Journal: Nature

Article Title: Early-life inflammation primes a T helper 2 cell–fibroblast niche in skin

doi: 10.1038/s41586-021-04044-7

Figure Lengend Snippet: a, Schematic of the anatomical layers of murine skin. dWAT, dermal white adipose tissue; PC, panniculus carnosus. b, Schematic of the experiment. FoxP3DTR mice were treated with either PBS (Ctrl) or DT at indicated postnatal days. c–e, Representative skin histology images of mice euthanized at 10 days (c, d) or 35 days (e), with fibrous bands outlined. Scale bars, 100 μm. f, g, Flow cytometry analysis (f) and quantification (g) of TH2 cells in skin from adult (P50) Il5Red5/+;FoxP3DTR mice following neoTreg cell reduction. n = 3–7 animals per data point (47 total), one experiment per time point. h, Quantification of skin T helper subsets by intracellular cytokine staining at P50. n = 17 animals. i, Lymphocyte integrin expression in ΔneoTreg mice at P50. j, k, Representative images (j) and quantification (k) of TH2 and ILC2 cell localization in skin of ΔneoTreg Il5Red5/+; Rosa26tdTomato/+;FoxP3DTR mice. Cells were segmented by tdTomato and CD3 expression. Dotted lines denote the boundary between the dermis and subcutis. Scale bar, 100 μm. n = 3 animals. Data are displayed as the mean ± s.d. from one independent experiment, representative of two to three repeats (b–j). NS, not significant. *P < 0.05, ***P < 0.001 (all two-sided); two-way analysis of variance (ANOVA) with Šidák’s multiple comparisons test (g, h) or Student’s t-test (k).

Article Snippet: Image analysis For quantification of T H 2 and ILC2 cell localization in skin, Imaris 9.3 (BitPlane) was used to analyse confocal imaging data from Il5 Red − 5/ + ;Rosa26 tdTomato ;Foxp3 DTR mice, in which the IL-5 reporter signal is boosted by additional Cre-mediated recombination and tdTomato expression from the Rosa26 locus.

Techniques: Flow Cytometry, Staining, Expressing

Journal: Nature

Article Title: Early-life inflammation primes a T helper 2 cell–fibroblast niche in skin

doi: 10.1038/s41586-021-04044-7

Figure Lengend Snippet: (a–b) Control and ΔneoTreg mice were aged to adulthood and then treated with two shots DT (identical to neonatal dosing regimen). Representative histology is shown with fibrous bands outlined. (c) Th2 and ILC2 cell numbers in adult ctrl (Il5Red5/+; FoxP3DTR + PBS), ΔneoTreg (Il5Red5/+; FoxP3DTR + DT), and ΔneoTreg/ΔTh2 (Il5Red5/+; Rosa26DTA/+; FoxP3DTR) mice. n = 16 animals. (d) Wound bed ILC2 numbers (gated as CD45+ CD3− CD4− CD8− Thy1+ IL5Red5+). n = 7–10 animals per data point in 2 pooled experiments per time point (108 total). (e–n) Control, ΔneoTreg, and ΔneoTreg/ΔTh2 mice were aged to adulthood and subjected to full-thickness cutaneous wounding. (f) Th2 numbers in skin 0 − 10 days post wounding (dpw). n = 4–11 animals per data point in 2 pooled experiments per time point (89 total). (g) Th2 frequency in paired skin biopsies taken at dpw0 and dpw7. n = 22 animals in one experiment. (h–i) Wound area was quantified daily, fit to a one-phase exponential decay model, and tested for equivalence of the rate constant (h). Rate constants of curves fit to each biological replicate are shown in (i). (j–k) Alternatively activated macrophage (AAM) and eosinophil frequency in dpw10 wounded skin. n = 42 animals in 2 pooled experiments. (l) TIFF abundance in wound beds at 0 −10 dpw. n = 4–16 animals in 2 pooled experiments (97 total). (m–n) Flow cytometric quantification (m) and IF microscopy (n) of CD26hi TIFFs in wounds at dpw10. n = 39 animals in 2 pooled experiments. (o–u) ΔneoTreg mice were aged to adulthood, wounded, and treated with FTY720 every other day. Wound closure (p-q) and flow cytometric quantifications of Th2 cells, alternatively activated macrophages (AAMs), eosinophils, and TIFFs are shown (r-u) are shown. n = 11 animals. (v-ab) Neonatal mice were immunized with OVA/papain, aged to adulthood, and wounded. Wound closure (w-x) and flow cytometric quantifications of Th2 cells, alternatively activated macrophages (AAMs), eosinophils, and TIFFs are shown (y-ab). n = 22 animals. Data are displayed as mean +/− SD (c-d, f, i-m, q-u, x-ab) or SEM (h, p, w). Results were reproduced over 2 independent experiments (a-d; o-ab) or 4 independent experiments pooled into two separate analyses (e-n). *p < 0.05, **p < 0.01, ***p < 0.001 (all two-sided); ANOVA with Dunnett Multiple Comparisons Test (c, i-k, m, x-ab); least-squares quadratic regression with extra sum-of-squares F test (f, l); mixed-effects analysis with Šídák multiple comparison test (g); nonlinear one-phase exponential decay regression (h, p, w); Student’s t-test (q-u).

Article Snippet: Image analysis For quantification of T H 2 and ILC2 cell localization in skin, Imaris 9.3 (BitPlane) was used to analyse confocal imaging data from Il5 Red − 5/ + ;Rosa26 tdTomato ;Foxp3 DTR mice, in which the IL-5 reporter signal is boosted by additional Cre-mediated recombination and tdTomato expression from the Rosa26 locus.

Techniques: Control, Microscopy, Comparison

Journal: The FASEB Journal

Article Title: Postprandial activation of leukocyte-endothelium interaction by fatty acids in the visceral adipose tissue microcirculation

doi: 10.1096/fj.201802637RR

Figure Lengend Snippet: Neutrophils accumulate in the VAT following acute fat overload. Epididymal VAT samples were collected before high-fat feeding [baseline (BSLN)] and at 2 and 4 h following a single HFM, as well as after 24 h of ad libitum HFD feeding. SVFs were isolated and stained with antibodies for neutrophils [Ly6G (A)] lymphocytes [CD4 (B)] and monocytes [F480 (C)]. Neutrophils were found to be the main leukocyte subpopulation to postprandially infiltrate VAT following consumption of HFMs. Values are means ± sem from 3 mice in each experimental group. Three samples were obtained from each mouse. NS, not significant; P-sel, P-selectin.

Article Snippet: The stromal vascular fraction (SVF) was isolated by centrifugation and fluorescently stained with antibodies to quantify CD11B + [Becton Dickinson, San Diego, CA, USA] innate immune cells, CD4 + lymphocytes (Becton Dickinson), F4/80 + monocytes (Abcam, Cambridge, United Kingdom), and lymphocyte antigen 6 complex locus G6D (Ly6g) + neutrophils (Becton Dickinson).

Techniques: Isolation, Staining

Journal: Frontiers in Immunology

Article Title: Characterization of Rat ILCs Reveals ILC2 as the Dominant Intestinal Subset

doi: 10.3389/fimmu.2020.00255

Figure Lengend Snippet: Tissue distribution of ILC populations between rat strains. (A) Frequencies of total Lin − CD127 + ILCs expressed as percentage of CD45 + leukocytes in different tissues from LEW (white bars), BN (gray bars) and SPD (black bars) rats (B–D) Frequencies of ILC1 (B) , ILC2 (C) , and ILC3 (D) among total ILCs within each rat strain in different tissues. n = 6 per each tissue and rat strain except lung and VAT ( n = 4). Numeric data represents means ± SEM, significance was determined at P < 0.05. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: For the ILC2 stimulation cocktail, human rTSLP, human rIL-33 and rat rIL-25 were purchased from Peprotech (Neuilly-sur-seine, France) and used at 50 ng/ml concentration.

Techniques:

Journal: Frontiers in Immunology

Article Title: Characterization of Rat ILCs Reveals ILC2 as the Dominant Intestinal Subset

doi: 10.3389/fimmu.2020.00255

Figure Lengend Snippet: Comparison of ILC2-derived IL-13 production between rat strains. (A) The percentage of constitutive ILC2-derived IL-13 production in the indicated tissues of LEW (white bars), BN (gray bars) and SPD (black bars) rats and (B) after 4 h of stimulation with ILC2 specific cocktail. ( n = 6) per each tissue and rat strain. Numeric data represents means ± SEM, significance was determined at P < 0.05. *** p < 0.001, **** p < 0.0001.

Article Snippet: For the ILC2 stimulation cocktail, human rTSLP, human rIL-33 and rat rIL-25 were purchased from Peprotech (Neuilly-sur-seine, France) and used at 50 ng/ml concentration.

Techniques: Comparison, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Characterization of Rat ILCs Reveals ILC2 as the Dominant Intestinal Subset

doi: 10.3389/fimmu.2020.00255

Figure Lengend Snippet: Sex-effect on ILC2 proportions in MLN, lung and VAT of rats. (A) Representative plots of Lin − GATA-3 + (Pregated on live CD45 + cells) ILC2 in the lung of SPD female vs. male rats. (B) ILC2 frequencies in MLN, lung and visceral adipose tissues (VAT) of different rat strains. ( n = 4), numeric data represents means ± SEM, statistical differences were determined by nonparametric Mann Whitney test with significance was determined. F, Female; M, Male. * p < 0.05, n.s, not significant.

Article Snippet: For the ILC2 stimulation cocktail, human rTSLP, human rIL-33 and rat rIL-25 were purchased from Peprotech (Neuilly-sur-seine, France) and used at 50 ng/ml concentration.

Techniques: MANN-WHITNEY

Journal: Cell reports

Article Title: Tumor-Derived Lactic Acid Contributes to the Paucity of Intratumoral ILC2s.

doi: 10.1016/j.celrep.2020.01.103

Figure Lengend Snippet: Figure 1. Rapidly Growing B16F10 Tumors Are Depleted of ILC2s (A) Representative plots and analysis of ILC2 populations in the skin (n = 6) and subcutaneous (n = 6) and mesenteric adipose tissue (n = 6) from naive and tumor- bearing mice by flow cytometry. (legend continued on next page)

Article Snippet: Retired female mice, defined as C57BL/6 mice older than four months of age and withdrawn from any further Cell Reports 30, 2743–2757.e1–e5, February 25, 2020 e2 breeding, were used for ILC2 isolation.Rag2 / (stock no. RAGN12) andRag2 / Il2rg / (stock no. 4111) mice were purchased from Taconic.

Techniques: Cytometry