Semaphorin 3E Alleviates Hallmarks of House Dust Mite–Induced Allergic Airway Disease
2017; Elsevier BV; Volume: 187; Issue: 7 Linguagem: Inglês
10.1016/j.ajpath.2017.03.008
ISSN1525-2191
AutoresHesam Movassagh, Lianyu Shan, Jonathan S. Duke‐Cohan, Andrew J. Halayko, Jude E. Uzonna, Abdelilah S. Gounni,
Tópico(s)Axon Guidance and Neuronal Signaling
ResumoSemaphorins are an essential family of guidance cues ubiquitously expressed in various organs, which play diverse developmental, homeostatic, and pathological roles. Semaphorin 3E (Sema3E), initially identified as a neuronal chemorepellent, is involved in the regulation of cell migration, proliferation, and angiogenesis. However, expression and function of Sema3E in allergic asthma has not been extensively investigated. We determined the expression of Sema3E in the airways and its effect on airway inflammation, hyperresponsiveness, and remodeling as pathological features of allergic asthma provoked by house dust mite in vivo. Our data indicate that exposure to house dust mite markedly reduces Sema3E expression in mouse airways. More important, replenishment of Sema3E by intranasal administration of exogenous Sema3E protects mice from allergic asthma by reducing eosinophilic inflammation, serum IgE level, and T helper cell 2/T helper cell 17 cytokine response. The regulatory effect of Sema3E on cytokine response was sustained on allergen recall response in the lymph nodes and spleen. Furthermore, goblet cell hyperplasia, collagen deposition, and airway hyperresponsiveness were significantly diminished on Sema3E treatment. The inhibitory effect of Sema3E was associated with a reduction of pulmonary CD11b+ conventional dendritic cells and regulation of CD4+ T-cell cytokine response. Collectively, our data represent a novel approach to treating allergic asthma via regulation of immune response to house dust mite. Semaphorins are an essential family of guidance cues ubiquitously expressed in various organs, which play diverse developmental, homeostatic, and pathological roles. Semaphorin 3E (Sema3E), initially identified as a neuronal chemorepellent, is involved in the regulation of cell migration, proliferation, and angiogenesis. However, expression and function of Sema3E in allergic asthma has not been extensively investigated. We determined the expression of Sema3E in the airways and its effect on airway inflammation, hyperresponsiveness, and remodeling as pathological features of allergic asthma provoked by house dust mite in vivo. Our data indicate that exposure to house dust mite markedly reduces Sema3E expression in mouse airways. More important, replenishment of Sema3E by intranasal administration of exogenous Sema3E protects mice from allergic asthma by reducing eosinophilic inflammation, serum IgE level, and T helper cell 2/T helper cell 17 cytokine response. The regulatory effect of Sema3E on cytokine response was sustained on allergen recall response in the lymph nodes and spleen. Furthermore, goblet cell hyperplasia, collagen deposition, and airway hyperresponsiveness were significantly diminished on Sema3E treatment. The inhibitory effect of Sema3E was associated with a reduction of pulmonary CD11b+ conventional dendritic cells and regulation of CD4+ T-cell cytokine response. Collectively, our data represent a novel approach to treating allergic asthma via regulation of immune response to house dust mite. Asthma is a multifaceted disease of the airways associated with chronic inflammation, bronchoconstriction, remodeling, and airway hyperresponsiveness (AHR).1Hekking P.P. Bel E.H. Developing and emerging clinical asthma phenotypes.J Allergy Clin Immunol Pract. 2014; 2 (quiz 681): 671-680Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar The prevalence of allergic asthma has been remarkably increasing for the past decades.2Schatz M. Rosenwasser L. The allergic asthma phenotype.J Allergy Clin Immunol Pract. 2014; 2 (quiz 649): 645-648Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar Therefore, development of new strategies to treat allergic asthma, in particular severe refractory form of the disease, is an urgent unmet clinical need. However, novel therapeutic approaches fail to significantly alleviate the cardinal features of asthma, especially AHR and remodeling.3Barnes P.J. Therapeutic approaches to asthma-chronic obstructive pulmonary disease overlap syndromes.J Allergy Clin Immunol. 2015; 136: 531-545Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 4Darveaux J. Busse W.W. Biologics in asthma: the next step toward personalized treatment.J Allergy Clin Immunol Pract. 2015; 3 (quiz 161): 152-160Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 5Fahy J.V. Type 2 inflammation in asthma: present in most, absent in many.Nat Rev Immunol. 2015; 15: 57-65Crossref PubMed Scopus (912) Google Scholar Continuous exposure with innocuous aeroallergens, such as house dust mite (HDM), deviates the immune system toward a granulocytic rich response. It perpetuates the airway inflammation that leads to a massive structural alteration of the airways and subsequently AHR as the hallmark of disease pathology.6Lauzon A.M. Martin J.G. Airway hyperresponsiveness: smooth muscle as the principal actor.F1000Res. 2016; 5: 306Crossref Scopus (46) Google Scholar However, the mechanisms underlying airway remodeling and AHR could spontaneously develop independent of inflammatory response.7Balenga N.A. Jester W. Jiang M. Panettieri Jr., R.A. Druey K.M. Loss of regulator of G protein signaling 5 promotes airway hyperresponsiveness in the absence of allergic inflammation.J Allergy Clin Immunol. 2014; 134: 451-459Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 8Balenga N.A. Klichinsky M. Xie Z. Chan E.C. Zhao M. Jude J. Laviolette M. Panettieri Jr., R.A. Druey K.M. A fungal protease allergen provokes airway hyper-responsiveness in asthma.Nat Commun. 2015; 6: 6763Crossref PubMed Scopus (90) Google Scholar Also, a considerable portion of asthmatic patients have a T helper cell (Th) 2-low noneosinophilic phenotype that poorly responds to current anti-inflammatory treatments (eg, corticosteroids).5Fahy J.V. Type 2 inflammation in asthma: present in most, absent in many.Nat Rev Immunol. 2015; 15: 57-65Crossref PubMed Scopus (912) Google Scholar, 9McGrath K.W. Icitovic N. Boushey H.A. Lazarus S.C. Sutherland E.R. Chinchilli V.M. Fahy J.V. Asthma Clinical Research Network of the National Heart, Lung, Blood InstituteA large subgroup of mild-to-moderate asthma is persistently noneosinophilic.Am J Respir Crit Care Med. 2012; 185: 612-619Crossref PubMed Scopus (388) Google Scholar, 10Peters M.C. Mekonnen Z.K. Yuan S. Bhakta N.R. Woodruff P.G. Fahy J.V. Measures of gene expression in sputum cells can identify TH2-high and TH2-low subtypes of asthma.J Allergy Clin Immunol. 2014; 133: 388-394Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar Thus, efficient therapeutic strategies for allergic asthma should be able to target various arms of the disease, including both inflammatory and structural compartments of the airways. Semaphorins are guidance cues that play diverse roles, such as immune regulation, angiogenesis, cell proliferation, and migration.11Kumanogoh A. Kikutani H. Immunological functions of the neuropilins and plexins as receptors for semaphorins.Nat Rev Immunol. 2013; 13: 802-814Crossref PubMed Scopus (148) Google Scholar, 12Roth L. Koncina E. Satkauskas S. Cremel G. Aunis D. Bagnard D. The many faces of semaphorins: from development to pathology.Cell Mol Life Sci. 2009; 66: 649-666Crossref PubMed Scopus (145) Google Scholar, 13Suzuki K. Kumanogoh A. Kikutani H. Semaphorins and their receptors in immune cell interactions.Nat Immunol. 2008; 9: 17-23Crossref PubMed Scopus (249) Google Scholar Various semaphorins have been reported to exert proinflammatory or anti-inflammatory responses in a context-dependent manner.14He M. Bian Z. Expression of hypoxia-induced semaphorin 7A correlates with the severity of inflammation and osteoclastogenesis in experimentally induced periapical lesions.Arch Oral Biol. 2017; 75: 114-119Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 15Mucka P. Levonyak N. Geretti E. Zwaans B.M. Li X. Adini I. Klagsbrun M. Adam R.M. Bielenberg D.R. Inflammation and lymphedema are exacerbated and prolonged by neuropilin 2 deficiency.Am J Pathol. 2016; 186: 2803-2812Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 16Xie J. Wang H. Semaphorin 7A as a potential immune regulator and promising therapeutic target in rheumatoid arthritis.Arthritis Res Ther. 2017; 19: 10Crossref PubMed Scopus (41) Google Scholar, 17Xue D. Kaufman G.N. Dembele M. Beland M. Massoud A.H. Mindt B.C. Fiter R. Fixman E.D. Martin J.G. Friedel R.H. Divangahi M. Fritz J.H. Mazer B.D. Semaphorin 4C protects against allergic inflammation: requirement of regulatory CD138+ plasma cells.J Immunol. 2017; 198: 71-81Crossref PubMed Scopus (12) Google Scholar For instance, Semaphorin 3E (Sema3E) is able to dualistically promote or suppress inflammation in the obesity model18Shimizu I. Yoshida Y. Moriya J. Nojima A. Uemura A. Kobayashi Y. Minamino T. Semaphorin3E-induced inflammation contributes to insulin resistance in dietary obesity.Cell Metab. 2013; 18: 491-504Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar or atherosclerotic plaques,19Wanschel A. Seibert T. Hewing B. Ramkhelawon B. Ray T.D. van Gils J.M. Rayner K.J. Feig J.E. O'Brien E.R. Fisher E.A. Moore K.J. Neuroimmune guidance cue Semaphorin 3E is expressed in atherosclerotic plaques and regulates macrophage retention.Arterioscler Thromb Vasc Biol. 2013; 33: 886-893Crossref PubMed Scopus (96) Google Scholar respectively, via regulation of macrophage functions. Sema3E is ubiquitously expressed in different cell types, including adipocytes,18Shimizu I. Yoshida Y. Moriya J. Nojima A. Uemura A. Kobayashi Y. Minamino T. Semaphorin3E-induced inflammation contributes to insulin resistance in dietary obesity.Cell Metab. 2013; 18: 491-504Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar macrophages,19Wanschel A. Seibert T. Hewing B. Ramkhelawon B. Ray T.D. van Gils J.M. Rayner K.J. Feig J.E. O'Brien E.R. Fisher E.A. Moore K.J. Neuroimmune guidance cue Semaphorin 3E is expressed in atherosclerotic plaques and regulates macrophage retention.Arterioscler Thromb Vasc Biol. 2013; 33: 886-893Crossref PubMed Scopus (96) Google Scholar tumor cells,20Luchino J. Hocine M. Amoureux M.C. Gibert B. Bernet A. Royet A. Treilleux I. Lecine P. Borg J.P. Mehlen P. Chauvet S. Mann F. Semaphorin 3E suppresses tumor cell death triggered by the plexin D1 dependence receptor in metastatic breast cancers.Cancer Cell. 2013; 24: 673-685Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar hepatocytes,21Yagai T. Miyajima A. Tanaka M. Semaphorin 3E secreted by damaged hepatocytes regulates the sinusoidal regeneration and liver fibrosis during liver regeneration.Am J Pathol. 2014; 184: 2250-2259Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar osteoblasts,22Hughes A. Kleine-Albers J. Helfrich M.H. Ralston S.H. Rogers M.J. A class III semaphorin (Sema3e) inhibits mouse osteoblast migration and decreases osteoclast formation in vitro.Calcif Tissue Int. 2012; 90: 151-162Crossref PubMed Scopus (36) Google Scholar and dendritic cells.23Holl E.K. Roney K.E. Allen I.C. Steinbach E. Arthur J.C. Buntzman A. Plevy S. Frelinger J. Ting J.P. Plexin-B2 and Plexin-D1 in dendritic cells: expression and IL-12/IL-23p40 production.PLoS One. 2012; 7: e43333Crossref PubMed Scopus (34) Google Scholar It is involved in essential cellular functions that are dysregulated in allergic asthma.24Choi Y.I. Duke-Cohan J.S. Ahmed W.B. Handley M.A. Mann F. Epstein J.A. Clayton L.K. Reinherz E.L. PlexinD1 glycoprotein controls migration of positively selected thymocytes into the medulla.Immunity. 2008; 29: 888-898Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 25Moriya J. Minamino T. Tateno K. Okada S. Uemura A. Shimizu I. Yokoyama M. Nojima A. Okada M. Koga H. Komuro I. Inhibition of semaphorin as a novel strategy for therapeutic angiogenesis.Circ Res. 2010; 106: 391-398Crossref PubMed Scopus (59) Google Scholar, 26Sabag A.D. Bode J. Fink D. Kigel B. Kugler W. Neufeld G. Semaphorin-3D and semaphorin-3E inhibit the development of tumors from glioblastoma cells implanted in the cortex of the brain.PLoS One. 2012; 7: e42912Crossref PubMed Scopus (53) Google Scholar, 27Sakurai A. Gavard J. Annas-Linhares Y. Basile J.R. Amornphimoltham P. Palmby T.R. Yagi H. Zhang F. Randazzo P.A. Li X. Weigert R. Gutkind J.S. Semaphorin 3E initiates antiangiogenic signaling through plexin D1 by regulating Arf6 and R-Ras.Mol Cell Biol. 2010; 30: 3086-3098Crossref PubMed Scopus (121) Google Scholar We have previously reported that in vitro treatment of human airway smooth muscle (ASM) cells with Sema3E inhibits growth factor–induced proliferation and migration considered as fundamental mechanisms contributing to airway remodeling.28Movassagh H. Shan L. Halayko A.J. Roth M. Tamm M. Chakir J. Gounni A.S. Neuronal chemorepellent Semaphorin 3E inhibits human airway smooth muscle cell proliferation and migration.J Allergy Clin Immunol. 2014; 133: 560-567Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar This effect was associated with suppression of mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and Ras-related C3 botulinum toxin substrate 1 GTPase signaling.28Movassagh H. Shan L. Halayko A.J. Roth M. Tamm M. Chakir J. Gounni A.S. Neuronal chemorepellent Semaphorin 3E inhibits human airway smooth muscle cell proliferation and migration.J Allergy Clin Immunol. 2014; 133: 560-567Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar Furthermore, we have recently revealed that genetic abrogation of Sema3E in mice leads to a hypergranulocytic phenotype, which is associated with enhanced AHR, remodeling, and Th2/Th17 inflammation.29Movassagh H. Shan L. Mohammed A. Halayko A.J. Gounni A.S. Semaphorin 3E deficiency exacerbates airway inflammation, hyperresponsiveness, and remodeling in a mouse model of allergic asthma.J Immunol. 2017; 198: 1805-1814Crossref PubMed Scopus (27) Google Scholar Sema3E was also shown to reduce neutrophil recruitment to the airways on HDM reexposure.30Movassagh H. Saati A. Nandagopal S. Mohammed A. Tatari N. Shan L. Duke-Cohan J.S. Fowke K.R. Lin F. Gounni A.S. Chemorepellent semaphorin 3E negatively regulates neutrophil migration in vitro and in vivo.J Immunol. 2017; 198: 1023-1033Crossref PubMed Scopus (30) Google Scholar However, the therapeutic potential of Sema3E in allergic asthma has remained to be understood. Our goal was first to determine the expression of Sema3E in the airways and then to address the in vivo effect of Sema3E on allergic airway inflammation, remodeling, and AHR using the HDM model of the disease. We also aimed to understand the mechanisms underlying Sema3E effects on the allergic asthma model. We found that Sema3E expression was down-regulated in murine airways on HDM sensitization and challenge. Administration of exogenous recombinant Sema3E protected mice from HDM-induced airway inflammation, mucus overproduction, collagen deposition, and AHR. The mechanism underlying the Sema3E effect on the HDM mouse model of allergic asthma was mediated, at least in part, via regulating cytokine response, pulmonary dendritic cell (DC) recruitment, and function. Taken together, our findings reveal a major regulatory role of Sema3E in allergic asthma. Female 6- to 8-week-old BALB/c mice were purchased from the Central Animal Care Services at the University of Manitoba (Winnipeg, MB, Canada). All of the mice were maintained at the Central Animal Care Services facility under specific pathogen-free conditions and used according to the guidelines stipulated by the Canadian Council for Animal Care. Lyophilized HDM protein extract was obtained from Greer Laboratories (Lenoir, NC), which was reconstituted in sterile saline as 2.5 mg/mL stock concentration before treatment. A working concentration (25 μg per mouse in 35 μL of saline) was freshly prepared, and an acute model of the disease was established via intranasal administration under gaseous anesthesia for 5 days per week during 2 consecutive weeks.31Hirota J.A. Budelsky A. Smith D. Lipsky B. Ellis R. Xiang Y.Y. Lu W.Y. Inman M.D. The role of interleukin-4Ralpha in the induction of glutamic acid decarboxylase in airway epithelium following acute house dust mite exposure.Clin Exp Allergy. 2010; 40: 820-830Crossref PubMed Scopus (23) Google Scholar Recombinant mouse Sema3E-Fc (10 μg/kg in sterile phosphate-buffered saline) was administered through the intranasal route 1 hour before each HDM exposure. The control group received sterile saline-Fc at the same time points. All experiments were performed 48 hours after the last HDM exposure, unless otherwise indicated. Murine Sema3E-Fc recombinant protein was produced as fusion protein N-terminal to a functional mouse γ2c Fc domain. Complete cDNA was amplified from total RNA of a mouse brain, ligated into pFUSE-mfc1 vector, and electroporated into CHO cells. Finally, secreted Sema3E-Fc protein was purified from the conditioned media by protein A-affinity chromatography, as described previously.24Choi Y.I. Duke-Cohan J.S. Ahmed W.B. Handley M.A. Mann F. Epstein J.A. Clayton L.K. Reinherz E.L. PlexinD1 glycoprotein controls migration of positively selected thymocytes into the medulla.Immunity. 2008; 29: 888-898Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar Formalin-fixed lung tissues were paraffin embedded, and sections (5 μm thick) were prepared, dewaxed in xylene, and rehydrated through graded concentrations of alcohol to water, and then boiled for 10 minutes in sodium citrate buffer. Then, sections were incubated with blocking buffer for 1 hour at room temperature. Rabbit anti-Sema3E antibody (2.5 μg/mL; Abcam, Cambridge, MA) or isotype control IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) was added, and sections were incubated overnight at 4°C. Slides were then washed twice with tris-buffered saline, followed by incubation with Alexa-488–conjugated anti-rabbit secondary antibody for 1 hour at room temperature away from light. After an extensive wash with tris-buffered saline, slides were finally mounted with a DAPI-containing antifade reagent (Life Technologies Inc., Burlington, ON, Canada) and visualized by Axioskop 2 mot plus microscope using AxioVision software version 4.8 (Carl Zeiss, Inc., Thornwood, NY). Integrated density of Sema3E immunostaining in the airways was compared between saline and HDM-challenged mice by ImageJ version 1.51j8 (NIH, Bethesda, MD; http://imagej.nih.gov/ij). Mice were anesthetized and cannulated via the trachea 48 hours after the last HDM challenge. An increasing gradient of methacholine dose was administered intratracheally, and AHR parameters, including airway resistance, tissue resistance, and tissue elastance, were measured by using a FlexiVent small animal ventilator system (SCIREQ, Montreal, QC, Canada). Bronchoalveolar lavage (BAL) was performed with two instillations of 1 mL of sterile saline containing 0.1 mmol/L EDTA. Red blood cells were lysed using ammonium-chloride-potassium buffer. Total cells in BAL fluid (BALF) were spun down at 300 × g/4°C for 5 minutes, and supernatants were stored at −80°C to measure cytokines. Cytospins from BALF cells were prepared and stained, and different inflammatory cell types were characterized morphologically and counted by two individuals in a blind manner (H.M. and L.S.). Dissected left lobes of mouse lungs were inflated, fixed, and embedded in paraffin. Then, sections were stained with hematoxylin and eosin, periodic acid–Schiff, and Sirius red for assessing the presence of airway inflammation, mucus overproduction, and collagen deposition, respectively. Slides were quantified by pathological scoring from 0 (the lowest staining intensity) to 5 (the highest staining intensity) in a blind manner (H.M. and L.S.). An enzyme-linked immunosorbent assay was performed to measure IL-4, IL-5, IL-9, IL-12, IL-17A, and interferon (IFN)-γ in BALF supernatants, according to the manufacturer's instructions. Plates were read with SpectraMax plate reader and analyzed with SoftMax Pro software version 5.4.1 (Molecular Devices, Sunnyvale, CA). All cytokine enzyme-linked immunosorbent assay kits were from BioLegend (San Diego, CA). A similar procedure was performed on supernatants obtained from the culture of mediastinal lymph node (MLN) cells, splenocytes, or co-culture of lung DC subsets and splenic T cells 72 hours after HDM or vehicle stimulation. Serum was obtained from the mice exposed intranasally with saline-Fc or HDM-Fc with or without Sema3E-Fc. Total and HDM-specific IgE and IgG1 levels were quantified using commercial kits, according to the manufacturer's instructions (Southern Biotech, Birmingham, AL), as we described previously.32Gounni A.S. Spanel-Borowski K. Palacios M. Heusser C. Moncada S. Lobos E. Pulmonary inflammation induced by a recombinant Brugia malayi gamma-glutamyl transpeptidase homolog: involvement of humoral autoimmune responses.Mol Med. 2001; 7: 344-354PubMed Google Scholar Briefly, MLN or spleen was collected and a single-cell suspension was prepared by using a cell strainer. The cells were resuspended at a concentration of 4 × 106 cells/mL in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mmol/L l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 5 × 10−5 mmol 2-mercaptoethanol, plated in 24-well tissue culture plates. Then, MLN or spleen cells were incubated with a freshly prepared cocktail containing 50 ng/mL phorbol myristate acetate, 500 ng/mL ionomycin, and 10 μg/mL brefeldin A, all from Sigma-Aldrich (Oakville, ON, Canada) for 4 hours at 37°C and 5% CO2. Extracellular staining was performed by using anti-mouse CD3 e-Fluor 450 (clone 17A2) and CD4–fluorescein isothiocyanate (clone RM4-5), both from eBioscience (San Diego, CA). Fixed and surface-stained MLN cells were permeabilized with 0.1% saponin in flow cytometry buffer and then stained with specific fluorochrome-conjugated monoclonal antibodies, including anti-mouse IFN-γ–phosphatidylethanolamine (clone XMG1.2) and IL-4–allophycocyanin (clone 11B11) from eBioscience. Samples were acquired on a FACSCanto II (BD Biosciences, San Jose, CA) and analyzed using FlowJo software version 10 (Tree Star Inc., Ashland, OR). Major pulmonary DC subsets from the lungs were characterized 3 days after intranasal exposure with a single high dose of HDM (100 μg/mouse).33Plantinga M. Guilliams M. Vanheerswynghels M. Deswarte K. Branco-Madeira F. Toussaint W. Vanhoutte L. Neyt K. Killeen N. Malissen B. Hammad H. Lambrecht B.N. Conventional and monocyte-derived CD11b(+) dendritic cells initiate and maintain T helper 2 cell-mediated immunity to house dust mite allergen.Immunity. 2013; 38: 322-335Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar Sema3E-Fc or saline-Fc was administered 1 hour before HDM sensitization. Briefly, lungs were minced and enzymatically digested by 1 mg/mL collagenase IV and 0.5 mg/mL deoxyribonuclease I (Worthington Biochemical, Lakewood, NJ), followed by red blood cell lysis and Fc blocking. Then, DCs were stained by anti-mouse F4/80–fluorescein isothiocyanate (clone BM8; eBioscience), anti-mouse CD11c-allophycocyanin (clone 418; eBioscience), MHCII eFluor 450 (clone M5/114.15.2; eBioscience), CD11b- phosphatidylethanolamine-Cy7 (clone M1/70; BioLegend), and CD103-PerCP-Cy5.5 (clone 2E7; BioLegend) antibodies. Finally, pulmonary CD11b+ or CD103+ DC subsets (pregated as nonautofluorescent F4/80− CD11c+ MHCIIhi) were sorted using a BD FACSAria-III Digital Cell Sorter (BD Biosciences) and co-cultured with splenic T cells in 1:10 ratios for 72 hours to measure the cytokine response. GraphPad Prism 5.0 software (Graphpad Software Inc., La Jolla, CA) was used for statistical analysis, and values were presented as the means ± SEM of at least three independent experiments. Depending on the number of groups and treatments, data were analyzed by unpaired t-test, one- or two-way analysis of variance, followed by the Bonferroni's multiple comparisons post hoc test. Differences were considered to be statistically significant at P ≤ 0.05. Because repeated exposure to HDM induces AHR, inflammation, and tissue remodeling, we exposed Balbc mice with this clinically relevant allergen for 2 consecutive weeks (Figure 1A).31Hirota J.A. Budelsky A. Smith D. Lipsky B. Ellis R. Xiang Y.Y. Lu W.Y. Inman M.D. The role of interleukin-4Ralpha in the induction of glutamic acid decarboxylase in airway epithelium following acute house dust mite exposure.Clin Exp Allergy. 2010; 40: 820-830Crossref PubMed Scopus (23) Google Scholar, 34Fattouh R. Al-Garawi A. Fattouh M. Arias K. Walker T.D. Goncharova S. Coyle A.J. Humbles A.A. Jordana M. Eosinophils are dispensable for allergic remodeling and immunity in a model of house dust mite-induced airway disease.Am J Respir Crit Care Med. 2011; 183: 179-188Crossref PubMed Scopus (65) Google Scholar, 35Johnson J.R. Wiley R.E. Fattouh R. Swirski F.K. Gajewska B.U. Coyle A.J. Gutierrez-Ramos J.C. Ellis R. Inman M.D. Jordana M. Continuous exposure to house dust mite elicits chronic airway inflammation and structural remodeling.Am J Respir Crit Care Med. 2004; 169: 378-385Crossref PubMed Google Scholar Then, Sema3E immunoreactivity was assessed in the allergic airways and compared with that of the saline-treated control group. Immunofluorescence staining on lung tissue sections revealed that Sema3E is highly expressed in saline-treated mice, especially in the airway epithelium (Figure 1B). In contrast, HDM sensitization and challenge dramatically decreased Sema3E expression (Figure 1C). Staining with isotype control antibody confirmed the specificity of Sema3E immunoreactivity (Figure 1D). Intensity of Sema3E immunostaining in the airways was significantly reduced on HDM exposure (Figure 1E). Decreased expression of Sema3E on HDM challenge suggests an unknown key role of this protein in airway homeostasis, which is impaired in asthmatic conditions. We previously reported an inhibitory effect of Sema3E on ASM cell proliferation and migration in vitro28Movassagh H. Shan L. Halayko A.J. Roth M. Tamm M. Chakir J. Gounni A.S. Neuronal chemorepellent Semaphorin 3E inhibits human airway smooth muscle cell proliferation and migration.J Allergy Clin Immunol. 2014; 133: 560-567Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar and also a protective role of this protein in the HDM model of asthma using Sema3e−/− mice.29Movassagh H. Shan L. Mohammed A. Halayko A.J. Gounni A.S. Semaphorin 3E deficiency exacerbates airway inflammation, hyperresponsiveness, and remodeling in a mouse model of allergic asthma.J Immunol. 2017; 198: 1805-1814Crossref PubMed Scopus (27) Google Scholar, 30Movassagh H. Saati A. Nandagopal S. Mohammed A. Tatari N. Shan L. Duke-Cohan J.S. Fowke K.R. Lin F. Gounni A.S. Chemorepellent semaphorin 3E negatively regulates neutrophil migration in vitro and in vivo.J Immunol. 2017; 198: 1023-1033Crossref PubMed Scopus (30) Google Scholar These findings combined with decreased expression of Sema3E (Figure 1) encouraged us to examine whether Sema3E has a potential therapeutic effect on allergic asthma in vivo. Therefore, we first treated mice with either Sema3E-Fc or saline-Fc, followed by intranasal HDM or saline exposure 1 hour later for 2 consecutive weeks (Figure 2A). We observed a significant decrease in HDM-induced airway resistance (Figure 2B) as a key measure of lung function in mice that received Sema3E-Fc before HDM exposure compared to those that received saline-Fc alone. Similarly, both tissue resistance (Figure 2C) and tissue elastance (Figure 2D) were decreased on Sema3E treatment. In addition, mice treated with Sema3E-Fc exhibited a reduced number of total inflammatory cells (Figure 2E), particularly eosinophils and, to some extent, neutrophils (Figure 2F) in BALF compared to HDM-Fc–treated controls. The inhibitory effect of Sema3E on HDM-induced lung leukocytic infiltration was confirmed by hematoxylin and eosin staining (Figure 3, A and B). Mucus secretion and collagen deposition, two critical facets of airway remodeling in allergic asthma, were significantly decreased by Sema3E administration, as evaluated by periodic acid–Schiff (Figure 3, C and D) and Sirius red staining (Figure 3, E and F) on lung tissue sections, respectively. Together, these data suggest that Sema3E treatment modulates airway inflammation, remodeling, and AHR provoked by HDM.Figure 3Sema3E reduces HDM-induced pulmonary inflammation and remodeling. Lung inflammation (A and B), mucus overproduction (C and D), and collagen deposition (E and F) were histologically evaluated in lung tissue sections by performing hematoxylin and eosin (H&E), periodic acid–Schiff (PAS), and Sirius red staining, respectively. Statistical comparison was performed between HDM plus saline-Fc and HDM plus Sema3E-Fc groups. All data represent two independent experiments. Data are expressed as means ± SEM. n = 3 to 5 mice per group (A–F). ∗P < 0.05, ∗∗P < 0.01. Scale bars = 50 μm (A, C, and E).View Large Image Figure ViewerDownload Hi-res image Download (PPT) We then investigated the effect of Sema3E-Fc treatment on cytokine response. Compared to the mice given only saline-Fc before HDM challenge, Sema3E-Fc–treated mice showed a significant decrease of IL-4, IL-5, IL-9, and IL-17A. However, IL-12 level in the BALF was not significantly altered on Sema3E-Fc treatment, whereas Sema3E-Fc treatment increased IFN-γ level in the BALF (Figure 4A). The effect of Sema3E-Fc treatment on Th1/Th2 cytokine response was further investigated by performing flow cytometry on lung-draining MLN ex vivo. Sema3E-Fc decreased HD
Referência(s)