Allergin-1 on mast cells suppresses house dust mite-induced airway hyperresponsiveness in mice

Abstract

Although airway hyperresponsiveness (AHR) is a prominent feature of asthma, how it is regulated remains incompletely understood. Allergin-1, an inhibitory immunoglobulin-like receptor containing an immunoreceptor tyrosine-based inhibitory motif (ITIM), is expressed on human and mouse mast cells (MCs) and inhibits high-affinity receptor for IgE (FcεRI)-mediated signaling. Using MC-deficient Kit^W-sh/W-sh mice and Mas-TRECK mice, which carries a diphtheria toxin (DT)-induced MC deletion system based on il4 enhancer elements, we demonstrate here that MCs are involved in the induction of house dust mite (HDM)-induced AHR. Further, we show that MCs deficient in Allergin-1 exacerbated HDM-induced AHR, but had no effect on airway inflammation. In vitro analysis demonstrated that Allergin-1 inhibited anti-HDM allergen antibody-dependent HDM allergen-mediated degranulation by MCs. Thus, Allergin-1 on MCs plays an important role in the regulation of HDM-induced AHR.

allergic asthma, immunoglobulin-like receptor, ITIM, FcεRI

Introduction

Airway hyperresponsiveness (AHR) causing airway obstruction is a prominent feature of asthma. Mast cells (MCs) infiltrated into the airways play an important role in AHR (1). MCs express the high-affinity receptor for IgE (FcεRI) that binds to an allergen through FcεRI-bound IgE. This interaction activates MCs, resulting in the secretion of a broad spectrum of pro-inflammatory cytokines, chemokines and chemical mediators (2). IgE production by B cells is promoted by T helper 2 (T_h2)-derived cytokines, IL-4, and anti-IgE therapy is beneficial in certain patients with severe asthma (3), suggesting that IgE-mediated signals in MCs play an important role in the pathogenesis of asthma. However, the role of MCs in the development of AHR is somewhat controversial (4), and the precise mechanisms by which MCs regulate AHR remain to be elucidated.

Allergin-1, which is encoded by Milr1, is an inhibitory immunoglobulin-like receptor expressed on both human and mouse MCs (5, 6). When Allergin-1 is co-ligated with IgE-bound FcεRI, tyrosine residues of immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic region of Allergin-1 are phosphorylated and recruit Src homology 2 domain-containing tyrosine phosphatase 1 (SHP-1) and SHP-2, thereby blocking an early step in the activation signal mediated by FcεRI (5). Allergin-1-deficient mice show significantly enhanced passive systemic and cutaneous anaphylaxis, indicating that Allergin-1 suppresses IgE-mediated MC activation in vivo (5). Allergin-1 also inhibits toll-like receptor (TLR) 2-mediated activation of MCs in the skin and suppresses dermatitis (7).

In this study, we examined the role of Allergin-1 on MCs in AHR and T_h2 responses in mice. We show that Allergin-1 regulated MC activation and suppressed AHR, but not T_h2 responses, induced by house dust mite (HDM).

Methods

Animals

Female BALB/cAJcl and C57BL/6N wild-type (WT) mice (8 to 12 weeks old) were purchased from Clea Japan (Tokyo, Japan). Allergin-1-deficient (Milr1^−/−) mice on the BALB/c background were generated by backcrossing Milr1^−/− C57BL/6N mice (5) to the BALB/cAJcl genetic background for 12 generations. Kit^W-sh/W-sh mice were provided by RIKEN BioResource Center (Tsukuba, Japan). Mas-TRECK mice on the BALB/c background have been previously described (8). All procedures were done according to the guidelines of the animal ethics committee of the University of Tsukuba (permit number 17-284).

MC engraftment

To generate bone marrow (BM)-derived cultured MCs (BMMCs), 2 × 10⁶ BM cells were cultured in a 10-cm dish in complete RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS (Life Technologies, Auckland, New Zealand) in the presence of 10 ng ml⁻¹ stem cell factor (R&D Systems, Minneapolis, MN, USA) and 4 ng ml⁻¹ IL-3 (R&D Systems) for 4–5 weeks, at which time the cell population was composed of >95% MCs, as assessed with flow cytometry. Mas-TRECK mice were injected with diphtheria toxin (DT, 250 ng; Sigma-Aldrich) intra-peritoneally on 5 consecutive days every 2 weeks to deplete endogenous MCs. On day 7 after the first injection of DT, 1 × 10⁷ WT or Milr1^−/− mice-derived BMMCs were injected intravenously into each DT-treated Mas-TRECK mouse. MC reconstitution was assessed by flow cytometric analysis of peritoneal exudate MCs at 3 months after BMMC injection.

HDM-induced allergic asthma model

HDM allergen whole body extract (LSL Co., Ltd, Tokyo, Japan), derived from Dermatophagoides pteronyssinus (Der p), was used to induce allergic asthma. Animals were treated intra-nasally with 10 μg of HDM extract on 5 consecutive days a week for 4 weeks. Mice were assessed for AHR, airway inflammation and serum IgE concentration 48 h after the last intra-nasal challenge.

Measurement of AHR to methacholine

Airway resistance to different doses of aerosolized methacholine was measured by the computer-controlled piston ventilator system (FlexiVent, Montreal, Quebec, Canada) in accordance with the manufacturer’s instructions. Briefly, 48 h after the last HDM challenge, mice were anesthetized, tracheotomized, and intubated intra-tracheally and exposed to an aerosol of 70 μl phosphate-buffered saline (PBS; basal readings) and then of 70 μl methacholine in PBS at the indicated concentration. Respiratory resistance was determined as the percentage change from the baseline index.

Antibodies

A monoclonal antibody (mAb) specific to mouse Allergin-1 (TX83; mouse IgG1) was generated in our laboratory, as described previously (5). TX83 was biotinylated with a Biotinylation Kit (Sulfo-OSu; DOJIN, Tokyo, Japan). MAbs to mouse CD45.2 (104), c-Kit (2B8), Ly6G (1A8), CD11b (M1/70), CD11c (HL3) and Siglec-F (E50-2440) were purchased from BD Biosciences (San Joes, CA, USA); mAbs against CD16/32 (2.4G2), FcεRI (MAR-1), CD3ε (145-2C11) and B220 (RA3-6B2) were purchased from TONBO Bioscience (San Diego, CA, USA); mAbs against Gr-1 (RB6-8C5) and CD107a (1D4B) were purchased from BioLegend (San Diego, CA, USA).

Flow cytometric analysis of the lung and bronchoalveolar lavage fluid cells

The lung tissues were perfused with 1 ml of PBS through the right ventricle, removed, and digested with collagenase II (300 U per sample; Worthington Biochemical, Lakewood, NJ, USA) at 37°C for 1 h and dissociated with a gentleMACS Dissociator (B.01 program; Miltenyi Biotec, Auburn, CA, USA). To assess the airway eosinophilia, bronchoalveolar lavage fluid (BALF) was harvested 48 h after the last HDM challenge and the trachea was cannulated with a 21-G needle and plastic tube and washed with 1 ml of PBS three times. The cells were treated with anti-CD16/32 mAb to avoid binding to FcγR on ice for 10 min prior to incubation with indicated combination of antibodies. Propidium iodide (PI, 1 μg ml⁻¹; Sigma-Aldrich) was used to gate out dead cells. Cells were analyzed on a BD LSR Fortessa cell analyzer (BD Biosciences) and the FlowJo software program (Tree Star, San Carlos, CA, USA).

Measurement of serum IgE concentration by ELISA

For quantitation of serum levels of IgE, 96-well ELISA plates (NUNC Thermoscientific, Yokohama, Japan) were coated with rat anti-mouse IgE (catalog no. R35-72; BD Biosciences) diluted in 50 mM sodium bicarbonate buffer (pH 9.6) as a capture antibody. IgE was detected by using biotinylated anti-mouse IgE (catalog no. R35-118; BD Biosciences), followed by streptavidin-conjugated horseradish peroxidase (GE Healthcare Biosciences, Little Chalfont, UK). Mouse IgE (catalog no. C38-2; BD Biosciences) was used as a standard. All of these assays were performed in accordance with the manufacturers’ instructions.

Histology

Forty-eight hours after the last HDM challenge, mice were anesthetized, and their lungs were harvested and fixed in 10% neutral buffered formalin. Fixed lungs were embedded in paraffin and stained with hematoxylin-eosin (HE) and periodic acid-Schiff (PAS). The goblet cell hyperplasia was assessed by measuring % area of the airway basement membrane that was positive for PAS staining in the total area of the airway basement membrane in three randomly selected bronchioles of each mouse. Three mice per group were analyzed for the goblet cell hyperplasia. All images were acquired using a BZ-X710 microscope (Keyence, Osaka, Japan), and the data were analyzed using a BZ-H3 analyzer and Hybrid Cell Count (Keyence).

Degranulation assay

Degranulation was evaluated by a flow cytometric analysis of the cell-surface exposure of CD107a (LAMP-1). LAMP-1 is an intracellular protein found on granule membranes that becomes exposed on the cell-surface upon degranulation (9). BMMCs were incubated for 20 h at 37°C with serum obtained from a naive mouse or from HDM-immunized mice (in which the serum IgE level was as high as 1 μg ml⁻¹) and then were stimulated for 30 min at 37°C with the indicated concentrations of HDM extract. BMMCs were then stained with allophycocyanin (APC)-conjugated anti-CD107 and analyzed for the proportion of CD107a⁺ cells among c-Kit⁺ FcεRI⁺ cells by using flow cytometry.

Statistics

Statistical analyses were performed with the GraphPad Prism 7 software (La Jolla, CA, USA) by using the unpaired Student’s t-test or one-way or two-way ANOVA followed by Bonferroni’s multiple comparison test as a post-test. A P value <0.05 was considered statistically significant.

Results and discussion

We first examined the involvement of MCs in HDM-induced AHR by using MC-deficient Kit^W-sh/W-sh mice. WT and Kit^W-sh/W-sh mice were immunized intra-nasally with 10 μg of HDM extract on 5 consecutive days a week for 4 weeks (Fig. 1A). The airway eosinophilia and serum IgE level were comparable between the two groups (Fig. 1B). However, AHR to methacholine was significantly decreased in Kit^W-sh/W-sh mice compared with that in WT mice (Fig. 1B), suggesting that MCs are responsible for HDM-induced AHR, but are dispensable for T_h2 responses. To further demonstrate the function of MCs in HDM-induced AHR, we used DT-treated Mas-TRECK mice, an MC deletion system that uses Il4 enhancer elements (8). Although basophils are also depleted in DT-treated Mas-TRECK mice transiently, they quickly recovered within 3 days after the last DT treatment (8). In agreement with a previous report (8), flow cytometry did not detect MCs in the peritoneal cavity 2 days after the last injection of DT in Mas-TRECK mice (Fig. 1C and D). The DT-treated Mas-TRECK mice were reconstituted with WT BMMCs, and then repeatedly immunized with 10 μg of HDM extract by intra-nasal injection (Fig. 1C). The reconstitution of MCs was confirmed by analyzing the MC population in the peritoneal cavity 3 months after the BMMC transfer (Fig. 1D). Although DT-treated Mas-TRECK mice, which lack MCs, were resistant to methacholine-induced AHR, the mice that had received WT BMMCs developed AHR in response to the challenge with methacholine (Fig. 1E). Collectively, these results indicate that MCs are responsible for HDM-induced AHR.

Fig. 1.

AHR in MC-deficient mice. (A and B) C57BL/6N WT (n = 4) and MC-deficient Kit^W-sh/W-sh mice (n = 4) were immunized intra-nasally with 10 μg of HDM extract on 5 consecutive days a week for 4 weeks. (A) The experimental design. (B) The proportion of eosinophils (CD45⁺CD11c⁻Siglec-F⁺) in the BALF, serum IgE level and AHR to methacholine were analyzed at 48 h after the last HDM immunization. (C–E) The experimental design (C). Three months after the BMMC injection, peritoneal exudate MCs in the naive state from DT-non-treated, DT-treated (DT) and BMMC-reconstituted DT-treated (DT + BMMC) Mas-TRECK mice were analyzed by flow cytometry (D). DT-treated Mas-TRECK mice given no BMMCs (DT; n = 4) or reconstituted with WT-derived BMMCs (DT + BMMCs; n = 5) were immunized intra-nasally with 10 μg of HDM extract. AHR to methacholine was analyzed at 48 h after the last HDM immunization (E). Rrs, respiratory resistance. An unpaired t-test was used to determine significant differences between the two groups. NS, not significant. *P < 0.05. Data are representative of at least two independent experiments (mean ± SEM).

We then examined the expression of Allergin-1 on MCs accumulated in the lungs after HDM treatment. We found that the lung MCs (identified as CD45.2⁺Lin⁻CD11b⁺Siglec-F⁻FcεRI⁺c-Kit⁺ cells) highly expressed Allergin-1. In contrast, the lung eosinophils (indicated as CD45.2⁺Siglec-F⁺CD11b^high cells) expressed undetectable level of Allergin-1 (Fig. 2A). The numbers of MCs in the lung were comparable between WT and Milr1^−/− mice after HDM treatment (Fig. 2A). To investigate the role of Allergin-1 on MCs in HDM-induced AHR, we reconstituted DT-treated Mas-TRECK mice with BMMCs derived from either WT or Milr1^−/− mice and treated them with HDM as shown in Fig. 1(C). The reconstitution efficiency was comparable between WT and Milr1^−/− BMMCs, as assessed by the MC population in the peritoneal cavity 3 months after the BMMC transfer (Fig. 2B). After the intra-nasal administration of HDM, the numbers of total cells (CD45⁺ cells) and eosinophils (CD45⁺Gr-1^lowSiglec-F⁺CD11c⁻ cells) in the BALF and the serum IgE levels were induced equally in both mouse populations, and no striking differences in goblet cell hyperplasia were observed between DT-treated Mas-TRECK mice that had been reconstituted with WT or Milr1^−/− BMMCs (Fig. 2C–E). However, those mice reconstituted with Milr1^−/− BMMCs showed more severe AHR to methacholine than did those reconstituted with WT BMMCs (Fig. 2F). Collectively, these results indicate that Allergin-1 on MCs is not involved in HDM-induced T_h2 responses but rather inhibits HDM-induced AHR.

Fig. 2.

Allergin-1 on MCs suppresses AHR. (A) Flow cytometric analysis of MCs (CD45.2⁺Lin⁻Siglec-F⁻c-Kit⁺FcεRI⁺) and eosinophils (CD45.2⁺Lin⁻Siglec-F⁺CD11b^high) in the lungs of WT and Milr1^−/− mice after intra-nasal injection of HDM extract (100 μg per mouse). Blue line, WT; red line, Milr1^−/−; (right) quantification of numbers of MCs. (B–F) DT-treated Mas-TRECK mice were injected with BMMCs derived from WT or Milr1^−/− mice. (B) Three months after the BMMC injection, the reconstitution capacity of the injected BMMCs was assessed in the peritoneal exudate cells by flow cytometry. (C–F) DT-treated MC-reconstituted Mas-TRECK mice were immunized with HDM extract as shown in Fig. 1(C). (C) BALF and (D) blood samples collected 48 h after the last HDM challenge were analyzed for the numbers of total cells (CD45⁺) and eosinophils (CD45.2⁺Gr-1^lowCD11c⁻Siglec-F⁺) and IgE concentration, respectively. (E) Lungs were harvested 48 h after the last HDM challenge and analyzed for histology after treatment with hematoxylin-eosin (HE) and periodic acid-Schiff (PAS) staining. Original magnification: ×200. Right, quantification of PAS-positive goblet cells. (F) AHR to methacholine. DT-treated Mas-TRECK mice given no BMMCs (n = 3) or reconstituted with BMMCs from WT (n = 4) or Milr1^−/− (n = 5) were immunized with HDM extract. Forty-eight hours after the last HDM challenge, AHR to methacholine was analyzed. Data are representative of three (C–E) or two (A, B and F) independent experiments (mean ± SEM). Each point represents a single mouse. NS, not significant. *P < 0.05 (WT versus Milr1^−/−, unpaired t-test).

AHR is considered to be triggered by chemical mediators, such as histamine, in the granules released from MCs (1). Therefore, we examined whether Allergin-1 inhibits HDM allergen-induced degranulation from MCs. Since HDM-specific mouse IgE or IgG antibodies were unavailable, we used sera from HDM-immunized mice, in which the serum IgE level was as high as 1 μg ml⁻¹ for HDM-induced degranulation from MCs. WT or Milr1^−/− BMMCs were sensitized overnight with the sera from either naive or HDM-immunized mice, stimulated with HDM extract for 30 min, and then analyzed for CD107a expression on MCs (a marker of degranulation) by flow cytometry. After sensitization, Milr1^−/− BMMCs had a larger population of CD107a⁺ cells than did WT BMMCs upon 1000 ng ml⁻¹ of HDM stimulation (Fig. 3A and B). In contrast, HDM-induced CD107a expression was scarcely detected on either genotype of BMMCs sensitized with naive serum, demonstrating HDM allergen-induced MC degranulation requires sensitization of MCs with HDM-immunized sera. These results suggest that Allergin-1 inhibited anti-HDM antibody-dependent degranulation of MCs, thus suppressing HDM allergen-induced AHR.

Fig. 3.

Allergin-1 on MCs inhibits anti-HDM antibody-dependent degranulation. WT or Milr1^−/− BMMCs were treated for 16 h with serum from naive or HDM-treated mice, followed by HDM extract stimulation for 30 min, and analyzed for degranulation by CD107a expression. (A) Representative results by flow cytometric analysis. (B) Statistical analysis. ***P < 0.005; ****P < 0.001 (WT versus Milr1^−/−, unpaired t-test). Data are representative of three independent experiments (mean ± SEM).

The contribution of MCs in AHR has been controversial (4), because most previous studies on this issue used MC-deficient Kit^W-sh/W-sh or Kit^W/W-v mice and a virtual allergen, such as ovalbumin (OVA) with or without aluminum salt adjuvant (4, 10). Those mice carry abnormalities due to c-Kit mutation in both hematopoietic and non-hematopoietic tissues as well as MCs (11). Mas-TRECK mice used in this study show only a defect of MCs and basophils, and we reconstituted Mas-TRECK mice with BMMCs, thus they were more appropriate than mice with the c-Kit mutation. Moreover, HDM is one of the commonest aeroallergens; >50% of allergic patients and up to 85% of asthmatic children are sensitized with HDM (12–14). Therefore, a mouse model using HDM as an allergen might be more appropriate for human asthma.

A recent study by using mice deficient in IgE or FcεRIα demonstrated that HDM-induced AHR is independent of IgE and FcεRIα (15). In the current study, we showed that MCs were required for HDM-induced AHR in vivo and that MC activation was dependent on the serum of HDM-immunized, but not naive, mice in vitro, suggesting that HDM-induced AHR was induced by MCs activated by anti-HDM antibody. However, we did not show the direct evidence for involvement of anti-HDM IgE antibody and FcεRIα. Since MC degranulation is also induced by IgG immune complexes (16), it is also possible that MC activation was mediated by the FcγRI, rather than FcεRIα, on MCs in our model. Further studies are required for understanding the mechanisms of HDM-induced AHR.

We demonstrated that MCs play an important role in HDM-induced AHR. However, previous reports demonstrated that treatment with cysteine proteases, including papain, induce AHR, for which basophils and type 2 innate lymphoid cells (ILC2) are responsible (17, 18). Cysteine proteases promote IL-4 production from basophils and activate ILC2, which augment inflammation via production of inflammatory cytokines and chemokines (17). Thus, basophil and ILC2-dependent AHR are involved in innate immune responses, whereas MC-dependent AHR requires antibodies against HDM as a result of adaptive immune responses.

Because AHR is a fundamental pathology in asthma, control of AHR should be a therapeutic goal in the management of patients with asthma. Our findings show that the MC is a key driver for AHR and that Allergin-1 on MCs plays a regulatory role in this process.

Funding

This work was supported in part by grants provided by Japan Society for the Promotion of Science (KAKENHI) (grant numbers 15H04862 to S.T.-H. and 16H06387 to A.S.) and was funded by ONO Pharmaceutical Co., Ltd.

Acknowledgements

We thank S. Tochihara for secretarial assistance. K.H. performed most of the experiments and analyzed the data; H.M. and K.I. did experiments; S.S. and M.K. contributed to experimental design; S.T.-H. designed and did experiments, analyzed data and wrote the paper; and A.S. supervised the overall project and wrote the paper.

Conflicts of interest statement: We are conducting research sponsored by ONO Pharmaceutical Co., Ltd. S.S. is an employee of ONO Pharmaceutical Co., Ltd. The sponsor had no control over the interpretation, writing or publication of this work.