Hypoxia Response in Asthma

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Hypoxia Response in Asthma Differential Modulation on Inflammation and Epithelial Injury Tanveer Ahmad1, Manish Kumar1, Ulaganathan Mabalirajan1, Bijay Pattnaik1, Shilpi Aggarwal1, Ranjana Singh1, Suchita Singh1, Mitali Mukerji1, Balaram Ghosh1, and Anurag Agrawal1 1

Centre for Excellence in Asthma & Lung disease, CSIR-Institute of Genomics and Integrative Biology, Delhi, India

Oxygen-sensing prolyl-hydroxylase (PHD)-2 negatively regulates hypoxia-inducible factor (HIF)1-a and suppresses the hypoxic response. Hypoxia signaling is thought to be proinflammatory but also attenuates cellular injury and apoptosis. Although increased hypoxic response has been noted in asthma, its functional relevance is unknown. The objectives of this study were to dissect the mechanisms and role of the hypoxic response in asthma pathophysiology. Experimental studies were conducted in mice using acute and chronic allergic models of asthma. The hypoxic response in allergically inflamed lungs was modulated by using pharmacologic PHD inhibitors (ethyl-3–4-dihydroxybenzoic acid [DHB], 1–10 mg/kg) or siRNA-mediated genetic knockdowns. Increased hypoxia response led to exacerbation of the asthma phenotype, with HIF-1a knockdown being beneficial. Chronically inflamed lungs from mice treated with 10 mg/kg DHB showed diffuse up-regulation of the hypoxia response, severe airway remodeling, and inflammation. Fatal asphyxiation during methacholine challenge was noted. However, bronchial epithelium restricted up-regulation of the hypoxia response seen with low-dose DHB (1 mg/kg) reduced epithelial injury and attenuated the asthmatic phenotype. Up-regulation of the hypoxia response was associated with increased expression of CX3CR1, a lymphocyte survival factor, and increased inflammatory cell infiltrate. This study shows that an exaggerated hypoxia response may contribute to airway inflammation, remodeling, and the development of asthma. However, the hypoxia response may also be protective of epithelial apoptosis at lower levels, and the net effects of modulating the hypoxia response may vary based on the context. Keywords: hypoxia; HIF-1a; PHD2; apoptosis

Increased activation of the hypoxia response pathway has been noted in bronchial biopsies of patients with asthma and chronic obstructive pulmonary disease (1, 2). Induction of hypoxiainducible factor (HIF)-1a is considered to be proinflammatory and leads to transcriptional activation of key genes implicated in airway remodeling, such as vascular endothelial growth factor (3–5). Furthermore, induction of the hypoxia response has been associated with increased collagen synthesis, fibrosis, and proliferation of fibroblasts (2, 6–8). Yet, in a cobalt-induced model of lung injury, conditional total deletion of HIF-1a in the lung epithelium enhanced eosinophilic inflammation and mucus cell metaplasia, suggesting that epithelial-derived HIF signaling has a critical role in establishing a tissue’s inflammatory response, and

CLINICAL RELEVANCE The hypoxic response mediated by hypoxia-inducible factor-1 promotes cell survival during inflammation. Depending on the context, namely epithelial cells versus inflammatory cell infiltrate, this may attenuate or enhance features of asthma, respectively. We found fatal bronchoconstriction in some mice with allergic airway inflammation additionally treated with high-dose prolyl hydroxylase inhibitors that induce hypoxic response and are being considered as drugs in cancer.

compromised HIF-1a signaling may bias the tissue toward a Th2mediated reaction (9). Thus, the effects of hypoxia response may be context sensitive, varying across tissue compartments of inflamed lungs as well as dependent upon other genes that regulate the hypoxia response. Hypoxic cell response, exemplified by increased HIF-1a, is critically regulated by oxygen-dependent hydroxylation of HIF-1a by prolyl hydroxylase 2 (PHD2, also known as “Egl nine homolog 1”), leading to HIF-1a being degraded (2, 3, 10–13). We and others have recently shown that common single-nucleotide polymorphisms in the first intron of PHD2 are associated with its altered expression and with a variant hypoxic response, high-altitude adaptation, and highaltitude pulmonary edema (14, 15). Further functional investigations of the role of hypoxic response in asthma are needed, and some aspects are clarified in this manuscript. Because PHD2 is the master regulator of HIF-1a and thereby the hypoxic response, we investigated the functional roles of PHD2 and HIF-1a in modulating the asthma phenotype using mouse models of acute and chronic allergic airway inflammation.

MATERIALS AND METHODS Animals Male BALB/c or C57B/6 mice (8–10 wk old; National Institute of Nutrition, Hyderabad, India) were acclimatized for 1 week before starting the experiments. All animals were maintained as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, and the protocols were approved by the Institutional Animal Ethics Committee.

Grouping of Mice (Received in original form June 15, 2011 and in final form January 26, 2012) This work was supported by the Swarnajayanti Fellowship (A.A.) and projects MLP 5501 and NWP0033 from the Institute of Genomics and Integrative Biology, Council of Scientific and Industrial Research, Government of India. Correspondence and requests for reprints should be addressed to Anurag Agrawal M.D., Ph.D., CSIR-Institute of Genomics and Integrative Biology, Mall Road, Delhi-110007. E-mail ID: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Cell Mol Biol Vol 47, Iss. 1, pp 1–10, Jul 2012 Copyright ª 2012 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2011-0203OC on February 2, 2012 Internet address: www.atsjournals.org

Mice were divided into two groups as acute and chronic according to the dose of allergen and duration of allergen challenge. Each group was further divided into three groups (n ¼ 6), which were named according to sensitization/challenge/treatment: DHB-treated mice without airway hyperresponsiveness (Sham)/vehicle (Veh) (normal controls), chicken egg ovalbumin (OVA)/Veh (allergic controls) (Grade V; Sigma, St. Louis, MO), and OVA/ethyl 3,4-dihydroxybenzoic acid (DHB) (TCI AMERICA, Portland, OR). DHB (1, 5, and 10 mg/kg in 200 ml volume) was administered by intraperitoneal injection for 7 consecutive days 2 hours before every local challenge in the acute model of asthma, and siRNA was given 2 hours before the challenge on the last 3 days. To further validate the effect of DHB treatment on potentiating asthma features, 1 or 10 mg was administered to mice in the chronic model of asthma for last 15 days of challenge.



Sensitization, Challenge, and Treatment of Mice Mice were sensitized and challenged as described previously (16). OVAsensitized mice had elevated specific IgE to ovalbumin compared with sham (mean 6 SEM; 1.7 6 0.5 vs. 0.7 6 0.2 in arbitrary units) as measured by ELISA (16). Vehicle or DHB was given by intraperitoneal injection from Day 21 to 27 in acute mice and from Day 36 to Day 50 in chronic mice, whereas as siRNA was given intranasally.

Airway Hyperresponsiveness Measurement Airway hyperresponsiveness (AHR) to methacholine (MCh) (Sigma, St. Louis, MO) was determined by flexivent (SCIREQ, Montreal, PQ, Canada), as described elsewhere with some modifications (17).

Lung Histopathology Formalin-fixed, paraffin-embedded lung tissue sections were stained with hematoxylin and eosin, periodic acid-Schiff, and Masson’s trichrome staining as described previously (16).

siRNA Delivery into the Lungs siRNA was delivered into the mouse lungs by a well established intranasal route. Briefly, 90 mg of siRNA in 60 ml volume (PHD2 or HIF-1a or scrambled siRNA; Dharmacon, Lafayette, CO), dissolved in ultrapure DNase and RNAse free water with in vivo-jetPEI as the transfection reagent (Polyplus Transfection, Illkirch France), was administered intranasally to isoflourene-anesthetized mice.

Measurement of CX3CR1 and TUNEL Apoptotic Assay CX3CR1 ELISA (USCNK, Wuhan, China) and TUNEL assay of apoptosis (Dead End Calorimetric TUNEL System; Promega, Madison, WI) were performed as per the manufacturers’ instructions.

Measurement of IL-5, IL-13, TGF-b1, and Total Collagen Levels IL-5, TGF-b1 (BD Biosciences, Sparks, MD), and IL-13 (R&D Systems, Minneapolis, MN) were measured in lung homogenates by ELISA as per the manufacturers’ protocols. Total lung collagen content was measured by

sircol assay (Biocolor Life Science Assays, Carrickfergus, UK) as described previously (18).

Statistical Analysis Data are expressed as means 6 SEM. Statistical significance was set at P < 0.05. ANOVA with post hoc correction was used to compare groupwise data.

RESULTS PHD2 and HIF-1a Are Increased in Experimental Models of Asthma, and Hypoxia Response Varies across Cell Compartments in the Lung

A graphical model of the experimental design is shown in Figure 1. To determine whether the PHD2/HIF-1a axis is altered in asthma, we estimated the expression of these proteins in normal and allergically inflamed mouse lungs. HIF-1a was increased during acute airway inflammation (AAI) (see Figure E1a in the online supplement), with the HIF-1a increase mostly seen in bronchial epithelium (Figure E1c). The rise in HIF-1a was limited by a compensatory rise in PHD2 (Figure E1b). To further determine the role of PHD2 or HIF-1a on asthma features and hypoxia responses, allergically inflamed mice were treated with specific siRNAs to PHD2 and HIF-1a or PHD inhibitor DHB (1, 5, and 10 mg/kg). HIF-1a levels were estimated in allergically inflamed lungs. PHD2 siRNA decreased PHD2 protein in lungs of mice with AAI to normal levels (Figure E2a). This was associated with a significant increase in HIF-1a compared with mice that received scrambled siRNA (Figures E2 and E3). In contrast, HIF-1a siRNA decreased HIF-1a expression to normal levels (Figure E3) and did not affect PHD2 expression compared with scrambled siRNA (Figure E2b). HIF-1a was dose-dependently increased by DHB treatment (Figure E4). Immunohistochemical studies showed that with a low dose of DHB (1 mg/kg body weight), HIF-1a was mostly localized to bronchial

Figure 1. Schematic diagram of experimental design. Induction of allergic asthma in male BALB/c mice. Mice were grouped, sensitized, and challenged with chicken egg ovalbumin (Ova) as allergen. Ethyl-3–4-dihydroxybenzoic acid (DHB) was given intraperitoneally from Day 21 to 27 in acute allergic model (A) or from Day 36 to 50 in the chronic allergic model (B). siRNA was given on Day 25 to 27. Animals were killed on Day 28, and measurements of lung function were performed on Day 28 in the acute model and on Day 51 in the chronic model. AHR ¼ airway hyperresponsiveness.

Ahmad, Kumar, Mabalirajan, et al.: Hypoxic Response Modulates Asthma


Figure 2. Measurement of airway mechanics and structural changes in DHB-treated mice in acute allergic model. (A) Dose-dependent increase in airway resistance with DHB treatment. Treatment with 1 mg of DHB shows a slight improvement in AHR, whereas treatment with 5 and 10 mg of DHB shows a dose-dependent increase in AHR. Similar results were found with lung histology with 1 mg showing a significant decrease, whereas 5 and 10 mg showed a dose-dependent increase in airway inflammation (B1, B2), mucus production (C1, C2), and collagen deposition (D1, D2). Data are shown as mean 6 SEM. *P , 0.05 versus Sham/Veh; yP , 0.05 versus OVA/Veh; zP . 0.05 versus OVA/Veh (n ¼ 6 per group). OVA ¼ chicken egg ovalbumin; Veh ¼ vehicle (control).



siRNA administration modestly improved AHR. Corresponding changes were observed in airway inflammation, mucus metaplasia, and collagen deposition. PHD2 knockdown increased airway inflammation, airway mucin content, and collagen deposition; HIF-1a knockdown attenuated airway inflammation and collagen deposition (Figures 3B–3D). Corresponding changes in inflammatory cytokine levels were also seen (Table 1).

epithelia, similar to the allergic control mice without an obvious increase in total lung nuclear lysate (Figures E4 and E5). In contrast, the highest dosage (10 mg/kg body weight) treatment was associated with a substantial and diffuse increase in HIF-1a, extending beyond the epithelium to subepithelial mesenchymal cells and peribronchial inflammatory cells, when compared with low-dose DHB. Thus, hypoxia response is increased in asthma but varies across cell compartments, being most prominent in the epithelial cells in control allergically inflamed lungs as well as low-dose DHB treatment but was seen predominantly in the subepithelium when strongly potentiated by high-dose DHB treatment. Increased subepithelial cellularity was noted in mice treated with high-dose DHB. Counterregulatory mechanisms, such as an increase in PHD2, limit the hypoxic response.

Chronic Inhibition of Prolyl Hydroxylase in a Chronic Model of Asthma Shows Dose-Related Variations in the Effect on Airway Hyperresponsiveness

To determine the effect of potentiation of hypoxic response during prolonged allergen exposure, analogous to human asthma, we tested the effect of 1 and 10 mg/kg DHB in a chronic mouse model of asthma. The effects were contrasting across these doses, with attenuation of the asthma phenotype seen at low dose and marked accentuation of asthma seen at high dose (Figure 4). High-dose chronic inhibition of PHD was associated with exquisite sensitivity to MCh, with asphyxiation being noted in few mice with progressive difficulty in ventilation with extremely high and unstable values of airway resistance. We carefully excluded technical artifacts, and this was reproducible on repetition in a fresh batch of mice. Figure 4 shows the flexivent data for each MCh dose using survivors.

Exaggerated Hypoxic Response Increases Airway Hyperresponsiveness, Airway Inflammation, and Airway Remodeling

Because high doses of DHB were associated with potentiation of hypoxia response, the effects of this potentiation on asthma phenotype during acute AAI were studied. Mice with AAI developed a dose-dependent increase of airway hyperresponsiveness to MCh upon treatment with DHB (Figure 2A). This effect was statistically and biologically significant at 5 and 10 mg/kg DHB treatment, being associated with increased baseline airway resistance and a nearly doubled response to MCh compared with vehicletreated OVA mice. Quantitative scoring and morphometry of lung sections showed that this was associated with increased cellular infiltration, mucus metaplasia of the bronchial epithelium, and increased subepithelial collagen deposition (Figures 2B–2D). Corresponding changes in inflammation and inflammatory cytokine levels were seen (Table 1 and Table E1 in the online supplement). A dosage of 1 mg/kg was associated with a trend toward improvement of the asthma phenotype. DHB treatment had no significant effect on naive mice even at a dose of 10 mg/kg, suggesting that this was not a direct toxic effect (Figure E6). A slight increase in collagen deposition could be seen. In confirmation, siRNA-mediated PHD2 knockdown was associated with increased AHR, similar to results from DHB treatment (Figure 3A). In contrast, HIF-1a

PHD2 Inhibition Changes the Pattern of Inflammation, Cellular Injury, and Apoptosis in the Lung

The initial increase in cellular inflammatory cells followed by a gradual decline during chronic allergen challenge is well known. Highdose DHB treatment, but not low-dose treatment, was associated with persistence of the inflammatory cell infiltrate at levels similar to that seen in the acute model (Figure 5A compared with Figure 2B1). Corresponding changes in inflammatory cytokine levels were also seen (Table 1). To determine the mechanisms of increased inflammation, we determined the expression of CX3CR1, which promotes T-helper lymphocyte survival in inflamed lungs (19). Treatment with highdose DHB, but not low-dose DHB, was associated with increased

TABLE 1. MEASUREMENT OF CYTOKINES AND LUNG COLLAGEN CONTENT Treatment Acute Sham/Veh Ova/Veh Ova/1 mg DHB Ova/5 mg DHB Ova/10 mg DHB Chronic Sham/Veh Ova/Veh Ova/1 mg DHB Ova/10 mg DHB siRNA treated Sham/Veh Ova/Veh Ova/HIF siRNA Ova/PHD 2siRNA Ova/Scram siRNA

IL-5 Levels in Lung (pg/100 mg protein)

IL-13 Levels in Lung (pg/100 mg protein)

TGF-b1 Levels in Lung (pg/100 mg protein)

Total Lung Collagen (mg/100 mg protein)

13.3 33.9 23.86 37.25 52.06

6 6 6 6 6

2.2 5.6* 2.5† 10.4† 3.7‡

10.3 30.1 24.24 37.66 40.4

6 6 6 6 6

1.06 2.6* 2.3† 3.2† 3.02‡

110.0 334.1 242.3 376.4 469.09

6 6 6 6 6

8.3 26.35* 24.0† 22.8† 37.6‡

4.09 12.3 7.4 11.5 16.2

6 6 6 6 6

1.03 1.02* 1.0‡ 1.0† 2.1‡

9.64 36.7 23.8 51.3

6 6 6 6

2 1.4* 2.7‡ 2.3‡

8.8 31.8 17.1 46.9

6 6 6 6

1 2.06* 2.4‡ 2.8‡

486.02 1,396.2 993.8 1,775.6

6 6 6 6

87.4 211.4* 164.8‡ 53.8‡

5.8 30.42 13.1 42.0

6 6 6 6

1.3 2.5* 2.3‡ 1.2‡

12.8 37.5 23.6 51.9 37.6

6 6 6 6 6

2.5 3.9* 3.5‡ 3.9‡ 5.8*

11.8 38.5 26.4 52.3 36.6

6 6 6 6 6

1.9 5.4* 3.5‡ 2.7‡ 3.6*

348.2 1,131 830.2 1,732.8 1,173.3

6 6 6 6 6

43.7 140* 78.7‡ 181.8‡ 148.4*

4.0 12.9 8.0 17.6 13.1

6 6 6 6 6

0.4 1.0* 0.9‡ 1.7‡ 1.3*

Definition of abbreviations: DHB ¼ ethyl-3–4-dihydroxybenzoic acid; HIF ¼ and hypoxia-inducible factor; PHD ¼ prolyl-hydroxylase; Scram ¼ scrambled; Sham ¼ DHB-treated mice without airway hyperresponsiveness; DHB-treated mice without AAI; Veh ¼ vehicle (normal control). Data are shown as mean 6 SEM. * P , 0.05 vs. Sham/Veh. y P , 0.05 vs. Sham/Veh and . 0.05 vs. Ova/Veh. z P , 0.05 vs. Ova/Veh or Ova/Scram/SiRNA.

Ahmad, Kumar, Mabalirajan, et al.: Hypoxic Response Modulates Asthma


Figure 3. Prolyl-hydroxylase (PHD)2 knockdown worsens and hypoxia-inducible factor (HIF)-1a knockdown improves asthma features in acute airway inflammation (AAI). Airway hyperresponsiveness to methacholine challenge (A), and histological studies; hematoxylin and eosin (B1, B2) for cellular infiltration, periodic acid-Schiff (C1, C2) for mucus and Masson’s trichome staining (D1, D2) for collagen are shown along with quantitative morphometry of inflammation. In mice with allergically inflamed lungs, AHR was attenuated by HIF-1a knock-down and is markedly increased by PHD2 knock-down (A). This is in consonance with histological changes (B–D). Data are shown as mean 6 SEM. * P , 0.05 versus Sham/Veh. y P , 0.05 versus Ova/Scram siRNA (n ¼ 6 per group).



Figure 4. Pharmacologic potentiation of hypoxic response worsens lung function in chronic AAI. Airway hyperresponsiveness in the form of airway resistance (R) (i), Newtonian resistance (Rn) (ii), and tissue damping (G) (iii), related to tissue resistance to methacholine challenge in ovalbumin-induced mice treated with DHB was estimated in the chronic AAI model. Treatment with 1 mg DHB showed a significant improvement in AHR, whereas treatment with 10 mg DHB was associated with increased AHR. Data are shown as mean 6 SEM. * P , 0.05 versus Sham/Veh; y P , 0.05 versus Ova/Veh (n ¼ 6 per group except for DHB-treated mice, where n ¼ 11).

CX3CR1 expression in lungs compared with relevant controls (Figure 5B). Up-regulation of CX3CR1 by hypoxic response could also be seen in the acute model, with HIF-1a siRNA being associated with reduced CX3CR1 expression but with PHD2 siRNA being associated with increased CX3CR1 expression (Figure 5C).

Because recruited inflammatory cells release proinflammatory mediators that injure structural cells, TUNEL assay was performed to determine whether the hypoxic response alters apoptosis of epithelial and mesenchymal cells. Acute and chronic models were associated with an increased number of TUNEL stain–positive cells. Although low-dose DHB treatment reduced

Figure 5. Pharmacologic potentiation of hypoxic response worsens airway inflammation and corresponds with CX3CR1 levels in lung homogenates. Histological studies: hematoxylin and eosin–stained lung sections (A1) are shown along with quantitative morphometry of inflammation (A2). Treatment with 10 mg/kg DHB was associated with inflammation compared with vehicle-treated control mice. DHB-treated mice without AAI (Sham) do not show significant pathology, but a slight increase in collagen can be seen (Figure E4). CX3CR1 levels were increased in AAI and further increased by DHB (B). PHD2 knockdown increased and HIF-1a knockdown decreased the CX3CR1 levels (C). Data are shown as mean 6 SEM. * P , 0.05 versus Sham/Veh; y P , 0.05 versus OVA/Veh or OVA/Scram siRNA (n ¼ 6 per group except for DHB-treated mice, where n ¼ 11).

Ahmad, Kumar, Mabalirajan, et al.: Hypoxic Response Modulates Asthma

only epithelial apoptosis, high-dose DHB treatment primarily reduced the percentage of TUNEL stain–positive cells in the subepithelial mesenchymal and peribronchial region where the inflammatory cell infiltrate was prominently seen (Figure 6A). siRNA to HIF-1a was associated with a significant decrease in epithelial apoptosis and an increase in mesenchymal cell apoptosis compared with the mice given scrambled siRNA, whereas PHD2 siRNA was associated with slightly decreased mesenchymal cell apoptosis and significantly increased epithelial cell apoptosis (Figure 6B). Confirmatory quantitative trends were observed in the levels of cytochrome c and caspase 9 in whole lungs, with DHB treatment reducing these apoptotic markers in the chronic model (Figure E7). However, in the acute model dominated by epithelial injury, siRNA to HIF-1a was associated with a significant decrease in apoptotic markers, whereas PHD2 siRNA was associated with increased apoptosis (Figures E7 and E8). Whereas low- and high-dose DHB treatments were associated with absolute reduction of apoptotic markers, TUNEL stains suggested that only low-dose DHB treatment reduced epithelial injury (Figures E7 and E9). To determine the net effect of DHB on airway remodeling in the chronic model of asthma, Masson’s trichrome and periodic acid-Schiff stainings were performed in lung sections. Histologic studies showed thickening of the subepithelial region with higher cellularity, excess collagen deposition, and increased mucus metaplasia during high-dose DHB treatment


(Figures 6C and 6D). Low-dose DHB treatment resulted in improvement of the asthma phenotype, possibly as a consequence of reduced epithelial injury.

DISCUSSION Modulators of hypoxic response are close to entering clinical practice, with inhibitors of HIF thought to have antitumor effects and inhibitors of PHDs thought to inhibit metastasis. Yet, this critical homeostatic pathway may have important functions in a variety of physiological and pathological settings that are not fully understood. In this study, we show for the first time that exaggeration of the hypoxic response by chronic high-dose inhibition of prolyl hydroxylase activity increases asthma severity in a mouse model to the extent that mice develop fatal asphyxia during MCh challenge. We relate this to the undesirable spread of the hypoxic response to inflammatory and mesenchymal cells, with increased cell survival and perpetuation of airway inflammation. At the same time, we find the opposite effects of 10-fold lower doses that maintain the epithelial localization of the hypoxic response. A synthesis of what is understood about regulation of the hypoxic response and how it may pertain to asthma is discussed further. It is well known that PHD2 is an oxygen sensor that regulates the hypoxic response. In physiological normoxic conditions, PHD2

Figure 6. Effect of pharmacologic and genetic potentiation of hypoxic response on airway epithelial injury and airway remodeling. TUNEL apoptotic assay done on paraffin-embedded sections and its quantitation as TUNEL positive (apoptotic) cells are shown. There was increased apoptotic epithelial and mesenchymal cells in OVA/Veh mice compared with Sham/Veh. DHB treatment or PHD2 knockdown reduced mesenchymal apoptosis without affecting epithelial apoptosis, whereas HIF-1a knockdown increased mesenchymal cells and reduced epithelial apoptosis (A and B). Decreased mucus production and collagen content with 1 mg of DHB was found, whereas 10 mg DHB treatment significantly increased mucus production and collagen content compared with the Ova mice (C1, C2 and D1, D2). Data are shown as mean 6 SEM. * P , 0.05 versus Sham/Veh; y P , 0.05 versus OVA/Veh or OVA/Scram/siRNA (n ¼ 6 per group except for DHB-treated mice, where n ¼ 11).



hydroxylates the constitutively expressed HIF-1a at two proline residues, leading to its polyubiquitination and subsequent degradation by the proteosomal machinery (20). Reduced oxygen binding to PHD2, due to hypoxia or competitiors such as DHB, or oxonitrosative attack of its ferrous core leads to its inability to hydroxylate HIF-1a and therefore to an increase in HIF-1a, which, after binding to HIF-1b, translocates to the nucleus, where it induces a number of hypoxia response genes, such as vascular endothelial growth factor, inducible nitric oxide synthase, erythropoietin, and MUC5AC (5, 21–23), which are implicated in asthma pathogenesis. HIF-1a also up-regulates PHD2, thereby creating a robust feedback system that maintains a critical balance in the PHD2/HIF-1a axis (24). In an experimental mouse model of asthma, accentuation of the hypoxic response to levels beyond a normal increase led to increased inflammatory cell infiltrate, increased fibrosis, and increased airway remodeling. This was seen in acute and chronic models of asthma but was not seen in normal uninflamed lungs. We speculate that induction of the hypoxia response in the bronchial epithelium, as seen in control mice with a mild asthma phenotype, is a protective mechanism to attenuate cell injury and apoptosis (3, 25, 26). This best explains the observed protective effect of low-dose DHB inhibition in the chronic model and is consistent with other reports where accentuation of the hypoxic response or loss of prolyl hydroxylases leads to increased cell survival (25). Similar opposite effects with low and high hypoxic conditions have been found in ischemic ventricular arrhythmic rats (27) and in pulmonary and systemic hypertension of mice (28). A schematic model fitting our data is shown in Figure 7. It is well known that epithelial injury promotes inflammatory cell infiltration, which in turn promotes epithelial injury, creating an amplification loop (29). We speculate that optimal levels of hypoxic response terminate this loop by attenuating epithelial injury and apoptosis. A probable undesirable consequence of the accentuated hypoxic response, seen most clearly with highdose DHB treatment or PHD2 knockdown, is increased survival

and activation of mesenchymal fibroblast like cells and inflammatory cells (30, 31). This is likely to lead to accentuated subepithelial fibrosis and persistent inflammation. Although this model fits current knowledge on the subject as well as our data, it cannot predict what the optimal level of the hypoxic response would be, and further investigation into the mechanisms for differential apoptosis with differing grades of hypoxia response is needed. Increased HIF-1–driven expression of CX3CR1, which promotes T-helper lymphocyte survival in inflamed lungs, was seen in our study, providing at least one possible new mechanism for proinflammatory effects of hypoxic response. Although it seems contradictory that siRNA knockdown of HIF-1a and low-dose inhibition of PHD had antiasthma effects in our study, this may be explained by differences in the experimental model. The acute model is predominantly an inflammatory model with secondary epithelial injury. Thus, the antiinflammatory effects of HIF-1a knockdown may attenuate epithelial injury. The chronic model has only low-grade inflammation, and the epithelium-restricted hypoxic response associated with low-dose DHB may enhance epithelial survival and facilitate resolution of asthma. In support of this contention, low-dose DHB was not effective in attenuating the acute asthma phenotype. Although no adverse effects of knocking down the hypoxic response were seen in our inflammation-predominant model, the role of this pathway should be confirmed in noninflammatory mechanisms of lung injury, where the balance between the protective and proinflammatory consequences should be different. Our findings are mostly consistent with previous and contemporaneous experimental literature. Most recently, it has been found that conditional bronchial epithelial HIF-1a knockout mice are resistant to allergic airway inflammation and that hypoxic response accentuates acute allergic inflammation (32). Yet, such knockouts have been found to be predisposed to Th2 inflammation and asthma in different models (9). Our work significantly advances the field by bringing out the complexity within the subject where the hypoxic response is neither friend nor foe as well as a mechanistic understanding of

Figure 7. Schematic model to illustrate the dual effects of the hypoxic response in asthma. Characteristic features of asthma, namely epithelial apoptosis, cellular inflammation, and mesenchymal cell proliferation, are shown graphically. Epithelial injury can cause inflammation by liberation of cytokines such as thymic stromal lymphopoietin (TSLP) (see Reference 29), and inflammation can cause epithelial injury. This represents an amplification loop that can be attenuated by hypoxic response– mediated survival of epithelial cells (top, attenuation of pathological features) or potentiated by hypoxic response–mediated survival and proliferation of inflammatory and mesenchymal cells (bottom, worsening of pathological features). Thus, a modest degree of hypoxic response may be protective in preventing epithelial injury in asthma, and large increases in hypoxic response lead to worsening of asthma-related pathological processes, such as inflammation and fibrosis. These scenarios correspond to the opposing effects of lowdose (1 mg/kg) and high-dose (10 mg/kg) DHB treatment in the chronic asthma model.

Ahmad, Kumar, Mabalirajan, et al.: Hypoxic Response Modulates Asthma

the effect of hypoxic response on asthmatic processes. Understanding that different doses of DHB in a chronic inflammation model with similarities to human asthma can lead to resolution of asthma or asphyxiation during MCh challenge is critical to the development of therapeutic strategies related to modulation of the hypoxic response. It is also interesting that common genetic variations, which were identified using normal endophenotypes of the traditional medical system Ayurveda, alter the physiological hypoxia response regulation and thereby the risk of diseases (14). Whether the hypoxic response is in response to hypoxia has been questioned by Semenza and colleagues because allergen provocation leads to hypoxic response in conditions where cells are exposed to ambient air concentrations of oxygen (33). They suggest that finding nonhypoxic mechanism of the hypoxic response is an important challenge in understanding asthma pathology. We speculate that oxidative stress drives the hypoxic response in the mouse model of asthma. Our model (or even true human asthma) is unlikely to be associated with substantial hypoxia, being very mild, but is associated with high levels of oxo-nitrosative stress in the airway epithelium, as shown by us previously (16). This is due to increased levels of the endogenous NOS uncoupler asymmetricdimethyl-arginine (ADMA), reduced function of eNOS, and increased inducible nitric oxide synthase. Using in vitro experiments, we have found that ADMA is sufficient to induce the hypoxic response in vitro (Figure E10). In this context, it is noteworthy that exogenous administration of ADMA leads to increased peribronchial fibrosis and AHR (34). This is being investigated further. It is possible that the hypoxic response will be important in other models of aberrant repair such as lung fibrosis, as indicated by preliminary results in a mouse model of bleomycin-induced fibrosis (Figure E11). Whether human genetic variations in prolyl hydroxylase 2 associated with hypoxia adaptation (14, 15) are related to risks of asthma or fibrosis needs to be further investigated. In summary, a hypoxic response may be induced during allergic inflammation without true hypoxia. The effects of the hypoxic response in asthma are complex and context dependent, with mild hypoxic response restricted to airway epithelium being protective and exaggerated diffuse hypoxic response being highly proinflammatory and proasthmatic. Author disclosures are available with the text of this article at www.atsjournals.org. Acknowledgments: The authors thank Mrs. Jyotirmoi Aich, Dr. Geeta Devi Leishangthem, Mrs. Kamiya Mehla, Mrs. Veda Krishnan, and Mr. Ankur Kulsheshtra for their kind help and Mr. Anirban Kar and Mr. Gunjan Purohit for their help in immunocytochemistry.

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