|Home | About | Journals | Submit | Contact Us | Français|
Neutrophilic corticosteroid-resistance asthma accounts for a significant proportion of asthma however, little is known about mechanisms that underlie the pathogenesis of the disease.
To address the role of autophagy in lung inflammation and the pathogenesis of corticosteroid-resistant Neutrophilic asthma.
We developed CD11c-specific Atg5-/- mice and used several murine models to investigate the role of autophagy in asthma.
We found, for the first time, that deletion of Atg5 gene specifically in CD11c+ cells which leads to the impairment of 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 (WT) mice. We further found that adoptive transfer of Atg5-/- but not WT bone marrow derived DCs augments lung inflammation with elevated IL-17A in the lungs. Our data indicates that neutrophilic asthma in Atg5-/- mice is glucocorticoid-resistance and IL-17A dependent.
Our results suggest that lack of autophagy in pulmonary CD11c+ cell induces neutrophilic airway inflammation and hyperreactivity.
Asthma is a heterogeneous disease with different phenotypes of lung inflammation involving the large and small airways and alveoli resulting in airway hyperreactivity (AHR), bronchoconstriction and airway remodeling1-3. Prevalence of allergic disease and asthma has increased dramatically over the past five decades. Whilst treatment of mild to moderate asthma has dramatically improved with combination therapy of inhaled corticosteroid and long acting β2 agonists, approximately 10% of patients with asthma are unresponsive to conventional treatment and suffer from severe refractory asthma4,5. A recent cohort analysis has revealed that non-eosinophilic inflammation is predominant in mild to moderate asthma and neutrophilic inflammation is dominant in severe refractory asthma6. In particular, patients with elevated neutrophils as well as eosinophils in the sputum have been found to suffer from declined lung function7. Severe allergic asthma starts with a Th2 mediated disease with the secretion of IL-4, IL-5 and IL-13 and as the severity of the disease increases, through unknown mechanisms, other cytokines such as IL-17 mediate the recruitment of inflammatory cell types such as neutrophils that further contribute to the pathogenesis of the disease8-10. Several reports suggest that IL-17 is increased in the lung of patients with severe asthma and correlates with the severity of AHR11-13. Dendritic cells (DCs) may play an essential role in regulating IL-17 production and greatly contribute to the pathogenesis of asthma14,15.
Autophagy is a critically important intracellular process by which damaged self-organelles are cleared, disassembled and their composing units are recycled16-18. Interestingly, genetic polymorphisms in autophagy related gene 5 (Atg5) have been associated with childhood asthma19,20. However, the role of autophagy in the development of allergic asthma remains unknown. In the present study, we demonstrate a critical role for autophagy in inducing neutrophilic lung inflammation in a murine model of allergic asthma by modulating CD11c+ cells through the axis of IL-23/IL-17 producing T cells. This study will provide new evidence to understand the biological mechanisms of asthma pathogenesis that may be fundamental for the development of novel treatment options.
We used Atg5 deficient mice to study the role of autophagy pathway in lung allergic inflammation since depletion of Atg5 has been shown to efficiently disrupts autophagy pathway16-18,26,27,28. Constitutively Atg5 deficient mice die soon after birth28, so we used inducible conditional Atg5 knockout mice in which injection of Tamoxifen deletes approximately 80-90% of Atg5 gene which we refer to as Atg5-/- mice (Supplementary Figure 1). WT and Atg5-/- mice were sensitized and intranasally challenged with house dust mite (HDM) extract according to the protocol shown in Figure 1A. One day after the last HDM challenge, lung function was evaluated by direct measurements of lung resistance (RL) and dynamic compliance (Cdyn) as described in Methods. The results showed that HDM challenged Atg5-/- mice had significantly higher RL and Cdyn compared to HDM challenged WT mice (Figure 1B). HDM challenged Atg5-/-mice showed significantly higher number of total cells, neutrophils and macrophages in bronchoalveolar lavage (BAL) compared to WT mice (Figure 1C). Histological examinations revealed marked peribronchial inflammatory responses in Atg5-/- mice with increased airway wall thickness and accumulation of inflammatory cells than WT mice (Figure 1D).
To explore the mechanism of autophagy dependent neutrophilic airway inflammation, the levels of IL-17A, IL-1β, IL-4 and IL-13 were assessed in whole-lung lysates by ELISA and at the intracellular level by flow cytometry. We found significantly higher levels of IL-17A and IL-1β, cytokine that can induce IL-17A production, in the lungs of HDM sensitized Atg5-/- than those in WT mice (Figure 1E). This finding is consistent with the role of IL-17A in the recruitment of neutrophils in the BAL (Figure 1C). Interestingly, there was no difference in the Th2 cytokines IL-4 and IL-13 between Atg5-/- and WT mice, which correlates with the comparable number of eosinophils in the BAL found in WT and Atg5-/- mice (Figure 1C, E). Intracellular cytokine staining assay also revealed a significantly increased frequency of CD3+CD4+CD44+ IL-17A+ pulmonary Teff cells in Atg5-/- compared to WT mice, whereas the frequency of the IL-4, IL-5, IL-13 and IFN-γ from the CD3+CD4+CD44+ population did not differ (Figure 1F, Supplementary figure 2). Since, TCR-γδ T cells, TCR-αβ and innate lymphoid cells can produce IL-17 in the lungs, we further identified the source of IL-17A production in the lungs. We found that the TCR-γδ T cells are the major source of IL-17A in the lungs of Atg5-/- mice (Supplementary Figure 3). We further examined whether disruption of autophagy leads to enhanced viability of neutrophils in the BAL and found no difference between Atg5-/- and WT (Supplementary Figure 5). To investigate whether disruption of autophagy influences the pulmonary DC repertoire we evaluated the number of different DCs subsets in the lungs of Atg5-/-and WT mice at the steady state and after HDM stimulation using an approached described previously24. The number of alveolar macrophages, plasmacytoid dendritic cells, CD103+ and CD11b+ DCs are comparable in Atg5-/- and WT mice at the steady state and after HDM stimulation (Supplementary Figure 4). These results demonstrate that lack of autophagy in Atg5-/- mice causes severe lung inflammation and AHR, which is mediated by increased neutrophilic airway inflammation through the secretion of IL-17A by T cells.
Since a recent study revealed that epithelial autophagy is involved in pathogenesis of cigarette smoke induced Chronic Obstructive Pulmonary Disease (COPD)29,30, we investigated whether lack of autophagy in the alveolar epithelial cells (AECs) may play a role in the development of severe AHR. We generated lung AECs specific autophagy deficient mice as described previously and in online repository methods31. Our data showed the levels of lung inflammation and AHR, IL-1β or IL-17A in SPC-Atg5-/- mice were similar to those of WT mice (Supplementary Figure 6), suggesting a non-epithelial cell involvement for the development of autophagy mediated severe lung inflammation.
Next, to examine the contribution of immune cells in autophagy mediated severe lung inflammation we injected bone marrow (BM) of Atg5-/- (CD45.2+) or WT (CD45.2+) mice into sub-lethally irradiated WT (CD45.1+) mice (Figure 2A) and that resulted in approximately 90% reconstitution of donor cells (Figure 2B). Compared with WT-BM recipient mice, Atg5-/- BM recipients displayed significantly higher AHR (Figure 2C-D) and significantly higher number of neutrophils in the BAL (Figure 2E). There is no significant induction of AHR in the recipient of WT BM. Lung histology show similar results to AHR and BAL findings (Figure 2F). These results indicate that BM derived immune cells rather than non- hematopoietic cells (including alveolar and bronchial epithelial cells) contribute to the severity of AHR induced by autophagy deficiency.
To confirm our findings and to exclude the role of autophagy in non-hematopoietic cells, we perform a new set of chimeric experiments where irradiated Atg5-/- hosts received either WT or Atg5-/- BM followed by intranasal sensitization (Figure 2G). This is an inverse experiment to the aforementioned experiment and enables us to address whether radio-resistant Atg5-/- host cells play an important role in the induction of neutrophilic lung inflammation. Donor BM cells reconstituted more than 80% of irradiated hosts CD45+ hematopoietic cells (Figure 2H). In agreement with our previous findings our results show that i.n. HDM administration increased neutrophilic lung inflammation and AHR only in Atg5-/- BM recipients but not in WT BM recipients (Figure 2I-L). Taken together, these data show that lack of autophagy in hematopoietic cells underlies the augmented lung inflammation and AHR.
Since it has been shown that IL-4 and IL-13 can reduce autophagy in macrophage32, we tested whether severe lung inflammation impairs autophagy in CD11c+ cells. To induce sever lung inflammation we sensitized mice by i.p. injection of HDM+Alum followed by 4 i.n. administration of HDM as shown in Figure 3A. Autophagy assessment was done according to recent guidelines for the use and interpretations of assays for monitoring autophagy33,34. Interestingly we observed that severe asthma impairs autophagy in pulmonary CD11c+ (Figure 3B and 3C). We confirmed these findings by analyzing LC3 foci using confocal microscopy and LC3-GFP mice. Severe asthma clearly decreased the number of LC3 foci in pulmonary CD11c+ cells (Figure 3D and 3E).
To investigate the mechanism by which impairment of autophagy in DCs contribute to Th17 differentiation, we isolated and analyzed WT and Atg5-/- BM-DCs for production of IL-1α, IL-1β, IL-6, and IL-239,11-13,35,36. Interestingly, BM-DCs from Atg5-/- mice displayed higher amounts of IL-1α, IL-1β, and IL-23 production upon LPS or HDM stimulation at both protein and mRNA level (Figure 4A-B, Supplementary Figure 7A-B). Atg5-/- BM-DCs significantly increased the levels of IL-17A in the co-culture with purified naïve DO11.10 or HDM-sensitized CD4+ T cells as compared to WT (Figure 4C, Supplementary Figure 7C). To explore the mechanism underlying the observed phenotype in Atg5-/- BM-DCs we evaluated the amount of Caspase-1 in Atg5-/- and WT BM-DCs cultured in the presences or absence of LPS for 24 hours and found that Atg5-/- BM-DCs express significantly higher amount of Caspase-1 in the presence or absence of LPS (Supplementary Figure 7D). Taken together, these results suggest that Atg5-/-BM-DCs enhance IL-17 production through elevated IL-1 and IL-23 pathway.
Since we found that impairment of autophagy increases the production of IL-1 and IL-23 by BM-DCs and IL-17 by T cells in vitro, we tested whether impairment of autophagy in DCs induces severe neutrophilic inflammation in vivo. Mice were immunized with HDM-loaded BM-DCs as shown in figure 5A. AHR was significantly higher in the mice immunized with HDM-loaded Atg5-/- BM-DCs compared to WT BM-DCs (Figure 5B-C). Analysis of BAL contents showed a high number of neutrophils but not eosinophils in Atg5-/- BM-DC immunized mice compared to WT-BM-DC immunized mice (Figure 5D). Analysis of cytokines in lung lysates showed a higher level of IL-17A but not IL-4, IL-1β or IL-13 production in the lungs of Atg5-/- BM-DCs immunized mice compared to WT BM-DCs immunized mice (Figure 5E).
To confirm these findings, we generated CD11c-specific ATG5-/- mice (CD11c-Atg5-/-) as described in online repository methods. Interestingly, we found that CD11c-Atg5-/- mice developed unprovoked asthma. Lung resistance is higher and dynamic compliance is lower in CD11c-Atg5-/- than WT mice at the steady state (Figure 5F-G). Our results further show that specific impairment of autophagy in CD11c+ cells leads to a substantially high number of neutrophils and to a lesser extend eosinophils in the BAL of mice at the steady state (Figure 5H).
The presence of neutrophils in the lungs is predominant in patients with severe refractory asthma and neutrophils do not respond to steroid treatment7,8,10. We tested whether AHR in Atg5-/- mice is IL-17 dependent and steroid resistant. Mice were immunized with HDM according to the protocol of Figure 1A. Dexamethasone or anti-IL-17A blocking antibody were administered one day before HDM challenge. In WT mice, steroid treatment significantly reduced AHR and the presence of eosinophils. However, the level of AHR and number of neutrophils in BAL of Atg5-/- mice were resistant to steroid treatment even though eosinophils counts were decreased (Figure 6A and 6B). Blockade of IL-17A significantly reduced AHR and neutrophilic inflammation in Atg5-/- mice, but had no obvious effects on AHR and BAL analyses of WT mice (Figure 6D and 6E). Histological analyses also revealed that steroid treatment decreased airway thickness and infiltrated cells in WT mice, but only had marginal effect on the Atg5-/- mice. In contrast, anti-IL-17A therapy reduced inflammatory response with Atg5-/- mice (Figure 6C and 6F). These results indicated that Atg5-/- mice developed steroid resistant IL-17-dependent AHR with enhanced neutrophilic inflammation.
In the present study, we examined the involvement of autophagy in the pathogenesis of neutrophilic lung inflammation. We found that lack of autophagy causes a severe IL17-mediated neutrophilic lung inflammation. We showed that impaired autophagy in BM-DCs significantly increased proinflammatory cytokines, in particular IL-1 and IL-23. Using BM-DCs immunization experiments and CD11c specific Atg5-/- mice we show that lack of autophagy in CD11c+ cells lead to sever neutrophilic airway inflammation and AHR. Importantly, we showed that lack of autophagy in CD11c+ cells leads to the development of spontaneous asthma. Moreover, impairment of autophagy causes glucocorticoid-resistance and IL-17A dependent lung inflammation.
Our findings that impaired autophagy is involved in the pathogenesis of asthma is supported by several lines of research that showed association of Atg5 polymorphisms with asthma in human studies19,20 and showed that Th2 cytokines17,32 and virus infection inhibit autophagy37,38, whilst Th1 cytokine induces autophagy. Importantly, impaired autophagy itself can further induce Th17 polarization resulting in refractory asthma. A randomized clinical trial shows that carbamazepine, an anticonvulsant drugs and autophagy inducer, is significantly efficacious in severe asthma patients39, underscoring the importance of the autophagy pathway in the pathogenesis of asthma. Taken together, autophagy plays a protective role in asthma and inducers of this pathway may represent novel therapeutic targets for asthma in particular corticosteroid resistance asthma.
Our findings also suggest that impaired autophagy induces IL-17 production by T cell subsets including TCR-γδ and TCR-αβ T cells and IL-17-mediated corticosteroid resistant asthma. The importance of IL-17 is highlighted in certain patients with severe asthma that have increased levels of IL-17A, which correlate with neutrophilic airway inflammation and AHR11,13.
Besides IL-6 and TGF-β, IL-1α, IL-1β, and IL-23 are secreted by BMDCs and play a crucial role in the differentiation of Th179,36. Secretion of IL-1β is controlled by caspase-1 through the inflammasome pathway and consequently promotes IL-17 production by T cells. This caspase-1 mediated IL-1β production is negatively regulated by autophagy40. The observed neutrophilic airway inflammation in Atg5-/- mice in our studies may be explained by the enhanced production of IL-1α, IL-1β, and IL-23 by DCs in Atg5-/- mice. Autophagy also regulates IL-1α secretion and is dependent upon ROS and the calpain pathway, but independent of the inflammasome pathway41.
Airway epithelial cells act as an external barrier against foreign environmental antigens by secreting mucous and interacting with innate and adaptive immune cells. Interestingly autophagy in epithelial cells plays a distinct role in the pathogenesis of COPD29,30, but is protective in cystic fibrosis, lung fibrosis, and acute lung injury16,42-46. As the pathogenesis of asthma and COPD are different, we found no evidence that non- hematopoietic cells including AECs and lung bronchial epithelial cells play a crucial role in the induction of IL17-mediated corticosteroid resistant asthma in Atg5-/- mice.
We found that severe asthma impairs autophagy in pulmonary DCs and that autophagy disrupted DCs more potently induced IL17 secretion in T cells. Autophagy deficiency in DCs affects antigen presentation and induces hyper-stable interactions and activation of T cells47-49. Furthermore, blockade of autophagy inhibits degradation of the adapter protein BCL-10, which plays a key role in transmitting signals from the TCR50. This may explain the increased number of effector T cells in Atg5-/- mice with AHR. Furthermore, adoptive transfer of autophagy deficient DCs enhanced AHR, and severe steroid resistant AHR in Atg5-/- mice was neutralized by anti-IL-17A antibody. The recipients of WT bone marrow fail to show an increased AHR in our adoptive transferred experiment suggesting that a more sever model of asthma is needed to induce AHR in adoptive transfer experiments.
In conclusion, the present study demonstrates that impaired autophagy causes severe neutrophilic inflammation, and this condition is mainly mediated by modulated DCs function. We suggest a model where initiation of asthma leads to impairment of autophagy in pulmonary CD11c+ cells through Th2 cytokines IL-4 and IL-13 and impairment of autophagy in turn causes neutrophilic lung inflammation and more severe asthma. Therefore, these findings suggest a protective role for autophagy in the pathogenesis of asthma and improving the autophagy pathway in the lung might be an effective therapeutic target for patients with refractory severe asthma, especially for IL-17A mediated neutrophilic asthma patients.
Female C57BL/6, BALB/c, CD45.1 and OT-II mice (6 to 8 weeks old) were purchased from Jackson Laboratory (Bar Harbor, ME). Atg5flox/flox and LC3-GFP mice are the gift from Dr. Noboru Mizushima (Tokyo Medical and Dental University). CD11c specific Atg5-/- mice were generated by crossing Atg5flox/flox mice to CD11c-Cre mice. Mice were screened by PCR and Atg5flox/flox homozygote CD11c-Cre hemizygote mice were selected for the experiments. Atg5flox/flox mice were backcrossed to rosa26Cre ERT mice and sftpc-cre mice (Jackson Laboratories) were bread in our facility at the Keck School of Medicine, University of Southern California under protocols approved by the Institutional Animal Care and Use Committee. To induce the deletion of Atg5 mice received tamoxifen (800 μg/mouse/day) for five consecutive days (Figure 1A).
Mice were sensitized intranasally (i.n.) with 200μg of house dust mite (HDM, Stallergenes, MA) on Day 1, and then followed by 100μg HDM (i.n.) on Day 8 and 15. In some experiments, mice were treated intraperitoneally (i.p.) with dexamethasone (Sigma-Aldrch, St Louis, MO) or PBS, and anti-mouse IL-17A antibody (clone 17F3, BioXcell, West Lebanon, NH) or IgG1 isotype control antibody before each HDM challenge. One day after the last HDM challenge lung resistance and dynamic compliance were measured using Fine Pointe RC System (Buxco Research Systems, Wilmington, NC) as previously described57,58.
After measurements of AHR, the trachea was cannulated and the lungs lavaged three times with 1ml ice cold PBS to collect BAL cells as previously described57,59. Transcardial perfusion of the lungs with cold PBS was then performed to remove red blood cells and the lungs fixed and harvested for histology with 4% paraformaldehyde in PBS as described previously58,60. In some experiments, the lungs were collected and homogenized in 3ml of RIPA Buffer (Millipore, Temecula, CA). The homogenates were analyzed for cytokines as described elsewhere58.
The composition of BAL cells were analyzed using flow cytometry as described previously60. The repertoire of pulmonary DCs were analyzed using flow cytometry as described elsewhere35.
To identify T cell subsets and innate lymphoid cells the following antibodies and reagent were used: FITC anti-CD45 (clone 30F11, Biolegend), PE anti-TCR-γδ (clone GL3, Biolegend), PerCP/Cy5.5 anti TCR-β (clone H57-597, eBiosciences), biotin anti- (Gr-1 (clone RB6-8C5, Biolegend), anti-B220 (clone RA3-6B2, Biolegend), anti-CD11c (clone N418, Biolegend), anti-TER119 (clone TER-119, Biolegend), anti-CD11b (clone M1/70, Biolegend), anti-FCεRI (clone MAR-1, Biolegend)), Brilliant Violet 510™ Streptavidin (Biolegen) and eFluor 780 dead cell discrimination dye (eBioscience).
For intracellular cytokine evaluation, lung single cell suspension was made using collagenase D (Worthington Biochemical corp., NJ, USA). Lung cells were then cultured were cultured in RMPI with 10% FCS in the presence of PMA/Ionomycin (50/500 ng/ml) for 5 hours. Cells were then fixed using BD Cytofix/Cytoperm kit (BD bioscience, San Jose, CA) according to the manufacture's instruction. IL-17A intracellular staining was performed and live CD45+ IL-17+ single cells were further analyzed for the expression of TCR-β, TCR-γδ and the lack of expression of CD11b, CD11c, Gr-1, Ter-119, B220 and FCεRI for ILCs. In some experiments the cell surface was first stained with APC-Cy7 labeled anti-CD3 (clone 145-2-C11, Biolegend), eFluor-450 labeled anti-CD44 (clone IM7, ebioscience), and Brilliant Violet-510 (BV510) labeled anti-CD4 (clone RM4-5, Biolegend). After fixation and permeabilization, cells were then stained with FITC labeled anti-IFN-γ (clone XMG1.2, ebioscience), PE labeled anti-IL-5 (clone TRFK5, ebioscience), PE labeled anti-IL-13 (clone eBio13A, ebioscience), Alexa Fluor-647 labeled anti-IL-4 (clone 11B11, ebioscience), and PE-Cy7 labeled anti-IL-17A (clone TC11-18H10.1, Biolegend). Flow cytometry was carried out on the FACSCanto II (BD Bioscience) and the data were analyzed with FlowJo version 8.6 software (TreeStar, Ashland, Oregon).
Mice were irradiated with 600 rad and injected the following day with 5 × 106 BM cells. Reconstitution of lung leukocytes was approximately 90% by the flow cytometry using PE-Cy7 conjugated anti-CD45.1 (clone A20, Biolegend).
BM cells were harvested and cultured as previously described57,58. After eight days in culture with GM-CSF, BM-DCs were harvested and incubated with HDM (80μg/ml) for 6 hours. BM-DCs (1 × 105) were then adoptively intravenously transferred into naïve C57BL/6 mice (day 1) and challenged intratracheally (i.t.) with 1 × 105 BM-DCs on day 8 and 15 and sacrificed on day 17.
Naïve CD4+ T cells were obtained from OT-II murine spleens using CD4-conjugated magnetic beads (Miltenyi Biotec, Auburn, CA) and positively sorted by magnetic cell sorting (MACS; Miltenyi Biotec). Purified CD4+ T cells (5 × 105/ml) were co-cultured with BM-DCs (1 × 105/ml) for three days in the presence of O VA323-339 peptide (10 mg/ml, Invivo Gen, San Diego, CA). In some experiments, BMDCs (2.5 × 106/ml) were incubated with HDM (50 μg/ml), LPS (1 mg/ml, Sigma-Aldrich, St Louis, MO) for one or two days.
For generating HDM-specific T cells WT mice were sensitized with HDM according to the protocol described in Figure 1A and one day after the sensitization, total CD4+ T cells were isolated from the lung using magnetic isolation (Miltenyi biotec). Purified CD4+ cells (1×101 cells/ml) were co-cultured with BM-DCs from CD11c-Atg5-/- or WT mice (2 × 105 cells/ml) in the presence or absence of HDM (10 μg/ml) for 3 days.
The levels of cytokines were measured by ELISA (eBioscience), according to the manufacture's instruction.
For measuring Atg5 or SPC-Cre expression, mRNA was extracted and converted to cDNA for each mouse and quantified by Real Time-Polymerase Chain Reaction (RT-PCR). Total RNA was extracted from BM-DCs incubated for one day with LPS (1 μg/ml) using the RNAasy mini kit (Qiagen, Valencia, CA) and cDNA generated with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). RT-PCR was performed using CFX96 thermal cycler (Bio-Rad, Hercules, CA) and the ΔΔCt method was used for data analysis.
BALB/c, C57BL/6 and LC3-GFP knock-in transgenic mice were immunized with HDM with Alum (Al(OH)3, Thermo, IL) on day 1, and then challenged with HDM or PBS (i.n.) on day 8, 10, 12 and 14. On day 15, the lungs were either isolated for lung lysates or for lung CD11c+ cells using magnetic beads and sorting by Auto-MACS (Miltenyi Biotec). Autophagy levels were quantified by Western blot and confocal microscopy analysis. For Western blot analysis, expression of p62 (MBL, Nagoya, Japan), LC3 (Cosmo, Tokyo, Japan), and Actb (Santa Cruz, CA) were assessed. Lung CD11c+ cells isolated from LC3-GFP mice and LC3 expression was analyzed by confocal microscopy (Nikon, Instruments, Melville, NY).
P-Values for lung function data were calculated by repeated measures analyses of variance, P-values for other data were calculated using student t test. P values of less than 0.05 were considered significant. All data are expressed as the mean ± SD. Statistical analyses were performed using JMP Start Statistics (SAS Institute Inc., NC, USA)
Our findings offer an explanation for pathogenesis of neutrophilic asthma. Severe asthma causes impairment of autophagy in pulmonary CD11c+ cells which in turn causes neutrophilic asthma.
Supplementary Figure 1. Confirmation of depletion of Atg5 in the lung and spleen of Atg5-/- mice
ROSA CRE ERT-Atg5 and WT mice received tamoxifen (800 μg/mouse/day) for five consecutive days. Six days after the last tamoxifen injection mice were euthanized and relative gene expression of Atg5 in lung and spleen of ROSA CRE ERT-Atg5-/- and WT were assessed by RT-PCR. Values are expressed as the mean ± SEM.
Supplementary Figure 2. Representative dot plot presentation of cytokine production by T cells in the lung. Atg5-/- and WT mice were sensitized to HDM as described in Figure 1A. One day after the last sensitization lung single cells were cultured in the presence of PMA/Ionomycin as described in the methods section followed by analysis of cytokine production by flow cytometry.
Supplementary Figure 3. T cells are the major source of IL-17 production in the lungs of Atg5-/-mice. Atg5-/- and WT mice were sensitized as mentioned in figure 1A. One day after the last sensitization lungs were harvested and cultured in the presence of PMA/Ionomycin for 5 hours followed by evaluation of IL-17 production by flow cytometry. Live IL-17+ CD45+ single cells were further gated to identify the source of IL-17A production. Bar graphs show the percent of each subsets within IL-17+ cells as indicated.
Supplementary Figure 4. The proportion of different subsets of antigen presenting cells in the lungs of Atg5-/- and WT mice is similar. Deletion of Atg5 gene was induced in ROSA-Atg5-/- mice using Tamoxifen subsequently Atg5-/- and WT mice were sensitized as mentioned in Figure 1A. One day after the last HDM sensitization lungs of HDM-sensitized or control mice were analyzed for different subsets of antigen presenting cells. A. Bar graph shows the number of Alveolar macrophages, plasmacytoid dendritic cells, CD103 and CD11b dendritic cells in the lungs. B. Dot plots show the gating strategy. Red rectangles and arrows show gating hierarchy. Data are expressed as the mean ± SEM. P-values were calculated with student t test *, P<0.05. (n=4-5 mice/ group)
Supplementary Figure 5. BAL neutrophils show similar viability in Atg5-/- and WT mice. BAL of HDM-sensitized or control Atg5-/- and WT mice were analyzed for the viability of neutrophils using flow cytometry.
Supplement Figure 6. Lack of autophagy in lung epithelial cells does not contribute to induction of AHR
A. Relative expression level of SPC-Cre gene were assessed by RT-PCR. B. WT and SPC-Atg5-/- mice were immunized i.n. with HDM as described in Figure 1A. The mice were subsequently assessed for AHR. Pooled data from two experiments are shown (n=8/group). C. Lung tissues from WT and Atg5-/- mice were stained with H&E. Original magnification × 200. D. Cytokine levels in whole-lung lysates of asthmatic WT and SPC-Atg5-/- mice immunized as in Figure 1A (n=5/group). Values are expressed as the mean ± SEM. P-values were calculated with student t test. *, P<0.05. RL, lung resistance; Cdyn ,dynamic compliance TCC, total cell number; MAC, Macrophages; Eos, Eosinophils; Lym, Lymphocytes; Neut, Neutrophils.
Supplementary Figure 7. 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 LPS (1μg/ml) for 1 or 2 days. The cytokine levels in the supernatants were measured by ELISA (n=6-8/group). B. Quantification of mRNA expression by RT-PCR (n=6/group). C. BM-DCs from WT or Atg5-/- mice were co-cultured with DO11.10 CD4 T cells in the presence of OVA peptide (OVA323-339) for 3 days. The cytokine levels in the supernatants were measured by ELISA. D. the level of Caspase-1 as measured by flow cytometry in Atg5-/- and WT BM-DCs in the presence or absence of LPS (1 μg/ml) for 24 hours. One representative experiment of two is shown (n=4/group). Data are expressed as the mean ± SEM. P-values were calculated with student t test. *, P<0.05.
Studies described in this article were financially supported by National Institutes of Health Public Health Service Grant R01 AI 066020, R01 ES 021801, R21 ES 024707 (O. A.) and R01 HL110609, R01 AI073099 (J.U.J.). Hadi Maazi is supported by American Association of Immunology (AAI) Careers in Immunology Fellowship.
The authors have declared that no conflict of interest exists.
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.