Journal: Nature Communications

Article Title: The arginine methyltransferase PRMT7 promotes extravasation of monocytes resulting in tissue injury in COPD

doi: 10.1038/s41467-022-28809-4

Figure Lengend Snippet: a Heat map of normalized enrichment score (NES) following GSEA of cell death pathway gene lists on array data from lung tissue of COPD patients (GSE47460-GPL14550; 145 COPD patients vs 91 healthy controls). b Expression of ferroptotic genes in the lungs of COPD patients relative to healthy controls taken from GSE47460-GPL14550, relative fold change and P value calculated using the GEO2R interactive web tool running limma R . c Western blot analysis of ACSL4 expression in lung core biopsies from healthy ( n = 3) and COPD patients ( n = 3), quantification relative to vinculin shown for individual patients. d Representative images of immunohistochemical analysis for ACSL4 (brown signal indicated by arrowhead, hematoxylin counterstained, scale bar 25 μm) in lung sections from COPD patients ( n = 4) and healthy controls ( n = 4). e Quantification of alveolar epithelial cells positive for ACSL4. f Heat map of NES following GSEA of cell death pathway gene lists on array data from mice exposed to 4 m chronic cigarette smoke (CS, n = 3) v filtered air (FA, n = 3). g Expression of ferroptotic genes in the lungs of mice exposed to 4 m chronic CS ( n = 3) relative to FA ( n = 3) taken from array data used in ( f ), relative fold change and P value calculated using the GEO2R interactive web tool running limma R. h , i Cells from whole lung suspensions of mice exposed to FA ( n = 3) or CS for 4 m ( n = 5), were analysed by scRNA-Seq (Drop-Seq). h UMAP of scRNA-Seq profiles (dots) colored by cell type. i UMAP plots showing expression of ACSL4 in scRNA-Seq profiles. j Representative images of immunohistochemical analysis for ACSL4 (red signal indicated by arrowheads, hematoxylin counterstained, scale bar 25 μm) in lung sections from WT and Prmt7 +/− mice exposed to FA or CS for 4 months ( n = 4–5 per group). k Quantification of alveolar epithelial cells positive for ACSL4 from ( j ) ( n = 5 for WT FA, Prmt7 +/ − FA, Prmt7 +/− CS and n = 4 for WT CS). l , m Mouse AT2 cells (MLE12) were treated with RSL3 (250 nM), or conditioned medium from control macrophages (MФ, RAW264.7 cells) or those polarized to a pro-inflammatory (IFNγ + LPS) or anti-inflammatory (IL4) phenotype for 48 h, representative data shown from a single experiment repeated twice. l Representative image showing PI uptake at 24 h (Scale bar 50 µm). m Number of dead AT2 cells at the time indicated, data shown from a single experiment repeated twice. n BODIPY oxidation in AT2 cells (MLE12) at 6 h after treatment with RSL3 (250 nM, n = 3) or conditioned medium from control macrophages ( n = 2) or those polarized to a pro-inflammatory ( n = 6) or anti-inflammatory ( n = 3) phenotype . o Number of dead AT2 cells (MLE12) by PI uptake at the time indicated following treatment with RSL3 (250 nM), or conditioned medium from macrophages (MФ, RAW264.7 cells) polarized to pro-inflammatory (IFNγ + LPS) plus 20 μM zVAD or 10 μM necrostatin-1s (Nec1-s), representative data shown from a single experiment repeated twice. p Western blot analysis of ACSL4 in AT2 cells (MLE12) treated for 24 h with conditioned medium from control macrophages ( n = 11) or those polarized to a pro-inflammatory ( n = 10) or anti-inflammatory ( n = 5) phenotype, normalized to β-actin and shown relative to control macrophages. q Western blot analysis of ALOX5 at 72 h in control macrophages ( n = 11) or those polarized to a pro-inflammatory ( n = 11) or anti-inflammatory ( n = 11) phenotype, normalized to β-actin and shown relative to control macrophages. r Western blot analysis of ACSL4 in AT2 cells (MLE12) treated for 24 h with conditioned medium from control macrophages or those polarized to a pro-inflammatory phenotype that were treated with siRNA against Alox5 , normalized to β-actin and shown relative to control macrophages, from individual experiments ( n = 3). Data shown mean ± SD, P values shown in charts determined by unpaired two-tailed Student’s t -test ( c , n ), two-tailed Mann–Whitney test ( e ), one-way ANOVA Bonferroni’s multiple comparisons test ( k , p , q ). Source data are provided as a Source Data file.

Article Snippet: The next day adherent cells were harvested, counted, and seeded at a density of 1 × 10 6 cells/ml in 24-well plates, and cultured for 24 h in fresh medium to obtain M0 cells, medium containing 1 μg/ml LPS (from Escherichia coli 0111:B4, Sigma-Aldrich) and 20 ng/ml recombinant murine IFNγ (ImmunoTools) for M1 or medium containing 20 ng/ml recombinant murine IL4 (ImmunoTools) for M2.

Techniques: Expressing, Western Blot, Immunohistochemical staining, Two Tailed Test, MANN-WHITNEY

Journal:

Article Title: Coccidioides releases a soluble factor that suppresses nitric oxide production by murine primary macrophages

doi: 10.1016/j.micpath.2010.11.006

Figure Lengend Snippet: Spherule initials of Coccidioides suppress NO production by activated, primary macrophages isolated from C57BL/6 mice. Peritoneal macrophages were activated with IFN-γ + LPS, and NO concentration was measured by fluorescence using DAF-2DA and expressed as relative fluorescence units (RFU). Spherule initials of Coccidioides were co-cultured with macrophages to yield a multiplicity of infection (MOI) of 2:1, 5:1 or 10:1 (macrophages:fungal cells), and incubated 1, 3, 6 or 12 h in a 5% CO2 incubator at 37° C. All experiments were performed at least three times. Results of a representative experiment are shown. Error bars indicate standard errors of the mean (SEM). Statistically significant differences in NO production between test groups was determined using the Student t-test for the following comparisons: # vs non-activated macrophages = P<0.001, and + or ¥ vs activated, non-infected macrophages = P<0.001 & P<0.05, respectively. Non-ifx., non-infected macrophages; Ctl., non-activated, control macrophages.

Article Snippet: Murine IFN-γ was purchased from BD Biosciences/Pharmingen (San Jose, CA), and ultrapure E. coli -derived lipopolysaccharide (LPS) was obtained from InvivoGen (San Diego, CA).

Techniques: Isolation, Concentration Assay, Fluorescence, Cell Culture, Infection, Incubation

Journal: PLoS Pathogens

Article Title: HIF-1α is a key regulator in potentiating suppressor activity and limiting the microbicidal capacity of MDSC-like cells during visceral leishmaniasis

doi: 10.1371/journal.ppat.1006616

Figure Lengend Snippet: Macrophages were derived for six days from the bone marrow of naïve Hif flox/flox - Cd11c-Cre + and Cre - mice. Cells were then activated with IFNγ or IL-4, or infected L . donovani . Polarization and infection were monitored for 24h. (A) Representative FACS plot for macrophages expressing CD38 + (left panels) and iNOS + ( right panels) in Hif-1α flox/flox Cd11c-cre - (WT) and Cre + (KO) mice. Frequency of (B) CD38 + and (C) iNOS + in different polarization conditions and following infection. (D—I) Real-time PCR analysis of mRNA expression levels in in vitro polarized and infected BMM. (D) Arg1 , (E) Fizz1 , (F) Il10 , (G) Hif1α , (H) Pgk1 and (I) Glut1 . All data represent mean ± SEM, n = 3. * denotes p <0.05, ** denotes p <0.01, and *** denotes p<0.001.

Article Snippet: Cells were classically activated (M1) with 20 ng/ml of murine IFNγ (Peprotec), alternatively activated (M2) with 20 ng/ml of murine IL-4 (Peprotec), or infected with L . donovani amastigotes at a MOI of 1:10.

Techniques: Derivative Assay, Infection, Expressing, Real-time Polymerase Chain Reaction, In Vitro