Journal: JCI Insight

Article Title: Vancomycin eliminates gut deoxycholic acid, restoring ER proteostasis in ILC2s and relieving colitis

doi: 10.1172/jci.insight.197470

Figure Lengend Snippet: ( A – C ) Large intestinal ILC2s were sorted from WT mice and cultured in the presence of IL-2, IL-7, IL-25, and IL-33 for 5 days. ILC2s were treated with DCA (100 μM) at the indicated concentrations for 24 hours and then subjected to RNA-Seq. ( A ) Experimental strategy, ( B ) volcano plot, and ( C ) heatmap of ILC2-characteristic genes. ( D – F ) Sorted large intestinal ILC2s from Il5 RFP-Cre mice were treated with DCA (100 μM) for 24 hours. ( D ) Experimental strategy. ( E ) Representative flow cytometry plots. ( F ) Percentages of RFP + ILC2s (CD45.2 + Lin – GATA3 + ) in large intestine. n = 6 wells per group. The experiment was repeated 3 times. ( G – I ) Rabbit reticulocyte lysate system. ( G ) Experimental strategy. Protein levels of Areg, IL-5, and IL-13 were analyzed by ( H ) Western blotting and ( I ) cytometric bead array in samples treated or untreated with DCA. n = 6 wells per group. The experiment was repeated twice. Data are shown as mean ± SD. Statistical analysis was performed using unpaired 2-tailed t test. *** P < 0.001, **** P < 0.0001.

Article Snippet: Il5 RFP-Cre mice (catalog R5/+) and CD45.1/CD45.1 mice (catalog 002014) were purchased from The Jackson Laboratory.

Techniques: Cell Culture, RNA Sequencing, Flow Cytometry, Western Blot

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

Article Title: Sensitization with fungal protease allergen establishes long-lived, allergenic helper T cell memory in the lung

doi: 10.4049/jimmunol.2300694

Figure Lengend Snippet: A) Asp f 13 protease treatment schedule for murine model of allergic inflammation. B) Representative micrographs of lungs from primary and recall mice stained with Periodic Acid Schiff and Hematoxylin. Scale bar = 50µm. Insert on left is magnified 2.5X. C) Flow cytometry plot (left) and enumerated eosinophils from the lungs of mice. Gated on Live, IV-, CD90-, CD19-, Ly6G-, Siglec F+. D&G) Enumerated eosinophils from the lungs of mice at indicated treatment/timepoint. E) Lung leukocytes from naïve and sensitized/challenged mice. Parental gate on Live, IV-, CD45+ cells. F) IL-5 measured from lungs lysates of mice. C, D & G are pooled and B, E, &F are representative from 2 independent experiments. Groups indicated as “ ⎴ “ are compared by Mann-Whitney U . p- value < 0.05 = * and n.s. = p- value > 0.05.

Article Snippet: Wild type (C56BL/6J), CD90.1 (B6.PL- Thy1 a /CyJ), OTII (B6.Cg-Tg(TcraTcrb)425Cbn/J), and IL-5 reporter (B6(C)- Il5 tm1.1(icre)Lky /J) mice were purchased from Jackson Laboratory (Bar Harbor, ME).

Techniques: Staining, Flow Cytometry, MANN-WHITNEY

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

Article Title: Sensitization with fungal protease allergen establishes long-lived, allergenic helper T cell memory in the lung

doi: 10.4049/jimmunol.2300694

Figure Lengend Snippet: A) Leukocytes extracted and pooled from 5 mice after sensitization with Asp f 13 through 14 days. Leukocytes were stimulated ex vivo with the indicated substances. Flow plots are proportion of IL-5+ T helper (Th) cells. B) Representative flow plots (left, proportion +/− SEM) and absolute numbers (right, mean +/− SEM) of Asp f 13 tetramer+ Th cells during sensitization (0–14d) and memory formation (21d+). C) Concatenated flow cytometry data (n=7) from TCRb+ CD4+ T cells from lungs of mice 120d post-sensitization. Plot on the left shows parental gates for the CD44hi, CD44low, and Tetramer+ plots of Foxp3 x GATA3 on the right. A & C are representative and B is pooled from at least 2 independent experiments.

Article Snippet: Wild type (C56BL/6J), CD90.1 (B6.PL- Thy1 a /CyJ), OTII (B6.Cg-Tg(TcraTcrb)425Cbn/J), and IL-5 reporter (B6(C)- Il5 tm1.1(icre)Lky /J) mice were purchased from Jackson Laboratory (Bar Harbor, ME).

Techniques: Ex Vivo, Flow Cytometry

Journal: Journal of Clinical Investigation

Article Title: Eosinophil pathogenicity mechanisms and therapeutics in neuromyelitis optica

doi: 10.1172/jci67554

Figure Lengend Snippet: Figure 5 Eosinophil-dependent NMO pathology in mice. (A) Mice were intraperitoneally administered anti–IL-5 or anti-Ly6G antibodies, alone or together, to deplete eosinophils and/or neutrophils, followed by a 3-day NMO-IgG/hc infusion (top). Peripheral neutrophil (Nφ) and eosinophil counts in control and treated mice (bottom) (SEM; 5 mice per group; P < 0.001). (B) Mice were infused for 3 days with 3.3 μg/day NMO-IgG and 16.7 μl/ day hc, and sacrificed on day 3. Immunofluorescence staining of AQP4 and MBP (scale bar: 2 mm); Siglec F and Ly6G (scale bar: 100 μm). (C) Lesion severity assessed by loss of AQP4, myelin, and numbers of eosinophils and neutrophils in lesions (SEM; 4–8 mice per group; *P < 0.05; **P < 0.01). (D) Control and hypereosinophilic IL-5 Tg mice were infused for 3 days with NMO-IgG (3.3 μg/day) and submaximal hc (3.4 μl/day). Immunofluorescence staining of AQP4 (scale bar: 2 mm), Siglec F, and Ly6G (scale bar: 100 μm). Summary of lesion scores (SEM; 4 mice per group; *P < 0.05). (E) Control and Tg hypoeosinophilic ΔdblGata1 mice were infused for 3 days with NMO-IgG (3.3 μg/day) and hc (16.7 μl/day). AQP4 immunofluorescence and loss of AQP4 immunofluorescence are shown (SEM; 5 mice per group; *P < 0.05). Scale bar: 2 mm.

Article Snippet: Some experiments were conducted using hypereosinophilic IL-5 Tg (Tg(Cd3d-Il5)NJ.1638Nal) mice (38) on a BALB/c background (provided by Richard Locksley, UCSF, San Francisco, California, USA) and ΔdblGata1 mice (C.Cg-Gata1tm6Sho/J mice; The Jackson Laboratory) that lack eosinophils because of Gata1 promoter mutation.

Techniques: Control, Immunofluorescence, Staining