ORIGINAL RESEARCH Chemical Chaperones Mitigate Experimental Asthma by Attenuating Endoplasmic Reticulum Stress Lokesh Makhija1, Veda Krishnan1, Rakhshinda Rehman1, Samarpana Chakraborty1, Shuvadeep Maity2, Ulaganathan Mabalirajan1, Kausik Chakraborty2, Balaram Ghosh1, and Anurag Agrawal1,2 1
Centre of Excellence for Translational Research in Asthma & Lung Disease; and 2Functional Genomics, CSIR-Institute of Genomics and Integrative Biology, Delhi, India
Abstract Endoplasmic reticulum (ER) stress and consequent unfolded protein response (UPR) are important in inflammation but have been poorly explored in asthma. We used a mouse model of allergic airway inflammation (AAI) with features of asthma to understand the role of ER stress and to explore potential therapeutic effects of inhaled chemical chaperones, which are small molecules that can promote protein folding and diminish UPR. UPR markers were initially measured on alternate days during a 7-day daily allergen challenge model. UPR markers increased within 24 hours after the first allergen challenge and peaked by the third challenge, before AAI was fully established (from the fifth challenge onward). Three chemical chaperones— glycerol, trehalose, and trimethylamine-N-oxide (TMAO)—were initially administered during allergen challenge (preventive regimen). TMAO, the most effective of these chemical chaperones and 4-phenylbutyric acid, a chemical chaperone currently in clinical trials, were further tested for potential therapeutic
Asthma is a chronic disease characterized by airway inflammation, injury, remodeling, and hyperresponsiveness to general constrictor stimuli (1). Despite scientific advances, a mechanistic understanding of the pathogenesis of asthma remains challenging (2, 3). Initial insights focused on the role of Th2 inflammatory cells and cytokines, but it now seems likely that airway epithelial stress is a critical orchestrator of the inflammation, possibly
activities after AAI was established (therapeutic regimen). Chemical chaperones showed a dose-dependent reduction in UPR markers, airway inflammation, and remodeling in both regimens. Our results indicate an early and important role of the ER stress pathway in asthma pathogenesis and show therapeutic potential for chemical chaperones. Keywords: asthma; chemical chaperones; endoplasmic
reticulum stress; glucose-regulated protein 94; glucoseregulated protein 78
Clinical Relevance The use of chemical chaperones is likely to be beneficial where unfolded protein response becomes maladaptive and leads to disease. Our data support the potential benefit of inhaled chemical chaperone therapy in asthma.
by modulating epithelial injury. Mitochondrial stress in airway epithelium is well recognized as a determinant of the extent of epithelial injury and of the consequent amplification of allergic inflammation (3). The endoplasmic reticulum (ER) has a central role in various cellular functions via protein folding, in calcium homeostasis, and in cell signaling; therefore, the role of the ER stress pathway in asthma pathogenesis requires further
investigation. “ER stress” refers to wide range of unfolded protein responses (UPRs), ranging from adaptive to maladaptive (4, 5). In the adaptive form, the presence of unfolded proteins in the ER triggers signaling that leads to a reduction of unfolded proteins by chaperone-assisted folding and inhibition of new protein synthesis. Maladaptive UPR can result from excessive or persistent stress due to the inability to restore protein folding or an
( Received in original form July 12, 2013; accepted in final form November 13, 2013 ) This work was supported by Council of Scientific and Industrial Research, India grant MLP5502, by a senior research fellowship (L.M.), and by the Department of Science and Technology, India through a Swarnjayanti Fellowship (A.A.). Correspondence and requests for reprints should be addressed to Anurag Agrawal, M.D., Ph.D., CSIR-Institute of Genomics and Integrative Biology, Mall Road, New Delhi, India 110007. E-mail: [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 50, Iss 5, pp 923–931, May 2014 Copyright © 2014 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2013-0320OC on December 3, 2013 Internet address: www.atsjournals.org
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ORIGINAL RESEARCH irrecoverable cause of the signaling (e.g., severe calcium depletion of ER), leading to apoptosis (4, 5). Such epithelial injury or apoptosis can amplify the inflammatory response, and it appears likely that maladaptive (excessive) UPR is proinflammatory. In many systems, inflammatory signals have been shown to induce UPR in target cells, and UPRinducing agents can induce inflammatory pathways (6–9). Some inflammatory diseases, including chronic obstructive pulmonary disease, are associated with increased ER stress (10–14). There is some evidence that this may also be true for asthma. ER-resident orosomucoid-like 3 (ORMDL3), which is the first asthma gene identified from genome-wide association studies, may be a modulator of the ER stress pathway (15, 16). However, the presence or role of UPR in asthma has not been directly shown, nor has a clear functional role of ORMDL3 in UPR and asthma been defined (17, 18). This is an important lacuna because it is possible to inhibit UPR using high concentrations of small molecules, called “chemical chaperones” without any major toxicity (19–24). Chemical chaperones are small molecules that accumulate in the ER and directly accelerate protein folding, thereby inhibiting ER stress and associated UPR (19, 20). These may be endogenous molecules, such as polyamines, or exogenous drugs, such as 4-phenylbutyrate (4-PBA). We hypothesized that airway injury in asthma relates to excessive maladaptive UPR and therefore administration of exogenous chemical chaperones could alleviate asthma by attenuating ER stress and UPR. Chemical chaperones, such as 4-PBA or tauroursodeoxycholic acid, were found to be extremely safe in preclinical studies and are now in clinical trials (21–24, 42–44) for diseases like diabetes. In this study we investigated the possibility of inhaled chemical chaperone as treatment for allergic airway disease.
Materials and Methods Reagents and Chemicals
Ovalbumin (OVA) (Grade V chicken egg), trimethylamine-N-oxide (TMAO), glycerol, methacholine, and staining reagents including horseradish peroxidase–labeled secondary antibodies were purchased from 924
Sigma (St. Louis, MO). 4-PBA and trehalose were obtained from Calbiochem (La Jolla, CA). All chemicals and reagents were molecular biology grade. Experimental Design
Male Balb/c mice (4–6 wk old) were used. Allergic airway inflammation (AAI) was induced by sensitization to OVA through a series of weekly intraperitoneal injections of 50 mg OVA/3 mg alum for 3 weeks followed by 1.5% OVA aerosol challenge daily for 7 days. For the time kinetics experiment, mice were killed 24 hours after the first, third, fifth, and seventh challenges (Figure 1A), referred to named as C1, C3, C5, and C7/OVA, respectively. All animal experiments in this study were approved by the Institution’s animal ethics committee. To observe the chemical chaperone effects, mice were administered one of the following aerosolized solutions for
30 minutes: saline, TMAO (0.375, 0.75, or 1.5 M), 1 M trehalose, 20% glycerol, or 4PBA (7.5, 15, and 30 mM). Further details on nebulization are provided in the online supplement. In the preventive model, chemical chaperone treatment was given 2 hours before every OVA challenge or to naive mice for 30 minutes in a whole body chamber every day for 7 days. In the therapeutic model, which was more analogous to human disease, AAI and remodeling was first established by four daily OVA challenges, followed by administration of various chemical chaperones as mentioned above, for the next 3 days. OVA challenge was continued during treatment because it mimics most practical scenarios. In both the preventive and therapeutic models, two independent experimental sets of three or four mice each were used per assay per group. Airway hyperresponsiveness (AHR)
Figure 1. Endoplasmic reticulum (ER) stress markers are elevated in allergically inflamed mouse lungs. (A) Experimental protocol used for inducing mouse allergic airway inflammation (AAI) and different experiments performed in this study. For the time course model, C1, C3, C5, and C7 data were collected 24 hours after one, three, five, and seven ovalbumin (OVA) challenges, respectively, while developing the AAI model. Colored boxes show the days when different drugs were administered under preventive and therapeutic regimes in the AAI model. (B) Representative Western blot of lung homogenate protein from SHAM and OVA-treated mice and (C) their quantitative densitometry score for n = 4. *Statistical significance (P , 0.05). AHR, airway hyperresponsiveness; BiP, binding immunoglobulin protein; CHOP, C/EBP homologous protein; i.p., intraperitoneal; NS, statistically nonsignificant relative to the SHAM group.
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ORIGINAL RESEARCH assays in ventilated mice were combined with BAL and serology tests. A minimum of five independent data points was ensured for each assay. AHR measurement
AHR to methacholine was determined in anesthetized mice ventilated on the Flexivent mouse ventilator system (SCIREQ, Montreal, Canada) as reported previously (25). Airway resistance measured at different methacholine doses was normalized to resistance measured with saline treatment in the same mouse before methacholine treatment. Lung Histopathology
Formalin-fixed, paraffin-embedded lung tissue sections were stained with hematoxylin and eosin, periodic acid-Schiff, Masson’s trichrome, and Van Geison’s staining as described previously (26, 27). Slides were examined under a light microscope (Nikon Eclipse 90i; Nikon, Tokyo, Japan). Quantitative blind scoring for different stains was performed as described previously (3). Briefly, in Image J software (National Institutes of Health, Bethesda, MD) the required color area was selected using color threshold and quantified using the measure tool. This area was then normalized by the bronchi perimeter for cross comparison among the groups. Statistical Analysis
Data are expressed as means 6 SEM. Statistical significance of the difference between groups was estimated using the unpaired student t test for two groups or ANOVA with post hoc correction for multiple groups. Significance was set at P < 0.05.
Results Allergically Inflamed Lungs Show ER Stress Markers
To determine whether AAI is associated with ER stress, we performed immunohistochemistry for the wellestablished UPR markers (glucose-regulated protein 78/binding immunoglobulin protein [GRP78/BiP], GRP94, C/EBP homologous protein [CHOP], and caspase12) in allergically inflamed mouse lungs (7-d challenge; Figure 1A, model C7). Significant up-regulation of stress markers
was seen (see Figure E2 in the online supplement). This was further confirmed by Western blot (Figure 1B), whose densitometry confirmed a significant increase of each marker except caspase-12 (Figure 1C). In all further experiments, we performed Western blots of GRP78/BiP, GRP94, and CHOP as markers of ER stress. ER Stress Is an Early Event in AAI and Airway Remodeling
Because ER stress was clearly well established by the seventh challenge, we examined mouse lungs from earlier time points during allergen challenge to understand the progression of UPR in AAI (Figure 1A; after Day 1, C1; after Day 3, C3; after Day 5, C5). We observed that asthmatic features, such as inflammation, mucus metaplasia, AHR, and collagen deposition, increased in a time-dependent manner (Figures 2A, 2B, and 2D). BiP, which is an early component of the UPR cascade, peaked after the first challenge and declined thereafter. GRP-94 was also maximally increased by a single allergen challenge, but CHOP showed a later increase (Figures 2C and 2E). Being chaperones, BiP and GRP-94 are considered adaptive, whereas CHOP is increased due to excessive or sustained ER stress during the maladaptive stage and can trigger apoptosis (28–31). For GRP94 and CHOP, we observed a second band lower to the main band (Figure 2C), which might be due to increased proteolytic calpain activity in asthma, as reported previously (32, 33). These results suggested that ER stress may have an early important role in asthma pathogenesis, possibly leading to cell injury, rather than being a nonspecific outcome of AAI. Attenuating ER Stress during Allergen Challenge Inhibits Airway Remodeling and Inflammation
To establish the role of maladaptive ER stress in the pathogenesis of allergic asthma, we used chemical chaperones, a class of compounds that promote protein folding, enhancing the adaptive UPR and potentially preventing maladaptive cellular injury. Because BiP and GRP-94 were increased within a day of allergen challenge, we initially screened three chemical chaperones (20% glycerol, 1 M trehalose, and 1.5 M TMAO) in a preventive protocol during each allergen challenge. Each of these chaperones was generally effective in attenuating CHOP expression at doses
Makhija, Krishnan, Rehman, et al.: Chemical Chaperones Alleviate Asthma
comparable to those reported previously (34–36). This was associated with a significant reduction in asthmatic features (Figures 3A–3D), such as AHR, inflammation, mucus metaplasia, and collagen deposition. BiP and GRP-94 showed more variable reduction (Figures 3 and E3). Among the chemical chaperones tested, TMAO was the most effective in attenuating CHOP and asthmatic features and was tested further in a therapeutic regimen at varying doses. Therapeutic Application of Chemical Chaperones in AAI in a Dose-Dependent Fashion
To assess the therapeutic potential of the chemical chaperones in asthma, TMAO was administered to mice with wellestablished AAI and features of asthma (fifth challenge; Figures 2A–2C). Three different doubling doses, below the dose found to be nontoxic in naive mice (Figure E1), were used. Allergen exposure was continued during treatment, mimicking the usual clinical scenario. CHOP was effectively inhibited by TMAO in a dose-dependent fashion (Figure 4C). This inhibition of maladaptive ER stress was associated with substantial attenuation of asthmatic features (Figures 4A, 4B, and 4D). To confirm that the effect is shared by other chaperones and is not unique to TMAO, we tested the efficacy of 4-PBA, a chemical chaperone that is currently in human clinical trials for diabetes. At doses comparable to those reported previously for acute lung injury (34), 4-PBA treatment attenuated asthmatic features such as inflammation, mucus metaplasia, and collagen deposition (Figures 5A, 5B, and 5D) in a dose-dependent manner. This was associated with inhibition of CHOP (Figures 5C and E4A), as seen for TMAO. Minor differences were seen in the effects of TMAO and 4-PBA on cytokine and immunoglobulin profiles at the doses used, with only 4-PBA reducing immunoglobulin levels (Figures E4B and E4C), but both TMAO and 4-PBA efficiently reduced IL13 (Figure E4C). Such differences may relate to other effects of these drugs, such as their antioxidant properties (20).
Discussion To the best of our knowledge, this is the first report showing that UPR/ER stress may be an early event in asthma pathogenesis 925
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Figure 2. Kinetic relationship between unfolded protein response (UPR) proteins and asthmatic features. (A) Representative micrographs of different histochemical stains to estimate inflammation (hematoxylin and eosin [H&E]), mucus metaplasia (periodic acid-Schiff [PAS]), and collagen deposition (red) (Van Geison’s [VG]) in time course experiment. (B and C) AHR (B) and representative Western blot (C) of ER markers in lung homogenates. (D) Morphometry score of different stains shown in A. (E) Densitometry score of Western blots shown in C. Each group had six to eight mice each from two independent experiments. *P , 0.05 relative to the SHAM group. GRP, glucose-regulated protein.
during allergic inflammation and that aerosolized chemical chaperones have preventive and therapeutic efficacy in attenuating asthmatic features. Previous 926
observations suggest that proinflammatory molecules can up-regulate UPR in nonimmune cells (4–9) and that UPR in such cells can modulate inflammation (13,
14). In the mouse model of asthma, UPR markers are increased in the lung within 24 hours of the first allergen challenge. Initially, the increase is of ER-resident
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Figure 3. Chemical chaperones (CC) attenuate asthmatic features when administered 2 hours before every allergen challenge. (A) Representative micrographs of different histochemical stains to estimate inflammation (H&E), mucus metaplasia (PAS), and collagen deposition (blue) (MT) where CC were treated preventively to AAI-induced mice. Insets (original magnification: 32) identify specific regions of mucus secretion in bronchial epithelial cells. (B) Quantitative morphometry score of different stains shown in A. (C and D) Representative Western blot for UPR proteins in lung homogenates (C) and airway hyperresponsiveness in the same groups (D). Each group had six to eight mice each from two independent experiments. *Statistically significant (P , 0.05) relative to OVA group. GLY, glycerol; TMAO, trimethylamine-N-oxide; TREH, trehalose.
protein chaperones (BiP, GRP94), which are markers of adaptive UPR response in response to the presence of unfolded proteins in the ER. The subsequent increase
in CHOP, by the third allergen challenge, indicates excessive ER stress and maladaptive UPR. The asthma-like phenotype, as indicated by histological or
Makhija, Krishnan, Rehman, et al.: Chemical Chaperones Alleviate Asthma
biochemical markers and AHR, becomes most severe at and beyond this time point. This indicates a possible role of maladaptive UPR in asthma pathogenesis, 927
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Figure 4. The chemical chaperone TMAO attenuates asthmatic features even when administered only on the last three allergen challenges. (A and B) Representative micrographs (A) and their quantitative morphometry score (B) for inflammation (H&E), mucus metaplasia (PAS), and collagen deposition (blue) (MT) for TMAO administration at three different doses (0.375, 0.75, and 1.5 M, named T1, T2, and T3, respectively) and vehicle (saline) to OVA mice. (C and D) Representative Western blot for UPR proteins in lung homogenates (C) and airway hyperresponsiveness in the same groups (D). Each group had five mice. *Statistically significant at P , 0.05. NS, statistically nonsignificant relative to the marked/previous group; VEH, vehicle.
which is further supported by the observation that a variety of chemical chaperones were able to markedly attenuate the features of asthma. Individual effects of the different molecules used, unrelated to chemical chaperone activity, cannot be completely excluded; however, such an explanation is highly unlikely because (1) 928
these molecules were chosen to be structurally unrelated (lipids, sugars, amines), (2) they are not known to bind to any receptor relevant to the current study, and (3) the ability of each of these to attenuate airway inflammation, mucin content, or collagen deposition was correlated with the reduction of CHOP
(composite r = 0.71; P , 0.05) (Figures 3–5). There was no relationship to nonspecific properties, such as antioxidant effects. TMAO, which was one of the most effective molecules, has no antioxidant properties (20), thereby excluding a significant role for a nonspecific free radical quenching effect. This is strong
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ORIGINAL RESEARCH evidence that diminution of excessive ER stress is the primary mechanism of antiasthma effects of the various molecules used in our study. The observation that diminution of ER stress has antiinflammatory effects in experimental asthma is consistent with
recent observations in other systems that UPRs regulate immune responses (35–39). However, the effects are highly context dependent (40, 41). For example, inducing ER stress in injured lungs results in exaggerated fibrotic remodeling, whereas there is no effect when ER stress is induced
in naive lungs (40). UPR is an adaptive pathway that should not be seen as a pathologic process. If unfolded proteins start accumulating, induction of protein chaperones like GRP-94 or GRP-78/BiP is essential for the maintenance of cellular health. Levels of such UPR markers did not
Figure 5. The chemical chaperone 4-phenylbutyric acid (4-PBA) was effective in reversing asthmatic features when administered therapeutically. (A and B) Representative micrographs (A) and the quantitative morphometry score for inflammation (H&E), mucus metaplasia (PAS), and collagen deposition (blue) (MT) (B) for 4-PBA administration at three different doses (7.5, 15, and 30 mM, named P1, P2, and P3, respectively) or vehicle (saline) to OVA mice. (C and D) Representative Western blot for UPR proteins in lung homogenates (C) and airway hyperresponsiveness (D) in the same groups. Each group had five mice. *Statistical significance with P , 0.05. NS, statistically nonsignificant relative to the marked/previous group.
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ORIGINAL RESEARCH correlate with the pathology in our model system. Conditions of excessive or sustained ER stress (too much, too long) may lead to irreversible damage, triggering induction of molecules like CHOP and ultimately Caspase-12, leading to apoptotic cell death. Chemical chaperones are important therapeutic options in such situations because they promote protein folding, assist the adaptive aspect of UPR, and prevent excessive or sustained ER stress, thereby preventing maladaptive UPR. Consistent with our observation that adaptive UPR is induced after the first allergen challenge, we found that chemical chaperones are much more effective in the preventive regimen in comparison to the
therapeutic regimen. This is true for most therapeutic interventions and therefore is not a limitation. The dose-response studies for TMAO and 4-PBA in the therapeutic model found biologically and statistically significant dose-dependent attenuation of inflammation and remodeling. Dosedependent effects were not seen for AHR. One possibility is that the highest concentrations may have some irritant effects that counterbalance the antiinflammatory effects. Because all doses were associated with significant attenuation of AHR, when compared with vehicle controls, such an effect is likely to be small. The doses used here are similar to those being used systemically in mice
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Centre of Excellence for Translational Research in Asthma & Lung Disease; and 2Functional Genomics, CSIR-Institute of Genomics and Integrative Biology, Delhi, India
Abstract Endoplasmic reticulum (ER) stress and consequent unfolded protein response (UPR) are important in inflammation but have been poorly explored in asthma. We used a mouse model of allergic airway inflammation (AAI) with features of asthma to understand the role of ER stress and to explore potential therapeutic effects of inhaled chemical chaperones, which are small molecules that can promote protein folding and diminish UPR. UPR markers were initially measured on alternate days during a 7-day daily allergen challenge model. UPR markers increased within 24 hours after the first allergen challenge and peaked by the third challenge, before AAI was fully established (from the fifth challenge onward). Three chemical chaperones— glycerol, trehalose, and trimethylamine-N-oxide (TMAO)—were initially administered during allergen challenge (preventive regimen). TMAO, the most effective of these chemical chaperones and 4-phenylbutyric acid, a chemical chaperone currently in clinical trials, were further tested for potential therapeutic
Asthma is a chronic disease characterized by airway inflammation, injury, remodeling, and hyperresponsiveness to general constrictor stimuli (1). Despite scientific advances, a mechanistic understanding of the pathogenesis of asthma remains challenging (2, 3). Initial insights focused on the role of Th2 inflammatory cells and cytokines, but it now seems likely that airway epithelial stress is a critical orchestrator of the inflammation, possibly
activities after AAI was established (therapeutic regimen). Chemical chaperones showed a dose-dependent reduction in UPR markers, airway inflammation, and remodeling in both regimens. Our results indicate an early and important role of the ER stress pathway in asthma pathogenesis and show therapeutic potential for chemical chaperones. Keywords: asthma; chemical chaperones; endoplasmic
reticulum stress; glucose-regulated protein 94; glucoseregulated protein 78
Clinical Relevance The use of chemical chaperones is likely to be beneficial where unfolded protein response becomes maladaptive and leads to disease. Our data support the potential benefit of inhaled chemical chaperone therapy in asthma.
by modulating epithelial injury. Mitochondrial stress in airway epithelium is well recognized as a determinant of the extent of epithelial injury and of the consequent amplification of allergic inflammation (3). The endoplasmic reticulum (ER) has a central role in various cellular functions via protein folding, in calcium homeostasis, and in cell signaling; therefore, the role of the ER stress pathway in asthma pathogenesis requires further
investigation. “ER stress” refers to wide range of unfolded protein responses (UPRs), ranging from adaptive to maladaptive (4, 5). In the adaptive form, the presence of unfolded proteins in the ER triggers signaling that leads to a reduction of unfolded proteins by chaperone-assisted folding and inhibition of new protein synthesis. Maladaptive UPR can result from excessive or persistent stress due to the inability to restore protein folding or an
( Received in original form July 12, 2013; accepted in final form November 13, 2013 ) This work was supported by Council of Scientific and Industrial Research, India grant MLP5502, by a senior research fellowship (L.M.), and by the Department of Science and Technology, India through a Swarnjayanti Fellowship (A.A.). Correspondence and requests for reprints should be addressed to Anurag Agrawal, M.D., Ph.D., CSIR-Institute of Genomics and Integrative Biology, Mall Road, New Delhi, India 110007. E-mail: [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 50, Iss 5, pp 923–931, May 2014 Copyright © 2014 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2013-0320OC on December 3, 2013 Internet address: www.atsjournals.org
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ORIGINAL RESEARCH irrecoverable cause of the signaling (e.g., severe calcium depletion of ER), leading to apoptosis (4, 5). Such epithelial injury or apoptosis can amplify the inflammatory response, and it appears likely that maladaptive (excessive) UPR is proinflammatory. In many systems, inflammatory signals have been shown to induce UPR in target cells, and UPRinducing agents can induce inflammatory pathways (6–9). Some inflammatory diseases, including chronic obstructive pulmonary disease, are associated with increased ER stress (10–14). There is some evidence that this may also be true for asthma. ER-resident orosomucoid-like 3 (ORMDL3), which is the first asthma gene identified from genome-wide association studies, may be a modulator of the ER stress pathway (15, 16). However, the presence or role of UPR in asthma has not been directly shown, nor has a clear functional role of ORMDL3 in UPR and asthma been defined (17, 18). This is an important lacuna because it is possible to inhibit UPR using high concentrations of small molecules, called “chemical chaperones” without any major toxicity (19–24). Chemical chaperones are small molecules that accumulate in the ER and directly accelerate protein folding, thereby inhibiting ER stress and associated UPR (19, 20). These may be endogenous molecules, such as polyamines, or exogenous drugs, such as 4-phenylbutyrate (4-PBA). We hypothesized that airway injury in asthma relates to excessive maladaptive UPR and therefore administration of exogenous chemical chaperones could alleviate asthma by attenuating ER stress and UPR. Chemical chaperones, such as 4-PBA or tauroursodeoxycholic acid, were found to be extremely safe in preclinical studies and are now in clinical trials (21–24, 42–44) for diseases like diabetes. In this study we investigated the possibility of inhaled chemical chaperone as treatment for allergic airway disease.
Materials and Methods Reagents and Chemicals
Ovalbumin (OVA) (Grade V chicken egg), trimethylamine-N-oxide (TMAO), glycerol, methacholine, and staining reagents including horseradish peroxidase–labeled secondary antibodies were purchased from 924
Sigma (St. Louis, MO). 4-PBA and trehalose were obtained from Calbiochem (La Jolla, CA). All chemicals and reagents were molecular biology grade. Experimental Design
Male Balb/c mice (4–6 wk old) were used. Allergic airway inflammation (AAI) was induced by sensitization to OVA through a series of weekly intraperitoneal injections of 50 mg OVA/3 mg alum for 3 weeks followed by 1.5% OVA aerosol challenge daily for 7 days. For the time kinetics experiment, mice were killed 24 hours after the first, third, fifth, and seventh challenges (Figure 1A), referred to named as C1, C3, C5, and C7/OVA, respectively. All animal experiments in this study were approved by the Institution’s animal ethics committee. To observe the chemical chaperone effects, mice were administered one of the following aerosolized solutions for
30 minutes: saline, TMAO (0.375, 0.75, or 1.5 M), 1 M trehalose, 20% glycerol, or 4PBA (7.5, 15, and 30 mM). Further details on nebulization are provided in the online supplement. In the preventive model, chemical chaperone treatment was given 2 hours before every OVA challenge or to naive mice for 30 minutes in a whole body chamber every day for 7 days. In the therapeutic model, which was more analogous to human disease, AAI and remodeling was first established by four daily OVA challenges, followed by administration of various chemical chaperones as mentioned above, for the next 3 days. OVA challenge was continued during treatment because it mimics most practical scenarios. In both the preventive and therapeutic models, two independent experimental sets of three or four mice each were used per assay per group. Airway hyperresponsiveness (AHR)
Figure 1. Endoplasmic reticulum (ER) stress markers are elevated in allergically inflamed mouse lungs. (A) Experimental protocol used for inducing mouse allergic airway inflammation (AAI) and different experiments performed in this study. For the time course model, C1, C3, C5, and C7 data were collected 24 hours after one, three, five, and seven ovalbumin (OVA) challenges, respectively, while developing the AAI model. Colored boxes show the days when different drugs were administered under preventive and therapeutic regimes in the AAI model. (B) Representative Western blot of lung homogenate protein from SHAM and OVA-treated mice and (C) their quantitative densitometry score for n = 4. *Statistical significance (P , 0.05). AHR, airway hyperresponsiveness; BiP, binding immunoglobulin protein; CHOP, C/EBP homologous protein; i.p., intraperitoneal; NS, statistically nonsignificant relative to the SHAM group.
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ORIGINAL RESEARCH assays in ventilated mice were combined with BAL and serology tests. A minimum of five independent data points was ensured for each assay. AHR measurement
AHR to methacholine was determined in anesthetized mice ventilated on the Flexivent mouse ventilator system (SCIREQ, Montreal, Canada) as reported previously (25). Airway resistance measured at different methacholine doses was normalized to resistance measured with saline treatment in the same mouse before methacholine treatment. Lung Histopathology
Formalin-fixed, paraffin-embedded lung tissue sections were stained with hematoxylin and eosin, periodic acid-Schiff, Masson’s trichrome, and Van Geison’s staining as described previously (26, 27). Slides were examined under a light microscope (Nikon Eclipse 90i; Nikon, Tokyo, Japan). Quantitative blind scoring for different stains was performed as described previously (3). Briefly, in Image J software (National Institutes of Health, Bethesda, MD) the required color area was selected using color threshold and quantified using the measure tool. This area was then normalized by the bronchi perimeter for cross comparison among the groups. Statistical Analysis
Data are expressed as means 6 SEM. Statistical significance of the difference between groups was estimated using the unpaired student t test for two groups or ANOVA with post hoc correction for multiple groups. Significance was set at P < 0.05.
Results Allergically Inflamed Lungs Show ER Stress Markers
To determine whether AAI is associated with ER stress, we performed immunohistochemistry for the wellestablished UPR markers (glucose-regulated protein 78/binding immunoglobulin protein [GRP78/BiP], GRP94, C/EBP homologous protein [CHOP], and caspase12) in allergically inflamed mouse lungs (7-d challenge; Figure 1A, model C7). Significant up-regulation of stress markers
was seen (see Figure E2 in the online supplement). This was further confirmed by Western blot (Figure 1B), whose densitometry confirmed a significant increase of each marker except caspase-12 (Figure 1C). In all further experiments, we performed Western blots of GRP78/BiP, GRP94, and CHOP as markers of ER stress. ER Stress Is an Early Event in AAI and Airway Remodeling
Because ER stress was clearly well established by the seventh challenge, we examined mouse lungs from earlier time points during allergen challenge to understand the progression of UPR in AAI (Figure 1A; after Day 1, C1; after Day 3, C3; after Day 5, C5). We observed that asthmatic features, such as inflammation, mucus metaplasia, AHR, and collagen deposition, increased in a time-dependent manner (Figures 2A, 2B, and 2D). BiP, which is an early component of the UPR cascade, peaked after the first challenge and declined thereafter. GRP-94 was also maximally increased by a single allergen challenge, but CHOP showed a later increase (Figures 2C and 2E). Being chaperones, BiP and GRP-94 are considered adaptive, whereas CHOP is increased due to excessive or sustained ER stress during the maladaptive stage and can trigger apoptosis (28–31). For GRP94 and CHOP, we observed a second band lower to the main band (Figure 2C), which might be due to increased proteolytic calpain activity in asthma, as reported previously (32, 33). These results suggested that ER stress may have an early important role in asthma pathogenesis, possibly leading to cell injury, rather than being a nonspecific outcome of AAI. Attenuating ER Stress during Allergen Challenge Inhibits Airway Remodeling and Inflammation
To establish the role of maladaptive ER stress in the pathogenesis of allergic asthma, we used chemical chaperones, a class of compounds that promote protein folding, enhancing the adaptive UPR and potentially preventing maladaptive cellular injury. Because BiP and GRP-94 were increased within a day of allergen challenge, we initially screened three chemical chaperones (20% glycerol, 1 M trehalose, and 1.5 M TMAO) in a preventive protocol during each allergen challenge. Each of these chaperones was generally effective in attenuating CHOP expression at doses
Makhija, Krishnan, Rehman, et al.: Chemical Chaperones Alleviate Asthma
comparable to those reported previously (34–36). This was associated with a significant reduction in asthmatic features (Figures 3A–3D), such as AHR, inflammation, mucus metaplasia, and collagen deposition. BiP and GRP-94 showed more variable reduction (Figures 3 and E3). Among the chemical chaperones tested, TMAO was the most effective in attenuating CHOP and asthmatic features and was tested further in a therapeutic regimen at varying doses. Therapeutic Application of Chemical Chaperones in AAI in a Dose-Dependent Fashion
To assess the therapeutic potential of the chemical chaperones in asthma, TMAO was administered to mice with wellestablished AAI and features of asthma (fifth challenge; Figures 2A–2C). Three different doubling doses, below the dose found to be nontoxic in naive mice (Figure E1), were used. Allergen exposure was continued during treatment, mimicking the usual clinical scenario. CHOP was effectively inhibited by TMAO in a dose-dependent fashion (Figure 4C). This inhibition of maladaptive ER stress was associated with substantial attenuation of asthmatic features (Figures 4A, 4B, and 4D). To confirm that the effect is shared by other chaperones and is not unique to TMAO, we tested the efficacy of 4-PBA, a chemical chaperone that is currently in human clinical trials for diabetes. At doses comparable to those reported previously for acute lung injury (34), 4-PBA treatment attenuated asthmatic features such as inflammation, mucus metaplasia, and collagen deposition (Figures 5A, 5B, and 5D) in a dose-dependent manner. This was associated with inhibition of CHOP (Figures 5C and E4A), as seen for TMAO. Minor differences were seen in the effects of TMAO and 4-PBA on cytokine and immunoglobulin profiles at the doses used, with only 4-PBA reducing immunoglobulin levels (Figures E4B and E4C), but both TMAO and 4-PBA efficiently reduced IL13 (Figure E4C). Such differences may relate to other effects of these drugs, such as their antioxidant properties (20).
Discussion To the best of our knowledge, this is the first report showing that UPR/ER stress may be an early event in asthma pathogenesis 925
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Figure 2. Kinetic relationship between unfolded protein response (UPR) proteins and asthmatic features. (A) Representative micrographs of different histochemical stains to estimate inflammation (hematoxylin and eosin [H&E]), mucus metaplasia (periodic acid-Schiff [PAS]), and collagen deposition (red) (Van Geison’s [VG]) in time course experiment. (B and C) AHR (B) and representative Western blot (C) of ER markers in lung homogenates. (D) Morphometry score of different stains shown in A. (E) Densitometry score of Western blots shown in C. Each group had six to eight mice each from two independent experiments. *P , 0.05 relative to the SHAM group. GRP, glucose-regulated protein.
during allergic inflammation and that aerosolized chemical chaperones have preventive and therapeutic efficacy in attenuating asthmatic features. Previous 926
observations suggest that proinflammatory molecules can up-regulate UPR in nonimmune cells (4–9) and that UPR in such cells can modulate inflammation (13,
14). In the mouse model of asthma, UPR markers are increased in the lung within 24 hours of the first allergen challenge. Initially, the increase is of ER-resident
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Figure 3. Chemical chaperones (CC) attenuate asthmatic features when administered 2 hours before every allergen challenge. (A) Representative micrographs of different histochemical stains to estimate inflammation (H&E), mucus metaplasia (PAS), and collagen deposition (blue) (MT) where CC were treated preventively to AAI-induced mice. Insets (original magnification: 32) identify specific regions of mucus secretion in bronchial epithelial cells. (B) Quantitative morphometry score of different stains shown in A. (C and D) Representative Western blot for UPR proteins in lung homogenates (C) and airway hyperresponsiveness in the same groups (D). Each group had six to eight mice each from two independent experiments. *Statistically significant (P , 0.05) relative to OVA group. GLY, glycerol; TMAO, trimethylamine-N-oxide; TREH, trehalose.
protein chaperones (BiP, GRP94), which are markers of adaptive UPR response in response to the presence of unfolded proteins in the ER. The subsequent increase
in CHOP, by the third allergen challenge, indicates excessive ER stress and maladaptive UPR. The asthma-like phenotype, as indicated by histological or
Makhija, Krishnan, Rehman, et al.: Chemical Chaperones Alleviate Asthma
biochemical markers and AHR, becomes most severe at and beyond this time point. This indicates a possible role of maladaptive UPR in asthma pathogenesis, 927
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Figure 4. The chemical chaperone TMAO attenuates asthmatic features even when administered only on the last three allergen challenges. (A and B) Representative micrographs (A) and their quantitative morphometry score (B) for inflammation (H&E), mucus metaplasia (PAS), and collagen deposition (blue) (MT) for TMAO administration at three different doses (0.375, 0.75, and 1.5 M, named T1, T2, and T3, respectively) and vehicle (saline) to OVA mice. (C and D) Representative Western blot for UPR proteins in lung homogenates (C) and airway hyperresponsiveness in the same groups (D). Each group had five mice. *Statistically significant at P , 0.05. NS, statistically nonsignificant relative to the marked/previous group; VEH, vehicle.
which is further supported by the observation that a variety of chemical chaperones were able to markedly attenuate the features of asthma. Individual effects of the different molecules used, unrelated to chemical chaperone activity, cannot be completely excluded; however, such an explanation is highly unlikely because (1) 928
these molecules were chosen to be structurally unrelated (lipids, sugars, amines), (2) they are not known to bind to any receptor relevant to the current study, and (3) the ability of each of these to attenuate airway inflammation, mucin content, or collagen deposition was correlated with the reduction of CHOP
(composite r = 0.71; P , 0.05) (Figures 3–5). There was no relationship to nonspecific properties, such as antioxidant effects. TMAO, which was one of the most effective molecules, has no antioxidant properties (20), thereby excluding a significant role for a nonspecific free radical quenching effect. This is strong
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ORIGINAL RESEARCH evidence that diminution of excessive ER stress is the primary mechanism of antiasthma effects of the various molecules used in our study. The observation that diminution of ER stress has antiinflammatory effects in experimental asthma is consistent with
recent observations in other systems that UPRs regulate immune responses (35–39). However, the effects are highly context dependent (40, 41). For example, inducing ER stress in injured lungs results in exaggerated fibrotic remodeling, whereas there is no effect when ER stress is induced
in naive lungs (40). UPR is an adaptive pathway that should not be seen as a pathologic process. If unfolded proteins start accumulating, induction of protein chaperones like GRP-94 or GRP-78/BiP is essential for the maintenance of cellular health. Levels of such UPR markers did not
Figure 5. The chemical chaperone 4-phenylbutyric acid (4-PBA) was effective in reversing asthmatic features when administered therapeutically. (A and B) Representative micrographs (A) and the quantitative morphometry score for inflammation (H&E), mucus metaplasia (PAS), and collagen deposition (blue) (MT) (B) for 4-PBA administration at three different doses (7.5, 15, and 30 mM, named P1, P2, and P3, respectively) or vehicle (saline) to OVA mice. (C and D) Representative Western blot for UPR proteins in lung homogenates (C) and airway hyperresponsiveness (D) in the same groups. Each group had five mice. *Statistical significance with P , 0.05. NS, statistically nonsignificant relative to the marked/previous group.
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ORIGINAL RESEARCH correlate with the pathology in our model system. Conditions of excessive or sustained ER stress (too much, too long) may lead to irreversible damage, triggering induction of molecules like CHOP and ultimately Caspase-12, leading to apoptotic cell death. Chemical chaperones are important therapeutic options in such situations because they promote protein folding, assist the adaptive aspect of UPR, and prevent excessive or sustained ER stress, thereby preventing maladaptive UPR. Consistent with our observation that adaptive UPR is induced after the first allergen challenge, we found that chemical chaperones are much more effective in the preventive regimen in comparison to the
therapeutic regimen. This is true for most therapeutic interventions and therefore is not a limitation. The dose-response studies for TMAO and 4-PBA in the therapeutic model found biologically and statistically significant dose-dependent attenuation of inflammation and remodeling. Dosedependent effects were not seen for AHR. One possibility is that the highest concentrations may have some irritant effects that counterbalance the antiinflammatory effects. Because all doses were associated with significant attenuation of AHR, when compared with vehicle controls, such an effect is likely to be small. The doses used here are similar to those being used systemically in mice
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