The Journal of Immunology

Chemorepellent 3E Negatively Regulates Neutrophil Migration In Vitro and In Vivo

Hesam Movassagh,* Abeer Saati,* Saravanan Nandagopal,*,† Ashfaque Mohammed,* Nazanin Tatari,* Lianyu Shan,* Jonathan S. Duke-Cohan,‡ Keith R. Fowke,x Francis Lin,*,† and Abdelilah S. Gounni*

Neutrophil migration is an essential step in leukocyte trafficking during inflammatory responses. , originally discov- ered as guidance cues in neural development, have been shown to regulate cell migration beyond the . However, the potential contribution of semaphorins in the regulation of neutrophil migration is not well understood. This study examines the possible role of a secreted chemorepellent, Semaphorin 3E (Sema3E), in neutrophil migration. In this study, we demonstrated that human neutrophils constitutively express Sema3E high-affinity , PlexinD1. Sema3E displayed a potent ability to inhibit CXCL8/IL-8–induced neutrophil migration as determined using a microfluidic device coupled to real-time microscopy and a transwell system in vitro. The antimigratory effect of Sema3E on human neutrophil migration was associated with suppression of CXCL8/IL-8–mediated Ras-related C3 botulinum toxin substrate 1 GTPase activity and polymerization. We further addressed the regulatory role of Sema3E in the regulation of neutrophil migration in vivo. Allergen airway exposure induced higher neutrophil recruitment into the lungs of Sema3e2/2 mice compared with wild-type controls. Administration of exogenous recombinant Sema3E markedly reduced allergen-induced neutrophil recruitment into the lungs, which was associated with allevi- ation of allergic airway inflammation and improvement of lung function. Our data suggest that Sema3E could be considered an essential regulatory mediator involved in modulation of neutrophil migration throughout the course of neutrophilic inflamma- tion. The Journal of Immunology, 2017, 198: 1023–1033.

eutrophils are the most abundant circulating leukocytes gered by chemokine milieu that promote neutrophil migration (3). in humans and the first cells to be recruited toward a site However, the precise regulatory mechanism underlying chemokine- N of inflammation. These cells are extremely important in mediated neutrophil migration is not fully understood. several inflammatory and infectious diseases and play a critical Emerging evidence suggests that proteins could be role in the maintenance of (1). Imbalanced neutrophil involved in the regulation of neutrophil migration (4, 5). Guidance recruitment combined with a deregulated activity contribute to cues have been primarily identified as essential mediators of de- various human diseases, as such neutrophil functions should be velopment, which direct the cells to reach their targets via exerting tightly regulated (2). Neutrophil chemotaxis toward inflammatory attracting or repulsive signals (6–8). Semaphorins are a versatile sites requires spatiotemporal control of signaling events trig- family of axon guidance molecules ubiquitously expressed in di- verse tissues (9), although their postdevelopmental functions have *Department of Immunology, Rady Faculty of Health Sciences, Max Rady College yet to be completely understood. Class 3 semaphorins comprise all of Medicine, University of Manitoba, Winnipeg, Manitoba R3E 0T5, Canada; vertebrate-secreted semaphorins that play a key role in cell migra- †Department of Physics and Astronomy, Faculty of Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada; ‡Department of Medical Oncology, Labora- tion in a context-dependent manner (10–12). Apart from their tory of Immunobiology, Dana-Farber Institute and Department of Medicine, classical chemorepulsive function in axon guidance, class 3 sem- Harvard Medical School, Boston, MA 02215; and xDepartment of Medical Microbi- ology, Rady Faculty of Health Sciences, Max Rady College of Medicine, University aphorin signaling has been shown to be indispensable for immune of Manitoba, Winnipeg, Manitoba R3E 0T5, Canada regulation (13). We have previously reported that human airway ORCIDs: 0000-0002-1787-3356 (S.N.); 0000-0001-8403-0175 (N.T.); 0000-0002- smooth muscle (ASM) cells express semaphorin 3E (Sema3E) high- 9478-9609 (J.S.D.-C.); 0000-0003-1265-6560 (A.S.G.). affinity receptor, PlexinD1, and their migration is mitigated upon Received for publication June 22, 2016. Accepted for publication November 11, ligation with Sema3E as a crucial component of airway remodeling 2016. (14). Also, in atherosclerotic plaque Sema3E has been shown to This work was supported by Canadian Institute of Health Research Grant 115115 and Natural Sciences and Engineering Research Council Grant RGPIN/386289-2011 (to have a repulsive effect on (15) However, Sema3E is a A.S.G.). H.M., N.T., and A.M. were financially supported by studentships from chemoattractant of macrophages in a mouse model of obesity, which Research Manitoba-Children’s Hospital Research Institute of Manitoba. is mediated by a holoreceptor complex including PlexinD1, Neu- Address correspondence and reprint requests to Dr. Abdelilah S. Gounni, Department of Immunology, Rady Faculty of Health Sciences, Max Rady College of Medicine, ropilin 1 (Nrp1), and vascular endothelial growth factor receptor 2 University of Manitoba, 419 Apotex Centre, 750 McDermot Avenue, Winnipeg, MB (VEGFR2) (16). However, whether Sema3E regulates neutrophil R3E 0T5, Canada. E-mail address: [email protected] migration during homeostatic and inflammatory conditions is un- The online version of this article contains supplemental material. known. It is believed that the Sema3E binding signal is transduced Abbreviations used in this article: ASM, airway smooth muscle; BALF, bronchoal- through the segmented GTPase-activating protein domain in the veolar lavage fluid; HDM, house dust mite; mKC, mouse keratinocyte-derived che- mokine; MLN, mediastinal lymph node; Nrp1, 1; Rac1, Ras-related C3 cytoplasmic tail of PlexinD1. In response to Sema3E treatment, they botulinum toxin substrate 1; Sema3E, Semaphorin 3E; VEGFR2, vascular endothe- interact with small Rho such as Ras-related C3 botulinum lial growth factor receptor 2; WT, wild-type. toxin substrate 1 (Rac1), which critically regulates actin cyto- Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 skeleton during the process of directional cell migration (17). In www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601093 1024 ROLE OF SEMAPHORIN 3E IN NEUTROPHILIC addition, Rac1 has been previously shown as a key signaling com- Flow cytometry ponent involved in regulation of neutrophil chemotaxis (3). Neutrophil preparations were incubated for 30 m on ice with anti-human The current study aimed to determine the role of Sema3E in PlexinD1 (R&D Systems) or respective IgG isotype control (Sigma) be- neutrophil migration by using in vitro and in vivo approaches. Our fore the addition of AlexaFluor 488 conjugated donkey anti-goat IgG results indicated that Sema3E inhibits chemokine-induced human (1:100) (R&D Systems) or Alexa Fluor-conjugated donkey anti-sheep Ab neutrophil migration via suppression of Rac1 GTPase activity and in the dark for 1 h on ice. Samples were washed between steps and ana- lyzed by FACScan (BD Biosciences, San Jose, CA). Results are presented F-actin polymerization. Analysis of microfluidic data revealed that as specific mean fluorescence intensity using FlowJo software (Tree Star, Sema3E is able to suppress the chemotactic movement of primary Ashland, OR). human neutrophils induced by CXCL8/IL-8, which was further confirmed in a standard transwell migration assay. Genetic ablation Neutrophil chemotaxis using a microfluidic device of Sema3E led to enhanced airway neutrophilic infiltration in mice, A simple Y-shaped microfluidic device was used for neutrophil migration which was heightened upon house dust mite (HDM) allergen en- experiments as described previously (19). The microfluidic device was counter. Furthermore, Sema3E treatment reduced HDM-induced designed and fabricated by standard photolithography and soft lithog- raphy (20). The gradient channel of microfluidic device was coated with airway neutrophilia and pathological features of experimental al- 0.25 mg/ml fibronectin (BD Bioscience) for 1 h at room temperature lergic asthma. Taken together, these data suggest that Sema3E could before adding cells to provide a substrate for cell adhesion and migra- be considered as a novel mediator involved in the modulation of tion, followed by blocking with 0.4% BSA in RPMI 1640 for another neutrophil extravasation with a potential therapeutic implication in hour at room temperature. Chemical gradients were created and con- trolled by the continuous infusion of the CXCL8/IL-8 (10 ng/ml) or neutrophilic inflammatory diseases. Sema3E (100 ng/ml). In the combined gradient, both CXCL8/IL-8 and Sema3E were added either on the same side or opposite sides. RPMI Materials and Methods 1640 containing 0.4% BSA and Sema3E 6 CXCL8/IL-8 solutions were Subjects infused into the device and controlled by syringe pumps through the tubing and inlets of the device. The total flow rate was 0.2 ml/min. 6 This study was approved by the Ethics Committee of the Faculty of Health Before applying the gradient, 0.5–1.0 3 10 cells were loaded into the Sciences, College of Medicine, University of Manitoba. Adult, non-smoker, device within the cell inlet and allowed to flow through the main channel non-allergic healthy subjects volunteered to donate blood; 40 ml of venous for 5–10 min until 1000–2000 of the cells attached to the channel. blood was taken from each donor, between 9:00 and 11:00 AM on weekdays Utilizing time-lapse microscopy, neutrophil migration was recorded at only. Written consent was obtained from each volunteer. six frames per min for 30 min using a charge-coupled device camera (Model No. 370 KL 1044; Optikon, Canada), and image acquisition was Human peripheral blood neutrophil isolation and purification controlled by using manual tracking NIH ImageJ (v.1.34s). In brief, ∼25–50 cells were tracked from three independent experiments and Neutrophils were separated from human peripheral blood as described analyzed for each experimental condition. The gradient was checked previously (18). Briefly, blood was collected in sterile heparin tubes from before and after each migration experiment. The chemotactic index (CI) the peripheral veins of healthy volunteer donors and neutrophils were [defined as the ratio of the displacement of cells toward the chemokine separated using dextran, Ficoll-Paque histopaque sedimentation (Amer- gradient (dy), to the total migration distance (d)] was quantified using the sham Pharmacia Biotech), and a hypotonic lysis method. Neutrophil purity equation CI = dy/d. was 95–98% using Wright–Giemsa staining. Trypan blue exclusion de- termined neutrophil viability was .98%. Measurement of Rac1 GTPase activity RNA isolation and RT-PCR analysis Rac1 GTPase activity was measured using a luminometric-based G-LISA Rac1 Activation Assay Kit (Cytoskeleton Denver, CO) as described pre- RNA was purified from primary human neutrophils using TRIzol (Invi- viously (14). Briefly, snap-frozen cell lysates were prepared from unsti- trogen) followed by measuring of RNA concentration and integrity. Reverse mulated or Sema3E (100 ng/ml) 6 CXCL8/IL-8 (10 ng/ml) stimulated transcription was performed with 2 mg of total RNA using a high capacity human neutrophils (5 3 106 cells/ml) at the indicated time points. Neu- reverse transcription kit (Applied Biosystems) to synthesize cDNA. The trophil lysates were added to a 96-well plate coated with Rho binding annealing temperature and size of the amplified fragment for PLXND1 domain of Rac1. Rac1-GTP was detected using anti-Rac1 primary Ab and were 58˚C and 156 bp, respectively, and the following specific primers subsequent incubation with HRP-conjugated secondary Ab (45 min, 400 were used: Forward 59-CCCCAACCCACAGTTCTCTA-39 and Reverse rpm, room temperature) followed by development with a chemilumines- 59-CAAGTAAGCTGCGACATCCA-39. In parallel, human PBMCs, used cent reagent. A constitutively active Rac1 provided in the kit was used as as a positive control, were cultured in RPMI 1640, and reverse transcrip- positive control in all experiments, and a blank well was also used as a tion was performed as mentioned above. Primers for housekeeping negative control. GAPDH and standard controls were developed in our laboratory. Product specificity was determined by a melting curve analysis and by visualization Phalloidin staining and F-actin measurements of the PCR products on agarose gel. Primary human neutrophils were stimulated with Sema3E (100 ng/ml) with Immunocytochemistry or without CXCL8/IL-8 for 0, 0.5, 1, and 5 min. Cells were then fixed, permeabilized, and stained with AlexaFluor 488 phalloidin (Life Tech- 3 5 Immediately, after neutrophil isolation, a total of 1 10 cells were de- nologies). Then the intracellular fluorescence was determined by flow posited on microscope slides by cytospin centrifugation (ThermoShandon), cytometry as we described previously (14). The effect of Sema3E 6 and fixed with 4% paraformaldehyde (Fisher Scientific) for 20 min at room CXCL8/IL-8 treatment on F-actin content was quantified as mean fluo- temperature. Then the cytopreparations were washed in TBS, treated for rescence intensity and compared at each time point by using FlowJo 20 min with peroxidase blocking solution (1%) for 10 min, and left to dry software. overnight at room temperature. Slides were blocked with normal serum blocking solution (5% rabbit and 5% human sera) for 1 h at room tem- Allergen-induced airway inflammation model perature. Afterward, slides were briefly washed with TBS and incubated overnight at 4˚C with goat anti-human PlexinD1 Ab (1 mg/ml; R&D The Sema3e2/2 mouse model was kindly provided by Dr. Chengua Gu Systems) or with the respective IgG control (Sigma-Aldrich) in Ab dilution (Department of Neurobiology, Harvard Medical School, Boston, MA). buffer (DakoCytomation) overnight at 4˚C. Then slides were washed ex- As previously described, this model was generated in the 129P2 back- tensively with Cyto-TBS and incubated with the biotinylated secondary Ab ground, which is healthy and fertile without any pathological issue after (rabbit–anti-Goat IgG, 1:200 dilution in PBS) for 1 h at room temperature. deletion of the gene encoding Sema3e (21). Six- to eight-week old fe- The slides were then incubated with streptavidin-alkaline phosphatase for male Sema3e2/2 and wild-type (WT) mice were anesthetized, and 25 mg 30 min at room temperature and developed with Fast Red dissolved in of HDM (Greer) per mouse was administered intranasally five times a the alkaline phosphatase substrate followed by counterstaining with week for two consecutive weeks. The control group received saline. Mayer’s hematoxylin (Fisher Scientific). Isotype-matched control mAb Then 48 h after the last HDM challenge, bronchoalveolar lavage fluid was used as negative control. Slides were visualized by using AxioVision (BALF), lung, and blood collection was performed as described previ- software (Carl Zeiss). ously (22). The Journal of Immunology 1025

In the therapeutic protocol, after two consecutive weeks of HDM ex- 2 mot plus microscope and Carl Zeiss AxioCam MRC 5 camera using posure, recombinant Sema3E-Fc (23, 24) was administered to BALB/c AxioVision Rel 4.8 software. mice three times over a week starting 48 h after the last HDM chal- lenge; mice were then re-exposed to HDM. An Fc fragment from the same Methacholine challenge test subclass (g2C) was used as the control (23). To measure airway resistance, 48 h after the last HDM challenge mice were LPS-induced neutrophilia anesthetized and cannulated via the trachea. Airway resistance in response to an increasing gradient of intratracheal methacholine was measured by Five milligram per kilogram of LPS from Escherichia coli strain 0111: B4 using a FlexiVent small animal ventilator system (SCIREQ, Montreal, QC, 2 2 (Sigma-Aldrich) was injected interperitoneally to either Sema3e / or WT Canada). mice.Then8hafterinjection,miceweresacrificed,andbloodsamples were collected. RBC lysis was performed by using an ammonium- Serum IgE quantitation chloride-potassium lysing buffer (three times) before staining with neutrophil-specific surface markers and flow cytometry. Total and HDM-specific IgE levels were quantified using commercial ELISA kits according to the manufacturer’s instructions as we described FACS analysis of mouse neutrophils previously (26). ELISA kits for measuring total and HDM-specific IgE levels in serum samples were purchased from Southern Biotech (Bir- At the end of the protocols mentioned above, mice were sacrificed and lung, mingham, Al). BALF, and blood samples were harvested. Lung tissues were minced and underwent enzymatic digestion using 1 mg/ml collagenase IV (Worthington H&E staining Biochemical Corporation, Lakewood, NJ) and 0.5 mg/ml of bovine pan- creas DNase in RPMI 1640 medium. After RBC lysis and Fc blocking, Dissected left lobes of mouse lungs were inflated, fixed in formalin, and neutrophil surface markers were stained by using specific monoclonal Abs embedded in paraffin. Then sections were stained with H&E for assessing including allophycocyanin-conjugated CD11b (Clone M1/70; eBioscience) airway inflammation. and Gr1-PE (Clone 1A8-Ly6g; eBioscience) (25). Samples were acquired by FACS Canto II and analyzed using FlowJo software. Intracellular staining Neutrophil elastase Mediastinal lymph nodes (MLN) were collected from HDM 6 Sema3E- treated mice, and single-cell suspensions prepared by using a cell strainer. Formalin-fixed tissues were paraffin embedded, and 5 mm-thick sections The cells were resuspended at a concentration of 4 3 106 cells/ml in were prepared, deparaffinized in xylene, and rehydrated through graded DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml concentrations of alcohol to water, then boiled in a microwave for 10 min penicillin, 100 mg/ml streptomycin, and 5 3 1025 M 2-ME, and plated in a sodium citrate buffer (pH 6). Sections were washed and then incu- in 24-well tissue culture plates. Then MLN cells were incubated with a bated with blocking solution (1% BSA, 0.1% cold fish skin gelatin, and freshly prepared mixture containing 50 ng/ml PMA, 500 ng/ml ionomycin, 5% second animal serum in TBS) for 45 min at room temperature. Sec- and 10 mg/ml brefeldin A, all from Sigma-Aldrich (Oakville, ON, Canada) tions were stained for neutrophil elastase using mouse anti-human neu- for 4 h at 37˚C and 5% CO2. Extracellular staining was performed using trophil elastase mAb (Mouse IgG1k, clone NP57; Dako, Denmark) anti-mouse CD3 e-Fluor 450 (Clone 17A2) and CD4-FITC, both from following 3 h incubation at room temperature. Mouse IgG was used as an eBioscience. Fixed and surface-stained MLN cells were permeabilized isotype control Ab. AlexaFluor 488 goat anti–mouse IgG (H+L) (Life with 0.1% saponin in flow cytometry buffer and then stained with specific Technologies, OR) was used as secondary Ab. Slides were then washed fluorochrome-conjugated mAbs including anti-mouse IFN-g-PE (Clone extensively for 3 3 5 min with TBS and counterstained with Prolong gold XMG1.2), IL-4-allophycocyanin (Clone 11B11), and IL-17A-PE (Clone antifade mountant with DAPI (Molecular Probes, Life Technologies). eBio17B7) all purchased from eBioscience. Samples were acquired on the Images were taken at 3200 magnifications using a digital Zeiss Axioskop FACSCanto II and analyzed using FlowJo software.

FIGURE 1. Human neutrophils constitutively express PlexinD1. RT-PCR analysis was performed using specific primers of PlexinD1 (PLXND1), GAPDH served as a housekeeping gene, and N refers to the negative control (A). FACS analysis was performed to examine the surface expression of PlexinD1 protein on human neutrophils (B) or PBMC (C) using either specific Ab or isotype-matched goat IgG as negative control. A representative experiment of 12 human samples is shown. PlexinD1 protein expression on human neutrophils was further confirmed by immunocytochemistry and the isotype control revealed no immunoreactivity (D). 1026 ROLE OF SEMAPHORIN 3E IN NEUTROPHILIC INFLAMMATION

Quantification of in BALF neutrophils from healthy donors (n = 12), similar to PBMC The levels of IFN-g, IL-17A, and mouse keratinocyte-derived chemokines (Fig. 1C). Then we sought to confirm PlexinD1 expression in pri- (mKC) in BALF samples obtained from either Sema3E-Fc treated or un- mary human neutrophils utilizing immunocytochemistry. PlexinD1 treated mice were measured by the Meso Scale Discovery system (Meso immunoreactivity was detected in 99% of human neutrophils and no Scale Diagnostics). cross-reactivity was observed in cells stained with isotype control Statistics Ab (Fig. 1D). Taken together, these data indicate that human neu- trophils express the PlexinD1 receptor and suggest these cells are a Data were analyzed by one-way ANOVA first to determine if any significant potential target of Sema3E. differences existed between the various experimental groups. Tukey’s test was then performed to detect statistically significant differences and Sema3E reduces CXCL8/IL-8–induced directional migration of compare all possible pairs of means. Statistical significance was considered p , 0.05, determined using the GraphPad Prism 5.0 statistical package human neutrophils (GraphPad, San Diego, CA). Microfluidic data were analyzed using Stu- It was previously reported that Sema3E binding to PlexinD1 on dent two-sample (two-tailed) t test. developing double positive (CD4+ CD8+) thymocytes releases activated b1 bonds permitting chemokine-mediated Results cortical-to-medullary translocation of thymocyte cells required Human neutrophils constitutively express PlexinD1 for correct development (23, 24). Considering that Sema3E is the The expression of PlexinD1 has been identified in a wide variety of only characterized ligand to date for PlexinD1, PlexinD1 is well cells including T lymphocyte (23), endothelial (27, 28), and ASM expressed on neutrophils, and Sema3E binding to PlexinD1 may (14) cells. However, no reports address the expression of PlexinD1 impact chemokine responses, we investigated whether Sema3E on human neutrophils. In this study, to verify the functional re- influences CXCL8/IL-8–induced migration of human PlexinD1+ sponse of neutrophils to Sema3E we first investigated the ex- neutrophils. pression of PlexinD1, the high-affinity receptor of Sema3E, on Utilizing a microfluidic device that is optimized mainly to test freshly isolated highly pure peripheral blood human neutrophils. cell chemotaxis in response to chemokine gradient (29–31), we As shown in Fig. 1A, human neutrophils from four healthy donors found that Sema3E reduced CXCL8/IL-8–mediated human neu- expressed PlexinD1 at the mRNA level as compared with PBMCs trophil migration. As shown in Fig. 2A and Supplemental Video 1, used as a positive control. neutrophils exhibited chemotactic activity toward the CXCL8/IL-8 Surface expression of PlexinD1 was then studied by FACS on gradient. In the Sema3E gradient, the percentage of migrated cells peripheral blood human neutrophils and in PBMC as a positive is reduced; interestingly, among the migrated cells we tracked, control using goat anti–human PlexinD1 PE-conjugated Ab. moremovedintheoppositedirectiontotheSema3Egradient As shown in Fig. 1B, PlexinD1 is highly expressed on human (Fig. 2B, Supplemental Video 2). However, this movement was not

FIGURE 2. Sema3E negatively regulates CXCL8/IL-8–induced migration and chemotaxis of human neutrophils in a microfluidic device. Cell tracks in CXCL8/IL-8 gradient (A), Sema3E gradient (B), at the baseline (C), a coexisting gradient of both CXCL8/IL-8 and Sema3E along the same (D) or opposite (E) directions. Black tracks are cells moving downward; gray tracks are cells moving upward. The direction of gradients and flow are indicated by arrows for each condition. Chemotactic index (CI) for different conditions has been compared (F) in which positive and negative CI indicate upward and downward cell migration, respectively. The error bars represent SEM, and the figure is representative of at least four replicates per condition. **p , 0.01, ***p , 0.001. The Journal of Immunology 1027 significantly different to the baseline migration (Fig. 2C, 2F, experiment (Fig. 2B, 2F). Recombinant human Sema3E was able to Supplemental Video 3). We then assessed neutrophil chemotaxis in significantly inhibit CXCL8/IL-8–induced neutrophil migration in response to coexisting gradients of Sema3E and CXCL8/IL-8. all doses tested (1, 10, 50, and 100 ng/ml) (Fig. 3A). We then in- When we configured the CXCL8/IL-8 and Sema3E gradients in vestigated neutrophil migration when recombinant human Sema3E the same side of the microfluidic channel, neutrophils randomly was added to the lower chamber mixed with 10 ng/ml of CXCL8/ moved within the microfluidic channel and negatively responded to IL-8 (Fig. 3B). Sema3E had a dose-dependent inhibitory effect on CXCL8/IL-8 Sema3E gradients compared with the experiment for human neutrophil migration when introduced in the lower cham- CXCL8/IL-8 alone (Fig. 2D, Supplemental Video 4). Then, com- ber with CXCL8/IL-8 (Fig. 3B), suggesting a negative correlation peting gradients were created in which 10 ng/ml of CXCL8/IL-8 between the number of migrated cells and Sema3E concentra- was configured on one side and 100 ng/ml of Sema3E in the op- tion. In line with our microfluidic data, Sema3E alone did not posite direction (Fig. 2E, Supplemental Video 5). Neutrophil mi- significantly alter steady-state neutrophil migration in the transwell gration toward the CXCL8/IL-8 gradient was significantly reduced assay (Fig. 3B). Altogether, Sema3E markedly impairs CXCL8/IL- in the presence of the Sema3E gradient (Fig. 2D, 2E, Supplemental 8–induced human peripheral blood neutrophil migration. Videos 4, 5) compared with the IL-8 gradient alone (Fig. 2A, Supplemental Video 1). In both configurations, Sema3E signifi- Sema3E treatment decreases CXCL8/IL-8–mediated Rac1 cantly decreased the IL-8–induced chemotactic index of neutrophils GTPase activity and F-Actin polymerization (Fig. 2F), which was associated with random cell migration in It has been demonstrated that CXCL8/IL-8 promotes neutrophil different directions (Supplemental Videos 4, 5). migration via activation of a small GTPase called Rac1 (32–34). We further confirmed the inhibitory effect of Sema3E on human We and others have previously reported that the repulsive effect of neutrophil chemotaxis by performing a traditional standard Sema3E on cell migration is partly mediated by the suppression of transwell migration assay (Fig. 3). In fact, administration of Rac1 GTPase activity as an early signaling component affected recombinant human Sema3E alone into the upper chamber of the upon Sema3E treatment (14, 15). Therefore, the potential role of transwell insert slightly reduced basal neutrophil migration, which Sema3E in the abrogation of CXCL8/IL-8–induced Rac1 GTPase was not significantly different compared with the negative control activity was assessed by G-LISA in primary human neutrophils. as shown in Fig. 3A and as observed with the microfluidic device As shown in Fig. 3C, Sema3E significantly decreased Rac1 GTPase

FIGURE 3. Sema3E inhibitory effect on CXCL8/IL-8–induced human neutrophil migration is mediated by regulation of Rac1 GTPase activity and actin polymerization. Human recombinant Sema3E with or without CXCL8/IL-8 was added to the bottom insert of a transwell system and freshly isolated human neutrophils were added to the top chamber. After 1 h incubation, cells migrated to the bottom chamber were collected and subjected to cell count using a hemocytometer (A). Recombinant human Sema3E and neutrophils were added to the top chamber, whereas CXCL8/IL-8 was added to the bottom well (B). After 1 h incubation, cell migration toward CXCL8/IL-8 was measured as explained in (A). The data represent five independent experiments repeated in duplicate. Rac1 GTPase activity was measured in snap-frozen human neutrophil cell lysates harvested from either unstimulated or Sema3E 6 CXCL8/IL-8 stimulated cells at the indicated time points by performing G-LISA. Constitutively active Rac1 was used as a positive control (P), and the negative control (N) refers to blank well in all experiments. **p , 0.01, ***p , 0.001 (C). Phalloidin staining was performed to detect the F-actin content of human neutrophils upon Sema3E 6 CXCL8/IL-8 treatment (D). The statistical significance of Sema3E effect on F-actin depolymerization was determined by measuring MFI, which was reported as a percentage of the unstimulated control groups (E). The data represent four independent experiments. *p , 0.05. MFI, mean fluorescence intensity; Rac1, Ras-related C3 botulinum toxin substrate 1. 1028 ROLE OF SEMAPHORIN 3E IN NEUTROPHILIC INFLAMMATION activity 5 and 15 min after stimulation with CXCL8/IL-8 without any was comparable to unstimulated neutrophils, along with its non- significant effect at the baseline. significant effect on neutrophil migration and Rac1 GTPase activ- Induction of F-actin polymerization has been shown as an es- ity. Therefore, our data suggest that the inhibitory effect of Sema3E sential cytoskeletal target to promote neutrophil migration (32, 35, on CXCL8/IL-8–induced migration of human neutrophils is asso- 36). However, the role of Sema3E in the regulation of F-actin ciated with a reduction of Rac1 GTPase activity and depolymer- dynamics has not been addressed in neutrophils so far. We stim- ization of actin filaments. ulated human peripheral blood neutrophils with Sema3E (100 ng/ml) in the presence or absence of CXCL8/IL-8 (10 ng/ml) and Homeostatic and allergen-induced pulmonary neutrophil then stained them with a fluorescent conjugated phalloidin to spe- accumulation are enhanced in the absence of Sema3E in vivo cifically quantify F-actin polymerization. As depicted in Fig. 3D Neutrophil influx into the lung is a key feature of inflammatory and 3E, stimulation of human neutrophils with CXCL8/IL-8 alone airway diseases in which sustained accumulation of these cells, robustly increased F-actin polymerization, which was significantly mainly due to deregulated migration, leads to perpetuating patho- reduced upon costimulation with Sema3E and CXCL8/IL-8. F-actin logical manifestations (37). Therefore, understanding the mechanisms content of human neutrophils after treatment with Sema3E alone regulating impaired neutrophil migration during inflammatory

FIGURE 4. Basal pulmonary neutrophil accumulation is enhanced in Sema3e2/2 mice. Lung samples from Sema3e2/2 and WT mice were processed and stained with specific Abs to detect neutrophil surface markers. Flow cytometry determined the frequency of CD11bbright Gr1bright neutrophil population at the baseline. Staining with isotype control Ab revealed no immunoreactivity (A). Percentage (B) and the absolute number (C) of CD11b bright Gr1bright lung neutrophils was compared between Sema3e2/2 and WT mice at the baseline. The frequency of neutrophils was investigated on BALF (D–F) and spleen (G– I) from naive Sema3e2/2 or WT mice. n = 4 per group. *p , 0.05, **p , 0.01. The Journal of Immunology 1029 processes is of great importance. We investigated the potential role Sema3e2/2 than those of WT controls (Fig. 5B). Our statistical of Sema3E in neutrophilic airway inflammation in homeostatic analysis demonstrated that both neutrophil percentage (Fig. 5C) and conditions and mouse model of allergic asthma. As a conventional absolute number (Fig. 5D) are significantly increased in Sema3e2/2 gating strategy, the CD11bbright Gr-1bright subset from a live singlet mice upon intranasal HDM challenge. We further confirmed the FSClo SSChi granulocyte population was considered a neutrophil enhanced neutrophilic airway inflammation by higher expression of in the following mouse flow cytometry experiments, and auto- elastase on Sema3e2/2 mice compared with WT littermates (Fig. 5E). fluorescent alveolar macrophages were eliminated from the analysis In addition, blood neutrophils were significantly higher at the baseline (data not shown) (25). First, we observed that the genetic deletion of (Fig. 6A), which was sustained higher 8 h after i.p. injection of LPS Sema3E led to a higher neutrophil accumulation in the lungs of naive in Sema3e2/2 versus WT mice (Fig. 6B). Taken together, our data mice compared with those of WT controls (Fig. 4A), which was suggest that Sema3E deficiency is associated with a hyper- statistically significant (Fig. 4B, 4C). Enhanced neutrophil accumu- neutrophilic phenotype in HDM allergic asthma model. lation in the absence of Sema3E at homeostatic conditions was fur- ther confirmed in the airways by performing the same experiment on Exogenous Sema3E treatment protects mice from mouse BALF (Fig. 4D–F). However, basal neutrophil accumulation allergen-induced neutrophilic airway inflammation was not significantly different between Sema3e2/2 and WT mice in To address the potential therapeutic relevance of this study, we treated the spleen (Fig. 4G–I). HDM-challenged WT mice with exogenous recombinant Sema3E-Fc We induced allergic airway inflammation by sensitization of intranasally then re-exposed them with HDM as demonstrated in Sema3e2/2 and WT mice with a clinically relevant allergen, Fig. 7A. Sema3E-Fc treated mice demonstrated significantly lower HDM, via an intranasal route as depicted in Fig. 5A. Along with CD11bbright Gr1bright pulmonary neutrophil influx compared with the exacerbation of other asthma deficits such as IgE synthesis and AHR saline-Fc treated control group (Fig. 7B–D). These results were fur- in Sema3e2/2 mice (data not shown), HDM exposure induced higher ther confirmed in the airways of the same mice (Fig. 7E–G). The recruitment of CD11bbright Gr1bright neutrophils into the airways of surface expression of PlexinD1 was also detected on pulmonary

FIGURE 5. HDM-induced neutrophil recruitment into the lung is exacerbated in Sema3e2/2 mice. Age- and sex-matched Sema3e2/2 and WT mice were exposed to HDM for two consecutive weeks via an intranasal route (A). Then, the enzymatically digested lung samples underwent flow cytometry to detect recruitment of CD11bbright Gr1bright neutrophils. Staining with isotype control Ab revealed no immunoreactivity (B). Percentage (C) and the absolute number (D) of CD11bbright Gr1bright lung neutrophils was compared between Sema3e2/2 and WT mice upon HDM exposure. Neutrophil elastase im- munoreactivity was studied on lung tissue sections from Sema3e2/2 and WT mice upon either saline or HDM exposure by performing immunofluorescence microscopy (E). Scale bars, 50 mM. n = 4 per group. *p , 0.05, **p , 0.01. 1030 ROLE OF SEMAPHORIN 3E IN NEUTROPHILIC INFLAMMATION

FIGURE 6. Blood neutrophils are higher in Sema3e2/2 mice than WT controls upon HDM challenge. Blood collection was performed 8 h after PBS (A and B) or LPS (C and D) injection in Sema3e2/2 and WT mice followed by RBC lysis. Extracellular staining was performed to determine the surface expression of CD11b and Gr-1. Percentage of CD11bbright Gr1bright circulating neutrophils was compared between Sema3e2/2 and WT mice upon PBS (B) or LPS (D) challenge. n = 4 mice per group. *p , 0.05, **p , 0.01.

neutrophils after HDM exposure, suggesting that the Sema3E thera- neutrophil-derived mediators, it should be tightly regulated to avoid peutic effect could be mediated by this receptor (Fig. 7H). tissue damage caused by an excessive inflammatory reaction (41). It has been previously shown that neutrophil depletion leads to the Understanding novel mediators and pathways implicated in this alleviation of pathological features in a mouse model of allergic process is an essential step toward developing better strategies to asthma (38). Therefore, we investigated the effect of Sema3E-Fc control neutrophilic inflammatory disorders. treatment on HDM-mediated AHR, IgE synthesis, and airway in- Originally discovered as neural chemorepellents, the role of flammation as the hallmarks of asthma. Interestingly, reduction of semaphorins in immune regulation has emerged in recent years HDM-induced neutrophil recruitment upon Sema3E-Fc treatment in (13). In the current study, we investigated the role of Sema3E in our experimental model was associated with decreased airway re- regulating neutrophil migration. Our in vitro studies of primary sistance as a measure of lung function (Fig. 8A). In addition, extrinsic human neutrophils and our in vivo model of allergic airway in- intranasal administration of Sema3E-Fc significantly lowered both flammation show the repulsive role of Sema3E in neutrophil mi- total (Fig. 8B) and HDM-specific (Fig. 8C) forms of IgE in the se- gration and its potential to diminish airway neutrophil influx. rum. Our H&E staining revealed an inhibitory effect of Sema3E-Fc Considering the pivotal role of neutrophils in airway inflammation, on HDM-induced airway inflammation (Fig. 8D). As demonstrated in reduction of their pulmonary influx could result in better control of Fig. 8E, in vivo Sema3E-Fc treatment does not significantly change pathological features such as airway hyper-responsiveness and IFN-g secretion in the airway. However, Sema3E-Fc significantly remodeling. modulates HDM-induced secretionofmKC(Fig.8F),amurineho- A growing body of evidence over the last decade illustrates that molog of CXCL8/IL-8 and IL-17A (Fig. 8G), which could account semaphorins and their receptors are involved in the regulation of for the reduced neutrophil accumulation in the airway (39, 40). neutrophil functions. For instance, an orthopoxviral semaphorin, Collectively, our findings suggest a regulatory role of Sema3E in A39R, was previously shown to inhibit neutrophil phagocytosis (42). neutrophilic airway inflammation and modulation of neutrophil re- More recent studies reveal a crucial pathological role for the Sema7A- cruitment to the lung most likely via reducing neutrophil migration. PlexinC1 axis in promoting neutrophil migration in acute lung injury (43) and hypoxia (44) models. In contrast, the Sema3C therapeutic Discussion effect on lung injury is mediated by decreased lung neutrophil influx, Neutrophil influx to a site of inflammation is a double-edged sword: on and silencing its expression leads to a significant increase in neutro- the one hand, it is required for the inflammatory insult to be sufficiently phil activity (4). However, to our knowledge, the role of Sema3E in undertaken; on the other hand, considering the destructive capacity of neutrophil migration has not been previously investigated. The Journal of Immunology 1031

FIGURE 7. Exogenous Sema3E treatment decreases HDM-induced pulmonary neutrophilia. The timeline of HDM challenge and Sema3E treatment model is depicted in (A). Intranasal treatment with recombinant murine Sema3E-Fc after inducing experimental allergic asthma by HDM challenge significantly decreased lung (B–D) and airway (E–G) neutrophilia upon HDM re-exposure compared with Fc-Ig treated control group. Surface expression of Sema3E high-affinity receptor, PlexinD1, was determined by FACS analysis (H). n =4.*p , 0.05, **p , 0.01.

We have shown that the Sema3E high-affinity receptor, Plex- mates, suggestive of a regulatory role for Sema3E in pulmonary inD1, is constitutively expressed on human neutrophils obtained neutrophil extravasation. In addition, a higher number of neutro- from healthy individuals and on mouse neutrophils. It should be phils in naive Sema3e2/2 mice indicates an intrinsic defect at mentioned that all secreted mammalian semaphorins bind neuro- homeostatic conditions, which could potentially make the animals pilins except Sema3E, which interacts directly with PlexinD1 as the more prone to airway neutrophilia upon allergen exposure. It was binding receptor (9, 12). According to previous studies, the re- recently reported that a deteriorative role of Sema7A receptor, pulsive functional outcome of Sema3E-PlexinD1 signaling could PlexinC1, in an acute lung injury model is mediated by induction be reversed via interaction with Nrp1 (45) or VEGFR2 (46). of pulmonary neutrophil migration and its genetic or pharma- However, the antimigratory effect of Sema3E on human neutro- ceutical inhibition could be a new therapeutic option (43). phils suggests this effect is independent of gating by Nrp1 or Endothelial-derived Sema7A has been reported to propagate the VEGFR2. In fact, our unpublished data revealed that Nrp1 is not extravasation of neutrophils from the vascular space during hyp- expressed in human neutrophils, signifying that antimigratory is oxia (44). However, to our knowledge, this is the first study mainly mediated via PlexinD1. reporting the role of a semaphorin family member, Sema3E, in Besides Sema3E, PlexinD1 can bind Sema4A, which is an allergen-induced airway neutrophilia. immune semaphorin (47, 48). Sema4A has been shown to play a Sema3E intranasal treatment reduced HDM-induced recruitment crucial role in Th1 differentiation and T-bet expression, which of neutrophils into the lungs, which was associated with im- leads to an impaired Ag-specific Th1 and Ab response in Sema4A- provement of lung function, decreased IgE synthesis, and airway deficient mice (49). Furthermore, Nkyimbeng-Takwi et al. (50) inflammation. It further suggests Sema3E is a potential treatment have demonstrated that Sema4A downregulates the severity of option for severe refractory asthma, which deserves more mech- allergic asthma in an OVA mouse model. There might be a po- anistic studies. The HDM model was chosen primarily because it is tential competition between Sema3E and Sema4A for binding to known as a clinically relevant common indoor aeroallergen (54). PlexinD1, which should be further investigated. However, it Phipps et al. (55) reported that HDM induced airway neutrophilia should be considered that, unlike secreted Sema3E, Sema4A is a and IL-17A production. In addition, HDM exposure releases membrane-bound semaphorin, which suggests different modes of CXCL8/IL-8 from the airway epithelium as a neutrophil che- activation and function (47). moattractant, and may also contribute to neutrophilic airway Neutrophil influx into the lungs is a hallmark of many airway inflammation in acute asthma exacerbations or severe asthma inflammatory diseases such as allergic asthma and chronic ob- (56, 57). It further highlights the importance of Sema3E’s en- structive pulmonary diseases. In fact, severe asthmatic patients, dogenous defect as a predisposing factor in various contexts who are refractory to glucocorticoid therapies, are characterized by regarding pulmonary neutrophilia, especially where bacterial a massive recruitment of neutrophils to their lungs due to higher endotoxin contributes to the inflammation. migration or survival compared with normal conditions (51–53). In In this study, we employed a microfluidic-based system to assess our HDM-induced model of allergic asthma, lung neutrophils directional neutrophil migration at a single-cell level in defined were significantly higher in Sema3e2/2 mice than in WT litter- stable gradient of CXCL8/IL-8, Sema3E, and their combinations. 1032 ROLE OF SEMAPHORIN 3E IN NEUTROPHILIC INFLAMMATION

FIGURE 8. Sema3E reduces HDM-induced AHR and airway inflammation. Sema3E or saline-treated mice underwent tracheotomy accompanied by methacholine challenge to measure airway resistance (A). Serum levels of total and HDM-specific IgE were measured by ELISA (B and C). Airway inflammation was studied by performing H&E staining (D). Effect of Sema3E treatment on HDM-induced secretion of IFN-g (E), mKC (F), and IL-17A (G) into the airways was studied by performing ELISA on BALF samples. All data are representative of four to six mice per group. All data are representative of four to five mice per group. *p , 0.05.

The results further confirmed the inhibitory effect of Sema3E on Indeed, other signaling pathways activated by CXCL8/IL-8 stimu- CXCL8/IL-8–induced neutrophil migration as we demonstrated lation could undergo negative regulation upon Sema3E treatment. by a traditional transwell assay. In both methods, the Sema3E As we previously reported, both MAPK and PI3K signaling could inhibitory effect was evident when neutrophils were stimulated be targeted by Sema3E, which might lead to the decrease of other with Sema3E and CXCL8/IL-8 as either opposite or same-side neutrophil functions such as phagocytosis and survival (14). gradients. The microfluidic-based results not only confirmed the Treatment with Sema3E alone did not affect basal Rac1 GTPase Sema3E inhibitory effect on CXCL8/IL-8 by the significantly activity or F-actin rearrangement in neutrophils. It may explain the reduced number of migrated cells seen in the transwell assay but non-significant effect of Sema3E on basal neutrophil migration in also showed the inhibitory effect of Sema3E on the directionality agreement with findings in ASM and endothelial cells (14). of cell migration by the significantly reduced chemotactic index. All in all, our data provide novel insights into the potential contri- Monitoring neutrophil chemotaxis by microfluidic devices has bution of Sema3E in the regulation of neutrophilic inflammatory re- been recently considered a novel approach for sensitive asthma sponses. This study provides evidence of a previously unknown diagnosis (58, 59). In this study, we provide evidence that this tech- mechanism modulating neutrophil migration that could be considered as nical approach could be applicable in mechanistic and translational a therapeutic approach in neutrophil-dominated inflammatory disorders. studies in which unknown repulsive potency of a therapeutic candi- date on neutrophil migration is aimed to be assessed. Disclosures From a mechanistic point of view, Sema3E inhibited CXCL8/IL- The authors have no financial conflicts of interest. 8–induced neutrophil migration at least in part via reduction of F-actin polymerization and Rac1 GTPase activity. These findings References are in line with previous studies on ASM cells (14), macrophages 1. Hajishengallis, G., T. Chavakis, E. Hajishengallis, and J. D. Lambris. 2014. (15) and endothelial cells (27), further supporting the involvement Neutrophil homeostasis and inflammation: novel paradigms from studying of these key elements in underlying the Sema3E repulsive roles. periodontitis. J. Leukoc. Biol. 98: 539–548. The Journal of Immunology 1033

2. Nauseef, W. M., and N. Borregaard. 2014. Neutrophils at work. Nat. Immunol. 32. Al-Omari, M., E. Korenbaum, M. Ballmaier, U. Lehmann, D. Jonigk, 15: 602–611. D. J. Manstein, T. Welte, R. Mahadeva, and S. Janciauskiene. 2011. Acute-phase 3. Mo´csai, A., B. Walzog, and C. A. Lowell. 2015. Intracellular signalling during protein a1-antitrypsin inhibits neutrophil calpain I and induces random migra- neutrophil recruitment. Cardiovasc. Res. 107: 373–385. tion. Mol. Med. 17: 865–874. 4. Vadivel, A., R. S. Alphonse, J. J. Collins, T. van Haaften, M. O’Reilly, F. Eaton, 33. Filippi, M. D., K. Szczur, C. E. Harris, and P. Y. Berclaz. 2007. Rho GTPase and B. The´baud. 2013. The axonal guidance cue semaphorin 3C contributes to Rac1 is critical for neutrophil migration into the lung. Blood 109: 1257–1264. alveolar growth and repair. PLoS One 8: e67225. 34. Hwaiz, R., M. Rahman, I. Syk, E. Zhang, and H. Thorlacius. 2015. Rac1-dependent 5. Ye, B. Q., Z. H. Geng, L. Ma, and J. G. Geng. 2010. Slit2 regulates attractive secretion of platelet-derived CCL5 regulates neutrophil recruitment via activation eosinophil and repulsive neutrophil chemotaxis through differential srGAP1 of alveolar macrophages in septic lung injury. J. Leukoc. Biol. jlb.4A1214-603R. expression during lung inflammation. J. Immunol. 185: 6294–6305. 35. Karlsson, T., F. Musse, K. E. Magnusson, and E. Vikstro¨m. 2012. 6. Adams, R. H., and A. Eichmann. 2010. Axon guidance molecules in vascular N-Acylhomoserine lactones are potent neutrophil chemoattractants that act via patterning. Cold Spring Harb. Perspect. Biol. 2: a001875. calcium mobilization and actin remodeling. J. Leukoc. Biol. 91: 15–26. 7. Pircher, A., J. Wellbrock, W. Fiedler, I. Heidegger, E. Gunsilius, and W. Hilbe. 36. Mazaki, Y., S. Hashimoto, T. Tsujimura, M. Morishige, A. Hashimoto, 2014. New antiangiogenic strategies beyond inhibition of vascular endothelial K. Aritake, A. Yamada, J. M. Nam, H. Kiyonari, K. Nakao, and H. Sabe. 2006. growth factor with special focus on axon guidance molecules. Oncology 86: 46–52. Neutrophil direction sensing and superoxide production linked by the GTPase- 8. Van Battum, E. Y., S. Brignani, and R. J. Pasterkamp. 2015. Axon guidance activating protein GIT2. Nat. Immunol. 7: 724–731. proteins in neurological disorders. Lancet Neurol. 14: 532–546. 37. Kolaczkowska, E., and P. Kubes. 2013. Neutrophil recruitment and function in 9. Worzfeld, T., and S. Offermanns. 2014. Semaphorins and plexins as therapeutic health and inflammation. Nat. Rev. Immunol. 13: 159–175. targets. Nat. Rev. Drug Discov. 13: 603–621. 38. Manni, M. L., J. B. Trudeau, E. V. Scheller, S. Mandalapu, M. M. Elloso, 10. Mecollari, V., B. Nieuwenhuis, and J. Verhaagen. 2014. A perspective on the role J. K. Kolls, S. E. Wenzel, and J. F. Alcorn. 2014. The complex relationship of class III semaphorin signaling in central nervous system trauma. Front. Cell. between inflammation and lung function in severe asthma. Mucosal Immunol. 7: Neurosci. 8: 328. 1186–1198. 11. Mishra, R., D. Kumar, D. Tomar, G. Chakraborty, S. Kumar, and G. C. Kundu. 39. Hosoki, K., T. Itazawa, I. Boldogh, and S. Sur. 2016. Neutrophil recruitment by 2015. The potential of class 3 semaphorins as both targets and therapeutics in allergens contribute to allergic sensitization and allergic inflammation. Curr. cancer. Expert Opin. Ther. Targets 19: 427–442. Opin. Allergy Clin. Immunol. 16: 45–50. 12. Nasarre, P., R. M. Gemmill, and H. A. Drabkin. 2014. The emerging role of 40. Matsuzaki, H., Y. Mikami, K. Makita, H. Takeshima, M. Horie, S. Noguchi, class-3 semaphorins and their neuropilin receptors in oncology. Onco Targets T. Jo, O. Narumoto, T. Kohyama, H. Takizawa, et al. 2015. Interleukin-17A and Ther. 7: 1663–1687. toll-like receptor 3 ligand Poly(I:C) synergistically induced neutrophil chemo- 13. Kumanogoh, A., and H. Kikutani. 2013. Immunological functions of the neuro- attractant production by bronchial epithelial cells. PLoS One 10: e0141746. pilins and plexins as receptors for semaphorins. Nat. Rev. Immunol. 13: 802–814. 41. Headland, S. E., and L. V. Norling. 2015. The resolution of inflammation: 14. Movassagh, H., L. Shan, A. J. Halayko, M. Roth, M. Tamm, J. Chakir, and Principles and challenges. Semin. Immunol. 27: 149–160. A. S. Gounni. 2014. Neuronal chemorepellent Semaphorin 3E inhibits human 42. Walzer, T., L. Galibert, and T. De Smedt. 2005. Poxvirus semaphorin A39R inhibits airway smooth muscle cell proliferation and migration. J. Allergy Clin. Immunol. phagocytosis by dendritic cells and neutrophils. Eur. J. Immunol. 35: 391–398. 133: 560–567. 43. Granja, T., D. Ko¨hler, V. Mirakaj, E. Nelson, K. Ko¨nig, and P. Rosenberger. 15. Wanschel, A., T. Seibert, B. Hewing, B. Ramkhelawon, T. D. Ray, J. M. van Gils, 2014. Crucial role of Plexin C1 for pulmonary inflammation and survival during K. J. Rayner, J. E. Feig, E. R. O’Brien, E. A. Fisher, and K. J. Moore. 2013. lung injury. Mucosal Immunol. 7: 879–891. Neuroimmune guidance cue Semaphorin 3E is expressed in atherosclerotic plaques 44. Morote-Garcia, J. C., D. Napiwotzky, D. Ko¨hler, and P. Rosenberger. 2012. and regulates retention. Arterioscler. Thromb. Vasc. Biol. 33: 886–893. Endothelial Semaphorin 7A promotes neutrophil migration during hypoxia. 16. Shimizu, I., Y. Yoshida, J. Moriya, A. Nojima, A. Uemura, Y. Kobayashi, and Proc. Natl. Acad. Sci. USA 109: 14146–14151. T. Minamino. 2013. Semaphorin3E-induced inflammation contributes to insulin 45. Chauvet, S., S. Cohen, Y. Yoshida, L. Fekrane, J. Livet, O. Gayet, L. Segu, resistance in dietary obesity. Cell Metab. 18: 491–504. M. C. Buhot, T. M. Jessell, C. E. Henderson, and F. Mann. 2007. Gating of 17. Gay, C. M., T. Zygmunt, and J. Torres-Va´zquez. 2011. Diverse functions for the Sema3E/PlexinD1 signaling by neuropilin-1 switches axonal repulsion to at- semaphorin receptor PlexinD1 in development and disease. Dev. Biol. 349: 1–19. traction during brain development. Neuron 56: 807–822. 18. Saffar, A. S., M. P. Alphonse, L. Shan, K. T. Hayglass, F. E. Simons, and 46. Bellon, A., J. Luchino, K. Haigh, G. Rougon, J. Haigh, S. Chauvet, and F. Mann. A. S. Gounni. 2007. IgE modulates neutrophil survival in asthma: role of mi- 2010. VEGFR2 (KDR/Flk1) signaling mediates axon growth in response to tochondrial pathway. J. Immunol. 178: 2535–2541. semaphorin 3E in the developing brain. Neuron 66: 205–219. 19. Mahmood, S., S. Nandagopal, I. Sow, F. Lin, and S. K. Kung. 2014. 47. Nkyimbeng-Takwi, E., and S. P. Chapoval. 2011. Biology and function of Microfluidic-based, live-cell analysis allows assessment of NK-cell migration in neuroimmune semaphorins 4A and 4D. Immunol. Res. 50: 10–21. response to crosstalk with dendritic cells. Eur. J. Immunol. 44: 2737–2748. 48. Toyofuku, T., M. Yabuki, J. Kamei, M. Kamei, N. Makino, A. Kumanogoh, and 20. Lin, F., and E. C. Butcher. 2006. chemotaxis in a simple microfluidic M. Hori. 2007. Semaphorin-4A, an activator for T-cell-mediated immunity, device. Lab Chip 6: 1462–1469. suppresses via Plexin-D1. EMBO J. 26: 1373–1384. 21. Gu, C., Y. Yoshida, J. Livet, D. V. Reimert, F. Mann, J. Merte, C. E. Henderson, 49. Kumanogoh, A., T. Shikina, K. Suzuki, S. Uematsu, K. Yukawa, S. Kashiwamura, T. M. Jessell, A. L. Kolodkin, and D. D. Ginty. 2005. Semaphorin 3E and plexin- H. Tsutsui, M. Yamamoto, H. Takamatsu, E. P. Ko-Mitamura, et al. 2005. Non- D1 control vascular pattern independently of . Science 307: 265–268. redundant roles of Sema4A in the : defective T cell priming and 22. Hirota,J.A.,A.Budelsky,D.Smith,B.Lipsky,R.Ellis,Y.Y.Xiang,W.Y.Lu,and Th1/Th2 regulation in Sema4A-deficient mice. Immunity 22: 305–316. M. D. Inman. 2010. The role of interleukin-4Ralpha in the induction of glutamic 50. Nkyimbeng-Takwi, E. H., K. Shanks, E. Smith, A. Iyer, M. M. Lipsky, L. J. Detolla, acid decarboxylase in airway epithelium following acute house dust mite exposure. H. Kikutani, A. D. Keegan, and S. P. Chapoval. 2012. Neuroimmune semaphorin 4A Clin. Exp. Allergy 40: 820–830. downregulates the severity of allergic response. Mucosal Immunol. 5: 409–419. 23. Choi, Y. I., J. S. Duke-Cohan, W. B. Ahmed, M. A. Handley, F. Mann, J. A. Epstein, 51. Linde´n, A. 2001. Role of interleukin-17 and the neutrophil in asthma. Int. Arch. L. K. Clayton, and E. L. Reinherz. 2008. PlexinD1 glycoprotein controls migration Allergy Immunol. 126: 179–184. of positively selected thymocytes into the medulla. Immunity 29: 888–898. 52. Saffar, A. S., S. Dragon, P. Ezzati, L. Shan, and A. S. Gounni. 2008. Phospha- 24. Choi, Y. I., J. S. Duke-Cohan, W. Chen, B. Liu, J. Rossy, T. Tabarin, L. Ju, J. Gui, tidylinositol 3-kinase and p38 mitogen-activated protein kinase regulate induc- K. Gaus, C. Zhu, and E. L. Reinherz. 2014. Dynamic control of b1integrinad- tion of Mcl-1 and survival in glucocorticoid-treated human neutrophils. J. hesion by the plexinD1-sema3E axis. Proc. Natl. Acad. Sci. USA 111: 379–384. Allergy Clin. Immunol. 121: 492–498 e410. 25. Blomgran, R., and J. D. Ernst. 2011. Lung neutrophils facilitate activation of 53. Trevor, J. L., and J. S. Deshane. 2014. Refractory asthma: mechanisms, targets, naive antigen-specific CD4+ T cells during Mycobacterium tuberculosis infec- and therapy. Allergy 69: 817–827. tion. J. Immunol. 186: 7110–7119. 54. Gregory, L. G., and C. M. Lloyd. 2011. Orchestrating house dust mite-associated 26. Gounni, A. S., K. Spanel-Borowski, M. Palacios, C. Heusser, S. Moncada, and allergy in the lung. Trends Immunol. 32: 402–411. E. Lobos. 2001. Pulmonary inflammation induced by a recombinant Brugia 55. Phipps, S., C. E. Lam, G. E. Kaiko, S. Y. Foo, A. Collison, J. Mattes, J. Barry, malayi gamma-glutamyl transpeptidase homolog: involvement of humoral au- S. Davidson, K. Oreo, L. Smith, et al. 2009. Toll/IL-1 signaling is critical for toimmune responses. Mol. Med. 7: 344–354. house dust mite-specific helper T cell type 2 and type 17 [corrected] responses. 27. Sakurai, A., J. Gavard, Y. Annas-Linhares, J. R. Basile, P. Amornphimoltham, Am. J. Respir. Crit. Care Med. 179: 883–893. T. R. Palmby, H. Yagi, F. Zhang, P. A. Randazzo, X. Li, et al. 2010. Semaphorin 56. King, C., S. Brennan, P. J. Thompson, and G. A. Stewart. 1998. Dust mite 3E initiates antiangiogenic signaling through plexin D1 by regulating Arf6 and proteolytic allergens induce cytokine release from cultured airway epithelium. J. R-Ras. Mol. Cell. Biol. 30: 3086–3098. Immunol. 161: 3645–3651. 28. Suda, K., C. Guo, A. Oishi, S. Ikeda, A. Uemura, and N. Yoshimura. 2014. 57. Osterlund, C., H. Gronlund, N. Polovic, S. Sundstrom, G. Gafvelin, and Therapeutic potential of semaphorin 3E for the treatment of choroidal neo- A. Bucht. 2009. The non-proteolytic house dust mite allergen Der p 2 induce NF- vascularization. Invest. Ophthalmol. Vis. Sci. 55: 4700–4706. kappaB and MAPK dependent activation of bronchial epithelial cells. Clin. Exp. 29. Halilovic, I., J. Wu, M. Alexander, and F. Lin. 2015. Neutrophil migration under Allergy 39: 1199–1208. spatially-varying chemoattractant gradient profiles. Biomed. Microdevices 17: 9963. 58. Sackmann, E. K., E. Berthier, E. A. Schwantes, P. S. Fichtinger, M. D. Evans, 30. Wu, D., and F. Lin. 2011. Modeling cell gradient sensing and migration in L. L. Dziadzio, A. Huttenlocher, S. K. Mathur, and D. J. Beebe. 2014. Char- competing chemoattractant fields. PLoS One 6: e18805. acterizing asthma from a drop of blood using neutrophil chemotaxis. Proc. Natl. 31. Wu, J., C. Hillier, P. Komenda, R. Lobato de Faria, D. Levin, M. Zhang, and Acad. Sci. USA 111: 5813–5818. F. Lin. 2015. A microfluidic platform for evaluating neutrophil chemotaxis in- 59. Sackmann, E. K., A. L. Fulton, and D. J. Beebe. 2014. The present and future duced by sputum from COPD patients. PLoS One 10: e0126523. role of microfluidics in biomedical research. Nature 507: 181–189.