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Chronic Asthma–induced Airway Remodeling Is Prevented by Toll-like Receptor-7/8 Ligand S28463

¹ Department of Experimental Medicine, McGill University, Montreal, Quebec, Canada; ² Department of Pulmonary Medicine, Tokyo Medical and Dental University, Tokyo, Japan; ³ Meakins-Christie Laboratories, McGill University, Montreal, Quebec, Canada; ⁴ Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts; ⁵ Research Institute of the McGill University Health Center, Montreal, Quebec, Canada; ⁶ Department of Pathology, Montreal Neurological Hospital, McGill University, Montreal, Quebec, Canada; and ⁷ Department of Human Genetics, McGill University, Montreal, Quebec, Canada

Correspondence and requests for reprints should be addressed to Danuta Radzioch, Ph.D., Department of Experimental Medicine, McGill University, Montreal, PQ, H3G 1A4 Canada. E-mail: danuta.radzioch{at}muhc.mcgill.ca

	ABSTRACT

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Rationale: Allergic asthma is a heterogeneous disease, the pathology of which is a result of improper immune responses to innocuous antigens. We and others have previously shown that one of the Toll-like receptor (TLR)-7/8 ligands, the synthetic compound S28463 (resiquimod, R-848), is able to inhibit acute allergic asthma in mice.

Objectives: Given that the efficiency of this pharmacologic compound against the smooth muscle mass increase and goblet cell hyperplasia that are characteristic of chronic allergic asthma has not been previously assessed, we investigated the ability of this compound to prevent these aspects of chronic airway remodeling.

Methods: The impact of S28463 treatment was assessed in a Brown Norway rat model of chronic asthma by histologic, morphometric, and molecular techniques.

Measurements and Main Results: We demonstrate that treatment with S28463 is able to prevent the development of goblet cell hyperplasia and increases in airway smooth muscle mass, and that this effect is at least partially mediated by inhibiting proliferation of goblet and smooth muscle cells, respectively. Furthermore, we show that the abrogation of airway remodeling is preceded by inhibition of the inflammatory reaction normally occurring in response to allergen challenge in sensitized animals. This inhibition was associated with a reduction of both helper T cell type 1 and type 2 cytokine protein expression in the lungs, demonstrating the potent antiinflammatory effect of this pharmaceutical compound in the context of allergic reactions.

Conclusions: Taken together, our results indicate great potential for the use of S28463 as an antiinflammatory therapeutic agent for the management of chronic asthma.

Key Words: resiquimod • R-848 • imidazoquinoline • airway remodeling • Toll-like receptor

AT A GLANCE COMMENTARY TOP ABSTRACT AT A GLANCE COMMENTARY METHODS RESULTS DISCUSSION REFERENCES   Scientific Knowledge on the Subject Treatment with TLR7 ligand is effective in preventing the inflammatory reaction in an acute asthma model. The effectiveness of this compound in preventing chronic airway remodeling had not been assessed previously. What This Study Adds to the Field Treatment with S28463, a TLR 7/8 ligand, inhibits the increase in airway smooth muscle mass and the development of goblet cell hyperplasia that are associated with chronic asthma.

 
Allergic asthma is a heterogeneous disease affecting the airways and is characterized by a chronic inflammatory response involving several cellular and humoral mediators. Prototypical asthmatic airway inflammation is believed to be triggered by IgE-dependent mast cell degranulation that results in the recruitment of monocytes, lymphocytes, and, particularly, large numbers of eosinophils as well as the induction of several helper T cell type 2 (Th2) cytokines. This chronic inflammatory response is believed to be the underlying cause of airway remodeling, characterized by subepithelial fibrosis, goblet cell hyperplasia, and increased smooth muscle mass (1), and has been associated with increased asthma severity (2).

Current asthma management strategies principally involve the use of bronchodilators, in the form of short- and long-acting ₂ agonists, as rescue medication during exacerbations and to prolong the time to exacerbation, respectively, and inhaled corticosteroids to minimize the underlying inflammation. Despite having been shown to be effective in preventing inflammation (3) by inhibiting cytokine transcription (4) and eosinophil survival (5, 6), inhaled corticosteroids have been relatively underprescribed because of concerns, on the part of primary care physicians and patients alike, over potential side effects, which may include oral candidiasis, dysphonia, growth retardation, skin thinning/bruising, and loss of bone mineral density (7). There is therefore a need for alternative approaches to the treatment of the inflammation underlying asthma pathology.

Considerable effort has been devoted to exploring the potential of immunomodulatory treatment of asthma by means of Toll-like receptor (TLR) activation (8). TLRs are a family of molecules that mediate pathogen-associated molecular pattern recognition and that play a crucial role in both innate and adaptive immunity (9, 10). These widely expressed proteins are some of the first molecules to be involved in generating immune responses to environmental stimuli, a process that is mediated by cellular activation and stimulation of cytokine production (11). Furthermore, several TLRs or TLR ligands have been associated with asthma. Elevated levels of lipopolysaccharides, which are TLR4 ligands, have been negatively correlated with the incidence of asthma in several studies (12, 13), and CpG oligodeoxynucleotides, which are TLR9 ligands, have been used, with various degrees of success, in the treatment of asthma in animal models (14–17).

The natural ligands of TLR7 are believed to be a subset of single-stranded RNA molecules of viral origin (18–20); however, this receptor, and TLR8, are also known to be ligated by members of the imidazoquinoline family of pharmaceutical compounds (21, 22). S28463 (also known as resiquimod and R-848) is a member of the imidazoquinoline family and has been shown to induce the expression of a wide range of cytokines in many species from rodents to primates (23). Our group (24, 25), and others (26, 27) have previously demonstrated that S28463 treatment can prevent the development of lung pathology, including airway hyperresponsiveness and eosinophilia, in mouse models of acute asthma. On the basis of studies using BALB/c mice, Quarcoo and colleagues suggested that treatment with S28463 causes a shift from an allergic Th2 to a nonallergic Th1-biased immune response (26). In two other strains of mice (C57BL/6, with low airway hyperresponsiveness, and A/J, with high airway hyperresponsiveness) treatment with S28643 resulted in a dramatic inhibition of the acute inflammatory reaction that results from allergen exposure in properly sensitized animals and this inhibition was accompanied by a concurrent inhibition of both Th1 and Th2 cytokines (24).

Given that S28463 treatment could prevent the acute inflammatory reaction associated with allergen challenge in several genetically diverse strains of mice, we postulated that this compound could also inhibit the acute inflammatory response in other species. Furthermore, we hypothesized that if treatment with S28463 was capable of preventing the acute inflammatory reaction caused by allergen exposure, it might also prevent the development of the chronic remodeling associated with repeated allergen exposure. The potential of Toll-like receptor 7 ligands in preventing changes associated with chronic asthma remains to be addressed.

The Brown Norway rat has been shown to develop airway remodeling consistent with asthma after only 2 weeks of allergen challenge (28, 29), making this species a convenient experimental model for the study of the chronic aspects of the disease. Using an acute ovalbumin (OVA)-sensitized and -challenged Brown Norway rat model of asthma, we demonstrate that S28463 treatment is able to prevent the development of allergen-induced inflammation and that this effect is associated with a reduction of both Th1 and Th2 cytokine levels and in a dramatic reduction of inflammatory cell infiltration into the lungs. Furthermore, using a chronic model of OVA-sensitized and -challenged rats, we demonstrate that continued treatment with S28463 is capable of preventing the increase in smooth muscle mass and goblet cell hyperplasia associated with airway remodeling in asthma. We also demonstrate that this effect is mediated, at least partially, by inhibiting the proliferation of epithelial and subepithelial airway cells.

	METHODS

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Animals
All animals used in this study were 8- to 10-week-old male Brown Norway rats (Harlan, Bicester, UK) and all procedures performed on the animals were in compliance with the Canadian Council of Animal Care (CCAC, Ottawa, ON, Canada) guidelines and were approved by the McGill University (Montreal, PQ, Canada) Animal Care Committee.

Sensitization and Challenge for Assessment of Acute Inflammatory Response
OVA sensitization was performed by concurrent subcutaneous injection of OVA adsorbed to aluminum hydroxide, and intraperitoneal injection of heat-killed Bordetella pertussis (provided by T. Issekutz, Dalhousie University, Halifax, NS, Canada). Fourteen days later, the animals were challenged by aerosol with an OVA solution (OVA-OVA and OVA/S28), or with phosphate-buffered saline (OVA-PBS and OVA-S28/PBS). Twenty-four hours before antigen challenge, two groups of rats (OVA-S28/OVA and OVA-S28/PBS) were administered an intraperitoneal injection of 1 mg of S28463 (S28463 was generously provided by R. Miller, 3M Pharmaceuticals, St. Paul, MN). All animals were killed 24 hours after antigen challenge. See the online supplement for additional details.

Sensitization and Challenge for Assessment of Chronic Airway Remodeling
OVA sensitization was induced as described above. Thereafter, three airway antigen challenges were performed on Days 14, 19, and 24. One group of rats received 1 mg of S28463 24 hours before each antigen challenge and all animals were killed 48 hours after the final antigen challenge. See the online supplement for additional details.

Bronchoalveolar Lavage Fluid and Cellular Analysis
On killing, lungs of the animals were rinsed with saline and total and differential cell counts were performed. Additional details can be found in the online data supplement.

Plasma IgE
On killing, plasma was recovered from the animals and total IgE levels were measured by ELISA. See the online supplement for additional details.

Cytokine Analysis
Cytokine analysis was performed on lung tissue homogenate samples, using a custom LINCOplex rat cytokine kit (LINCO Research/Millipore, St. Charles, MO). See the online supplement for additional details.

Preparation of Histologic Samples
Lung samples were prepared for histology by formalin fixation and paraffin embedding. See the online supplement for additional details.

Measurement of Chronic Inflammation
Chronic inflammation was assessed by histologic analysis of hematoxylin and eosin–stained lung sections. See the online supplement for additional details.

Measurement of Airway Smooth Muscle Mass
Airway smooth muscle (ASM) mass was estimated from measurements of the area of smooth muscle–specific -actin (-SMA) immunoreactivity as previously described (30). See the online supplement for additional details.

Goblet Cell Hyperplasia
Goblet cell hyperplasia was assessed by histologic analysis of periodic acid–Schiff (PAS)–stained lung sections. See the online supplement for additional details.

Proliferating Airway Epithelial Cell and Subepithelial Cell Quantification
Proliferation of airway epithelial and subepithelial cells was investigated by immunostaining for proliferating cell nuclear antigen (PCNA). See the online supplement for additional details.

Statistical Analysis
All data were analyzed with GraphPad Prism 4 (version 4.03; GraphPad Software Inc., San Diego, CA), using a parametric one-way analysis of variance method followed by a Bonferroni post-test procedure. Data were normalized before analysis by logarithmic or square root transformation where appropriate. A p value of 0.05 or less was considered significant.

	RESULTS

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Treatment with S28463 Prevents the Influx of Inflammatory Cells into Bronchoalveolar Lavage Fluid
Having previously established that S28463 prevents acute airway inflammation in a mouse model of allergic asthma (24), in this study we investigated whether this compound had an effect on the development of airway remodeling in a rat model of chronic asthma. We first established the effectiveness of S28463 treatment in preventing the acute inflammatory reaction observed after a single allergen challenge. A prototypical finding in allergic asthma and its models is an influx of inflammatory cells, composed primarily of eosinophils but often also including neutrophils and macrophages, into the bronchoalveolar space. As expected (Figure 1A), there was a dramatic increase in the number of cells recovered in the bronchoalveolar lavage fluid (BALF) of OVA-sensitized and -challenged (OVA) rats when compared with rats that were challenged with vehicle only (PBS). Treatment with S28463 (S28/OVA) significantly reduced the number of inflammatory cells recovered in the BALF of OVA-challenged rats. Furthermore, treatment of sham-challenged animals (S28/PBS) did not lead to significant changes in BALF cell numbers. Although both macrophages and lymphocytes are increased in the BALF of OVA-challenged animals (Figure 1B), eosinophils, and to a lesser extent neutrophils, account for the majority of the increase. The dramatic increase in eosinophil numbers was significantly inhibited by treatment with S28463, and neutrophil and lymphocyte numbers trended downward, but the observed decrease did not reach statistical significance.

View larger version (24K): [in this window] [in a new window]   Figure 1. Analysis of inflammatory cells found in bronchoalveolar lavage fluid (BALF) after imidazoquinoline treatment (S28/OVA and S28/PBS) and OVA (OVA and S28/OVA) or PBS (PBS and S28/PBS) challenge. (A) Total number of inflammatory cells recovered in the BALF of animals. (B) Differential count of the inflammatory cells recovered. Results are represented as means ± SEM. *Significance between PBS and OVA groups; **significance between OVA and S28/OVA groups. n = 5 for S28/PBS group; n = 6 for all other groups. PBS = phosphate-buffered saline; OVA = ovalbumin; S28 = S28463.

View larger version (105K): [in this window] [in a new window]   Figure 2. Representative hematoxylin-and-eosin–stained lung sections collected 24 hours after challenge from animals acutely sensitized with OVA and challenged with PBS (A and B) or OVA (C and D) with prior treatment with (B and D) or without (A and C) S28463.

View larger version (7K): [in this window] [in a new window]   Figure 3. Total blood plasma IgE levels in rats after imidazoquinoline treatment (S28/OVA and S28/PBS) and OVA (OVA and S28/OVA) or PBS (PBS and OVA-S28/PBS) challenge. Data are represented as medians ± interquartile range (n = 6 for all groups).

Th2 cytokines IL-4 (Figure 4A), IL-5 (Figure 4B), and IL-13 (Figure 4D) and Th1 cytokine IFN- (Figure 4E) were strongly induced (16-, 85-, 10-, and 32-fold relative to PBS control, respectively) 48 hours after OVA challenge (OVA), compared with mock-challenged animals (PBS; Figures 4A, 4B, 4D, and 4E, respectively). The inflammatory cytokines IL-1 (Figure 4F), IL-1 (Figure 4G), and IL-6 (Figure 4C) were also found to be induced to a much greater extent in the OVA-challenged animals than in the PBS group (Figures 4C, 4F, and 4G). Furthermore, the chemokines CXCL-1 (Figure 4H) and macrophage inflammatory protein-1 (Figure 4I) were also elevated in OVA-challenged animals, but to a lesser extent than the other cytokines.

View larger version (17K): [in this window] [in a new window]   Figure 4. Cytokine levels in lung homogenates of OVA-sensitized animals after challenge with PBS (PBS and S28/PBS) or OVA (OVA and S28/OVA) with (S28/PBS and S28/OVA) or without (PBS and OVA) prior imidazoquinoline treatment. Data are presented as means ± SEM (*p < 0.05, ***p < 0.001; n = 6 for all groups). MIP-1 = macrophage inflammatory protein-1.

The cytokine profile (Figure 4) of animals that received a mock challenge (PBS) was similar to the profile of animals that received both S28463 treatment and mock challenge (S28/PBS) as evidenced by the fact that none of the cytokine levels tested was significantly different. IL-9 and eotaxin were not found to be significantly modulated by any treatment (data not shown).

Treatment with S28463 Prevents the Airway Inflammation Associated with Repeated Allergen Challenge
After characterizing acute inflammatory responses to allergic stimulation with OVA, we assessed the ability of S28463 to prevent the development of airway remodeling and inflammation induced by repeated allergen challenge. The presence of inflammatory cells surrounding the airways of animals after repeated allergen challenge was evaluated by histologic analysis (Figure 5). Sensitization with OVA followed by repeated allergen (OVA) challenges resulted in significant inflammatory cell infiltration in the area surrounding airways (Figure 5B). Comparatively few inflammatory cells could be found surrounding the airways of animals that had been sensitized with OVA but sham challenged with PBS (PBS) (Figure 5A). Similarly, the airways of OVA-sensitized and -challenged animals that had received S28463 treatment (S28/OVA) displayed markedly lower numbers of inflammatory cells (Figure 5C).

View larger version (90K): [in this window] [in a new window]   Figure 5. Representative hematoxylin and eosin–stained lung sections collected 48 hours after challenge from animals sensitized with OVA and chronically challenged with PBS (A) or chronically challenged with OVA (B and C) with prior S28463 treatment (C) or without prior S28463 treatment (A and B). Peribronchial inflammation was quantified (D) by counting the number of inflammatory cells, or eosinophils, surrounding airways and normalizing for airway size by dividing by the square of the perimeter of the basement membrane (Pbm2). Data are presented as means ± SEM (*p < 0.01 between PBS and OVA; **p < 0.01 between OVA and S28; ***p < 0.001 between OVA and S28; n = 6 for all groups).

Treatment with S28463 Prevents the Increase in Airway Smooth Muscle–specific -Actin–positive Cell Mass
One of the key structural changes observed in asthmatic lungs is an increase in ASM mass. Sensitization and repeated challenge of an animal led to more ASM-specific -actin staining (Figure 6B) in lung sections than could be observed in animals that were mock challenged (Figure 6A). The lungs of animals that were treated with S28463 (Figure 6C) were not visually distinguishable from the mock-challenged lung sections and showed less -actin immunoreactivity than the OVA-sensitized and -challenged lung sections (Figure 6B).

View larger version (76K): [in this window] [in a new window]   Figure 6. Representative airway smooth muscle–specific -actin staining (red stain) of lung sections from OVA-sensitized animals chronically challenged with PBS (A), OVA (B), or OVA after S28463 treatment (C). Smooth muscle–specific -actin–staining area was standardized for airway size by dividing by the square of the perimeter of the basement membrane of the airway (Pbm2). Data are presented as means ± SEM (n = 5 for OVA group; n = 4 for all other groups).

Goblet Cell Hyperplasia Is Prevented by S28463 Treatment
A second alteration of airway structure characteristic of the chronic remodeling changes in asthma is goblet cell hyperplasia. Goblet cells can be easily identified by the PAS method, which stains mucus glycoproteins. Only a relatively few and isolated PAS⁺ cells can be observed in the airway epithelium of OVA-sensitized and mock-challenged lungs (Figure 7A). OVA challenge leads to an easily observable increase in airway PAS⁺ cells (Figure 7B), which was inhibited in animals that were treated with S28463 (Figure 7C).

View larger version (89K): [in this window] [in a new window]   Figure 7. Representative periodic acid–Schiff (PAS) staining of lung sections from OVA-sensitized animals chronically challenged with PBS (A), OVA (B), or OVA after S28463 treatment (C). Goblet cells stain magenta in color. Goblet cell hyperplasia was determined by counting the number of PAS+ cells per airway and standardized for airway size by dividing by the perimeter of the basement membrane (Pbm). Data are presented as means ± SEM (n = 6 for all groups).

S28463 Treatment Prevents Proliferation of Airway Cells in Response to Repeated Allergen Challenge
One of the mechanisms believed to contribute to ASM mass increases in asthma is ASM cell proliferation (35, 36). Similarly, proliferation may account for the increase in the number of goblet cells found in the airways. We therefore evaluated the expression of PCNA in epithelial cells and in the subepithelial cells corresponding to the smooth muscle zone to determine whether proliferation might be involved in the goblet cell hyperplasia or ASM mass increase, respectively.

The airways of PBS-challenged rats contain only a relatively low number of PCNA-positive epithelial (Figure 8A, red arrows) and subepithelial (Figure 8A) cells scattered along the airway wall. OVA challenge led to a clear increase in the number of PCNA-positive airway epithelial cells (Figure 8B, red arrows) visible in the histologic samples. Furthermore, OVA challenge led to a dramatic increase in the number of subepithelial PCNA⁺ cells that could be observed in the ASM zone (Figure 8B, blue arrow). Treatment with S28463 before allergen challenge prevents the increase in both epithelial and subepithelial PCNA⁺ cells, as can be observed by inspection of the lung sections, which reveal relatively few isolated epithelial PCNA⁺ cells and even fewer PCNA⁺ subepithelial cells (Figure 8C).

View larger version (80K): [in this window] [in a new window]   Figure 8. Representative proliferating cell nuclear antigen (PCNA)–specific stain from OVA-sensitized animals chronically challenged with PBS (A), OVA (B), or OVA after S28463 treatment (C). The nuclei of PCNA-positive cells stain dark blue. PCNA-positive epithelial cells are indicated by red arrows and PCNA-positive subepithelial cells by a blue arrow. (D) Quantification of PCNA+ cells in the airway epithelium and subepithelium (smooth muscle zone). PCNA+ cells were counted in each airway and standardized for airway size by dividing by the square of the perimeter of the basement membrane (Pbm2, subepithelial cells) or the perimeter of the basement membrane (Pbm, epithelial cells). Data are presented as means ± SEM (*p < 0.01 between PBS and OVA; **p < 0.01 between OVA and S28/OVA; n = 6 for PBS, n = 4 for OVA, n = 5 for S28).

Cytokine Levels in Lungs of Chronically Challenged Animals
As illustrated in Figure 4 and Table 1, we found a significant induction of both Th1 and Th2 cytokines in the lungs of OVA-sensitized and -challenged rats 24 hours after the first allergen challenge. Forty-eight hours after the final allergen challenge, however, we did not detect elevated levels of any cytokine (Table 1). Levels of the cytokines in rats that were chronically challenged with allergen (OVA) did not remain significantly elevated, when compared with mock-challenged animals (PBS), when assessed at the 48-hour time point. Both of these groups expressed the measured cytokines at levels comparable to those found in the lungs of mock-challenged animals 24 hours after the initial exposure.

	DISCUSSION

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Sensitization and challenge of Brown Norway rats resulted in significant recruitment of cells, composed mainly of eosinophils and to a lesser extent neutrophils, into the airway tissue and alveolar space. Animals that were treated with S28463 one day before allergen challenge in this acute model of allergic asthma showed greatly reduced eosinophilic inflammation, consistent with previous observations in murine models. Furthermore, we found that, during the initial phase of the allergic response to allergen exposure, total IgE levels were lowered by S28463 treatment, which is also consistent with the published results of experiments performed in murine models (24, 26). We also observed an increase in the Th2 cytokines IL-4, IL-5, and IL-13, the Th1 cytokine IFN-, as well as an increase in IL-6 after allergen challenge that was prevented by S28463 treatment.

Previous studies employing TLR7 ligands have demonstrated that signaling through this receptor can stimulate the production of the Th1 cytokines IL-12, IFN-, and IFN- in a CD4⁺ lymphocyte and macrophage coculture (34). The cell type responsible for the bulk of the IL-12 production was determined to be the macrophage and, in addition, S28463 treatment of these cultures resulted in an inhibition of IL-5 production that was only partially mediated by IFN- (34). More recently, studies conducted with human cells have shown that S28463 can directly induce proliferation of, and upregulate IFN- production in, effector memory CD4⁺ T cells (37). Neither of these mechanisms, however, would fully account for the available data.

The natural resistance–associated protein-1 (Nramp1, now SLC11A1) gene is macrophage restricted, involved in modulating macrophage responsiveness to TLR7 agonists (38), and has been shown to be necessary for the TLR7 ligand S28463-induced activation of macrophages in a murine model of Mycobacterium bovis BCG infection (39). In the BCG model, S28463 treatment allows wild-type Nramp1^r mice to clear infection with the intracellular parasite; on the other hand, Nramp1^–/– or Nramp1^s (susceptible) mice did not respond to S28463 treatment. Our previous results demonstrated that S28463 treatment in an acute model of allergic asthma was effective in both Nramp1^r and Nramp1^s mice (24). These findings suggest that prevention of the acute allergic inflammation of the airways seen in asthma is not dependent on macrophage responsiveness to TLR7 ligands.

Whereas treatment with S28463 in a model of allergic asthma in the BALB/c mouse appears to result in an increase in Th1 cytokines, particularly IFN- (26), we have previously shown that, in both A/J and C57BL/6 mice, a similar treatment that successfully prevents the development of airway inflammation and hyperresponsiveness does not cause an increase in IFN- or IL-12 p70 (24). The present study indicates that treatment in the Brown Norway rat produces a similar result. In fact, in both the present study and in our previous work (24) we have found that IFN- is elevated after allergen challenge and that TLR7 ligand treatment prevents this induction. These findings suggest that it is unlikely that S28463 exerts its effect simply by redirecting the Th2 immune response typical of allergic asthma toward a Th1 response.

Th2 responses and activated CD4⁺ cells generated by the acute inflammatory reaction have both been implicated in the development of chronic airway remodeling (29, 40). It is therefore not surprising, but nonetheless important, that TLR7 ligand treatment can suppress the development of airway remodeling given the effectiveness with which it prevents the development of the initial inflammatory events.

Goblet cell hyperplasia may contribute to airway obstruction through excessive mucus production (41). We have demonstrated in this study that continued treatment with the TLR7 ligand S28463 prevents the development of goblet cell hyperplasia. Another important aspect of airway remodeling, which has been linked to asthma severity and is assumed to contribute to the development of airway hyperresponsiveness (2), is the increase in smooth muscle mass in asthmatic airways. Given that eosinophils have been shown to play a crucial role in the disease process leading to the marked increase in smooth muscle area (42), preventing the development of eosinophilic inflammation after allergen challenge would be expected to prevent the increase in smooth muscle mass observed in allergen-sensitized and -challenged animals. TLR7 ligand treatment behaved as expected in this respect, as we found no evidence of an increase in smooth muscle mass in S28463-treated animals.

The increase in ASM -actin–positive cell mass can occur through hyperplasia or hypertrophy. We have observed an increase in PCNA⁺ cells in the subepithelial smooth muscle zone of OVA-sensitized and -challenged rats. We therefore believe that an increase in proliferation of ASM -actin–positive cells is at least partially responsible for the ASM mass increase in our model. Similarly, we observed an increase in epithelial PCNA⁺ cells, which leads us to believe that the goblet cell hyperplasia we observed in chronically challenged animals is also at least partially caused by an increase in the proliferation of goblet cells or their precursors.

In summary, our data demonstrate that treatment with S28463, a TLR7/8 ligand, can prevent the development of the inflammatory reaction that results from allergen exposure in sensitized rats and that this was accomplished through inhibition of both Th1 and Th2 cytokines. Our data also demonstrated that continued treatment with S28463 can prevent the development of chronic airway remodeling, including goblet cell hyperplasia and airway smooth muscle hyperplasia. The ability of TLR7 signaling to modulate both acute and chronic asthma pathology makes this receptor a prime target for a new class of therapies aimed at minimizing the pathology associated with chronic inflammation in asthma. Furthermore, elucidation of the mechanism by which TLR7 modulates the immune response in the context of asthma should provide us with invaluable insight into the processes that lead to the development of the disease.

	Acknowledgments

 
The authors gratefully acknowledge the contributions of Dr. Yasuyuki Yoshizawa from the Department of Geriatric and Pulmonary Medicine, Tokyo Medical and Dental University, Tokyo.

	FOOTNOTES

 
Supported by a grant from the Sandler Program for Asthma Research (SPAR) awarded to Dr. D. Radzioch and by a grant from the Canadian Institutes of Health Research (MT 10381) to Dr. J. Martin. S28463 (resiquimod, R-848) was kindly provided by Dr. Richard Miller (3M Pharmaceuticals). Pierre Camateros is a recipient of a Natural Sciences and Engineering Council (NSERC) Postgraduate Scholarship and a Canadian Graduate Scholarship Doctoral Award from the Canadian Institutes of Health Research (CIHR). Dr. Jacques Moisan was a recipient of a Doctoral Research Award from the CIHR.

^* These investigators contributed equally to this article.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200701-054OC on March 30, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form January 10, 2006; accepted in final form March 28, 2007

	REFERENCES

TOP ABSTRACT AT A GLANCE COMMENTARY METHODS RESULTS DISCUSSION REFERENCES

 

1. Bai TR, Knight DA. Structural changes in the airways in asthma: observations and consequences. Clin Sci (Lond) 2005;108:463–477.[Medline]
2. Benayoun L, Druilhe A, Dombret MC, Aubier M, Pretolani M. Airway structural alterations selectively associated with severe asthma. Am J Respir Crit Care Med 2003;167:1360–1368.[Abstract/Free Full Text]
3. Barnes PJ. Anti-inflammatory actions of glucocorticoids: molecular mechanisms. Clin Sci (Lond) 1998;94:557–572.[Medline]
4. Mori A, Kaminuma O, Suko M, Inoue S, Ohmura T, Hoshino A, Asakura Y, Miyazawa K, Yokota T, Okumura Y, et al. Two distinct pathways of interleukin-5 synthesis in allergen-specific human T-cell clones are suppressed by glucocorticoids. Blood 1997;89:2891–2900.[Abstract/Free Full Text]
5. Lamas AM, Leon OG, Schleimer RP. Glucocorticoids inhibit eosinophil responses to granulocyte-macrophage colony-stimulating factor. J Immunol 1991;147:254–259.[Abstract]
6. Wallen N, Kita H, Weiler D, Gleich GJ. Glucocorticoids inhibit cytokine-mediated eosinophil survival. J Immunol 1991;147:3490–3495.[Abstract]
7. Irwin RS, Richardson ND. Side effects with inhaled corticosteroids: the physician's perception. Chest 2006;130:41S–53S.[CrossRef][Medline]
8. Camateros P, Moisan J, Henault J, De Sanctis J, Skamene E, Radzioch D. Toll-like receptors, cytokines and the immunotherapeutics of asthma. Curr Pharm Des 2006;12:2365–2374.[CrossRef][Medline]
9. Medzhitov R. Toll-like receptors and innate immunity. Nat Rev Immunol 2001;1:135–145.[CrossRef][Medline]
10. Schnare M, Barton GM, Holt AC, Takeda K, Akira S, Medzhitov R. Toll-like receptors control activation of adaptive immune responses. Nat Immunol 2001;2:947–950.[CrossRef][Medline]
11. Takeda K, Kaisho T, Akira S. Toll-like receptors. Annu Rev Immunol 2003;21:335–376.[CrossRef][Medline]
12. Braun-Fahrlander C, Riedler J, Herz U, Eder W, Waser M, Grize L, Maisch S, Carr D, Gerlach F, Bufe A, et al.; Allergy and Endotoxin Study Team. Environmental exposure to endotoxin and its relation to asthma in school-age children. N Engl J Med 2002;347:869–877.[Abstract/Free Full Text]
13. Douwes J, Pearce N, Heederik D. Does environmental endotoxin exposure prevent asthma? Thorax 2002;57:86–90.[Abstract/Free Full Text]
14. Broide D, Schwarze J, Tighe H, Gifford T, Nguyen MD, Malek S, Van Uden J, Martin-Orozco E, Gelfand EW, Raz E. Immunostimulatory DNA sequences inhibit IL-5, eosinophilic inflammation, and airway hyperresponsiveness in mice. J Immunol 1998;161:7054–7062.[Abstract/Free Full Text]
15. Kline JN, Waldschmidt TJ, Businga TR, Lemish JE, Weinstock JV, Thorne PS, Krieg AM. Modulation of airway inflammation by CpG oligodeoxynucleotides in a murine model of asthma. J Immunol 1998;160:2555–2559.[Abstract/Free Full Text]
16. Broide DH, Stachnick G, Castaneda D, Nayar J, Miller M, Cho JY, Roman M, Zubeldia J, Hayashi T, Raz E, et al. Systemic administration of immunostimulatory DNA sequences mediates reversible inhibition of Th2 responses in a mouse model of asthma. J Clin Immunol 2001;21:175–182.[CrossRef][Medline]
17. Sur S, Wild JS, Choudhury BK, Sur N, Alam R, Klinman DM. Long term prevention of allergic lung inflammation in a mouse model of asthma by CpG oligodeoxynucleotides. J Immunol 1999;162:6284–6293.[Abstract/Free Full Text]
18. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, Lipford G, Wagner H, Bauer S. Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8. Science 2004;303:1526–1529.[Abstract/Free Full Text]
19. Lund JM, Alexopoulou L, Sato A, Karow M, Adams NC, Gale NW, Iwasaki A, Flavell RA. Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc Natl Acad Sci USA 2004;101:5598–5603.[Abstract/Free Full Text]
20. Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 2004;303:1529–1531.[Abstract/Free Full Text]
21. Hemmi H, Kaisho T, Takeuchi O, Sato S, Sanjo H, Hoshino K, Horiuchi T, Tomizawa H, Takeda K, Akira S. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol 2002;3:196–200.[CrossRef][Medline]
22. Gorden KK, Qiu XX, Binsfeld CC, Vasilakos JP, Alkan SS. Cutting edge: activation of murine TLR8 by a combination of imidazoquinoline immune response modifiers and poly(T) oligodeoxynucleotides. J Immunol 2006;177:6584–6587.[Abstract/Free Full Text]
23. Wu JJ, Huang DB, Tyring SK. Resiquimod: a new immune response modifier with potential as a vaccine adjuvant for Th1 immune responses. Antiviral Res 2004;64:79–83.[CrossRef][Medline]
24. Moisan J, Camateros P, Thuraisingam T, Marion D, Koohsari H, Martin P, Boghdady ML, Ding A, Gaestel M, Guiot MC, et al. TLR7 ligand prevents allergen-induced airway hyperresponsiveness and eosinophilia in allergic asthma by a MYD88-dependent and MK2-independent pathway. Am J Physiol Lung Cell Mol Physiol 2006;290:L987–L995.[Abstract/Free Full Text]
25. Moisan J, Camateros P, Martin P, Khossari H, Boghdady ML, Guiot MC, Martin J, Skamene E, Radzioch D. Toll-like receptor 7 signalling prevents allergen-induced inflammation and lung hyperrespnsiveness in C57BL/6 mice [extended abstract]. Proceedings of the 12th International Congress of Immunology and 4th Annual Conference of FOCIS, Montreal, Canada, July 18–23, 2004, pp. 131–143.
26. Quarcoo D, Weixler S, Joachim RA, Stock P, Kallinich T, Ahrens B, Hamelmann E. Resiquimod, a new immune response modifier from the family of imidazoquinolinamines, inhibits allergen-induced Th2 responses, airway inflammation and airway hyper-reactivity in mice. Clin Exp Allergy 2004;34:1314–1320.[CrossRef][Medline]
27. Weixler S, Quarcoo D, Lode HN, Hamelmann E. Imidazoquinoline derivative inhibits allergen-induced T-helper cell 2 responses, eosinophilic airway inflammation and airway hyperreactivity in mice [extended abstract]. Proceedings of the 12th International Congress of Immunology and 4th Annual Conference of FOCIS, Montreal, Canada, July 18–23, 2004, pp. 549–553.
28. Palmans E, Kips JC, Pauwels RA. Prolonged allergen exposure induces structural airway changes in sensitized rats. Am J Respir Crit Care Med 2000;161:627–635.[Abstract/Free Full Text]
29. Ramos-Barbon D, Presley JF, Hamid QA, Fixman ED, Martin JG. Antigen-specific CD4⁺ T cells drive airway smooth muscle remodeling in experimental asthma. J Clin Invest 2005;115:1580–1589.[CrossRef][Medline]
30. Panettieri RA Jr, Murray RK, Eszterhas AJ, Bilgen G, Martin JG. Repeated allergen inhalations induce DNA synthesis in airway smooth muscle and epithelial cells in vivo. Am J Physiol 1998;274:L417–L424.[Medline]
31. Flood-Page PT, Menzies-Gow AN, Kay AB, Robinson DS. Eosinophil's role remains uncertain as anti-interleukin-5 only partially depletes numbers in asthmatic airway. Am J Respir Crit Care Med 2003;167:199–204.[Abstract/Free Full Text]
32. Wong CK, Ho CY, Ko FW, Chan CH, Ho AS, Hui DS, Lam CW. Proinflammatory cytokines (IL-17, IL-6, IL-18 and IL-12) and Th cytokines (IFN-, IL-4, IL-10 and IL-13) in patients with allergic asthma. Clin Exp Immunol 2001;125:177–183.[CrossRef][Medline]
33. Kuo ML, Huang JL, Yeh KW, Li PS, Hsieh KH. Evaluation of Th1/Th2 ratio and cytokine production profile during acute exacerbation and convalescence in asthmatic children. Ann Allergy Asthma Immunol 2001;86:272–276.[Medline]
34. Wagner TL, Ahonen CL, Couture AM, Gibson SJ, Miller RL, Smith RM, Reiter MJ, Vasilakos JP, Tomai MA. Modulation of T_H1 and T_H2 cytokine production with the immune response modifiers, R-848 and imiquimod. Cell Immunol 1999;191:10–19.[CrossRef][Medline]
35. Ebina M, Takahashi T, Chiba T, Motomiya M. Cellular hypertrophy and hyperplasia of airway smooth muscles underlying bronchial asthma: a 3-D morphometric study. Am Rev Respir Dis 1993;148:720–726.[Medline]
36. Woodruff PG, Dolganov GM, Ferrando RE, Donnelly S, Hays SR, Solberg OD, Carter R, Wong HH, Cadbury PS, Fahy JV. Hyperplasia of smooth muscle in mild to moderate asthma without changes in cell size or gene expression. Am J Respir Crit Care Med 2004;169:1001–1006.[Abstract/Free Full Text]
37. Caron G, Duluc D, Fremaux I, Jeannin P, David C, Gascan H, Delneste Y. Direct stimulation of human T cells via TLR5 and TLR7/8: flagellin and R-848 up-regulate proliferation and IFN- production by memory CD4⁺ T cells. J Immunol 2005;175:1551–1557.[Abstract/Free Full Text]
38. Moisan J, Thuraisingam T, Henault J, De Sanctis J, Radzioch D. Role of SLC11A1 (formerly NRAMP1) in regulation of signal transduction induced by Toll-like receptor 7 ligands. FEMS Immunol Med Microbiol 2006;47:138–147.[CrossRef][Medline]
39. Moisan J, Wojciechowski W, Guilbault C, Lachance C, Di Marco S, Skamene E, Matlashewski G, Radzioch D. Clearance of infection with Mycobacterium bovis BCG in mice is enhanced by treatment with S28463 (R-848), and its efficiency depends on expression of wild-type Nramp1 (resistance allele). Antimicrob Agents Chemother 2001;45:3059–3064.[Abstract/Free Full Text]
40. Komai M, Tanaka H, Masuda T, Nagao K, Ishizaki M, Sawada M, Nagai H. Role of Th2 responses in the development of allergen-induced airway remodelling in a murine model of allergic asthma. Br J Pharmacol 2003;138:912–920.[CrossRef][Medline]
41. Elias JA, Zhu Z, Chupp G, Homer RJ. Airway remodeling in asthma. J Clin Invest 1999;104:1001–1006.[Medline]
42. Wills-Karp M, Karp CL. Biomedicine: Eosinophils in asthma: remodeling a tangled tale. Science 2004;305:1726–1729.[Abstract/Free Full Text]

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