Journal: Cell reports

Article Title: B Cells Produce the Tissue-Protective Protein RELMα during Helminth Infection, which Inhibits IL-17 Expression and Limits Emphysema

doi: 10.1016/j.celrep.2018.11.038

Figure Lengend Snippet: (A) B cell numbers in the lung at different time points after N. brasiliensis inoculation were assessed by flow cytometry. (B) At day 5 after Nb inoculation, sort-purified B cells were analyzed for mRNA expression of candidate regulatory factors. (C) Sort-purified B cells from Il4ra −/− and WT BALB/c mice were analyzed for Retnla mRNA expression at day 5 after Nb inoculation. (D–F) Sort-purified B cells (D), alveolar macrophages (E), or non-alveolar macrophages (F) at different time points after Nb infection were analyzed for Retnla mRNA expression. (G and H) RELMα mean fluorescence intensity (MFI) of lung B cells (CD19+) (G) and alveolar macrophages (H) was determined by cytoplasmic staining and FACS at day 5 after N. brasiliensis inoculation. (I and J) Sort-purified B cells from either untreated Retnla −/− or WT mice were transferred to recipient Jh −/− mice at days −3, 0, and +1 after N. brasiliensis inoculation. Lungs were collected for analysis 7 days after inoculation, emphysematous pathology was digitally imaged as described in (I), and lung tissues were analyzed for the expression of Il17a by qPCR (J). (K) Retnla −/− and WT mice were inoculated with Nb, and 2 days later, γδ T cells were assessed for IL-17A production by intracellular staining and flow cytometric analyses. (L and M) FACS analysis of lung B cell subsets at day 5 after N. brasiliensis inoculation, showing expression of IgM and IgD (L) and CD21, CD23, and CD24 (M) on gated CD19 + lymphocytes. Each symbol represents an individual mouse. Small horizontal lines indicate the mean (A and D–I), or data shown are the mean and SEM from five individual mice per group (B–F and J) or a pool of five mice per group (K and L). Data shown are representative of at least two independent experiments (**p < 0.01). See also and .

Article Snippet: Cells were blocked with Fc Block (BD Biosciences, San Jose, CA), directly stained with fluorochrome-conjugated Abs against Ly6G (1A8), MHCII, CD11c, Siglec-F, F4/80, CD19, B220 (BD Biosciences), CD21, CD23, CD24, CD5, Cd1, CD138 (Biolegend), RELMα (Biorbyt), and analyzed by flow cytometry.

Techniques: Flow Cytometry, Purification, Expressing, Infection, Fluorescence, Staining

Journal: Mucosal Immunology

Article Title: Blockade of IL-33 release and suppression of type 2 innate lymphoid cell responses by helminth secreted products in airway allergy

doi: 10.1038/mi.2013.123

Figure Lengend Snippet: HES strongly suppresses early IL-33 production. ( a – d ) At 1, 24, and 48 h after Alternaria administration with or without HES, cell-free bronchoalveolar lavage (BAL) fluid was tested for levels of IL-5 ( a ), RELM-α ( b ), Ym1 ( c ), and IL-33 ( d ). Significance compares Alternaria level to Alternaria +HES level. ( e ) Alternaria , HES, and heat-treated HES (HT-HES) were administered to BALB/c mice and 1 h later levels of IL-33 in cell-free BAL fluid were measured. ( f ) Mean fluorescence intensity (MFI) of IL-33-Citrine within CD326 + CD45.2 – epithelial cells 12 h after Alternaria ±HES administration. ( g ) MFI of IL-33-Citrine within CD326 + CD45.2 – epithelial cells 12 h after the final OVA administration in the Alternaria /OVA model shown in . Results are pooled from two repeat experiments, 3–5 mice per group. Unless otherwise indicated differences are not significant. *** P <0.01, ** P <0.01, * P <0.05. HES, H. polygyrus excretory-secretory; OVA, ovalbumin.

Article Snippet: RELM-α and Ym-1 were measured by ELISA, using rabbit anti-mouse RELM-α, and biotinylated rabbit anti-mouse RELM-α, compared with a recombinant RELM-α standard (Peprotech, London, UK), or the Chitinase 3-like/ECF-L (Ym1) DuoSet kit (R&D Systems) according to the manufacturer's instructions.

Techniques: Fluorescence

Journal: Immunity

Article Title: Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells

doi: 10.1016/j.immuni.2018.05.003

Figure Lengend Snippet: OX40L on ILC2s Orchestrates Adaptive Type 2 Immunity to Allergens (A–D) Mice of the specified genotypes were treated with PBS or papain (Pap) (i.n., day 0 and 1), followed by quantification of lung Th2, GATA3 + Treg, and GATA3 − Treg cells on day 5 (A–C). Whole lung cell suspensions were re-stimulated with PMA and ionomycin, followed by quantification of IL-13 + Th2 cells by intracellular staining (D). (E–G) Mice were treated with papain on days 0, 1, 14, and 21, followed by quantification on day 24 of lung eosinophils (E), and detection (F) and quantification (G) of RELMα + M2 alveolar (CD45 + SiglecF + CD11c + CD11b − F4/80 + ) macrophages (MΦ). (H and I) Mice were treated with A. alternata ( A.alt ) (i.n., days 0 and 1), followed by quantification on day 9 of lung Th2, GATA3 + Treg, and GATA3 − Treg cells (H) and serum IgE concentration (I). Bar graphs indicate mean (±SEM). (A)–(D), ANOVA, two repeat experiments; (E), ANOVA, three repeat experiments; (F) and (G), ANOVA, two repeat experiments, representative gate shown in (F); (H) and (I), ANOVA, two repeat experiments. ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001. See also Figure S7 .

Article Snippet: Anti-mouse RELMα , Peprotech , 500-p214; RRID: AB_1268707.

Techniques: Staining, Concentration Assay

Journal: Immunity

Article Title: Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells

doi: 10.1016/j.immuni.2018.05.003

Figure Lengend Snippet: ILC2-Expressed OX40L Is Essential for Airway Adaptive Type 2 Immune Response to Helminth Infection Mice of the specified genotypes were infected with Nippostrongylus brasiliensis ( N.b. ) on day 0, followed by analysis on day 28 (or day 5) of: (A–C) Lung Th2, GATA3 + Treg, and GATA3 − Treg cell numbers. (D) Representative lung histology (Mason’s trichrome). (E and F) Lung (E) and bronchoalveolar lavage (F) eosinophil numbers. (G) Lung RELMα + M2 macrophage (MΦ) numbers. (H) Bronchoalveolar lavage IL-4 and IL-5 cytokine concentrations. (I) Whole lung cell suspensions were re-stimulated with PMA and ionomycin, followed by quantification of lung IL-13 + Th2 cell numbers by intracellular staining. (J–L) Mediastinal lymph node Th2, GATA3 + Treg, and GATA3 − Treg cell numbers. (M) Concentration of IgE present in lung homogenate, normalized for total protein content. (N) Intestinal worm burden of indicated mouse genotypes 5 days post infection. Bar graphs indicate mean (±SEM). (A)–(C), ANOVA, three repeat experiments; (D), two repeat experiments; (E)–(I), ANOVA, two repeat experiments; (J)–(L), ANOVA, three repeat experiments; (M), ANOVA, two repeat experiments; (N), two-tailed Student’s t test, two pooled experiments. ns = not significant, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001. See also Figure S7 .

Article Snippet: Anti-mouse RELMα , Peprotech , 500-p214; RRID: AB_1268707.

Techniques: Infection, Staining, Concentration Assay, Two Tailed Test

Journal: Immunity

Article Title: Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells

doi: 10.1016/j.immuni.2018.05.003

Figure Lengend Snippet:

Article Snippet: Anti-mouse RELMα , Peprotech , 500-p214; RRID: AB_1268707.

Techniques: Control, Recombinant, Staining, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Microarray, Software

Journal: PLOS Pathogens

Article Title: Choline metabolism underpins macrophage IL-4 polarization and RELMα up-regulation in helminth infection

doi: 10.1371/journal.ppat.1011658

Figure Lengend Snippet: A) Schematic of 7-day in vivo choline kinase inhibition. Mice were treated intraperitoneally with vehicle (40% DMSO in PBS) or RSM-932a (3 mg/kg) every other day for 6 days and sacrificed on day 7. B) UMAP of peritoneal cell populations. n = 9–10, representing 2 independent experiments. C) Intracellular RELMα expression in live CD11b + F4/80 hi large (LPM) and CD11b + F4/80 lo small (SPM) peritoneal macrophages. Two-way ANOVA with Šídák’s test for multiple comparisons (* p < 0.05, **** p < 0.001). D-E) PD-L1 or PD-L2 expression (gMFI) in live CD11b + F4/80 hi LPM and CD11b + F4/80 lo SPM. Two-way ANOVA with Šídák’s test for multiple comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001). F) Intracellular RELMα expression in LPM and SPM isolated from peritoneal cavity aggregates or suspended lavage cells. Two-way ANOVA with Šídák’s test for multiple comparisons (* p < 0.05, **** p < 0.0001). Schematics were created using BioRender.

Article Snippet: Tissue sections were incubated with polyclonal rabbit anti-CD86 (ThermoFisher), RELMα (Peprotech), APC-conjugated anti-RELMα (DS8RELM; Invitrogen), anti-F4/80 (CI:A3-1), and biotinylated anti-CD206 (C068C2; BioLegend) antibodies overnight at 4°C.

Techniques: In Vivo, Inhibition, Expressing, Isolation

Journal: PLOS Pathogens

Article Title: Choline metabolism underpins macrophage IL-4 polarization and RELMα up-regulation in helminth infection

doi: 10.1371/journal.ppat.1011658

Figure Lengend Snippet: A) Schematic of primary infection with 200 H . polygyrus ( Hp ) L3 larvae through oral gavage. Mice were treated intraperitoneally with vehicle (40% DMSO in PBS) or RSM-932a (3 mg/kg) every other day for 8 days starting on day 8 and sacrificed on day 17. B-C) Percentage of weights at start of vehicle or RSM injections at 8 DPI with H . polygyrus (B) or final weights at day 17 (C). Mixed-effects analysis with Tukey’s test for multiple comparisons (* p < 0.05). D-E) Eggs in feces were counted at multiple time points after infection, and E) adult worms were isolated from the small intestine and enumerated on the day of sacrifice. Values represent means ± SEM (n = 3–5 mice per group), representative of 3 experiments. Two-way ANOVA with Šídák’s test for multiple comparisons and unpaired t test (ns). F-G) Detection of serum and G) peritoneal fluid RELMα by ELISA in naïve and H . polygyrus -infected mice. n = 3–5 per group. Two-way ANOVA with Šídák’s test for multiple comparisons (* p < 0.05, *** p < 0.001 for differences between treatment and ## p < 0.01 for differences between naïve and infected vehicle-treated mice). H-L) Immunofluorescent staining of intestinal tissue for CD206 and RELMα against DAPI counterstain. Scale bar 50 μm. Quantification of CD206 + (I) or RELMα + (J) per DAPI + cell. Unpaired t test (* p < 0.05). Quantification of F4/80 + (K) and RELMα + F4/80 + (L) per DAPI + cell. n = 6. Two-way ANOVA with Šídák’s test for multiple comparisons (**** p < 0.0001). Schematics were created using BioRender.

Article Snippet: Tissue sections were incubated with polyclonal rabbit anti-CD86 (ThermoFisher), RELMα (Peprotech), APC-conjugated anti-RELMα (DS8RELM; Invitrogen), anti-F4/80 (CI:A3-1), and biotinylated anti-CD206 (C068C2; BioLegend) antibodies overnight at 4°C.

Techniques: Infection, Isolation, Enzyme-linked Immunosorbent Assay, Staining