allergens comprising alternaria alternata, aspergillus fumigatus, dermatophagoides pteronyssinus  (Greer Laboratories)

Journal: bioRxiv

Article Title: Neuron-dependent tuft cell expansion initiates sinonasal allergic Type 2 inflammation

doi: 10.1101/2023.07.04.547596

Figure Lengend Snippet: (A) Alternaria alternata and Aspergillus fumigatus -Fungal allergen mix (FAM) was administered intranasally (i.n.) to C57BL/6 (WT) mice every other day (D0, D2, D4) and on D5, the sinonasal wash fluid and respiratory tissue were collected. (B) Levels of interleukin (IL)-5, IL-13, and IL-33 in the sinonasal wash fluid. (C-D) The number of (C) eosinophils and (D) Group 2 Innate Lymphoid Cells (ILC2s) in respiratory tissue as determined by flow cytometry. (E) Top: immunofluorescent staining of respiratory epithelium from PBS or FAM-treated mice (D5) to identify DCLK1+ cells (green with yellow asterisks). Bottom: co-immunostaining for DCLK1 (green) and keratin 5+ cells (white) lining the airway (AW), scale bars = 100μm. (F) Left: flow cytometry gating strategy to identify DCLK1+CD45-Tuft-1 and DCLK1+CD45+ Tuft-2 cells (PBS-treated at top and FAM D5-treated at bottom). Right: total cell numbers recovered from respiratory epithelium of individual mice. (G) Sinonasal wash levels of IL-25 (top) and cysteinyl leukotrienes (CysLT) (bottom). (H) Respiratory epithelium of PBS (left) or FAM-treated mice (D5, right) depicting immunofluorescence staining to identify cells co-expressing keratin 5 (white) and Ki-67+ (magenta) lining the airway (AW) of sinonasal respiratory tissue (scale bar = 100μm). (I) Left: gating strategy used to identify Integrin β4+ Nerve Grow Factor Receptor+ (NGFR) basal cells that express Ki-67 from PBS or FAM D5-treated mice. Right: quantification of cell numbers in respiratory epithelium of individual mice. (J) Model for administration of Dermatophagoides pteronyssinus -House Dust Mite (HDM) extract using the protocol in (A). (K) Levels of IL-5 and IL-13 in sinonasal fluid. (L-N) Total number of (L) eosinophils, (M) ILC2s, (N) Tuft-1 and Tuft-2 cells as determined by flow cytometry. (O) Levels of IL-25 and CysLT in sinonasal fluid. (P) Cell numbers of Ki67+ Integrin β4+ NGFR+ basal cells from the respiratory epithelium of PBS or HDM-treated mice at D5. Data are mean ± SEM, representative of 3-4 experiments (4-5 mice/treatment), T-Test. *** p<0.001, **** p<0.0001. See also .

Article Snippet: To induce sinonasal allergic Type 2 inflammation, mice were anesthetized with isoflurane (1-4%) and treated intranasally (i.n.) with a fungal allergen mix (FAM) consisting of A. alternata allergen (Greer Laboratories, Cat# NC1620293) and A. aspergillus allergen (Greer Laboratories, Cat# NC1677927) 3 times a week for 3 weeks ( ).

Techniques: Flow Cytometry, Staining, Immunostaining, Immunofluorescence, Expressing

Journal: bioRxiv

Article Title: Neuron-dependent tuft cell expansion initiates sinonasal allergic Type 2 inflammation

doi: 10.1101/2023.07.04.547596

Figure Lengend Snippet: (A) PBS or Alternaria alternata and Aspergillus fumigatus -Fungal allergen mix (FAM) was administered intranasally (i.n.) every other day to Pou2f3 -/- mice, lacking mature tuft cells, or their WT littermate controls. Respiratory tissue and sinonasal wash were harvested and analyzed on D5. (B) Immunofluorescence staining of respiratory epithelium from FAM-treated WT or Pou2f3-/- mice to identify DCLK1+ cells (green with yellow asterisks) lining the airway (AW), scale bar = 100 μm. (C) Tuft 1 (left) and tuft 2 (right) cell numbers in the respiratory epithelium as determined by flow cytometry. PBS-treated WT (white), FAM D5-treated WT (Blue), PBS-treated Pou2f -/- (gray), and FAM D5-treated Pou2f -/- (red). (D) Levels of IL-25 (left) and cysteinyl leukotrienes (CysLT, right). (E) Number of total basal cell population (left) and Ki67+ basal cell population (right). (F) Sinonasal fluid levels of IL-5 and IL-13. (G-I) Total numbers of (G) ILC2, (H) CD4+ TH2 cells, and (I) eosinophils in respiratory tissue. Data are mean ± SEM, representative of 1 experiment (4 mice/treatment), one-way ANOVA with Tukey’s post-hoc tests. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. See also and .

Article Snippet: To induce sinonasal allergic Type 2 inflammation, mice were anesthetized with isoflurane (1-4%) and treated intranasally (i.n.) with a fungal allergen mix (FAM) consisting of A. alternata allergen (Greer Laboratories, Cat# NC1620293) and A. aspergillus allergen (Greer Laboratories, Cat# NC1677927) 3 times a week for 3 weeks ( ).

Techniques: Immunofluorescence, Staining, Flow Cytometry