House dust mite (HDM) allergens are leading causes of allergic asthma characterized by Th2 responses. The lung-resident CD11b+ dendritic cells (DCs) play a key role in Th2 cell development in HDM-induced allergic asthma. However, the regulatory mechanism of HDM-induced CD11b+ DC activation remains incompletely understood. In this study, we demonstrate that mice deficient in an inhibitory immunoreceptor, Allergin-1, showed exacerbated HDM-induced airway eosinophilia and serum IgE elevation. By using bone marrow–chimeric mice that were sensitized with adoptively transferred HDM-stimulated wild-type or Allergin-1–deficient CD11b+ bone marrow–derived cultured DCs (BMDCs), followed by challenge with HDM, we show that Allergin-1 on the BMDCs suppressed HDM-induced allergic airway inflammation. We also show that Allergin-1 suppressed HDM-induced PGE2 production from CD11b+ BMDCs by inhibiting Syk tyrosine kinase activation through recruitment of SHP-1, subsequently leading to negative regulation of Th2 responses. These results suggest that Allergin-1 plays an important role in regulation of HDM-induced allergic airway inflammation.

House dust mites (HDMs) contain several allergens, such as the Dermatophagoides pteronyssinus–associated family of Der p, D. farina–associated family of Der f, and HDM-associated bacterial and fungal products, including proteolytic enzymes, LPS, fecal pellets, fungal spores, and chitin (1). The Th2 responses induced by HDMs have been extensively studied in murine models of allergic diseases. Der p 2, a member of the Der p family, shows structural and functional similarities to MD-2, an LPS-binding component of TLR4 signaling complex (2). HDM extract stimulate TLR4 expressed on lung epithelial cells and induce the production of epithelium-associated cytokines such as IL-33, thymic stromal lymphopoietin (TSLP), IL-25, and GM-CSF, which activate lung-resident dendritic cells (DCs) to induce Th2 responses (3). TLR4 on lung-resident DCs also is involved in the Th2 responses to inhaled low-dose LPS (4, 5). The lung-resident CD11b+ DCs are responsible for HDM-induced Th2 cell differentiation (3). However, the signaling pathway mediated by TLR4 in DCs for the induction of Th2 responses and its regulatory mechanism remain incompletely understood.

Allergin-1 is an Ig-like receptor that contains ITIMs in the cytoplasmic portion and is highly expressed on mast cells (MCs) in both humans and mice (6, 7). Allergin-1 recruits the Src homology 2 domain–containing tyrosine phosphatases 1 (SHP-1) and SHP-2, which inhibit high-affinity receptor for IgE (FcεRI)–mediated signaling in MCs and suppress IgE and MC-dependent systemic and cutaneous anaphylaxis (6). Allergin-1 on MCs also inhibits TLR2-mediated activation of MCs in the skin and suppresses dermatitis (8). We have recently reported that Allergin-1 on MCs is involved in HDM-induced airway hyperresponsiveness (AHR) but dispensable for airway inflammation and Th2 responses by using MC-deficient KitW-sh/W-sh mice and Mas–TRECK mice, which carry a diphtheria toxin (DT)–induced MC-deletion system based on il-4–enhancer elements (9).

In addition to its expression on MCs, Allergin-1 is expressed on myeloid-lineage cells, including DCs, macrophages, splenic granulocytes in mice, and peripheral blood granulocytes in humans (6). However, the functional role of Allergin-1 in the activation of myeloid cells such as DCs and macrophages has not yet been defined. In this study, we investigated the role of Allergin-1 on myeloid cells in HDM-induced allergic asthma and show that Allergin-1 expressed on CD11b+ bone marrow–derived cultured DCs (BMDCs) plays important roles in allergic airway inflammation.

Mice (BALB/cAJcl) 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 (6) to the BALB/cAJcl genetic background for more than 12 generations. CD11c–DT receptor (DTR) on the BALB/c background and Myd88−/− mice on the BALB/c background were purchased from Oriental Yeast (Tokyo, Japan). Myd88−/−Milr1−/− double-deficient mice were generated in our laboratory. OVA peptide (OVA323–339)–specific, MHC class II I-Ad–restricted αβ TCR–transgenic (DO11.10 transgenic) mice crossed with recombination-activating gene–deficient 2 (RAG-2−/−) mice were kindly distributed by Dr. M. Kubo (RIKEN, Yokohama, Japan). For generation of bone marrow (BM)–chimeric mice, CD11c-DTR mouse–derived BM cells (2 × 106 cells) were i.v. injected into sublethally irradiated (two doses of 450 rad, 4 h apart) wild-type (WT) recipient mice, as described (10). After BM transfer, the recipients were allowed to rest for 8 wk before use. All experiments used female mice (age, 8–11 wk) that were bred under specific pathogen-free conditions at the animal facilities of the University of Tsukuba. All procedures followed the guidelines of the animal ethics committee of University of Tsukuba (permit number 19-238).

BMDCs were prepared by culturing BM cells in the presence of IL-4 (10 ng/ml) and GM-CSF (10 ng/ml) for 6 d, as described previously (11); CD11c+ cells were then purified by using anti-CD11c–conjugated MACS beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Naive CD4+ T cells (CD3+CD4+CD44CD62Lhigh) were sorted by flow cytometry (FACSAria; BD Biosciences, Franklin Lakes, NJ). The purities of CD11c+ DCs and naive CD4+ T cells exceeded 98%, as determined by flow cytometry.

Anti-mouse Allergin-1 (TX83; mouse IgG1) has been described previously (6) and was used to stain lung-resident hematopoietic cells. TX83 was biotinylated by using Biotinylation Kit (Sulfo-OSu; DOJIN, Tokyo, Japan). Anti-mouse Allergin-1 mAbs (TX98, mouse IgG1; EX36, rat IgG2a) were generated in our laboratory and used for immunoprecipitation and immunoblotting of Allergin-1, respectively. mAbs against Siglec-F (E50-2440), CD45.2 (104), CD11c (HL3), Ly-6G (1A8), CD11b (M1/70), I-A/I-E (M5/114.15.2), CD24 (M1/69), CD4 (RM4-5), CD44 (IM7), CD49b (DX5), CD62L (MEL-14), CD90.2 (53-2.1), IL-4 (11B11), IFN-γ (XMG1.2), IL-5 (TRFK5), IL-13 (JES10-5A2), I-A/I-E (M5/114.15.2), CD80 (16-10A1), and CD86 (GL1) were purchased from BD Biosciences; mAbs against FcεRI (MAR-1), CD25 (PC61.5), CD3ε (145-2C11), and B220 (RA3-6B2) were purchased from Tonbo Biosciences (San Diego, CA). mAbs against CD103 (2E7), CD64 (X54-5/7.1), CD40 (1C10), and mouse TCR DO11.10 (KJ1-26) were purchased from BioLegend (San Diego, CA).

Mice were treated with 10 μg of HDM extract (catalog number LG-5449, Mite Extract D. pteronyssinus; Cosmo Bio, Tokyo, Japan) intranasally 5 d/wk for 4 consecutive wk. Mice were assessed for AHR, airway inflammation, and serum IgE concentration 48 h after the last intranasal challenge. Bronchoalveolar lavage fluid (BALF) was collected by using three washes of 1 ml PBS containing 2% FBS; the washes were combined and then analyzed for differential cell count by flow cytometry. For adoptive transfer of HDM-stimulated BMDCs, CD11c-DTR BM-chimeric mice were injected with DT (8 ng/g body weight; Sigma-Aldrich) i.p. on day 0 and 1 d before HDM challenge. Sensitization of DT-treated naive mice by adoptive transfer of HDM-stimulated BMDCs was carried out as described previously (12, 13). Briefly, on day 1, DT-treated mice were sensitized intranasally with 2 × 106 of CD11c+ BMDCs, which were stimulated with HDM (100 μg/ml for 16 h). Then, mice were challenged with 10 μg of HDM extract intranasally on days 13, 15, 19, and 21. For papain, mice were treated intranasally with 50 μl of 50 μg papain (catalog no. 164-00172; Wako Pure Chemicals Industries, Osaka, Japan) in PBS or PBS alone for 3 consecutive d. At 24 h after the last challenge, BALF was collected.

Lung tissues were perfused with 1 ml of PBS through the right ventricle, removed, and treated with collagenase II (300 U/sample; Worthington Biochemical, Lakewood, NJ) at 37°C. Then lungs were homogenized by using gentleMACS Dissociator (B.01 program; Miltenyi Biotec). The cells were treated with anti-CD16/32 mAb (2.4G2; Tonbo Biosciences) to avoid binding to FcγR on ice for 10 min prior to incubation with indicated combination of Abs. Propidium iodide (1 μg/ml; Sigma-Aldrich, St. Louis, MO) or Zombie Aqua Fixable Viability Kit (BioLegend) was used to gate out dead cells. Allergin-1 expression on lung-resident cells was analyzed by flow cytometry using a gating protocol, as described previously (14). For intracellular staining of cytokines expression, cells were stained with mAbs against CD3 and CD4, treated with Cell Fixation and Cell Permeabilization Kit (Thermo Fisher Scientific, Waltham, MA), and then stained with mAbs against IL-4, IL-5, IL-13, or IFN-γ for 30 min on ice. Cells were analyzed on a BD LSRFortessa cell analyzer (BD Biosciences) and the FlowJo software program (Tree Star, San Carlos, CA).

For quantitation of serum levels of IgE, 96-well ELISA plates (Nunc; Thermo Fisher Scientific, 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 Ab. IgE was detected by using biotinylated anti-mouse IgE (catalog no. R35-118; BD Biosciences), followed by streptavidin-conjugated HRP (GE Healthcare Biosciences, Little Chalfont, U.K.). Mouse IgE (catalog no. C38-2; BD Biosciences) was used as a standard. IL-4 and IFN-γ were analyzed by using OptEIA Kits (BD Biosciences). PGE2 Express ELISA Kit (catalog no. 500141) was purchased from Cayman Chemical (Ann Arbor, MI). All of these assays were performed in accordance with the manufacturers’ instructions.

Airway resistances to different doses of aerosolized methacholine was measured by the computer-controlled piston ventilator system (flexiVent, Montreal, QC, Canada) in accordance with the manufacturer’s instructions. Briefly, 48 h after the last HDM challenge, mice were anesthetized, tracheotomized, and intubated intratracheally and exposed to an aerosol of 70 μl PBS (basal readings) and then of 70 μl methacholine in PBS at a concentration of 0.75, 1.5, 3, 6, or 12 mg/ml. Respiratory resistances were determined as the percentage change from the baseline index.

Whole-lung lobes were fixed with 10% formalin and embedded in paraffin. Sections were stained with H&E and with periodic acid–Schiff. The goblet cell hyperplasia was assessed by measuring the percentage of area of the airway basement membrane that was positive for periodic acid–Schiff staining in the total area of the airway basement membrane in randomly selected three bronchioles of each mice. Six 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). Images were manipulated in Adobe Photoshop CC 2018 (Adobe Systems, San Jose, CA).

Total RNA was extracted from sorted CD4+ T cells from mediastinal lymph nodes, sorted DCs from the lung, or purified CD11c+ BMDCs stimulated with HDM extract (100 μg/ml) or indicated concentration of PGE2 (Tokyo Chemical, Tokyo, Japan). Reverse (R) transcription was performed by using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Carlsbad, CA). Real-time PCR was performed by using Power SYBR Green PCR Master Mix (Applied Biosystems) and an ABI 7500 sequence detector (Applied Biosystems). The PCR primers were as follows: Gata3–forward (F), 5′-TTATCAAGCCCAAGCGAAGG-3′; Gata3-R, 5′-CATTAGCGTTCCTCCTCCAGAG-3′; Tbx21-F, 5′-AGCAAGGACGGCGAATGTT-3′; Tbx21-R, 5′-GGGTGGACATATAAGCGGTTC-3′; Rorc-F, 5′-GGAGGACAGGGAGCCAAGTT-3′; Rorc-R, 5′-CCGTAGTGGATCCCAGATGACT-3′; Foxp3-F, 5′-CCCATCCCCAGGAGTCTTG-3′; Foxp3-R, 5′-ACCATGACTAGGGGCACTGTA-3′; Il-6-F, 5′-GAGGATACCACTCCCAACAGACC-3′; Il-6-R, 5′-AAGTGCATCATCGTTGTTCATACA-3′; Tnf-F, 5′-GGGCCACCACGCTCTTC-3′; Tnf-R, 5′-GGTCTGGGCCATAGAACTGATG-3′; Il-12b-F, 5′-GGAGACCCTGCCCATTGAACT-3′; Il-12b-R, 5′-CAACGTTGCATCCTAGGATCG-3′; Ifng-F, 5′-ACA GCA AGG CGA AAA AGG ATG-3′; Ifng-R, 5′-TGG TGG ACC ACT CGG ATG A-3′; Il-10-F, 5′-GCTGGACAACATACTGCTAACC-3′; Il-10-R, 5′-ATTTCCGATAAGGCTTGGCAA-3′; Ccl2-F, 5′-TTAAAAACCTGGATCGGAACCAA-3′; Ccl2-R, 5′-GCATTAGCTTCAGATTTACGGGT-3′; Ptges-1-F, 5′-GGATGCGCTGAAACGTGGA-3′; Ptges-1-R, 5′-CAGGAATGAGTACACGAAGCC-3′; Tnfsf4-F, 5′-GCTAAGGCTGGTGGTCTCTG-3′; Tnfsf4-R, 5′-ACCGAATTGTTCTGCACCTC-3′; Ptgs2-F, 5′-ACCCGGACTGGATTCTAT-3′; Ptgs2-R, 5′-GCTTCCCACAGCTTTTGTAA-3′; Jag1-F, 5′-AGAAGTCAGAGTTCAGAGGCGTCC-3′; Jag1-R, 5′-AGTAGAAGGCTGTCACCAAGCAAC-3′; Jag2-F, 5′-AGCCACGGAGCAGTCATTTG-3′; Jag2-R, 5′-TCGGATTCCAGAGCAGATAGCG-3′; Dll4-F, 5′-AGGTGCCACTTCGGTTACACAG-3′; Dll4-R, 5′-CAATCACACACTCGTTCCTCTCTTC-3′; Actb-F, 5′-ACTGTCGAGTCGCGTCCA-3′; and Actb-R, 5′-GCAGCGATATCGTCATCCAT-3′.

Values were normalized according to the expression of the housekeeping gene β-actin.

WT or Milr1−/− BMDCs were stimulated with HDM extract (100 μg/ml) in the presence or absence of Syk inhibitor IV (BAY61-3606, catalog no. 574714 [Calbiochem, San Diego, CA]) for indicated time period. To detect tyrosine phosphorylation of Allergin-1 and recruitment of signaling molecules, BMDCs (3 × 107 cells) were lysed in digitonin extraction buffer (0.5% digitonin [Calbiochem], 0.12% Triton-X [Sigma-Aldrich], 150 mM NaCl, 20 mM triethanolamine [pH 7.8], and protease and phosphatase inhibitors), and lysates were immunoprecipitated by anti–Allergin-1 mAb (TX98)–binding Protein G Magnetic Beads (Dynabeads Protein G; Thermo Fisher Scientific). For the detection of MyD88-dependent signaling proteins, sorted CD11c+ BMDCs (5 × 104 cells) were lysed with 1% Nonidet P-40 containing protease inhibitors (1 mM PMSF and 10 U/ml aprotinin) and phosphatase inhibitors (1 mM EGTA, 10 mM NaF, 1 mM Na4P2O7, 0.1 mM β-glycerophosphate, and 1 mM Na3VO4). Whole-cell lysates and immunoprecipitates were separated by SDS-PAGE in an 8.5% Tris–hydrochloric acid gel and transferred (100 V, 1 h in 25 mM Tris, 195 mM glycine, and 20% methanol) to polyvinylidene difluoride membranes (Immobilon-P; MilliporeSigma, Carrigtwohill, Cork, Ireland). Membranes were incubated for 1 h at room temperature in blocking solution (TBST [10 mM TBS containing 0.1% Tween 20, 0.5 g/l MgCl2 (pH 8)] containing 5% BSA [Wako Pure Chemicals Industries]) and incubated overnight at 4°C in blocking solution containing primary Abs. Immunoblotting Abs against phospho–SHP-1 (clone: D11G5), IL-1R–associated kinase 1 (IRAK-1) (catalog no. H-273), phospho-Syk (Tyr525/526, catalog no. 2711), Syk (catalog no. 2712), phospho-p38 (D3F9), p38 (catalog no. 9212), phospho-Erk (D13.14.4E), Erk (catalog no. 9102), phospho-Jnk (81E11), Jnk (catalog no. 9252), and β-actin (8H10D10) were purchased from Cell Signaling Technology (Danvers, MA). Abs against SHP-1 (catalog no. sc-287) and SHP-2 (catalog no. sc-280) were purchased from Santa Cruz Biotechnology (Dallas, TX). HRP-conjugated phospho-tyrosine (4G10) was purchased from MilliporeSigma (Billerica, MA). Protein bands were detected by using HRP-conjugated anti-rabbit IgG (catalog no. 406401; BioLegend), anti-goat IgG (catalog no. sc-2768; Santa Cruz Biotechnology), or anti-rat IgG (NA935; GE Healthcare, Chicago, IL) and enhanced with SuperSignal West Pico Luminol/Enhancer Solution (Thermo Fisher Scientific). Chemiluminescence was detected by using ImageQuant LAS 4000 Mini and analyzed by using ImageQuant TL 8.1 (both from GE Healthcare). Stripping and reblotting were performed with Restore Western Blot Stripping Buffer (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions.

CD4+ T cells were prepared from the spleens of WT mice by using a magnetic bead–based cell sorting system (Miltenyi Biotec) by using anti-mouse CD4 Ab. Naive CD4+ T (CD4+CD44loCD62Lhigh) cells were then purified from CD4+ T cells by using FACSAria II cell sorter (BD Biosciences). The purity of naive CD4+ T cells exceeded 98%. Naive CD4+ T cells (2 × 104 per well in 96-well plates) were cocultured with CD11c+ BMDC or CD11b+ DCs sorted from the lung of HDM-immunized mice (10 μg/mouse for 5 consecutive d, 2 × 104 per well) in the presence anti-CD3 mAb (100 ng/ml) and HDM extract (100 μg/ml) for 5 d. For Th2-promoting conditions, IL-4 (50 ng/ml) and anti-mouse IL-12 Ab (10 μg/ml; catalog no. C17.8; BD Biosciences) were added, whereas the neutral condition lacked both. After 5 d of coculture, cells were collected, washed, and stimulated with PMA (50 ng/ml; Sigma-Aldrich) and ionomycin (500 ng/ml; Sigma-Aldrich) for 24 h. The culture supernatant was analyzed for IL-4 production from T cells by using a cytometric bead array (CBA) according to the manufacturer’s instructions. Briefly, culture supernatants (10 μl) from stimulated cells were mixed with 10 μl of mixed capture beads from mouse CBA Flex Set (BD Biosciences, San Diego, CA); then, IL-4 was detected by using PE-detection reagents (BD Biosciences). Flow cytometric analysis was performed by using an LSRFortessa cell analyzer (BD Biosciences) and CBA analysis FCAP software (BD Biosciences). For T cell proliferation assay, naive CD4+ T cells from DO11.10 RAG1−/− were labeled with CellTrace Violet (Thermo Fisher Scientific), according to the manufacture’s instruction, and cocultured with WT or Milr1−/− CD11c+ BMDC, which had been stimulated with HDM extract at 100 μg/ml for 16 h, in the presence of OVA peptide323–339 for 3 d. The reduction in CellTrace Violet fluorescence of DO11.10 T cells was analyzed by using an LSRFortessa cell analyzer. Celecoxib (10 μM; catalog no. C2816; Tokyo Chemical Industry, Tokyo, Japan) was used as a selective COX2 inhibitor. Recombinant mouse IL-33 was purchased from R&D Systems (Minneapolis, MN). The culture supernatant was analyzed for IL-6 production from CD11c+ BMDC by using a mouse CBA Flex Set (catalog no. 558301; BD Biosciences) according to the manufacturer’s instructions.

Control scrambled short hairpin RNA and Ptges–short hairpin RNA retroviral vectors were purchased from OriGene Technologies (Rockville, MD). Retroviruses were produced by transfecting 293GP-packaging cell line (a gift from H. Miyoshi) along with the vesicular stomatitis virus G expression vector, p-CMV–vesicular stomatitis virus G (RIKEN BioResource Research Center, Ibaraki, Japan). BMDCs were transduced with retroviral supernatants supplemented with polybrene (8 μg/ml; Sigma-Aldrich) at days 2 and 4. The culture plate was centrifuged at 1100 × g for 2 h at 32°C, then virus was removed and replaced with culture medium containing IL-4 and GM-CSF. Two days after the last transduction, transduced cells were selected by treating with puromycin (3 μg/ml) for 24 h, followed by stimulation with HDM extract (100 μg/ml) for 20 h. Culture supernatants were analyzed for PGE2 production by ELISA.

Statistical analyses were performed with the GraphPad Prism 7 software (La Jolla, CA) by using the unpaired Student t test or one-way or two-way ANOVA, followed by Bonferroni multiple comparison test as a posttest. A p value < 0.05 was considered statistically significant.

To investigate the role of Allergin-1 on myeloid cells in HDM-induced allergic airway responses, WT and Allergin-1–deficient (Milr−/−) mice were treated intranasally with HDM extract 5 consecutive d/wk for 4 wk (Fig. 1A). Consistent with our previous results (9), AHR to methacholine was significantly increased in Milr1−/− mice compared with WT mice (Fig. 1B, Supplemental Fig. 1A). However, we also found that Th2 responses, as determined by eosinophil number (Siglec-F+ CD11c) in BALF and the serum levels of IgE, were significantly increased in Milr1−/− mice compared with WT mice at 48 h after the final treatment (Fig. 1C, 1D). Histologic analyses revealed more severe cell infiltration and mucus production in the lung of Milr1−/− mice than in WT mice after HDM challenge (Fig. 1E, 1F). The proportions of IL-4+, IL-5+, and IL-13+ CD4+ T cells, but not IFN-γ+ CD4+ T cells, in the lungs of Milr1−/− mice were significantly larger than those in WT mice on day 28 after the start of the treatment (Fig. 1G).

FIGURE 1.

Allergin-1 suppresses HDM-induced asthma. WT and Milr1−/− mice were immunized with intranasal (i.n.) injection of HDM extract. (A) Experimental protocol. (B) AHR to methacholine (WT naive versus WT HDM or WT HDM versus Milr1−/− HDM, one-way ANOVA with post hoc Bonferroni t test). (C) The numbers of total cells (CD45.2+), alveolar macrophages (AM), neutrophils (Neutro), and eosinophils (Eosino) in BALF were assessed by cell counting and flow cytometry. (D) Serum IgE concentration. Data are pooled from more than three independent experiments (mean ± SEM). (E and F) Representative of H&E staining (E) and periodic acid–Schiff (PAS) staining (F) of formalin-fixed, paraffin-embedded lung sections. Scale bar, 100 μm. Right, quantification of PAS-positive goblet cells. Data are pooled from two independent experiments (mean ± SEM). (G) Flow cytometric analysis of cytokine expression in CD4+ T cells from the lung. Data are pooled from more than three independent experiments (mean ± SEM). Each point represents a single mouse. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 (WT versus Milr1−/−, unpaired t test).

FIGURE 1.

Allergin-1 suppresses HDM-induced asthma. WT and Milr1−/− mice were immunized with intranasal (i.n.) injection of HDM extract. (A) Experimental protocol. (B) AHR to methacholine (WT naive versus WT HDM or WT HDM versus Milr1−/− HDM, one-way ANOVA with post hoc Bonferroni t test). (C) The numbers of total cells (CD45.2+), alveolar macrophages (AM), neutrophils (Neutro), and eosinophils (Eosino) in BALF were assessed by cell counting and flow cytometry. (D) Serum IgE concentration. Data are pooled from more than three independent experiments (mean ± SEM). (E and F) Representative of H&E staining (E) and periodic acid–Schiff (PAS) staining (F) of formalin-fixed, paraffin-embedded lung sections. Scale bar, 100 μm. Right, quantification of PAS-positive goblet cells. Data are pooled from two independent experiments (mean ± SEM). (G) Flow cytometric analysis of cytokine expression in CD4+ T cells from the lung. Data are pooled from more than three independent experiments (mean ± SEM). Each point represents a single mouse. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 (WT versus Milr1−/−, unpaired t test).

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HDM contains a cysteine protease Der p 1 that induces basophils and/or type-II innate lymphoid cells (ILC2)–dependent, T cell–independent eosinophilia and mucus production through the release of IL-33 and TSLP from epithelial cells (15). To analyze whether the augmented Th2 responses induced by HDM in Milr1−/− mice is dependent on basophils or ILC2, we treated WT and Milr1−/− mice with a cysteine protease papain. However, both genotypes of mice showed comparable levels of airway eosinophilia (Supplemental Fig. 1B, 1C), suggesting that Allergin-1 on basophils and ILC2 were unlikely to be involved in the suppression of Th2 responses. Moreover, Allergin-1 is highly expressed on myeloid cells such as DCs (Fig. 2A), but not on lymphocytes, including ILC2, in the lung (Fig. 2B, 2C, Supplemental Fig. 1D). We have recently reported that Allergin-1 on MCs suppresses AHR, but not Th2 responses, after HDM treatment (9). Therefore, these results suggest a possibility that Allergin-1 on myeloid cells, rather than lymphocytes or basophils, might be involved in suppression of HDM-induced allergic airway inflammation and Th2 responses.

FIGURE 2.

Expression of Allergin-1 on lung-resident cells. (AC) Flow cytometry analysis of Allergin-1 expression in the lung of naive mice. Cells were stained with Abs against indicated lineage markers and Allergin-1 (TX83), and the expression of Allergin-1 on each cell subset of WT cells (thick line) was compared with Milr1−/− cells (thin line). Lineage markers contain CD3, B220, Ly-6G, CD49b, and CD11b. Data shown are representative of four independent experiments.

FIGURE 2.

Expression of Allergin-1 on lung-resident cells. (AC) Flow cytometry analysis of Allergin-1 expression in the lung of naive mice. Cells were stained with Abs against indicated lineage markers and Allergin-1 (TX83), and the expression of Allergin-1 on each cell subset of WT cells (thick line) was compared with Milr1−/− cells (thin line). Lineage markers contain CD3, B220, Ly-6G, CD49b, and CD11b. Data shown are representative of four independent experiments.

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To explore how Allergin-1 suppresses HDM-induced airway inflammation and Th2 responses, we first examined whether Allergin-1 on DCs directly stimulated with HDM extract suppresses DC activation for Th2 responses. Naive CD4+ T cells (CD4+, CD44Llo, and CD62high) purified from the spleen were cocultured with WT or Milr1−/− CD11c+ BMDCs (Supplemental Fig. 2A) in the presence of HDM extract for 5 d either under neutral or Th2-polarizing conditions. IL-4 production was significantly greater in the coculture of naive CD4+ T cells with Milr1−/− BMDCs than in that with WT BMDCs under the Th2-polarizing conditions (Supplemental Fig. 2B). We next examined whether Allergin-1 was involved in Ag-presenting function in DCs. When HDM extract–stimulated WT or Milr1−/− CD11c+ BMDCs pulsed with the OVA peptide were cocultured with naive OVA-specific DO11.10 T cells, which had been labeled with CellTrace Violet fluorescent dye, T cell proliferations were comparable between two genotypes of BMDCs (Supplemental Fig. 2D). Because the expression levels of MHC class II and the costimulatory molecules including CD40, CD80, and CD86 were comparable between two genotypes of BMDC before and after stimulation with HDM extract (Supplemental Fig. 2C), these results suggest that, although Allergin-1 on BMDCs suppressed augmentation of Th2 responses, it was not involved in Ag presentation function of BMDCs.

To examine the role of Allergin-1 on DCs, we generated BM-chimeric mice by injecting BM cells derived from mice transgenic for the CD11c-DTR (16) i.v. into sublethally irradiated WT mice (Fig. 3A). These mice were treated i.p. with DT, resulting in the efficient depletion of CD11c+MHC class II+ DCs from the lung of the BM-chimeric mice (Supplemental Fig. 2E). These mice were then adoptive transferred intranasally with HDM-stimulated WT or Milr1−/− BMDCs. The reconstitution efficiency of the BMDCs in the lung after HDM-stimulated BMDC transfer was comparable between mice that received WT and Milr1−/− BMDCs (Supplemental Fig. 2E). These mice showed increased numbers of eosinophils in BALF and an elevated serum IgE concentration after intranasal challenge with HDM, compared with DT-treated BM-chimeric mice that had not received BMDCs (Fig. 3B, 3C). Of note, the airway inflammation was more severe in the DT-treated BM-chimeric mice that had received HDM-stimulated Milr1−/− BMDCs than in those that had received HDM-stimulated WT BMDCs (Fig. 3B, 3C); this finding suggests that Allergin-1 on BMDCs suppressed HDM-induced allergic airway inflammation, although we could not exclude the possibility that this was caused by the different survival between WT and Milr1−/− BMDCs after transfer in vivo. Consistent with these results, CD4+ T cells purified from the mediastinal lymph nodes of Milr1−/− mice showed significantly higher expression of Gata3 than did those of WT mice, whereas the expression of Tbx21, Rorc, and Foxp3 did not differ between the two types of chimeric mice (Fig. 3D).

FIGURE 3.

Allergin-1 suppresses HDM-stimulated DC activation for Th2 responses. Sublethally irradiated WT mice were injected i.v. with BM cells from CD11c-DTR mice, then were treated with DT and reconstituted with HDM-stimulated CD11c+ BMDCs from the indicated genotypes of mice. (A) Experimental protocol. (B and E) The numbers of total cells (CD45.2+) and eosinophils in BALF were assessed by cell counting and flow cytometry. (C and F) Serum IgE concentration. (D) Quantitative PCR analysis of the expression of transcription factors in sorted CD4+ T cells from mediastinal lymph nodes (LN) at day 17. Each point represents a single mouse. Data are pooled from three independent experiments (mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 (one-way ANOVA with post hoc Bonferroni t test or unpaired t test).

FIGURE 3.

Allergin-1 suppresses HDM-stimulated DC activation for Th2 responses. Sublethally irradiated WT mice were injected i.v. with BM cells from CD11c-DTR mice, then were treated with DT and reconstituted with HDM-stimulated CD11c+ BMDCs from the indicated genotypes of mice. (A) Experimental protocol. (B and E) The numbers of total cells (CD45.2+) and eosinophils in BALF were assessed by cell counting and flow cytometry. (C and F) Serum IgE concentration. (D) Quantitative PCR analysis of the expression of transcription factors in sorted CD4+ T cells from mediastinal lymph nodes (LN) at day 17. Each point represents a single mouse. Data are pooled from three independent experiments (mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 (one-way ANOVA with post hoc Bonferroni t test or unpaired t test).

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Because HDM contains Der p 2 allergen that shares structural and functional similarity with MD-2, an LPS-binding component of TLR4 signaling complex, we examined whether TLR4 signaling in DC is involved in increased Th2 responses induced by HDM in Milr1−/− mice. Although the numbers of total cells and eosinophils in BALF and the serum IgE level were greater in DT-treated BM-chimeric mice reconstituted with WT BMDCs than in those that received Myd88−/− BMDCs, BALF cell numbers and serum IgE were similar between those reconstituted with Myd88−/− and Myd88−/−Milr1−/− BMDCs (Fig. 3E, 3F), indicating that the MyD88-signaling pathway in DC is involved in HDM-induced allergic airway inflammation, in which Allergin-1 was suggested to be involved in the regulatory role.

Allergin-1 contains the ITIMs that is tyrosine phosphorylated upon stimulation with protein tyrosine phosphatase inhibitor (pervanadate) in MCs (6). We found that HDM stimulation also induced tyrosine phosphorylation of Allergin-1 (Fig. 4A) and physical association of SHP-1 with Allergin-1 (Fig. 4B). Furthermore, Syk activation was augmented in Milr1−/− BMDCs compared with in WT BMDCs after HDM stimulation (Fig. 4C, Supplemental Fig. 3A). Moreover, stimulation with HDM activated the MAPK signaling molecules p38, Erk, and Jnk to a greater extent in Milr1−/− BMDCs than in WT BMDCs (Fig. 4D, Supplemental Fig. 3A). In addition, the degradation of IRAK-1 was enhanced in Milr1−/− BMDCs compared with WT BMDCs (Fig. 4D, Supplemental Fig. 3A). The addition of an Syk inhibitor suppressed phosphorylation of Erk in HDM-stimulated Milr1−/− BMDCs. In contrast, the Syk inhibitor did not affect the degradation of IRAK-1 and the phosphorylation of p38, and rather enhanced the phosphorylation of JNK probably because of the limitation of the pharmacological inhibitor (Supplemental Fig. 3B, 3C). Nevertheless, these results suggest that, although how HDM stimulation induced tyrosine phosphorylation of and recruitment of SHP-1 to Allergin-1 remains unclear, Allergin-1 inhibits Syk activation through SHP-1, thereby downregulating TLR4–MyD88-Erk signaling in BMDCs after stimulation with HDM in BMDCs.

FIGURE 4.

Allergin-1 suppresses Syk activation via SHP-1 recruitment. (A and B) Representative immunoprecipitation analyses of Allergin-1 from BMDC derived from mice of the indicated genotypes after stimulation with HDM extract. Allergin-1 was immunoprecipitated (IP) with TX98 mAb, then immunoblotted with Abs against the proteins indicated on the left of each panel. (C and D) Representative immunoblotting analyses of CD11c+ BMDCs whole-cell lysate derived from mice of the indicated genotypes after stimulation with HDM extract. Cell lysates were immunoblotted with Abs against the proteins indicated on the left of each panel.

FIGURE 4.

Allergin-1 suppresses Syk activation via SHP-1 recruitment. (A and B) Representative immunoprecipitation analyses of Allergin-1 from BMDC derived from mice of the indicated genotypes after stimulation with HDM extract. Allergin-1 was immunoprecipitated (IP) with TX98 mAb, then immunoblotted with Abs against the proteins indicated on the left of each panel. (C and D) Representative immunoblotting analyses of CD11c+ BMDCs whole-cell lysate derived from mice of the indicated genotypes after stimulation with HDM extract. Cell lysates were immunoblotted with Abs against the proteins indicated on the left of each panel.

Close modal

To finally examine how Allergin-1 on DCs suppressed MyD88-mediated signaling in DCs to induce Th2 responses, we analyzed whether Allergin-1 suppressed the TLR4–MyD88 signaling directly mediated by a TLR4 ligand contained in HDM. We found that, among the genes reported to be induced by TLR4 activation (17), mRNA transcripts of Ptges (which encodes PG E synthase [PGES]) and Jag1 (which encodes Jagged 1) in Milr1−/− BMDCs were upregulated most prominently compared with those in WT BMDCs after stimulation with HDM (Fig. 5A). However, the expression of these genes and Ptgs2 and production of PGE2 were significantly decreased in both Myd88−/− and Myd88−/−Milr1−/− BMDCs to the comparable level after stimulation with HDM in vitro (Fig. 5B, 5C). These results suggest that the enhanced expressions of Jag1 and PGE2 in Milr1−/− BMDCs in response to HDM were dependent on MyD88. In addition, HDM-induced PGE2 production in Milr1−/− BMDCs was also suppressed by addition of an Syk inhibitor (Fig. 5D), suggesting that the increased PGE2 production in Milr1−/− BMDCs was also dependent on Syk. Stimulation with IL-33, which binds to ST2 receptor and mediates a signal through MyD88 (18), showed no significant effect on PGE2 production but comparable levels of IL-6 production from WT or Milr1−/−BMDCs (Supplemental Fig. 4A). These results suggest that Allergin-1 specifically inhibits the TLR4–MyD88 signaling pathway for PGE2 production. We also found that treatment with PGE2 upregulated Jag1 to similar levels in both WT and Milr1−/− BMDCs in a dose-dependent manner, suggesting that Allergin-1 may not inhibit the PGE2R-mediated signaling pathway (Fig. 5E). Moreover, treatment of Milr1−/− BMDCs with a selective COX2 inhibitor (celecoxib) decreased HDM-induced Jag1 expression to a level comparable to that of WT BMDCs (Fig. 5F). Together, these results demonstrate that Allergin-1 suppresses Jag1 expression in BMDCs by inhibiting HDM-induced TLR4–MyD88-dependent PGE2 production.

FIGURE 5.

Allergin-1 suppresses the COX2–PGE2 axis in HDM-stimulated DCs. (A, B, E, and F) Quantitative PCR analyses of the expression of indicated genes in CD11c+ BMDC derived from mice of the indicated genotypes in the absence or presence of celecoxib (COXi) at 2 h (A, B, and E) or 4 h (F) after HDM (A, B, and F) or PGE2 (E) stimulation. (C, D, and H) ELISA analysis of the production of PGE2 by CD11c+ BMDCs (C and D) or Ptges–knocked down CD11c+ BMDCs (H) derived from the indicated genotypes of mice in the absence or presence of an Syk inhibitor (Syk-I) at 16 h after HDM stimulation. (G) Production of cytokines by naive CD4+ T cells cocultured with sorted lung CD11b+ DCs (Supplemental Fig. 4B) pooled from indicated genotypes of mice (n = 3) after immunization with HDM (10 μg/mouse for 5 consecutive d) under neutral (with no added cytokines) or Th2-promoting (with IL-4 and anti–IL-12) conditions in the absence or presence of celecoxib (10 μM). After 5 d of coculture, cells were restimulated with PMA and ionomycin for 24 h. The culture supernatant was analyzed by using a CBA. (I) DT-treated CD11c-DTR BM-chimeric mice were reconstituted with control or Ptges–knocked down CD11c+ BMDCs derived from the indicated genotypes of mice and analyzed as shown in Fig. 3E. Data are representative of three (A, B, E, and F) and two (C, D, H, and G) independent experiments with similar results (mean ± SEM). Each point represents a single mouse. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 (one-way or two-way ANOVA with post hoc Bonferroni t test).

FIGURE 5.

Allergin-1 suppresses the COX2–PGE2 axis in HDM-stimulated DCs. (A, B, E, and F) Quantitative PCR analyses of the expression of indicated genes in CD11c+ BMDC derived from mice of the indicated genotypes in the absence or presence of celecoxib (COXi) at 2 h (A, B, and E) or 4 h (F) after HDM (A, B, and F) or PGE2 (E) stimulation. (C, D, and H) ELISA analysis of the production of PGE2 by CD11c+ BMDCs (C and D) or Ptges–knocked down CD11c+ BMDCs (H) derived from the indicated genotypes of mice in the absence or presence of an Syk inhibitor (Syk-I) at 16 h after HDM stimulation. (G) Production of cytokines by naive CD4+ T cells cocultured with sorted lung CD11b+ DCs (Supplemental Fig. 4B) pooled from indicated genotypes of mice (n = 3) after immunization with HDM (10 μg/mouse for 5 consecutive d) under neutral (with no added cytokines) or Th2-promoting (with IL-4 and anti–IL-12) conditions in the absence or presence of celecoxib (10 μM). After 5 d of coculture, cells were restimulated with PMA and ionomycin for 24 h. The culture supernatant was analyzed by using a CBA. (I) DT-treated CD11c-DTR BM-chimeric mice were reconstituted with control or Ptges–knocked down CD11c+ BMDCs derived from the indicated genotypes of mice and analyzed as shown in Fig. 3E. Data are representative of three (A, B, E, and F) and two (C, D, H, and G) independent experiments with similar results (mean ± SEM). Each point represents a single mouse. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 (one-way or two-way ANOVA with post hoc Bonferroni t test).

Close modal

To assess whether the PGE2–Jagged 1 axis is involved in allergic airway inflammation, we added celecoxib to cocultures of naive CD4+ T cells with CD11b+ DCs sorted from the lung of either WT or Milr1−/− mice on day 7 after HDM immunization (Supplemental Fig. 4B) in the presence of HDM under neutral or Th2-promoting conditions. We found that the amount of HDM-induced IL-4 production was greater in Milr1−/− lung CD11b+ DCs than in WT lung CD11b+ DCs under Th2 conditions (Fig. 5G). However, treatment with celecoxib decreased IL-4 production from T cells cocultured with either WT or Milr1−/− lung CD11b+ DCs to comparable levels (Fig. 5G). We also observed that, although Milr1−/− BMDCs produced PGE2 greater than did WT BMDCs in response to HDM stimulation, the production of PGE2 by Ptges–knocked down Milr1−/− BMDCs decreased to a level comparable to those by Ptges–knocked down WT BMDCs (Fig. 5H). Moreover, intranasal transfer of HDM-stimulated Ptges–knocked down Milr1−/− BMDCs to DT-treated CD11c-DTR BM-chimeric mice alleviated airway inflammation to a level comparable to that of mice that received Ptges–knocked down WT BMDCs (Fig. 5I). Taken together, these results demonstrate that Allergin-1 inhibits HDM-induced PGE2 production by CD11b+ BMDCs, leading to suppression of allergic airway inflammation.

Because HDM extract contains Der p 2 and endotoxin, HDM inhaled in the lung is capable of activating TLR4 expressed on DCs as well as on lung epithelial cells. Lung DCs have been reported to play an important role in HDM-induced Th2 responses, as demonstrated by using DT-treated CD11c-DTR mice (10, 19, 20). However, a previous report showed that TLR4 on epithelial cells, but not DCs, was essential for HDM-induced Th2 responses (21), in which TLR4 signaling induced the secretion of alarmins, such as IL-33, TSLP, and IL-25, from epithelial cells and instructed DCs to promote HDM-induced Th2 responses (2224). Our current study demonstrates that HDM not only indirectly stimulates lung DCs by means of alarmins secreted from epithelial cells, but also directly activates lung DCs for Th2 responses through the production of PGE2. In addition, Allergin-1 is an inhibitory receptor on CD11b+ DCs, in which Allergin-1 specifically inhibits Th2 responses by downregulating PGE2 production from DCs, although we cannot formally exclude the possibility that Allergin-1 expressed on CD11b+ interstitial macrophages were also involved in the suppression of PGE2 production as well.

Lung conventional DCs (cDCs) can be classified into two functionally distinct subsets CD11b CD103+ cDC1 and CD11b+ CD103 cDC2 (2527), of which transcription factors IRF8 and IRF4 are critical for the development, respectively (2831). Similarly to CD11b+ cDC2, in vitro generation of BMDCs in the presence of GM-CSF depends on IRF-4 (29, 31), suggesting that the CD11b+ BMDCs generated in the presence of GM-CSF used in the current study may have similar characteristics to CD11b+ cDC2. Previous reports demonstrated that CD11b+ cDC2, rather than CD103+ cDC1, mediates Th2 priming in response to HDM (3236). However, how CD11b+ DCs initiate or amplify Th2 cells in response to HDM remains undetermined (37). PGE2 was reported to induce Th2 immunity by suppressing the production of cytokines from Th1 cells, macrophages, and DCs (38, 39). PGE2 can be produced by various cells including epithelia, fibroblasts, and immune cells in a COX2-dependent manner. In this study, we showed that PGE2 from CD11b+ BMDCs promoted Th2 immune responses under Th2-promoting environmental conditions. We demonstrated that HDM upregulated Jag1 expression, which was dependent on PGE2. Several findings indicate that Notch, a Jagged 1 ligand, on CD4+ T cells trans-activates Gata3, resulting in the amplification of Th2 cells (4044). In addition, the Notch ligand, Jagged 1, but not Jagged 2 or Δ 4, on APC promotes Th2 responses (4548). Therefore, PGE2-induced Th2 responses likely are dependent on the interaction of Jagged 1 on CD11b+ DCs with Notch on CD4+ T cells. However, further investigations are required to clarify the molecular mechanisms how PGE2 derived from CD11b+ DCs induces Th2 responses. Of note, addition of COX2 inhibitor reduced the production of IFN-γ from T cells cocultured with BMDCs after stimulation with HDM in vitro (Fig. 5G), whereas IFN-γ expression in T cells was not affected in the draining lymph nodes of Milr1−/− mice in HDM-induced allergic airway inflammation (Fig. 1G). This discrepancy might be due to the effect of the COX2 inhibitor on the intrinsic expression of PGE2 in T cells, because the COX2 inhibitor reduced the expression of T-bet via PGE2 without affecting the expression of GATA3 in T cells (49).

Previous reports demonstrated that Syk interacts with TLR4 upon LPS stimulation in BM-derived cultured macrophages (50) or is constitutively associated with TLR4 in the human monocytic cell line THP-1 and regulates MyD88-dependent signaling by transiently recruiting IRAK-1 to the complex containing the adaptor molecule MyD88, TLR4, and Syk after LPS engagement (51). Furthermore, Syk phosphorylates MyD88 and promotes MyD88-mediated signaling upon IL-1α stimulation (52), and SHP-1 inhibits the phosphorylation of Syk and, thus, MyD88, thereby suppressing downstream signaling (52). However, the details of mechanism by which Syk regulates TLR signaling remains obscure. In this study, we showed that HDM stimulation induced tyrosine phosphorylation of Allergin-1 and SHP-1, leading to prevention of the phosphorylation of Syk and inhibition of the MAPK-mediated signaling necessary for PGE2 production. We also showed that treatment with the Syk inhibitor IV efficiently canceled Allergin-1–dependent suppression of PGE2 production and enhanced phosphorylation of Erk in HDM-stimulated Milr1−/− BMDCs. The Syk inhibitor IV decreased only Erk phosphorylation, which might be due to a limitation of pharmacological approach as reported previously (50, 53). Previous reports demonstrated that Src family kinase Lyn is primarily responsible for tyrosine phosphorylation of ITIM (54, 55). Because Der p extract stimulation phosphorylates Lyn (56), we assume that Lyn may be involved in tyrosine phosphorylation of Allergin-1 upon HDM stimulation. Our results, together with previous reports (5052), support a model in which, upon HDM stimulation of lung CD11b+ DCs, Syk augments TLR4–MyD88 phosphorylation and downstream signaling through IRAK1 and the MAPK pathways, leading to increased expressions of COX2 and PTGES for PGE2 production. By activating SHP-1, Allergin-1 inhibits Syk phosphorylation, thus suppressing PGE2 production and Th2 responses. Further analyses are required to clarify the molecular mechanisms through which HDM allergens directly activate DCs to promote Th2 responses and to elucidate the involvement of Allergin-1 in this DC activation.

We thank M. Kubo (Tokyo University of Science and RIKEN Center of Institutive Medical Sciences) and S. Tochihara for discussions and secretarial assistance, respectively.

This work was supported by, in part, grants provided by Japan Society for the Promotion of Science (KAKENHI) grants (to A.S. [18H05022 and 16H06387] and to S.T.-H. [15H04862 and 15K15319]) and was funded by Ono Pharmaceutical Co., Ltd.

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • AHR

    airway hyperresponsiveness

  •  
  • BALF

    bronchoalveolar lavage fluid

  •  
  • BM

    bone marrow

  •  
  • BMDC

    bone marrow–derived cultured DC

  •  
  • CBA

    cytometric bead array

  •  
  • cDC

    conventional DC

  •  
  • DC

    dendritic cell

  •  
  • DT

    diphtheria toxin

  •  
  • DTR

    DT receptor

  •  
  • F

    forward

  •  
  • HDM

    house dust mite

  •  
  • ILC2

    type-II innate lymphoid cell

  •  
  • IRAK-1

    IL-1R–associated kinase 1

  •  
  • MC

    mast cell

  •  
  • R

    reverse

  •  
  • SHP-1

    Src homology 2 domain–containing tyrosine phosphatase 1

  •  
  • TSLP

    thymic stromal lymphopoietin

  •  
  • WT

    wild-type.

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S. Shibayama is an employee of Ono Pharmaceutical Co., Ltd. This research was supported in part by Ono Pharmaceutical Co., Ltd. The sponsor had no control over the interpretation, writing, or publication of this work. The other authors have no financial conflicts of interest.

Supplementary data