C57BL/6J (B6), GRK2^fl/fl, and CD4 Cre⁺ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). GRK2^+/− mice were kindly provided by Dr. Narayanan Parameswaran [Michigan State University (MSU)]. Mice were bred and housed under specific pathogen-free conditions. The CD4 Cre⁺ GRK2^fl/fl (Cre⁺) mouse strain that lacks GRK2 in T cells were generated by crossing the CD4 Cre⁺ mice with the GRK2^fl/fl mice. Littermate CD4 Cre⁻ GRK2^fl/fl (Cre⁻) mice were used as controls. Six to eight-week-old male and female mice used for all the experiments that were approved by the Institutional Animal Care and Use Committee at MSU.

Lung samples from deceased subjects that died of asthma or other causes were obtained from either the International Institute for the Advancement of Medicine or National Disease Resource Interchange. All lung samples were harvested anonymously and de-identified. The use of these samples was approved by Institutional Review Board at Rutgers University. The available information for individual donors is provided in Supplemental Table 1.

Mice were instilled intranasally (i.n) with either 25μl of HDME (50 μg of protein, Greer Laboratories, Lenoir, NC) or 25 μl of PBS on alternate days for a total of seven injections. The endotoxin level of the HDME preparation was 38.4 EU per injection. Twenty-four hours after the final HDME challenge, mice were anesthetized for measurement of AHR and then sacrificed and the blood, bronchoalveolar lavage fluid (BALF) and lung tissue were collected for various endpoint analysis.

Lung tissues were fixed for 48 h in 4% paraformaldehyde (PFA) and embedded in paraffin. Tissue sections (5-μm) were cut and adhered to a positively charged slides. Xylene was used to deparaffinize the tissues and 100% alcohol, 95% alcohol, and normal saline (pH 7.4) was used for rehydration followed by antigen retrieval by 10 mM sodium citrate buffer, pH 6.0. Endogenous peroxidase was inactivated by 3% hydrogen peroxide following which the tissue sections were the probed with the isotype control IgG antibody or the anti- GRK2 antibody (1:200, #3982, Cell Signaling Technology, Danvers, MA) overnight at 4°C, and anti-rabbit secondary antibody (1:500, Vector Laboratories, Burlingame, CA) for 1 h at room temperature. The slides were washed and treated with the DAB substrate (Vector laboratories) for 5 min. Hematoxylin and eosin (H&E) were used as the counter stains for color development. A representative image of the isotype control antibody staining is shown in Supplemental Figure 1. Six images for each specimen were taken at 20× magnification and the images were analyzed by ImageJ software (NIH, Bethesda, MD). The determination of staining intensity for GRK2 (relative density) were performed in a blinded fashion.

Mice were anesthetized with intraperitoneal (i.p.) injection of 100 mg/kg ketamine (Henry Schein Animal Health, Dublin, OH), 10 mg/kg xylazine (Akorn, Lake Forest, IL) and 3 mg/kg acepromazine (Henry Schein Animal Health, Dublin, OH) and tracheostomized. Airway mechanics was measured using forced oscillation technique by flexiVent (SCIREQ®, Quebec, Canada). Airway resistance (Rrs) and elastance (Ers) were assessed following exposure to varying concentrations methacholine (MCh; Sigma-Aldrich, St. Louis, MO).

The lungs were infused via the trachea with 10% buffered formalin, excised and stored in fresh 10% formalin overnight. Samples were then embedded in paraffin, cut into 5-μm-thick sections and stained with H&E (for lung inflammation) or Periodic acid-schiff (PAS, for goblet cell hyperplasia). Digital images of sections were obtained using a Nikon Eclipse 50i microscope (Nikon, Japan) equipped with an INFINITY-3 digital color camera (Lumenera Corporation, Canada), and INFINITY ANALYZE 6.5.4 software.

Slides were scored in a blinded fashion using the scoring system described below. Six pictures covering the whole lung section at 4× magnification were collected and the total number of inflamed bronchioles (surrounded by infiltrated cells) in each picture was counted. A value from 0 to 4 was adjudged to each bronchiole scored. A value of 0–1 was decided when no inflammation or occasional cuffing with inflammatory cells was detected, a value of 2–4 for bronchi surrounded by a thin layer (2–4 cells) of inflammatory cells and a value of >4 when bronchi were surrounded by a thick layer (more than five cells) of inflammatory cells. Number of bronchioles within each category (0–1, 2–4, or >4) was then divided by the total number of inflamed bronchioles to obtain percent bronchioles within each category of inflammation. For calculating the percent severity of inflammation, the number of inflamed bronchioles that received a score of 4 or more was divided by the total number of inflamed bronchioles.

Goblet cell hyperplasia was evaluated on PAS-stained lung sections. Each lung sample was divided into 6–8 imaginary sections and digitally imaged at 10× magnification. The intensity of PAS staining in the images was assessed using ImageJ software to determine PAS positive cells as well as the percent area of PAS positive cells in each section.

Blood was drawn from the superior mesenteric vein of the mouse and left at 4°C overnight. Serum was collected the next day and analyzed for total IgE and IgG1 using commercially available ELISA kits from Invitrogen (Carlsbad, CA).

Cell-free BALF fluid was collected from euthanized mice after inflating the lungs with 0.5 ml of sterile PBS three times and centrifuging it at 400 × G for 5 min to separate the cellular components from the supernatants. BALF supernatants were analyzed for cytokines including, IL-4 (BD Biosciences, San Diego, CA) and IL-13 (Invitrogen) by ELISA. The BALF cellularity was determined using a hemocytometer following differential staining of the cellular fraction. Briefly, 50,000 cells were cytospun onto a clean glass slide, air- dried and then stained with Giemsa Wright stain (Sigma-Aldrich, St. Louis, MO) for 3 min. The stained slides were washed with distilled water, dried, dipped in xylene (Avantor, Radnor Township, PA) for 2 s and a cover slip was placed immediately over the cells. For each sample, a total of 200 cells were counted at 40× magnification and the number of monocytes/macrophages, lymphocytes, and eosinophils was enumerated.

Spleens were harvested from mice and single cell suspension was prepared. CD4 T cells were purified from the spleen cells by positive selection with the mouse naïve CD4 T cell isolation kit from Miltenyi Biotech (San Diego, CA) as per the manufacturer's recommendations. The purity of the cells as determined by flow cytometry was ≥95%.

CD4 T cells (2 × 10⁶ cells) were plated in a 48-well plate coated with anti-mouse CD3 antibody (BioLegend, 10 μg/ml) and anti-mouse CD28 antibody (BioLegend, 5 μg/ml) and incubated for 72 h at 37°C/5% CO₂. The supernatant was harvested and analyzed for IL-2 by ELISA.

CD4 T cells purified from the spleen were washed twice in PBS and lysed with radio-immunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1.0% Triton X-100, 0.5% sodium-deoxycholate, 0.1% sodium dodecyl sulfate, 25 mM Tris [pH 8.0], 5 mM EDTA) with protease inhibitor cocktail (Roche Applied Sciences; Mannheim, Germany). Twenty μg of protein was loaded in a 10% polyacrylamide gel for electrophoretic separation. Proteins were then transferred to nitrocellulose membranes (GE Healthcare). Membranes were blocked in 5% milk solution for 2 h, washed in Tris-buffered saline [pH 7.6] with 0.1% Tween-20 (TBST), then probed with the anti-GRK2 antibody (Cell Signaling Technology, Cat# 3982). The following day, blots were washed in TBST and probed with LiCor IRDye® 680RD or IRDye® 800CW conjugated secondary antibodies for 2 h in the dark. Blots were imaged using LiCor Odyssey Imaging Systems (Lincoln, NE) and analyzed using ImageJ software. β-Actin antibody (Cell Signaling Technology, Cat# 3700) was used as the loading control.

RNA was isolated from mouse lungs using the TRIzol reagent (Invitrogen) and was reverse transcribed into cDNA using the High-Capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). Real-time quantitative PCR was performed using Quant Studio™ 3 system (Applied Biosystems) with validated Taqman primers and Fast Advanced Master Mix. Relative gene expression data (fold change) between samples was accomplished using the 2^−ΔΔCt method. GAPDH was used as the internal control.

Lungs were harvested from mice subjected to the asthma model and single cells were isolated using the Percoll density method. Briefly, lung samples were minced and digested with collagenase P (1 mg/ml, Roche Diagnostics, Indianapolis, IN) at 37°C for 30 min. Single cell suspension was obtained by passing the digested tissue through a 70-μm cell strainer (Alkali Scientific Inc., Fort Lauderdale, FL) with a plunger. Lung mononuclear cells were then isolated using density centrifugation with 70% Percoll (GE, Piscataway, NJ). The isolated cells were washed and resuspended in RPMI media (Life Technologies, Carlsbad, CA) conditioned with 10% FBS (Atlanta Biologicals, Flowery Branch, GA) and 1% penicillin-streptomycin (Mediatech Inc., Manassas, VA). The cells (2 × 10⁵ cells/well/200 μL) were plated in a 96 well plate and stimulated with HDME (3 μg/well). After 72 h the supernatant was harvested and was analyzed for cytokines (IL-4 and IL-13) by ELISA.

Statistical significance was determined using GraphPad Prism software (GraphPad, San Diego, CA). Student's unpaired t-test assuming equal standard deviation or two-way ANOVA was used for analysis of data. Significance is shown as ^*p < 0.05 and ^**p < 0.01.

GRK2 is a ubiquitously expressed adapter protein that is present in most cell types of the airways including AECs, ASMCs and other immune cells. Its expression however is altered during pathological conditions; for e.g., the expression levels of GRK2 is enhanced in cystic fibrosis patients (25). To determine if GRK2 is similarly upregulated in the lungs during asthma, we performed qRT-PCR and IHC staining on lung tissue sections from deceased asthmatic and non-asthmatic (control) individuals. GRK2 expression (both at the mRNA and protein levels) was significantly elevated in the lungs of asthmatic individuals (Figures 1A–C). To test if GRK2 levels are affected in a mouse model of human asthma, we treated wild-type B6 mice with HDME on alternative days (for a total of seven exposures) and harvested the lung tissue 24 h following the final exposure. Consistent with our data from human lung samples, GRK2 expression (mRNA and protein) was increased in the lung tissues of mice exposed to HDME as compared to PBS controls (Figures 1D–F).

Because GRK2 expression was elevated in the lungs of HDME-challenged mice, we hypothesized that it would regulate the asthmatic response in a mouse model. To test this, we used GRK2^+/− mice (GRK2^−/− is embryonically lethal) and GRK2^+/+ (control) mice and exposed them to PBS or HDME. AHR parameters such as central airway resistance (Rrs) and lung elastance (Ers) were assessed in response to varying concentrations of Mch (6.25–100 mg/ml). There was no difference in the baseline Rrs and Ers were between GRK2^+/+ and GRK2^+/− mice injected with PBS (Figures 2A,B). HDME-exposed GRK2^+/+ and GRK2^+/− mice demonstrated elevation in Rrs and Ers measurements in a dose-dependent manner to Mch. However, there was no difference in HDME-induced Rrs and Ers values between the two cohorts of mice. Allergic asthma is characterized by elevated levels of serum IgE. We observed that serum IgE was higher in HDME-treated GRK2^+/+ mice as compared to PBS controls (Figure 2C). Although, GRK2^+/− mice had lower serum IgE as compared to HDME exposed GRK2^+/+ mice, the values were not significantly different.

Airway inflammation is accompanied by an influx of inflammatory cells such as monocytes/macrophages, eosinophils and lymphocytes into the lungs and these cells are consistently detected in the BALF of allergic individuals. Since GRK2 has been previously reported to affect migration of immune cells (23, 26–28), we next determined the cellularity in BALF of GRK2^+/+ and GRK2^+/− mice. Following exposure to HDME, an increase in immune cell numbers was seen in the BALF of GRK2^+/+ mice. The inflammatory infiltrate mainly consisted of eosinophils along with lower numbers of monocytes/macrophages and lymphocytes. Surprisingly, there was no significant difference in the total leukocyte, monocyte/macrophage, eosinophil or lymphocyte counts between HDME treated GRK2^+/− and GRK2^+/+ mice (Figures 2D–G).

A previous study from our laboratory showed that repeated HDME treatment results in increased levels of Th2 cytokines in the lungs (29), so we next analyzed Th2 cytokines in the lung tissues and BALF of different cohorts of mice (Figure 3). As expected the mRNA (Figures 3A,B) and protein levels of IL-4, and IL-13 (Figures 3C,D) were elevated in the lungs and BALF of HDME-exposed GRK2^+/+ mice as compared to PBS controls. Interestingly, the expression levels of these cytokines were significantly reduced in GRK2^+/− HDME mice (Figures 3A–D).

Given that the Th2 cytokines (IL-4 and IL-13) are reduced in the lungs of GRK2^+/− mice, it is possible that GRK2 modulates T cell responses during asthma. To determine the role of T cell-specific GRK2 in affecting the asthma pathology, we generated a mouse stain [CD4 Cre⁺ GRK2^fl/fl (Cre⁺)] that lacks GRK2 in T cells. We then used the Cre⁺ and littermate CD4 Cre⁻ GRK2^fl/fl (Cre⁻) mice (as controls) for our experiments. First, we purified CD4 T cells from the spleens of these mice and analyzed for GRK2 expression in these cells using Western Blotting. As shown in Figure 4A, GRK2 expression was reduced by ~80% in CD4 T cells from Cre⁺ mice. A previous report had demonstrated that GRK2 promotes T cell activation and is required for production of cytokines such as IL-2 (24). To test this in our model, we stimulated T cells isolated from the Cre⁺ and Cre⁻ with anti-CD3 and anti-CD28 antibodies and assessed for IL-2 secretion as a measure of T cell activation. Our data suggests that IL-2 production was significantly reduced in Cre⁺ cells that had reduced levels of GRK2 (Figure 4B). We then subjected the Cre⁻ and Cre⁺ mice to the HDME asthma model, performed AHR experiments and determined serum IgE and IgG1 levels in allergic mice. In contrast to the results obtained with GRK2^+/− mice, both the AHR (Rrs and Ers values) (Figures 4C,D) and serum IgE levels (Figure 4E) were significantly reduced in Cre⁺ mice when compared to Cre⁻ mice following HDME exposure. This suggests that GRK2 is required in the T cell compartment to modulate AHR and serum IgE levels in our asthma model. Interestingly, though IgG1 was unaltered between the Cre⁺ and Cre⁻ mice treated with HDME, Cre⁺ mice had higher basal levels of this antibody as compared to Cre⁻ mice (Figure 4F).

To determine if GRK2 expression in T cells regulates airway inflammation, we harvested the lungs from Cre⁺ and Cre⁻ mice exposed to HDME and performed H&E staining on these tissues. We also enumerated the numbers of monocytes/macrophages, eosinophils and lymphocytes in the BALF. HDME treatment resulted in a significant increase in airway inflammation in Cre⁻ mice as shown by the intense H&E staining especially near the peri-bronchial and perivascular areas of the lung tissue. However, this inflammation was severely attenuated in the Cre⁺ mice exposed to HDME (Figures 5A–C). In addition, Cre⁺ HDME mice exhibited reduced total BALF cell counts (Figure 5D). Specifically, while there was no change in monocyte/macrophage numbers (Figure 5E), eosinophil and lymphocyte counts were significantly lower in the BALF of Cre⁺ HDME mice (Figures 5F,G).

It is well established that the Th1-Th2 axis is skewed during asthma pathogenesis and that the Th1 cytokines can modulate the expression levels of Th2 cytokines and vice-versa (30, 31). To determine if T cell-specific GRK2 expression modulates Th1 and Th2 cytokines, we estimated the mRNA levels of Th1 (Ifng) and Th2 (Il4 and Il13) cytokines in lungs of mice exposed to HDME. We observed a significant reduction in Ifng in the Cre⁺ mice as compared to the control Cre⁻ mice (Figure 6A). Consistent with the results obtained with GRK2^+/− mice (Figure 3), and reduced inflammation observed in the Cre⁺ HDME mice (Figure 5), Il4 and Il13 levels were significantly reduced in the lungs of Cre⁺ HDME mice (Figures 6B,C). Recent reports have suggested that the cytokines of the IL-17 family are increased in BALF samples from patients with asthma and IL-17A has been predicted to be a risk factor for severe asthma (32, 33). Accordingly, we observed higher levels of Il17a in the lungs of HDME-exposed Cre⁻ mice (Figure 6D). However, this was reduced in the Cre⁺ mice. Furthermore, epithelial cell-derived cytokines such as IL-33 play a major role in the initiation of allergic asthma (34). We observed an increase in Il33 levels in HDME-treated Cre⁻ mice that was significantly attenuated in Cre⁺ HDME mice (Figure 6E). Another important immune cell type that has been implicated in the pathology of asthma are regulatory T cells (Tregs) that express the transcription factor Foxp3. Specifically, several reports have shown that functional defects in Tregs leads to enhancement of Th2 responses and the pathogenesis of asthma (35–38). We observed an increase in Foxp3 expression following HDME exposure in Cre⁻ mice that was significantly decreased in Cre⁺ mice (Figure 6F). Consistent with the mRNA expression data (Figures 6B,C), we also observed a reduction in both IL-4 and IL-13 protein levels in the BALF of Cre⁺ HDME mice as compared to allergen-challenged Cre⁻ mice (Figures 6G,H). Additionally, this defect in IL-4 and IL-13 cytokine production was also observed when lung mononuclear cells from HDME-treated Cre⁺ mice were re-exposed to the allergen in vitro (Figures 6I,J).

Goblet cell hyperplasia (an increase in the numbers of goblet cells) is a characteristic feature of allergic asthma that results in excessive mucus production and airflow obstruction in the lungs. Increased level of Th2 cytokines such as IL-13 has been frequently associated with mucus secretion and is consequently believed to be a major contributor of goblet cell hyperplasia during asthma (39). We analyzed goblet cell hyperplasia through PAS staining of lung sections from Cre⁻ and Cre⁺ mice. Consistent with the diminished IL-13 levels (Figures 6C,H), HDME-treated Cre⁺ mice exhibited reduced PAS staining (Figure 7A). We also quantified percent area of PAS+ staining and PAS+ cell count and observed a significant decrease in these parameters in the HDME-challenged Cre⁺ mice (Figures 7B,C). The polymeric mucins, Muc5ac and Muc5b are primarily responsible for mucus production in mouse models of asthma (40, 41). We observed that the mRNA levels of both Muc5ac and Muc5b were significantly upregulated in the lungs of Cre⁻ HDME-exposed mice (Figures 7D,E), however, they were significantly decreased in Cre⁺ mice.