Therapeutic and prophylactic deletion of IL‐4Ra‐signaling ameliorates established ovalbumin induced allergic asthma

Abstract Background Allergic asthma is a chronic inflammatory airway disease driven predominantly by a TH2 immune response to environmental allergens. IL‐4Rα‐signaling is essential for driving TH2‐type immunity to allergens. Anti‐TH2 therapies have the potential to effectively reduce airway obstruction and inflammation in allergic asthma. Objective We investigated potential therapeutic effects of selective inhibition of this pathway in mice with established allergic airway disease. We further investigated whether IL‐4Rα disruption in systemically sensitized mice can prevent the onset of the disease. Methods We used RosacreERT2IL‐4Rα−/lox mice, a tamoxifen (TAM)‐inducible IL‐4Rα knockdown model to investigate the role of IL‐4/IL‐13 signaling prior to the onset of the disease and during the effector phase in the ovalbumin‐induced allergic airway disease. Results Inducible deletion of IL‐4Rα demonstrated therapeutic effects, on established allergic airway disease, and prevented the development of ovalbumin‐induced airway hyperreactivity, eosinophilia, and goblet cell metaplasia in allergen‐sensitized mice. Interestingly, IL‐4Rα knockdown after allergic sensitization did not induce TH17, a neutrophilic inflammatory response as observed in global IL‐4Rα‐deficient mice after intranasal allergen challenge. Conclusion Abrogation of IL‐4Rα signaling after allergic sensitization would have significant therapeutic benefit for TH2‐type allergic asthma.


| INTRODUC TI ON
Airway hyperresponsiveness (AHR), pulmonary inflammation, eosinophilia, and mucus hyperplasia are hallmarks of allergic asthma. Indeed, chronic disease is driven predominantly by T H 2 immune responses with pathology largely caused by IL-4 and IL-13 signaling, which share a common pleiotropic receptor subunit, interleukin 4 receptor alpha (IL-4Rα). [1][2][3][4] The IL-4/IL-13 axis has been a target for allergic asthma treatment; however, anti-T H 2-based therapies for asthma have seen limited success in clinical trials. [4][5][6] The therapy conundrum is further compounded by phenotypic heterogeneity among asthma cases, limited treatment options for steroid-resistant cases, and possible non-T H 2 inflammatory mechanisms involved in asthma pathogenesis. [7][8][9][10][11] However, targeting T H 2-type inflammation still remains a promising therapeutic approach for a large proportion of carefully stratified asthma patients. 9 Key in the IL-4/IL-13 axis is the IL-4Rα signaling which has been identified as a potential target for asthma therapies. [12][13][14] Deficiency of IL-4Rα in allergen-sensitized mice shows reduced allergen-induced symptoms such as AHR, eosinophilia, and mucus hyperplasia in vivo. [15][16][17] We and others have also illustrated various cell-specific roles of IL-4Rα signaling during the development of allergic disease pathology. 11,[18][19][20][21] However, limited successful anti-T H 2-based treatments for asthma calls for better understanding of the mechanisms involved in successful therapy. Hence, we investigated in vivo requirements of IL-4Rα signaling after allergic sensitization prior to the onset of disease as well as after T H 2 pulmonary allergic airway lung inflammation. We hypothesized that temporal significance of IL-4Rα signaling might highlight its prophylactic and therapeutic relevance, especially as most patients are diagnosed only after established exacerbations. 22,23 Resurgence of side effects such as T H 17 responses has been a concern for anti-T H 2 targeted therapies. [4][5][6][24][25][26][27][28] We developed a TAM-induced, conditional IL-4Rα knockdown mouse model (Rosa creERT2 IL-4Rα −/lox ) and sought to investigate the temporal role of IL-4Rα receptor signaling during the effector phase (therapeutic) and priming/sensitization phase (prophylactic) of allergic asthma. Rosa creERT2 IL-4Rα −/lox , IL-4Rα −/lox , and IL-4Rα −/− mice were sensitized with OVA/alum complex intraperitoneally and challenged intranasally with OVA to induce allergic airway disease. Temporal deletion of IL-4Rα chain after sensitization phase prevented disease development, whereas temporal deletion during the effector phase reduced most disease parameters, such as AHR, eosinophilia, as well as goblet cell metaplasia. Unexpectedly, temporal deletion of IL-4Rα signaling in both prophylactic and therapeutic models did not develop T H 17-type and neutrophilic airway inflammation, a phenotype completely different to global deletion of IL-4Rα. We, thus, conclude that abrogation of IL-4Rα signaling after allergic sensitization would have significant therapeutic benefit for T H 2 type asthma without inducing potentially detrimental T H 17 responses.

G R A P H I C A L A B S T R A C T
Interleukin 4 receptor alpha (IL-4Rα) is central in the initiation and maintenance of the T H 2 allergic airway disease. Temporal genetic deletion of the IL-4Rα after sensitization reduced T H 2 disease by abrogating effector mechanisms such as CD11b + migratory DCs, T H 2-associated cytokines, eosinophilia, mucus production, and AHR. Temporal genetic deletion of the IL-4Rα in an established disease reduced CD11b + migratory DCs, eosinophilia, IgE, mucus production, AHR, but not T H 2-associated cytokines. Global deletion of the IL-4Rα induces CD103 + DCs, neutrophilia, and TH17 cytokines, underscoring the importance of temporal genetic deletion. Abbreviations: AHR, airway hyperresponsiveness; IL-4Rα, interleukin 4 receptor alpha; RORt, RAR-related orphan receptor gamma; STAT6, signal transducer and activator of transcription 6 Rosa creERT2 IL-4Rα −/lox C57BL/6 mice 29 with IL-4Rα lox/lox BALB/c mice ( Figure S1). IL-4Rα −/lox30 and IL-4Rα −/−31 mice on a BALB/c background were used as control animals. Eight-to twelve-week-old mice were

| Lung function measurements
Airway resistance and elastance of the whole respiratory system (airways, lung chest wall) after intranasal challenge was determined by forced oscillation measurements as described previously 32 with the Flexivent system (SCIREQ) by using the single compartment ("snapshot") perturbation. Measurements were carried out on mice with increasing doses of acetyl-ß-methylcholine (methacholine, Sigma-Aldrich) treatment. Differences in the dose-response curves were analyzed by repeated-measures ANOVA with the Bonferroni post-test. Only mice with acceptable measurements for all doses (coefficient of determination > 0.90) were included in the analysis.

| Analysis of cell populations by flow cytometry
Bronchoalveolar lavage (BAL) cells were obtained as previously described. 19 Single-cell suspensions were prepared from lymph nodes in Iscove's modified Dulbecco's medium (IMDM) (Gibco) by passing them through 40-µm filter. To obtain single-cell suspensions from lung tissues, a left lobe lung was digested for 1 hour at 37°C in RPMI (Gibco, Paisley, United Kingdom) containing 13 mg/mL DNase I (Roche) and 50 U/mL collagenase IV (Gibco) and passed through 70-µm filter. Single cells were then blocked with 24G2 for 30 minutes at 4°C, followed by surface staining with fluorophore-conjugated antibodies for 30 minutes at 4°C in the dark.

| Histology and immunohistochemistry
Lungs were fixed in 4% formaldehyde/PBS and embedded in paraffin. Tissue sections were stained with periodic acid-Schiff (PAS) for mucus secretion, hematoxylin and eosin (H&E) staining for inflammation. Image analysis was performed on NIS Elements (Nikon Instruments). Mucus quantification was carried out using the auto-

| Statistical analysis
P values were calculated in GraphPad Prism 6 (GraphPad Software, Inc) by using nonparametric Mann-Whitney Student's t test or twoway ANOVA with Bonferroni's post-test for multiple comparisons, and results are presented as SE of the mean. Differences were considered significant if P was <.05.

| Characterization of IL-4Rα expression in Rosa creERT2 IL-4Rα −/lox mice
Inducible Rosa creERT2 IL-4Rα −/lox mouse strain has been previously characterized on a C57BL/6 background strain by our laboratory and shown to have impaired expression of IL-4Rα on lung and lymph node tissue upon TAM treatment. 33 Here, we characterized IL-4Rα expression in Rosa creERT2 IL-4Rα −/lox mouse strain on the BALB/c background under OVA-induced allergic asthma. The tamoxifen-induced deletion of IL-4Rα lasts for more than 16 weeks after 4 days of tamoxifen instillation by oral gavage with no homeostatic imbalance. 33 We induced deletion of IL-4Rα in two models, prophylactic (where mice were fed TAM orally after sensitization before acute challenge with OVA) and therapeutically (where mice were fed TAM orally after sensitization and acute challenge with OVA) ( Figure 1A). Efficient knockdown of IL-4Rα in both lung and mediastinal lymph node (mLN) tissue after TAM treatment compared to vehicle-treated mice was observed ( Figure 1B,C).
We further evaluated cell type-specific deletion of IL-4Rα and found significantly reduced expression in inflammatory cells such as CD4 +

T cells, B cells, and dendritic cells (DCs), but not macrophages in lung
tissue of TAM-treated mice compared to the OIL-treated littermate controls ( Figure 1D,E). Expression levels of IL-4Rα after TAM knockdown were similar to those observed in IL-4Rα global knockout mice ( Figure 1B-E). We therefore concluded that IL-4Rα was efficiently knocked down upon treatment with TAM.

| Knockdown of the IL-4Rα signaling prophylactically prevented the development of AHR and airway inflammation
Mice deficient in IL-4Rα are protected from OVA-induced allergic airway disease. 1 Here, we explored the temporal requirement of this signaling pathway postsensitization with OVA/alum. We assessed AHR after OVA challenge and found TAM-Rosa creERT2 IL-4Rα −/lox mice to have significantly reduced airway resistance and elastance compared to the OIL-Rosa creERT2 IL-4Rα −/lox littermate controls ( Figure 2A). We also observed significantly increased resistance and elastance in OVA-challenged and OIL-Rosa creERT2 IL-4Rα −/lox when compared to PBS-challenged control mice. This demonstrated that deletion of IL-4Rα after sensitization prevented the development of AHR following allergen challenge.
We measured total numbers of infiltrating cells, eosinophils, and neutrophils in the BAL fluid and lung tissue and found that total cells and eosinophils were reduced in TAM-Rosa creERT2 IL-4Rα −/lox mice compared to OIL-Rosa creERT2 IL-4Rα −/lox mice ( Figure 2B-D). We observed increased neutrophil infiltration in IL-4Rα −/− mice, which was absent in TAM-Rosa creERT2 IL-4Rα −/lox mice ( Figure 2D). Furthermore, we observed reduced mucus hypersecretion (Figure2E,F) and lung inflammation ( Figure 2E) in TAM-Rosa creERT2 IL-4Rα −/lox mice compared to OIL-Rosa creERT2 IL-4Rα −/lox mice. Taken together, these data demonstrate that IL-4Rα-dependent signaling postsensitization is important in regulating airway inflammation and that IL-4Rα-dependent signaling prior to sensitization may contribute to airway neutrophilia.

| IL-4Rα signaling is necessary for inducing a T H 2 allergic airway immune response in a prophylactic model
We measured T H 2 cytokines after stimulation of mLNs with anti-CD3 ( Figure 3A

| Deletion of IL-4Rα signaling after established allergic airway disease reduces AHR and airway inflammation
We assessed the temporal requirement of IL-4Rα signaling in a therapeutic model as described in Figure 1A. Firstly, we assessed lung function and observed a significant reduction in airway resistance and elastance in TAM-Rosa creERT2 IL-4Rα −/lox compared to OIL-Rosa creERT2 IL-4Rα −/lox littermate controls ( Figure 4A). We also observed significantly increased resistance and elastance in OVAchallenged OIL-treated Rosa creERT2 IL-4Rα −/lox when compared to PBS-challenged control mice. This demonstrated a protective therapeutic effect of IL-4Rα temporal deletion in established allergic airway disease.
We then measured total infiltrating cells in BAL fluid and found that they were reduced in TAM-Rosa creERT2 IL-4Rα −/lox mice compared to OIL-Rosa creERT2 IL-4Rα −/lox mice ( Figure 4B), although the difference in lung tissue was not significant ( Figure 4C). This correlated with a reduction in airway eosinophilia in BAL fluid of TAM-Rosa creERT2 IL-4Rα −/lox mice compared to the OIL-Rosa creERT2 IL-4Rα −/ lox mice ( Figure 4D). Global IL-4Rα-deficient mice had significantly higher neutrophilic infiltration in the BAL fluid when compared to TAM-Rosa creERT2 IL-4Rα −/lox mice, which correlated with increased total cell counts in this group ( Figure 4B-D). When analyzing lung pathology by histology, we observed reduced mucus hypersecretion ( Figure 4E,F) and lung inflammation ( Figure 4E) in TAM-Rosa creERT2 IL-4Rα −/lox mice compared to OIL-Rosa creERT2 IL-4Rα −/lox . These data, together with AHR, suggested that IL-4Rα signaling is crucial in maintaining allergic airway inflammation in established disease.

| T H 2 immune response and antibody production is maintained after temporal deletion of IL-4Rα signaling in established allergic airway disease
We demonstrated that temporal deletion of IL-4Rα during the effector stage reduced AHR and lung inflammation. We then measured T H 2 cytokines in mLNs stimulated with anti-CD3 ( Figure 5A) or OVA ( Figure 5B). Interestingly, T H 2 cytokine (IL-4 and IL-5) levels were similar between TAM-Rosa creERT2 IL-4Rα −/lox and OIL-Rosa creERT2 IL-4Rα −/lox mice in both anti-CD3 ( Figure 5A) and OVA-stimulated ( Figure 5B) mLNs. We then assessed humoral responses by measuring levels of OVA-specific antibodies and total IgE. We observed no reduction in OVA-specific IgG 1 and IgG 2a (Figure 5C), although a significant reduction in total IgE was observed ( Figure 5D) along with a slight reduction in OVA-specific IgE ( Figure 5C) when comparing TAM-Rosa creERT2 IL-4Rα −/lox mice and OIL-Rosa creERT2 IL-4Rα −/lox mice.
Taken together, these data suggested that the reduced signs of allergic airway inflammation and AHR during temporal deletion of IL-4Rα were unlikely to be caused by reduced type-2 cytokines.
Functionally distinct DCs are central in not only inducing, but also suppressing T H 2 or T H 17 immune response through secretion of T H cell-polarizing cytokines. [34][35][36][37][38] We investigated the DC compartment in lung draining LNs ( Figure 6A,B). We observed a reduction in migratory CD11b + DCs in both prophylactic ( Figure 6C) and therapeutic ( Figure 6D) IL-4Rα temporarily deleted TAM-Rosa creERT2 IL-

| T H 17-induced neutrophilia is dependent on IL-4Rα deletion prior to sensitization
We observed airway neutrophilia in IL-4Rα −/− mice, but not in TAM-Rosa creERT2 IL-4Rα −/lox mice in both prophylactic and therapeutic deletion of IL-4Rα signaling ( Figures 2D and 4D). We showed previously that increased production of IL-17, after disruption of IL-4Rα expression on CD4 + T cells, was responsible for increased IL-17 production and airway neutrophilia.

| D ISCUSS I ON
The critical importance of IL-4/IL-13 axis as a driver of T H 2 immunity in allergic asthma has been established with IL-4Rα being central mediator of disease pathology. 1,4 We and others have previously demonstrated cell-specific function of IL-4Rα signaling in the development of allergic disease. 1,11,16,[18][19][20][21]40 However, the temporal role of IL-4Rα signaling in vivo, during sensitization and effector phases of allergic disease, is not completely clear. In this study, we developed an inducible IL-4Rα deletion model, allowing us to conditionally delete receptor signaling. Using this approach, we were able to investigate the role of IL-4/IL-13 signaling during onset and effector phase of OVA-induced allergic airway disease in systemically sensitized animals. Our findings show that temporal deletion of IL-4Rα signaling postsensitization prevents development of OVA-induced AHR, eosinophilia, and goblet cell hyperplasia (summarized in Table   S1). Hence, abrogation of IL-4Rα signaling should have significant therapeutic benefit for T H 2-type allergic asthma. Our study contributes to already existing literature on the central role of IL-4Rα subunit in allergic asthma and may guide currently ongoing phase III clinical trials targeting IL-4Rα in various asthma endotypes.
IL-4Rα signaling is important in establishing a T H 2-driven allergic airway pathology, and its absence, for example, IL-4Rα-deficient mice, leads to amelioration of IL-4/IL-13-dependent allergic airway disease. [41][42][43] We observed a reduction in AHR, eosinophilia, mucus hypersecretion, and pathology during temporal deletion of IL-4Rα after sensitization phase or during effector phase. This indicates that anti-IL-4Rα therapies may be suitable for therapeutic treatment of acute allergic disease patients and possibly prophylactic intervention in asymptomatic, but sensitized patients, who represent 50%-60% of asthma patients 44,45 and are highly at risk of developing asthma or eczema. 46,47 We further showed that the diminished T H 2 cell response in our study is not caused by an increased IFN-γ response as previously suggested. 48 The reduction in T H 2 responses in our study correlated with reduced STAT6 phosphorylation. 43 Of significant note is the potential requirement for IL-4Rα signaling in migration of CD11b + DCs to secondary lymphoid tissues for antigen sensitization and priming of a T H 2 cell-mediated immune response. This would be consistent with previous studies showing ILC2-derived IL-13 being important in priming migration of DC to mediastinal lymph nodes through CCL21 chemoattractant. 49 It is also possible that IL-4Rα in DC is crucial for recruitment of memory T H 2 cells to the lung through production of chemokines such as CCL17. 38 The successful utility of targeting IL-4Rα signaling has led to clinical trials for dupilumab, a human monoclonal antibody for anti-T H 2 therapy in allergic asthma patients and atopic dermatitis. 12,13,58 Concern arises on unprecedented T H 1/T H 17 polarizing inflammation with anti-T H 2 interventions in humans and mice. 4,24,25,27,59,60 Our mouse model clears a T H 2 response without inducing any po- Development of T H 17 responses has been suggested as a potential detrimental side-effect of anti-allergic therapies targeting the IL-4/IL-13/IL-4Rα signaling pathway. 65 The exact mechanism of how the T H 17 response is potentiated is elusive. In our previous work, we have shown that disruption of IL-4Rα-signaling specifically on CD4 + T cells results in antigen-specific T H 17 responses after allergic sensitization. 11 Similar to the global IL-4Rα knockout mice, deficiency of IL-4Rα subunit on CD4 + T cells was prior to sensitization. In this study, T H 17 responses were only observed in global IL-4Rα-deficient mice, but not in TAM-Rosa creERT2 IL-4Rα −/ lox mice, where IL-4Rα signaling was disrupted only after sensitization. Additionally, with the observed long-lasting effects of embryonic deletion of IL-4Rα and that of "conditional" knockout of IL-4Rα at presensitized period, the protective effect against challenge with allergen could be due to the protective effect against IL-13 because of the deleted IL-4Rα signaling. This is further seen in the therapeutic model where in the presence of a residual IL-13 production, the protective effect against IL-13 still remains with an observed diminished airway hyperresponsiveness. Thus, our study supports the concept that IL-4Rα signaling directly prevents differentiation of CD4 + T cells into T H 17 cells during allergen sensitization and reveals a temporal IL-4Rα dependency in development of a T H 17 airway inflammation. Strikingly, with the absence of IL-4Rα signaling prior to sensitization, we observed a modulation of a DC compartment in lymphoid tissue. A predominant increase migratory CD103 + DC profile and antigen-specific IL-23 secretion were observed and are both known to be responsible for maintenance of memory T H 17 cells. 66  In conclusion, we provide further evidence for a therapeutic potential for blocking IL-4Rα signaling in acute disease cases as well as prophylactic possibility in cases of asymptomatic atopic patients in T H 2-type allergic airway inflammatory responses.

ACK N OWLED G M ENTS
We thank the UCT Research Animal Facility for maintaining mice, Munadia Ansari for genotyping mice, and Amkele Ngomti for assistance. We are grateful to Lizette Fick for excellent histology services.
We thank Michelle Epstein (Medical University Vienna) for critically reading the manuscript.

CO N FLI C T S O F I NTE R E S T
The authors declare that they have no conflicts of interest.