Journal: Gut Microbes

Article Title: Myeloid-derived suppressor cells prevent disruption of the gut barrier, preserve microbiota composition, and potentiate immunoregulatory pathways in a rat model of experimental autoimmune encephalomyelitis

doi: 10.1080/19490976.2022.2127455

Figure Lengend Snippet: Phenotypic and functional characterisation of MDSCs. MDSCs were differentiated from bone marrow of DA rats in the presence of FLT3/GM-CSF/IL-6 (MDSCs, blue) or FLT3/GM-CSF/IL-6 and PGE2 (MDSC-PGE2, green) for 4 days. A-C . Representative flow cytometry histograms are showing the expression of indicated markers from one experiment, out of three with similar results (for summarised results see Supplementary Figure 1). Common markers expressed by rat MDSCs (CD11b, SIRP-α, HIS48) ( A ); myeloid lineage markers (OX62, CD68 and RP1) ( B ); functional markers (MHCII, CD86 and CD80) are shown ( C ). D . The proliferation of ConA-stimulated Cell Trace Far Red-labelled splenocytes (3 × 10 5 /well) is shown, in which the splenocytes were cultivated without (wo) MDSCs (dashed line) or in the presence of MDSCs or MDSC-PGE2 (each at 6 × 10 4 , 3 × 10 4 , 1.5 × 10 4 or 0.75 × 10 4 /well, providing 1:5–1:40 MDSCs: splenocytes ratio, respectively) for 4 days. A representative analysis of 1:10 and 1:40 MDSC/splenocyte ratios is shown along with summarised data. E . Relative mRNA levels of COX-2, iNOS, ARG1 are shown relative to β-actin expression, as well as NO production as detected by Griess reaction method. D-E . The summarized data is shown as mean ± SD of three independent experiments. Student’s t -test was used for comparison (* p < 0.05).

Article Snippet: Intracellular staining of cells was conducted after surface staining using a flow cytometry fixation and permeabilization kit I (R&D Systems).

Techniques: Functional Assay, Flow Cytometry, Expressing, Comparison

Journal: Gut Microbes

Article Title: Myeloid-derived suppressor cells prevent disruption of the gut barrier, preserve microbiota composition, and potentiate immunoregulatory pathways in a rat model of experimental autoimmune encephalomyelitis

doi: 10.1080/19490976.2022.2127455

Figure Lengend Snippet: Immune response in animals treated with MDSCs and MDSC-PGE2 at the peak of EAE. A . The percentages of interferon (IFN)-γ-producing NK (CD161 + IFN-γ + ), Th17 (CD4 + IL-17 + ) and (IFN)-γ-producing T (CD3 + IFN-γ + ) lymphocytes in the spleen and spinal cord samples are shown, after their analysis upon isolation from animals (n = 5 animals in each group) at the peak of EAE (15dpi) in the control (ctl_EAE, red) group, and groups treated with either MDSCs (MDSC_EAE, blue) or MDSC-PGE2 (MDSC-PGE2_EAE, green). Data was obtained by flow cytometry analysis as shown in Supplementary Figure 4. The percentages of regulatory T cells (Tregs) (CD25 + FoxP3 + ) and IL-10-producing Tregs (CD25 + FoxP3 + IL-10 + ) were shown from the total gated CD4 + lymphocytes (the gating strategy is shown in Supplementary Figure 5). The results on total cell numbers are shown in Supplementary Figure 6. B . The levels of cytokines (interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-4, IL-17, IFN-γ and IL-10) were measured from ex vivo splenocytes isolated from each animal (as in A ), and cultivated (2 × 10 6 /mL/well of 24-wells plate) for 24 h in the complete RPMI medium in the presence of ConA. C . The relative mRNA levels of claudin and occludin in intestinal samples of animals treated as in (A ), were calculated relative to β-actin and presented as fold change expression relative to healthy animals D . The activity of alkaline phosphatase in faecal material collected at the peak of EAE, as well as from healthy animals were analysed as a measure of intestinal barrier integrity. A-D . Data is shown as mean ± SD (n = 5) from one experiment, out of two with similar results. One-way ANOVA followed by Tukey post hoc test was used for comparison (* p < 0.05, ** p < 0.01) to control EAE (ctl_EAE).

Article Snippet: Intracellular staining of cells was conducted after surface staining using a flow cytometry fixation and permeabilization kit I (R&D Systems).

Techniques: Isolation, Control, Flow Cytometry, Ex Vivo, Expressing, Activity Assay, Comparison

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of leukotriene A 4 hydrolase aminopeptidase in the pathogenesis of emphysema 1

doi: 10.4049/jimmunol.1400452

Figure Lengend Snippet: Assessment of the LTA4H protein expression in 129sv wild type mice post exposure to cigarette smoke over 5 months. Panel A: The levels of LTA4H protein in the whole lung BALF assessed by ELISA. Panel B: The levels of LTA4H protein in the whole lung protein soup assessed by ELISA. Panel C: Flow cytometry of the whole lung single cell suspension. Air = mice exposed to ambient air for 20 weeks. SM = mice exposed to cigarette smoke for 20 weeks. Panel D: Immunohistochemistry with antibody specific to murine LTA4H protein in lung tissues from mice exposed to cigarette smoke for 20 weeks (63× oil magnification). Positive signals appear brown and counterstained with blue. Arrowheads show nuclei with positive counterstain (blue) indicating absence of LTA4H protein. Arrows show nuclei with positive stain (grown) indicating presence of LTA4H protein. Gray bars in Panels A & B = ambient-air exposed mice with ages 26 – 28 weeks. * represents analysis by ANOVA, ** by Bonferroni subgroup comparison, and *** by nonparametric t-Test. N = 5 per group.

Article Snippet: Next, all CD45 high cells were gated into Ly6G high and CD11b high cells (neutrophils) as previously described. ( 3 ) For the purpose of detecting cells containing the LTA 4 H in their intra-cellular space, cells were permeabilized with flow cytometry permeabilization buffer (R&D) after stained with CD45 antibody.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Immunohistochemistry, Staining