Lack of autophagy induces steroid-resistant airway inflammation

Abstract

Background: Neutrophilic corticosteroid-resistant asthma accounts for a significant proportion of asthma; however, little is known about the mechanisms that underlie the pathogenesis of the disease.

Objective: We sought to address the role of autophagy in lung inflammation and the pathogenesis of corticosteroid-resistant neutrophilic asthma.

Methods: We developed CD11c-specific autophagy-related gene 5 (Atg5)(-/-) mice and used several murine models to investigate the role of autophagy in asthmatic patients.

Results: For the first time, we found that deletion of the Atg5 gene specifically in CD11c(+) cells, which leads to impairment of the autophagy pathway, causes unprovoked spontaneous airway hyperreactivity and severe neutrophilic lung inflammation in mice. We found that severe lung inflammation impairs the autophagy pathway, particularly in pulmonary CD11c(+) cells in wild-type mice. We further found that adoptive transfer of Atg5(-/-), but not wild-type, bone marrow-derived dendritic cells augments lung inflammation with increased IL-17A levels in the lungs. Our data indicate that neutrophilic asthma in Atg5(-/-) mice is glucocorticoid resistant and IL-17A dependent.

Conclusion: Our results suggest that lack of autophagy in pulmonary CD11c(+) cells induces neutrophilic airway inflammation and hyperreactivity.

Keywords: Autophagy; asthma; corticosteroid-resistance asthma; lung inflammation; neutrophilic asthma.

Figure 1. Lack of Autophagy augments neutrophilic airway inflammation

A. Experimental timeline. B. Lung resistance (R_L, left panel) and dynamic compliance (C_dyn, right panel) measured on day 16. C. Differential cell counts in BAL (TCC, total cell number; MAC, Macrophages; Eos, Eosinophils; Lym, Lymphocytes; Neut, Neutrophils.). D. H&E stained lung sections(× 200). E. Cytokine levels in whole-lung lysate. F. Frequency of IL-17A, IL-4, IL-5, IL-13 and IFN-γ producing CD3⁺CD4⁺CD44⁺ T_eff cells. Data are representative of 2-3 experiments and shown as the mean ± SEM (n=5-10/group). *: P<0.05, ***: P<0.001.

Figure 2. Lack of Autophagy in immune cells contributes to the induction of lung inflammation

A. WT or Atg5^-/- BM cells was adoptively transferred into irradiated CD45.1⁺ WT mice and then immunized and challenged with HDM as indicated. B. The reconstitution rate of transferred cells as assessed by the frequency of CD45.1⁺ and CD45.2⁺ neutrophils in BAL. C. Lung resistance R_L and D. dynamic compliance C_dyn. E. Differential cell counts in the BAL. F. H&E stained lung sections(× 200). G. Atg5^-/- mice were irradiated and received either WT or Atg5^-/- bone marrow followed by HDM sensitization as indicated. H. The reconstitution rate of transferred cells. I. Lung resistance and J. dynamic compliance after sensitization as shown in G. K. Differential cell counts in the BAL. L. H&E stained lung sections(× 200). TCC: Total cell count, MAC: macrophages, Lym: Lymphocytes, Eos: Eosinophils, Neut: Neutrophils (n=5/group). Data are shown as the Mean ± SEM. *: P<0.05, ***: P<0.001.

Figure 3. Induction of asthma impairs autophagy in lung CD11c⁺ cells

A. Timeline for sensitization and challenges. WT or LC3-GFP mice were immunized with HDM as indicated. B. Western blot analysis of the expression levels of p62, LC3-I, and LC3-II in purified pulmonary CD11c⁺ cells. C. The levels of the indicated proteins are expressed relative to Actb. D. Confocal microscopy of purified lung CD11c⁺ cells from LC3-GFP (×600). E. The mean fluorescence intensity of LC3. Data are expressed as the mean ± SEM (n=3-9/group). *, P<0.05.

Figure 4. Lack of autophagy in DCs induces IL-1 and IL-23, and Th17 polarization

A. BM-DCs from WT and Atg5^-/- mice were stimulated with or without HDM (50 μg/ml) for 36 hours. The cytokine levels in the supernatants were measured by ELISA. B. Quantification of mRNA expression by RT-PCR. C. BM-DCs from WT or Atg5^-/- mice were co-cultured with CD4⁺ T cells isolated from the lungs of HDM sensitized mice in the presence of HDM (10 μg/ml) for 3 days. HDM sensitization was performed according to Figure 1A. The cytokine levels were measured by ELISA. Data are representative of two experiments and expressed as the mean ± SEM (n=4-8/group). *: P<0.05, **: P<0.01.

Figure 5. Lack of Atg5 in CD11⁺ c cells causes unprovoked asthma and induces severe neutrophilic lung inflammation

A. Experimental time line. Cultured BM-DCs from WT or Atg5^-/- mice were stimulated with HDM for six hours, and then intravenously injected to WT mice. Then mice were intratracheally challenged with HDM stimulated BMDCs on day 8 and 15. B. Lung resistance (R_L) and C. dynamic compliance (C_dyn). D. Differential cell count in the BAL. E. Cytokine levels in whole-lung lysate. Pooled data from two experiments are shown (n=7-8/group). F. Lung resistance in CD11c-Atg5^-/- and wild type mice was measured at the steady state. G. Dynamic compliance. H. Differential cell counts in BAL. Values are expressed as the mean ± SEM. *: P<0.05, **: P<0.01, ***: P<0.001. TCC, total cell number; MAC, Macrophages; Eos, Eosinophils; Lym, Lymphocytes; Neut, Neutrophils.

Figure 6. Atg5^-/- mice develop IL-17A dependent steroid resistant airway inflammation A and D

WT and Atg5^-/- mice were immunized and challenged with HDM as described in Figure 1A and treated with dexamethasone (1mg/Kg) or anti-IL17ab (300μg) one day before HDM challenge. The mice were subsequently assessed for AHR by measuring R_L. B and E. BAL cells from the mice in Figure 6A and 6D were analyzed after AHR measurements. C and F. Lung tissues sections from WT and Atg5^-/- mice after dexamethasone or anti-IL17ab treatment were stained with H&E (× 200). Data are expressed as the mean ± SEM Data are representative of two independent experiments (n=5-6/group). *: P<0.05, ***: P<0.001.