macrophage percentage (normalized to vehicle-treated mice) and ( L ) MerTK + CD206 + percentage of Ly6c low macrophages (normalized to vehicle-treated mice). Data is pooled from six independent experiments and shown as mean ± SEM. ( G : 30 mice/group **p<0.01, ****p<0.00001, Student’s t-test; H : 3-10 mice/group, **p<0.01, Mann-Whitney test; K: 15 (skin) and 30 (lung) mice/group, **p<0.01, Student’s t-test; L : 10 mice/group, *p<0.05, ****p<0.0001, Student’s t-test).

Article Snippet: Primers were obtained from Thermo Fisher: Tgf1 (Mm01227699_m1), MMP2 (Mm00439498_m1), MMP9 (Mm00442991_m1), TIMP1 (Mm01341361_m1), TIMP2 (Mm00441825_m1), Arg1 (Mm00475988_m1), Lyve1 (Mm00475056_m1), Spp1 (Mm00436767_m1), Ctgf (Mm01192933_g1), Vegf (Mm00437306_m1), Egf (Mm00438696_m1), Tnf (Mm00443258_m1), Il1b (Mm00434228_m1), Ccl2 (Mm00441242_m1), Mertk (Mm00434920_m1), Sirpa (Mm00455928_m1), Slc2a1 (Mm00441480_m1), Il6 (Mm00446190_m1), Il10 (Mm01288386_m1), Adam17 (Mm00456428_m1), Lrp1 (Mm00464608_m1), Cd36 (Mm01135198_m1), Axl (Mm00437221_m1), Tyro3 (Mm00444547_m1), Gas6 (Mm00490378_m1), Elmo1 (Mm00519109_m1), Elmo2 (Mm01248046_m1), and Rac1 (Mm01201653_m1).

Techniques: Injection, Comparison, MANN-WHITNEY, Staining, Flow Cytometry

Journal: The American journal of pathology

Article Title: Macrophages are critical inducers of bleomycin-induced fibrosis in a systemic scleroderma model

doi: 10.1016/j.ajpath.2025.09.009

Figure Lengend Snippet: ( A ) Glycolytic activity was assessed by extracellular acidification rate (ECAR) in BMDM. ( B ) Glycolytic activity was further shown as basal glycolysis (left) and glycolytic capacity (right) by ECAR. ( C ) Oxidative phosphorylation was assessed by oxygen consumption rate (OCR) in BMDM. ( D ) Mitochondrial respiration in BMDM was assessed by OCR, shown as basal respiration (left, OCR pmol/min), maximal respiration (middle, OCR pmol/min), and spare respiratory capacity (right, % OCR). ( E ) Nutrient uptake in BMDM was assessed by flow cytometry, shown as 2NBDG (glucose analog) MFI (left) and BODIPY FL C16 (fatty acid analog) MFI (right). ( F ) Mitochondrial stress in BMDM was assessed by flow cytometry, shown as MTG (mitotracker green, left) and MTO (mitotracker orange, right). ( G ) Reactive oxygen species (ROS) levels in BMDM were measured by flow cytometry and are shown as percentage of ROS + cells (left) and geometric mean fluorescence intensity (MFI, right). ( H ) MerTK mRNA expression was measured by qPCR in BMDM. ( I ) Soluble MER (sMer) in the conditioned medium of BMDM was measured by Western blot. After 72 hours of bleomycin (MacroBLM) or vehicle (MacroCTRL) treatment, cells were washed and incubated for 2 hours in serum-free medium. The conditioned medium was then collected and TCA-precipitated prior to analysis. ( J ) BMDM survival after 72 hours of bleomycin or vehicle treatment was assessed. Cell survival is shown as total cell number (left), Annexin V/7-AAD staining (middle), and calculated viability based on the number of cells plated versus the number of cells remaining after 72 hours (right). ( K ) ADAM17 (sheddase) expression in BMDM was assessed by qPCR (right) and flow cytometry, shown as percentage of ADAM17 + cells (middle) and mean fluorescence intensity (MFI, right). ( L ) Actin cytoskeleton in BMDM was assessed by phalloidin staining and flow cytometry, shown as geometric mean fluorescence intensity (MFI). Panels A–D and I are representative of two independent experiments (n = 4/group for A–D; n = 3–4/group for I; n=4-5/group for L). Panels E, F, G, H, and K are from one experiment (n = 4-5/group). Panel J is from two independent experiments (n = 4–5/group). Data are shown as mean ± SEM. Statistical analysis was performed using Student’s t test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Primers were obtained from Thermo Fisher: Tgf1 (Mm01227699_m1), MMP2 (Mm00439498_m1), MMP9 (Mm00442991_m1), TIMP1 (Mm01341361_m1), TIMP2 (Mm00441825_m1), Arg1 (Mm00475988_m1), Lyve1 (Mm00475056_m1), Spp1 (Mm00436767_m1), Ctgf (Mm01192933_g1), Vegf (Mm00437306_m1), Egf (Mm00438696_m1), Tnf (Mm00443258_m1), Il1b (Mm00434228_m1), Ccl2 (Mm00441242_m1), Mertk (Mm00434920_m1), Sirpa (Mm00455928_m1), Slc2a1 (Mm00441480_m1), Il6 (Mm00446190_m1), Il10 (Mm01288386_m1), Adam17 (Mm00456428_m1), Lrp1 (Mm00464608_m1), Cd36 (Mm01135198_m1), Axl (Mm00437221_m1), Tyro3 (Mm00444547_m1), Gas6 (Mm00490378_m1), Elmo1 (Mm00519109_m1), Elmo2 (Mm01248046_m1), and Rac1 (Mm01201653_m1).

Techniques: Activity Assay, Phospho-proteomics, Flow Cytometry, Fluorescence, Expressing, Western Blot, Incubation, Staining

Journal: Translational Neurodegeneration

Article Title: Attenuating α-synuclein pathology in mice with in situ engineered astrocytes

doi: 10.1186/s40035-025-00518-0

Figure Lengend Snippet: Expression of CAR-A and effect on α-synO phagocytosis and digestion. a Design of the CAR-expression plasmid. CAR was expressed in fusion with 3A scFv and enhanced green fluorescent protein (EGFP) under the control of the astrocyte-specific promotor GfaABC1D. SP, signal peptide; Poly(A), polyadenylation signal; ORI, origin of replication; KanR, kanamycin resistance gene. b Representative image of CAR expression on an astrocyte. The co-localization of 3A, MerTK and EGFP was assessed by confocal microscopy. Scale bars, 10 μm. c Flow cytometry analysis of the binding of CAR-A and ns-CAR-A to α-syn monomers and oligomers (α-synOs). The astrocytes were transfected with CAR or ns-CAR lipoplexes for 48 h. After 2 h-incubation with 1 μmol/L α-syn monomers (α-syn) or α-synOs, cells were stained with PE-labeled anti-α-syn antibody. d PE fluorescence in EGFP-positive astrocytes. n = 3 independent experiments. e Flow cytometry analysis of the amount of α-synO engulfed by CAR-A, ns-CAR-A and NC-A in the presence of different α-synO concentrations. n = 3 independent experiments. f Representative images depicting the phases of engulfment and digestion of α-synO by CAR-A. CAR-A was treated with 1 μmol/L α-synO, and the medium was changed after 1 h incubation. α-SynO and Lamp1 in CAR-A were stained with respective antibodies at different time points and imaged by confocal microscopy. Scale bars, 5 μm. g The kinetic curves of α-synO digestion in CAR-A, ns-CAR-A and NC-A. n = 3 independent experiments. h Statistical analysis of the proportion of α-syn colocalized with Lamp1 in digestion stage in ( f ) by Image J. n = 4 independent experiments. i Intracellular α-syn in Triton X-100-soluble and -insoluble fraction detected by Western blotting at different time points post astrocytic phagocytose of α-synOs. β-actin was used as a control. j Quantification of α-syn ( i ) using Image J. n = 3 independent experiments. k Representative images depicting the binding of ns-CAR-A, NC-A and CAR-A to α-syn monomers and oligomers. Scale bars, 5 μm. Data are mean ± S.E.M. One-way ANOVA ( d ) or Two-way ANOVA ( e ) followed by Tukey’s multiple comparison test was used for statistical analysis. * P < 0.05, ** P < 0.01, **** P < 0.0001 indicate significance compared to respective groups

Article Snippet: In the first construct, the enhanced CMV promotor sequence in the MerTK expression plasmid (purchased from Sino Biological Inc., #MG50514-ACG) was replaced with GfaABC1D promotor sequence synthesized from Sangon.

Techniques: Expressing, Plasmid Preparation, Control, Confocal Microscopy, Flow Cytometry, Binding Assay, Transfection, Incubation, Staining, Labeling, Fluorescence, Western Blot, Comparison