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Steroid resistance of airway type 2 innate lymphoid cells from patients with severe asthma: The role of thymic stromal lymphopoietin  Sucai Liu, PhD,

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Presentation on theme: "Steroid resistance of airway type 2 innate lymphoid cells from patients with severe asthma: The role of thymic stromal lymphopoietin  Sucai Liu, PhD,"— Presentation transcript:

1 Steroid resistance of airway type 2 innate lymphoid cells from patients with severe asthma: The role of thymic stromal lymphopoietin  Sucai Liu, PhD, Mukesh Verma, PhD, Lidia Michalec, PhD, Weimin Liu, PhD, Anand Sripada, PhD, Donald Rollins, MD, James Good, MD, Yoko Ito, PhD, HongWei Chu, PhD, Magdalena M. Gorska, MD, PhD, Richard J. Martin, MD, Rafeul Alam, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 141, Issue 1, Pages e6 (January 2018) DOI: /j.jaci Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Effect of dexamethasone on IL-5+ ILC2s. PBMCs from a patient with allergic asthma were cultured with Aspergillus species, IL-2/IL-33, or IL-2/IL-7/IL-33 for 5 days with and without dexamethasone. A, PBMCs were stained with labeled antibodies against the lineage markers, CRTH2, IL-7Rα and their isotype antibody controls (top). Live and Lin− cells were gated for CRTH2+ and IL-7Rα+ cells. Dual CRTH2+IL-7Rα+ cells were then analyzed for IL-5 expression. B, Expression of IL-5 in Lin−CRTH2+IL-7Rα+ cells in cultures with Aspergillus species, IL-2/IL-33, or IL-2/IL-7/IL-33 in the presence or absence of dexamethasone. C, Effect of dexamethasone on BAL fluid Lin−CRTH2+IL-7Rα+IL-5+ cells. BAL fluid cells from a patient with allergic asthma were cultured with and without dexamethasone for 5 days and then analyzed for live CD45+Lin−CRTH2+IL-7Rα+IL-5+ cells. D and E, Cumulative data on the effect of dexamethasone (Dex) on blood (Fig 1, D) and BAL fluid (Fig 1, E) Lin−CRTH2+IL-7Rα+IL-5+ cells from 14 patients with allergic asthma. FS, Forward scatter; SS, side scatter; V, vehicle. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 A, Effect of cytokines on development of steroid resistance. PBMCs from patients with allergic asthma were cultured with a combination of ILC2-stimulating cytokines in the presence or absence of dexamethasone for 5 days. The cells were then stained for flow cytometry (FCM) and gated for IL-5+ ILC2s, as described in Fig 1, A. *P < .05 (n = 6). B, Kinetics of development of resistance against inhibition of IL-5+ ILC2s by dexamethasone. PBMCs were cultured with IL-2/TSLP plus vehicle or dexamethasone for 1, 3, and 5 days and then analyzed for IL-5+ ILC2s by using FCM. *P < .05 (n = 4). C and D, Lin− cells (0.4 × 106 per culture) from peripheral blood obtained from 3 patients with allergic rhinitis (nonasthmatic) were cultured with IL-2/IL-33 or IL-2/TSLP with and without dexamethasone (10−7 mol/L) for 5 days. Cell-free supernatants were analyzed for IL-5 by means of ELISA. Cells were analyzed by using FCM for intracellular IL-5. Dexamethasone inhibition of IL-5 secretion and intracellular IL-5 expression are presented in Fig 2, C and D, respectively. P values for data on Fig 2, C, were calculated by using paired t tests because of low numbers. E, Comparison of the effect of dexamethasone on IL-5+ ILCs (Lin−CRTH2+IL-7Rα+) and IL-5+Lin+ cells in BAL fluid. BAL fluid cells were cultured with vehicle or dexamethasone for 5 days and stained and first gated for CD45+ cells and then for Lin−, CRTH2+, IL-7Rα+, and IL-5+ cells (IL-5+ ILCs) and Lin+IL-5+ cells. Each data point is an individual asthmatic patient (n = 9). Dex, Dexamethasone; V, vehicle. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 A, TSLP Levels in BAL fluid from asthmatic patients and control subjects with disease. TSLP levels were measured by means of ELISA (n = 20). B, Correlation between BAL fluid TSLP levels and dexamethasone (Dex) inhibition of BAL fluid IL-5+ ILC2s. C, Effect of select cytokines and Toll-like receptor ligands on TSLP production by airway epithelial cells. Human airway epithelial cells cultured at the air-liquid interface were stimulated with IL-13, IFN-γ, LPS, or poly-IC for 24 hours, and then expression of TSLP mRNA was analyzed by using real-time PCR. *P < .05, paired t test (n = 5), compared with medium (Med). D, Effect of IL-13 and human rhinovirus-16 (RV) infection on TSLP production by airway epithelial cells. Human airway epithelial cells cultured as above were stimulated with IL-13, human rhinovirus-16, or a combination of both for 48 hours. Supernatants were assayed for TSLP by means of ELISA. *P < .05, paired t test (n = 3 replicates), compared with medium (Med). Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 A-D, Effect of dexamethasone on CRTH2 and IL-7Rα expression on ILC2s. PBMCs were cultured with vehicle or dexamethasone for 5 days and then stained for flow cytometry. Live Lin− cells were gated for CRTH2 and IL-7Rα. A representative flow cytogram is shown in Fig 4, A. Cumulative data from 14 asthmatic patients for the frequency of CRTH2+ and IL-7Rα+ cells are shown in Fig 4, B and C, respectively. Fig 4, D, shows the mean fluorescence intensity (MFI) of IL-7Rα+ expression from the same study subjects. E, Comparison of the frequency of IL-7Rα+ ILCs between blood and BAL fluid. PBMCs and BAL fluid cells from asthmatic patients were studied ex vivo for IL-7Rα on gated live Lin−CRTH2+ cells. Each symbol represents a donor (n = 24). F, Effect of dexamethasone on TSLP signaling. PBMCs from patients with allergic asthma were cultured with vehicle or dexamethasone for 3 days and stimulated with IL-2/TSLP for an increasing period of time. The frequency of pSTAT5+ cells on a gated live Lin−CRTH2+ cell population was measured. *P < .05, paired t test (n = 4). Dex, Dexamethasone; V, vehicle. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 A and B, Effect of dexamethasone on cytokine-induced MEK1. PBMCs from an asthmatic patient were cultured with medium or IL-2 plus one of the 2 epithelial ILC–inducing cytokines IL-33 and TSLP with and without dexamethasone for 5 days. The expression of MEK on gated live Lin−CRTH2+ cells was analyzed by using flow cytometry. Representative flow cytograms are shown in Fig 5, A, and cumulative data from 7 asthmatic patients are shown in Fig 5, B. C and D, PBMCs were cultured as under Fig 5, A, and then the frequency of c-Fos+ and pSTAT5+ cells in the live Lin−CRTH2+ cell population was assessed. *P < .05, paired t test (n = 4), compared with vehicle-treated cells. E, Effect of the MEK inhibitor trametinib on pSTAT5 in ILCs. PBMCs from patients with allergic asthma were cultured with medium, IL-2/IL-33, or IL-2/TSLP with and without trametinib (10 μmol/L) for 5 days and then assessed for pSTAT5. *P < .05, paired t test (n = 3). F, Expression of GR in cytokine-treated cells. PBMCs were cultured with medium, IL-2/TSLP, or IL-2/IL-33 for 5 days. GR expression was studied in gated live Lin−CRTH2+ cells. A representative flow cytogram from one of 3 similar experiments is shown. Dex, Dexamethasone; V, vehicle. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 Effect of dexamethasone on select signaling molecules in BAL fluid ILC2s. BAL fluid cells were cultured with vehicle or dexamethasone for 5 days, and then expression of MEK+ (A), pSTAT5+ (B), pSTAT3+ (C), and ID3+ (D) cells was analyzed on gated live Lin−CRTH2+ cells by using flow cytometry. Each symbol represents a single asthmatic patient (n = 5-7). Dex, Dexamethasone; V, vehicle. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig 7 Effect of MEK, JAK, and STAT5 inhibitors on steroid resistance of BAL fluid ILC2s. BAL fluid cells were cultured for 3 days with vehicle, dexamethasone, the MEK inhibitor trametinib (Tram) with and without dexamethasone (A), the JAK3 inhibitor tofacitinib (Tofa) with and without dexamethasone (B), or the STAT5 inhibitor pimozide (Pim) with and without dexamethasone (C and D). The effect of dexamethasone and that of the inhibitors with and without dexamethasone on BAL fluid IL-5+ ILC2s (Fig 7, A, B, and D) is shown. The flow cytogram in Fig 7, C, shows the effect of pimozide on pSTAT5. Each symbol represents a single asthmatic patient (n = 5-7). P values were calculated by using the nonparametric Kruskal-Wallis test. Dex, Dexamethasone; V, vehicle. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E1 A, Effect of dexamethasone (Dex) and cytokines on live and dead cells, as demonstrated by forward scatter (FS) and side scatter (SS). B, Live cells were then further analyzed for dead cells by using the fluorescent vital dye eFluor 780 (FVD e-Fluor 780, eBioscience). The boxed area in the flow cytogram shows the number of dead cells. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E2 A, Mean fluorescence intensity (MFI) of intracellular IL-5 staining of BAL fluid ILC2s cultured with vehicle or dexamethasone. BAL fluid cells were obtained from allergic asthmatic patients. Each symbol represents a single donor. B and C, Cell viability of BAL fluid cells on culture with vehicle or dexamethasone. Dead cells were analyzed by using 2 different dead cell dyes: eFluor 780 (Fig E2, B) and Zombie aqua (BioLegend; Fig E2, C). Flow cytograms are representatives of 3 separate experiments. Dex, Dexamethasone; V, vehicle. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Fig E3 Effect of dexamethasone (Dex) on CRTH2+ and IL-5+ ILC2s from healthy subjects. PBMCs were cultured with IL-2/IL-33 or IL-2/TSLP with and without dexamethasone for 5 days. A, Gated Lin− cells were analyzed for CRTH2/IL-7Rα. B, Gated Lin−CRTH2+ cells were analyzed for IL-5/IL-13. C, Cumulative data on the effect of dexamethasone on IL-5+ ILC2s obtained from 6 healthy control subjects. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 Fig E4 Effect of dexamethasone (Dex) with and without IL-7/TSLP on expression of the receptors for IL-2 (CD25), IL-33 (ST2), and IL-25. PBMCs were cultured with dexamethasone with and without IL-7 or TSLP or medium alone for 5 days. Expression of CD25, ST2, and IL-25 receptor on gated live Lin−CRTH2+ cells was analyzed by using flow cytometry. Representative flow cytograms from one of 3 similar experiments are shown. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

13 Fig E5 Effect of dexamethasone on expression of c-Fos by blood ILCs and of pSTAT3 and ID3 by BAL fluid ILCs. PBMCs or BAL fluid cells from patients with allergic asthma were cultured with vehicle or dexamethasone for 3 days and analyzed by using flow cytometry. Representative flow cytograms for c-Fos in blood Lin− cells (A) and pSTAT3 (B) and ID3 (C) in BAL fluid Lin− cells are shown. Dex, Dexamethasone; FS, forward scatter; V, vehicle. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

14 Fig E6 Comparison of MEK expression by blood and BAL fluid ILCs. A and B, Lin− cells from BAL fluid cells and PBMCs obtained from an asthmatic patient were stained and analyzed ex vivo for MEK expression. Representative flow cytograms are shown in Fig E6, A. Fig E6, B, shows cumulative data on MEK expression in Lin− cells from 4 asthmatic patients. C, PBMCs from the patient shown in Fig E6, A, were cultured in medium alone or with IL-2/TSLP for 5 days. Lin− cells were gated and analyzed for MEK expression. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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