Semaphorin 7A Contributes to West Nile Virus Pathogenesis Through TGF- Β1/Smad6 Signaling

Semaphorin 7A Contributes to West Nile Virus Pathogenesis through TGF- β1/Smad6 Signaling

This information is current as Hameeda Sultana, Girish Neelakanta, Harald G. Foellmer, of September 24, 2021. Ruth R. Montgomery, John F. Anderson, Raymond A. Koski, Ruslan M. Medzhitov and Erol Fikrig J Immunol 2012; 189:3150-3158; Prepublished online 15 August 2012;

doi: 10.4049/jimmunol.1201140 Downloaded from http://www.jimmunol.org/content/189/6/3150

Supplementary http://www.jimmunol.org/content/suppl/2012/08/08/jimmunol.120114 Material 0.DC1 http://www.jimmunol.org/ References This article cites 49 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/189/6/3150.full#ref-list-1

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Semaphorin 7A Contributes to West Nile Virus Pathogenesis through TGF-b1/Smad6 Signaling

Hameeda Sultana,* Girish Neelakanta,* Harald G. Foellmer,† Ruth R. Montgomery,‡ John F. Anderson,x Raymond A. Koski,{ Ruslan M. Medzhitov,‖,# and Erol Fikrig*,#

Semaphorin 7A (Sema7A) is a membrane-associated/secreted protein that plays an essential role in connecting the vertebrate neuronal and immune systems. However, the role of Sema7A has not been elucidated in viral pathogenesis. In this study, we show that abrogation of Sema7A protects mice from lethal West Nile virus (WNV) infection. Mice lacking Sema7A showed increased survival, reduced viral burden, and less blood–brain barrier permeability upon WNV infection. Increased Sema7A levels were evident in murine tissues, as well as in murine cortical neurons and primary human macrophages upon WNV infection. Treatment with Sema7A Ab blocked WNV infection in both of these cell types. Furthermore, Sema7A positively regulates the production of TGF- b1 and Smad6 to facilitate WNV pathogenesis in mice. Collectively, these data elucidate the role of Sema7A in shared signaling Downloaded from pathways used by the immune and nervous systems during viral pathogenesis that may lead to the development of Sema7A- blocking therapies for WNV and possibly other ﬂaviviral infections. The Journal of Immunology, 2012, 189: 3150–3158.

est Nile virus (WNV) is a mosquito-borne ﬂavivirus tween these two disparate systems to provide novel insights into

that can infect humans and cause fever, encephalitis, WNV pathogenesis. http://www.jimmunol.org/ W and death (1, 2). Treatment of WNV is supportive, Semaphorins are a large family of phylogenetically conserved because vaccines or therapeutics have not been approved for use soluble and membrane-bound proteins that function in both the in humans. A murine model characterized the kinetics of WNV, nervous and immune systems (14–18). Semaphorins are divided a neurotrophic virus that replicates in peripheral organs, crosses into eight classes based on sequence similarities and distinct the blood–brain barrier (BBB), and infects the CNS, leading to structural features (19, 20). Class 1 and 2 semaphorins are found death (3–6). Studies using genetically modified mice that lack in invertebrates (14–18), class 3 to 7 semaphorins are found in specific immune molecules provided insight into the immunopa- vertebrates, and class 8 semaphorins are found in viruses (16, 19). thogenesis of WNV (7–13). However, studies that characterize The characteristic feature of all semaphorins is the presence of the molecules involved in cross-talk between the nervous and immune ∼500-aa Sema domain at the N terminus (16, 19). The Sema by guest on September 24, 2021 system upon WNV infection are lacking. Therefore, we undertook domain is implicated in stimulating various signaling pathways in the current study to understand the molecular relationships be- both nervous and immune systems (14–18). Numerous studies have elucidated that semaphorins act as axon-guidance molecules and are involved in functions ranging from axon pruning to synaptic formation, specificity, and plasticity (14, 19–22). Sem- *Section of Infectious Diseases, Department of Internal Medicine, Yale University aphorins are also implicated in the regulation of blood vessel School of Medicine, New Haven, CT 06520; †Section of Endocrinology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520; development, modulation of the immune system, regulation of ‡Section of Rheumatology, Department of Internal Medicine, Yale University School organogenesis, angiogenesis, apoptosis, and neoplasia (19, 20, of Medicine, New Haven, CT 06520; xDepartment of Entomology, Connecticut Ag- { 23–25). Abnormalities of semaphorins are associated with tumor ricultural Experiment Station, New Haven, CT 06504; L2 Diagnostics, New Haven, CT 06511; ‖Department of Immunobiology, Yale University School of Medicine, progression and neurologic diseases (19, 20). New Haven, CT 06520; and #Howard Hughes Medical Institute, Chevy Chase, MD Semaphorin 7A (Sema7A), also called CDw108, was identified 20815 in a search for vertebrate homologs of viral semaphorins (16, 26, Received for publication April 20, 2012. Accepted for publication July 19, 2012. 27). Sema7A is expressed broadly by lymphoid cells, myeloid This work was funded by National Institutes of Health Grants AI 50031 and AI cells, bone cells, the nervous system, epidermal keratinocytes, 070343. fibroblasts, and endothelial cells of blood vessels (25, 28, 29). In H.S., G.N., and E.F. conceived and designed the experiments; H.S., G.N., and H.G.F. performed the experiments; H.S., G.N., R.A.K., R.M.M., and E.F. analyzed the data; addition, human T lymphocytes and NK cells express Sema7A at H.S., G.N., R.M.M., J.F.A., and R.R.M. contributed reagents/materials/analysis tools; high levels (30). Sema7A is unique among the semaphorins for and H.S. and E.F. wrote the manuscript. two reasons: it is the only member of the family that is GPI linked, Address correspondence and reprint requests to Dr. Hameeda Sultana, Section of and it enhances axonal growth and proper axon track formation Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, S210, 300 Cedar Street, New Haven, CT 06520-8022. E-mail address: during embryonic development (19, 20, 23, 27). Mice deficient [email protected] in Sema7A show diminished axonal tracts, which can be restored The online version of this article contains supplemental material. by treatment with Sema7A to increase axonal growth (20, 31). Abbreviations used in this article: AHV, Alcelaphine herpes virus; BBB, blood–brain Sema7A can induce monocyte chemotaxis and cytokine produc- barrier; MOI, multiplicity of infection; p.i., postinfection; Q-PCR, quantitative RT- tion (25) and is a negative regulator of T cell responses (23). The PCR; Sema7A, semaphorin 7A; siRNA, small interfering RNA; WNV, West Nile effects of Sema7A in both the nervous and immune system are virus. believed to be mediated via at least two receptors: plexin C1 Freely available online through The Journal of Immunology Author Choice option. and b1 integrin (17, 20, 24). However, the significance of these Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 interactions in various tissues is poorly understood. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201140 The Journal of Immunology 3151

DNA viruses that infect humans, such as vaccinia and alcelaphine six-well plates (BD Falcon), and nonadherent cells were removed after 2 h. herpesvirus, encode secreted semaphorin A39R and Alcelaphine Adherent cells were cultured for 7 d and washed once with prewarmed herpes virus (AHV)-sema (16, 26, 27). These viral semaphorins fresh medium. Differentiated macrophages were treated with 2.5, 5, 10, or 20 mg Sema7A mAb or the isotype-matched negative control rat IgG Ab show high similarity to vertebrate Sema7A (16, 26, 27). Similarly for 2 h prior to WNV infection at an MOI of 1. Macrophages were then to Sema7A, these semaphorins bind plexin-like receptors (32). harvested in duplicates on days 1–3 for extraction of total RNA, followed Upon vaccinia and AHV infection, A39R and AHV-sema are be- by Q-PCR. lieved to play immunomodulatory role(s) by mimicking Sema7A Q-PCR analysis (16, 26, 27). Indeed, A39R induces CD54 expression and stim- b ulates production of IL-6 and IL-8 by cultured monocytes (16, To determine the viral burden and expression of Sema7A, TGF- 1, SMAD genes, and several cytokine mRNA levels, total RNA was extracted from 33). No study has reported the presence of Sema7A homologs in either frozen tissues or cell cultures using an RNeasy Mini kit (QIAGEN). RNA viral genomes. Therefore, we postulated that RNA viruses cDNA was synthesized using the iScript cDNA kit (Bio-Rad). Q-PCR was may use vertebrate Sema7A during infection and assessed the performed using previously published primers for WNV E, TNF-a, IL-6, role of vertebrate Sema7A using the murine model of WNV in- IL-12p40, IFN-a, IFN-b, TGF-b1, and the SMAD genes (7, 34, 35). The b fection. mRNA levels of these genes were normalized to -actin mRNA levels. The ratio of WNV E gene copy/b-actin gene copy was used as an index to determine the infection rate of each sample. Materials and Methods Murine infection and survival studies Western blotting All WNV-challenge experiments were performed with 6–8-wk-old female Total brain lysates or HEK 293 cells were resuspended in SDS sample buffer mice in a Biosafety level 3 animal facility, according to the regulations of containing 2-ME, boiled for 5 min, and resolved on a 4–15%-gradient SDS- Downloaded from Yale University. C57BL/6 wild-type mice were purchased from Charles PAGE gel. Blots were probed with specific Abs (Sema7A Ab used in Fig. River Laboratories, and Sema7A knockout mice (34) were a kind gift from 1D was obtained from Abcam [Cambridge, MA] and Abs against SMAD 2 the Dr. Jack Elias laboratory (Yale University) and were bred at the Yale or 6 used in Fig. 6G were obtained from Cell Signaling Technologies), Animal Resources Center, under pathogen-free conditions, to the C57BL/6 followed by the respective HRP-conjugated secondary Abs (obtained from background by backcrossing for 10 successive generations. For each in- Sigma, St. Louis, MO) and detected with an ECL system from GE fection study, mice groups were rigorously matched for age and sex. Healthcare. Quantification of Western blots was performed as described (37). 3 Groups of 10–20 mice were inoculated i.p. with a lethal dose of 10 PFU http://www.jimmunol.org/ wild-type WNV strain CT 2741 (provided by J.F.A.) in PBS with 1% Confocal microscopy gelatin. Upon WNV infection, mice typically died at 6–14 d postinfection Cortical neurons were isolated from the brains of embryonic day-16 C57BL/ (p.i.) as the result of CNS invasion by the virus. Mice were monitored daily 6 mouse embryos and infected with WNV (MOI 2). Twenty-four hours p.i., for WNV-associated clinical signs (including lethargy, anorexia, and dif- neurons were fixed with 4% paraformaldehyde and processed for immu- ficulty ambulating), as well as for survival until days 21–25 p.i., as de- nofluorescence using Sema7A Ab (Abcam), followed by treatment with scribed (35). Mice survival data summarize the results of two independent FITC-conjugated secondary Ab (Sigma). WNV was detected with anti- experiments. Surviving mice were either euthanized to end the experiment WNV Ab (Chemicon, Billerica, MA), followed by treatment with tetra- or, in selected experiments, mice were euthanized for harvesting of tissues methyl rhodamine isothiocyanate-conjugated secondary Ab (Sigma). Con- (brain, blood, and spleen). For Ab-protection studies, mice were inoculated focal microscopy was performed, as described (35). i.p. with 100 mg Sema7A mAb or the isotype-matched negative control rat by guest on September 24, 2021 IgG Ab (obtained from R&D Systems) 1 d prior to WNV challenge; ELISA a second dose of either of these Abs was given at 2 d p.i. All mice experiments were approved and performed in accordance with regulations ELISA was performed to detect Sema7A protein levels in the total lysates of the Institutional Animal Care and Use Committee at Yale University. prepared from uninfected or WNV-infected wild-type mice brain tissue. Ten micrograms (100 ml) of total lysates from uninfected or WNV- Infection of in vitro cell cultures infected wild-type mice brain tissue was coated onto ELISA plates (Nunc, Human HEK 293 and mouse cerebrospinal microvascular endothelial cell Rochester, NY) and incubated at 4˚C overnight. Following incubation, lines were purchased from the American Type Culture Collection and wells were blocked and incubated with Sema7A Ab diluted in PBS-Tween propagated according to their protocols. Primary cultures of cortical neu- 20 (0.05%) buffer. Wells were then incubated with secondary Ab conju- rons were isolated and established from the brains of embryonic day-16 gated to HRP, followed by treatment with TMB Microwell Peroxidase C57BL/6 or Sema7A-deficient mouse embryos (obtained from pregnant (KPL) for color development. The reactions were stopped after 15 min female mice). Primary cultures of neurons and other cell types were seeded using TMB Stop Solution (KPL), and OD was read at 450 nm. 5 5 on 6- or 12-well plates at cell densities of 2 3 10 or 10 cells/ml, re- Statistical analysis spectively; incubated for 24 h at 37˚C; and infected with WNV at multiplicity of infection (MOI) of 2. Cell lysates were collected for analysis Error bars represent mean (+ SD) values. We used a log-rank test (Prism 4; by quantitative RT-PCR (Q-PCR). For Sema7A Ab in vitro cell line- GraphPad Software) to assess the statistical differences between the sur- protection assays, 2.5, 5, 10, or 20 mg Sema7A mAb or the isotype- vival rates. For calculating statistical significance between two means, matchednegativecontrolratIgGAbwastreatedfor2hpriortoWNV nonpaired two-tailed Student t test was used. A p value , 0.05 was infection at an MOI of 1. Primary cultures of neurons or cerebrospinal considered statistically significant in our analyses. microvascular cells were harvested in duplicates on days 1–3 for extraction of total RNA and Q-PCR. For silencing studies, small interfering RNAs (siRNAs) for TGF-b1, Smad6, or control (scrambled) were obtained from Results Santa Cruz Biotechnologies. Human HEK 293 cells were transfected with Sema7A is upregulated upon WNV infection the respective siRNA, per the manufacturer’s instructions. Cells were infected Sema7A functions in the nervous and immune systems (14, 16–18). with WNV at an MOI of 2 and collected in duplicate from days 1–3 for total RNA extraction, followed by Q-PCR and Western blot analysis. Therefore, we analyzed its role during neurotrophic WNV infection. We first determined whether WNV influences sema7A ex- Human macrophage isolation and Sema7A Ab-protection pression in mice. RNA samples were extracted from different assays tissues of mice infected with WNV on days 2, 4, and 7 p.i. Q-PCR Heparinized blood from healthy human donors (Subjects A–D), with no showed that sema7A mRNA levels were significantly upregulated acute illness and not taking antibiotics or nonsteroidal anti-inflammatory at days 6 and 7 p.i. in blood (p , 0.05) and brain (p , 0.05) drugs, was obtained from the blood bank in New York City, as described compared with the uninfected controls (Fig. 1A–C, Supplemental (36). Human PBMCs were isolated using Ficoll-Hypaque (GE Healthcare) ∼ density-gradient centrifugation and suspended in RPMI 1640 medium Fig. 1A). Immunoblots further showed that Sema7A protein ( 75 containing 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 1000 mg/ kDa) levels in the brain were induced 2–3-fold at day 7 p.i. (Fig. ml streptomycin (Invitrogen). Cells were plated at 5 3 106 PBMCs/well in 1D), the time point at which WNV invades the CNS. Higher levels 3152 ROLE OF Sema7A IN WNV PATHOGENESIS

FIGURE 1. WNV induces Sema7A expression in mice. Q-PCR results showing expression levels of sema7A mRNA in blood (A), brain (B), and spleen (C) on days 2, 4, and 7 in uninfected and WNV-infected mice (n = 5 mice per group and per data point). Levels of sema7A mRNA were normalized to the levels of actin mRNA. Error bars represent + SD from the mean value. Statistical signiﬁcance was calculated using the Student t test. (D) Immunoblotting results showing expression of Sema7A in brain tissue lysates from uninfected and infected (day-7 p.i.) mice. Levels of total actin were used as loading control. Representative gel images from two independent experiments are shown. (E) ELISA to quantify total amount of Sema7A in uninfected and infected (day 7 p.i.) mice brain tissue Downloaded from lysates used in (D). Data from two independent experiments are shown in (A–C)and(E); Q-PCR was performed in triplicate. http://www.jimmunol.org/

of the glycosylated form of Sema7A protein (∼100 kDa) were also (∼5-fold, p , 0.05; Fig. 2C). Decreased viral loads were also evident in WNV-infected brain samples in comparison with con- evident in both spleen (2–3-fold, p , 0.05) and brains (2–3-fold, trols (Fig. 1D). ELISA conﬁrmed a signiﬁcant increase (p , 0.05, p , 0.05) of sema7A2/2 mice at day 4 p.i. compared with the 2–3-fold) in Sema7A protein expression in the brain at day 7 p.i. controls (Fig. 2D). At days 6 and 7 p.i., sema7A2/2 mice had

(Fig. 1E). Together, these data show that Sema7A mRNA and a significantly reduced (2–3-fold, p , 0.05) viral burden com- by guest on September 24, 2021 protein levels are induced upon WNV infection. pared with control mice in all three tissues (Fig. 2E, Supplemental Fig. 1B). These results suggest that the increased survival of Sema7A deficiency decreases murine lethality from WNV sema7A2/2 mice is due to both diminished viremia during early infection infection and decreased viral neuroinvasion. Because Sema7A expression was dramatically upregulated upon Sema7A-deficient mice infected with WNV have less WNV infection, we characterized its role in vivo in the absence of inflammation Sema7A by infecting either Sema7A-deficient (sema7A2/2)or Sema7A Ab-treated mice. When animals were infected with 1000 To identify a mechanism for reduced neuroinvasion of WNV in 2/2 PFU of WNV, survival rates of sema7A2/2 mice were consider- sema7A mice, we assessed BBB permeability by quantifying ably higher compared with the wild-type controls (Fig. 2A). IgG levels (indicative of BBB permeability) in the brain tissue. 2/2 sema7A2/2 mice were significantly more resistant to death caused Immunoblots showed that sema7A mice had less IgG in the by WNV than were wild-type animals (56 versus 20% survival brain compared with the control mice, indicating less BBB per- rate, p , 0.05, log-rank test) (Fig. 2A). Similarly, Sema7A Ab- meability (Fig. 3A, Supplemental Table I). Increased production treated mice showed substantial resistance to the lethality caused of inflammatory and antiviral cytokines, such as TNF-a, IL-6, IL- by WNV compared with IgG isotype-matched negative control 12, IFN-a, and IFN-b, facilitate WNV neuroinvasion into the Ab-treated mice (60 versus 20% survival rate, p , 0.05) (Fig. 2B). CNS by altering the BBB permeability (3, 7). Therefore, we 2/2 Collectively, these results show that Sema7A is an important measured mRNA levels of these cytokines in sema7A mice factor in murine resistance to WNV infection. compared with the controls. Consistent with the measurement of viral loads (Fig. 2E) and BBB permeability (Fig. 3A), mRNA Sema7A influences WNV neuroinvasion levels of TNF-a, IL-6, IL-12, IFN-a, and IFN-b cytokines were During the initial stage of murine infection (days 2–4 p.i.), WNV significantly decreased in brain tissue of sema7A2/2 mice at day 7 replicates in peripheral organs (3–6). By day 5 p.i., WNV crosses p.i. compared with the control mice (3–8-fold, p , 0.05, Fig. 3B– the BBB and invades the CNS, leading to death of the animals F). In addition, blood from sema7A2/2 mice showed reduced starting at day 7 p.i (3–6, 38). Because Sema7A deficiency mRNA levels of IFN-a (9-fold, p , 0.05) and IFN-b (3-fold, p , resulted in enhanced survival after WNV infection in mice, we 0.05) compared with controls at day 7 p.i (Fig. 3E, 3F), sug- analyzed the kinetics of WNV infection to determine whether this gesting an important role for Sema7A in facilitating the WNV was due to reduced viral invasion into the brain. Indeed, when we infection from blood to brain. Furthermore, at day 7 p.i., TNF-a quantified WNV E-gene transcripts normalized to murine b-actin and IL-6 levels were significantly diminished (4- and 2-fold, 2 2 by Q-PCR, the sema7A / mice had a significantly reduced viral respectively, p , 0.05) in spleens of sema7A2/2mice (Fig. 3B, 3C). burden in the blood at day 2 p.i. compared with the control mice Overall, these results show that Sema7A contributes to cytokine The Journal of Immunology 3153

FIGURE 2. Abrogation of Sema7A renders resistance to WNV-induced lethality in mice. (A) Survival of wild-type and sema7A2/2 mice challenged i.p. with 103 PFU of wild-type WNV. Survival was recorded daily until day 21 p.i.; data were pooled from two independent experiments and represent a total of 25 animals/ group. (B) Survival of C57BL/6 wild-type mice inoculated with WNV and treated with anti- Sema7A mAb or isotype-matched negative control Ab on days 21 and +2. Data are pooled from two independent experiments and represent a total of 17 animals/group. Statistical Downloaded from significance (p , 0.05) was calculated using Prism 4 software. WNV is less neuroinvasive in sema7A2/2 mice than in wild-type mice. Viral loads in selected tissues (blood, spleen, and brain) of wild-type and sema7A2/2 mice inoculated i.p. with 103 PFU of WNV. Each group contained fie animals examined at day 2 http://www.jimmunol.org/ (C), day 4 (D), or day 7 (E) after viral challenge. Data are representative of results obtained in two independent experiments, performed in triplicate. Horizontal bars represent mean of the values. Statistical significance was calculated using the Student t test. UD, Undetectable. by guest on September 24, 2021

responses that are associated with inflammation during WNV isolated from wild-type mice were treated with either Sema7A or infection. isotype-matched negative control Abs for 2 h, followed by infection with WNV. Viral replication was analyzed 48 h p.i. Q-PCR Abrogation of Sema7A blocks WNV infection of cortical showed that neurons treated with 5–20 mg of Sema7A mAb neurons in vitro showed significantly (p , 0.05) reduced WNV E-gene transcripts To assess the role of sema7A in viral infection of neural tissue, we compared with the control (Fig. 4C). Similar results were obtained assessed viral replication in cortical neurons, which is a pathogenic when we performed experiments with a mouse cerebral micro- feature of WNV during CNS invasion (3–6, 39). We analyzed vascular endothelial in vitro cell line (Supplemental Fig. 1D). whether abrogation of Sema7A leads to altered viral replication by These results support a role for Sema7A in neuroinvasion and determining the sema7A mRNA levels in cortical neurons infected replication of WNV in the CNS. with WNV (35). Q-PCR results showed that sema7A mRNA levels were significantly elevated in WNV-infected cortical neurons Abrogation of Sema7A blocks WNV infection of human compared with uninfected controls at all time points (24, 48, and macrophages in vitro 72 h) p.i. (2–5-fold, p , 0.05, Fig. 4A). Coassociation of Sema7A In addition to the role for Sema7A in enhancing WNV replication with WNV was also evident in cultured cortical neurons (Sup- in neurons, we analyzed whether it is involved in immune-mediated plemental Fig. 1C). We then determined whether the absence of pathways. Therefore, we investigated whether abrogation of Sema7A has any impact on viral infection in cortical neurons. Sema7A in human macrophages also influences WNV replication. Cortical neurons from wild-type and sema7A2/2 mice were iso- PBMCs were isolated from healthy human volunteers and allowed lated, and WNV infection kinetics were analyzed at different time to differentiate into macrophages (36). We first analyzed sema7A points (24, 48, and 72 h) p.i. (Fig. 4B). The results showed a sig- mRNA levels in macrophages infected with WNV and compared nificantly reduced viral burden in cortical neurons isolated from the expression levels with uninfected controls. We found that sema7A2/2 mice compared with cortical neurons isolated from sema7A mRNA levels were significantly elevated in WNV- wild-type mice at all time points (p , 0.05, Fig. 4B). We next infected human macrophages isolated from all four volunteers determined whether treatment of cortical neurons with Sema7A compared with uninfected controls at all time points (24, 48, and Ab leads to reduced viral replication. Uninfected cortical neurons 72 h) p.i. (3–8-fold, p , 0.05, Fig. 5A, Supplemental Fig. 2A–C). 3154 ROLE OF Sema7A IN WNV PATHOGENESIS

FIGURE 3. sema7A2/2 mice have reduced cytokine responses that are associated with inflammation during WNV infection. (A) Immunoblot of IgG (H chain) in WNV- infected wild-type or sema7A2/2 mice brain tissue lysates. Actin served as the loading control. Representative gel images from two independent experiments are shown. TNF-a (B)), IL-6 (C), IL-12 (D), IFN-a (E), and IFN-b (F) mRNA levels in the blood, spleen, and brain of WNV-infected wild- type or sema7A2/2 mice at day 7 p.i. Both Downloaded from groups of mice were infected with 103 PFU of WNV. Results are from five mice/group and two independent experiments, performed in triplicate. Statistical significance (p , 0.05) was calculated using the Student t test. Error bars represent + SD from the http://www.jimmunol.org/ mean value. by guest on September 24, 2021

We determined whether treatment of human macrophages with increase in plexin C1 mRNA expression upon WNV infection Sema7A Ab has any influence on viral replication. Uninfected (Supplemental Fig. 3A) and found no differences in plexin C1 human macrophages were treated with either Sema7A mAb or levels from sema7A2/2 mice (Supplemental Fig. 3B). A recent isotype-matched negative control Ab for 2 h, followed by infec- study showed that Sema7A plays a critical role in TGF-b1–in- tion with WNV. The Q-PCR results showed that macrophages duced pulmonary fibrosis (34). To gain further insight into the from all four volunteers treated with 10–20 mg of Sema7A Ab involvement of Sema7A in WNV pathogenesis, we tested the showed significantly reduced WNV E-gene transcripts compared hypothesis that Sema7A uses TGF-b1 signaling to mediate WNV with the controls at 48 h p.i. (p , 0.05, Fig. 5B, Supplemental Fig. pathogenesis. When we analyzed tgf-beta1 mRNA levels in cor- 2D–F). We next determined whether overexpression of Sema7A tical neurons p.i. with WNV, tgf-beta1 mRNA levels were sig- influences WNV replication in an in vitro human cell line. We nificantly elevated in WNV-infected cortical neurons compared used HEK 293 cells because of their extreme transfection effi- with uninfected controls at all time points (24, 48, and 72 h) p.i. ciency. Constructs carrying Sema7A-Fc and Fc alone, which was (Fig. 6A). We then determined whether the absence of Sema7A used previously (23), were first transfected into HEK 293 cells and has any impact on tgf-beta1 mRNA levels upon WNV infection in infected with WNV. At 24 h post-WNV infection, HEK 293 cells cortical neurons. The results showed significantly reduced levels transfected with Sema7A-Fc showed significantly increased WNV of tgf-beta1 mRNA in cortical neurons isolated from sema7A2/2 E-gene transcripts compared with cells transfected with Fc control mice compared with controls (Fig. 6B). Because these results (2–3-fold, p , 0.05, Fig. 5C). Taken together, these results further suggested involvement of TGF-b1 in Sema7A-mediated WNV validate the role of Sema7A as a host factor that enhances viral pathogenesis, we analyzed the kinetics of WNV infection upon replication and supports the development of Sema7A as a thera- silencing of tgf-beta1 expression using HEK 293 cells. The ex- peutic target for WNV infection. pression of tgf-beta1 mRNA was also induced upon WNV in- fectioninHEK293cells(Supplemental Fig. 3C) and was b Sema7A facilitates WNV pathogenesis through TGF- 1/Smad6 significantly reduced in tgf-beta1 siRNA-transfected cells com- signaling pared with control siRNA (scrambled)-transfected cells at differ- To dissect the mechanisms underlying Sema7A’s enhancement ent time points (Fig. 6C). In addition, tgf-beta1–silenced cells of viral replication, we assessed signaling pathways involved in showed a significant reduction in viral burden that was also evi- Sema7A activity. Because Sema7A signaling in both the nervous dent at all time points (24, 48, and 72 h) p.i. in tgf-beta1–silenced and immune systems is believed to be mediated via plexin C1 cells compared with the control siRNA-transfected cells (Fig. 6D, (32), we first analyzed whether infection of WNV induces plexin Supplemental Fig. 3D). These studies highlight a previously un- C1 mRNA expression in wild-type mice. We found no significant recognized role for TGF-b1 in enhancing WNV infection. The Journal of Immunology 3155 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. Abrogation of Sema7A blocks WNV infection of cortical FIGURE 5. Abrogation of Sema7A blocks WNV infection of human neurons in vitro. (A) Q-PCR analysis showing expression of sema7A macrophages. (A) Q-PCR analysis showing expression of sema7A mRNA mRNA levels at 24, 48, and 72 h p.i. with WNV in cortical neurons iso- levels at 24, 48, and 72 h p.i. with WNV in macrophages isolated from lated from wild-type mice. Uninfected cortical neurons were used as a representative healthy human volunteer (Subject A). Uninfected mac- controls. Levels of sema7A mRNA were normalized to the levels of actin rophages were used as controls. Levels of sema7A mRNA were normalized mRNA. (B) Kinetics of WNV infection in cortical neuronal cells isolated to the levels of actin mRNA. (B) Q-PCR analysis showing WNV burden in from wild-type or sema7A2/2 mice at 24, 48, and 72 h p.i. (C) Q-PCR Sema7A mAb-treated or isotype-matched negative control Ab-treated analysis showing WNV burden in Sema7A mAb-treated or isotype- human macrophages from Subject A at 48 h p.i. (C) Q-PCR analysis matched negative control Ab-treated cortical neurons at 48 p.i. All results showing viral burden in Fc or Fc-Sema7A–transfected HEK 293 cells. are from three independent experiments, performed in triplicate. Error bars Infected, but untransfected, cells were used as controls. Data are repre- represent + SD from the mean value. Statistical signiﬁcance was calculated sentative of results performed in triplicates. Error bars represent + SD from using the Student t test. the mean value. Statistical signiﬁcance was calculated using the Student t test.

TGF-b1 signals through Smad transcription factors that mediate various cellular events (40, 41). Therefore, we determined mRNA siRNA-transfected cells (Fig. 6F). In addition, we found that HEK levels of several smads upon WNV infection in cortical neurons. 293 cells transfected with tgf-beta1 siRNA showed reduced Smad6 Only smad6 was significantly elevated upon WNV infection in protein levels upon WNV infection compared with the uninfected cortical neurons isolated from wild-type animals (Fig. 6E). We controls at both 48 and 72 h p.i. (Fig. 6G, Supplemental Table I). found no Sema7A-dependent differences in the expression of other To examine whether Smad6 controls viral replication, we analyzed smads upon WNV infection (Supplemental Fig. 3E). To test WNV infection upon silencing of smad6 expression in HEK 293 whether Sema7A mediates signaling through smad6 during WNV cells. Q-PCR analysis showed a significant reduction in smad6 infection, we analyzed smad6 expression in cortical neurons from mRNA levels in smad6 siRNA-transfected cells (Fig. 6H). A sig- sema7A2/2 mice. Results showed that, in the absence of Sema7A, nificant reduction in viral burden was also evident at tested time no induction of smad6 was observed in sema7A2/2 cortical neu- points (48 and 72 h p.i.) in smad6-silenced cells compared with the rons infected with WNV at 48 h p.i. (Fig. 6E). Reduced levels of control siRNA-transfected cells (Fig. 6I). Taken together, these smad6 mRNA were also evident in HEK 293 cells transfected with results suggest that Sema7A contributes to WNV pathogenesis tgf-beta1 siRNA at 72 h p.i. with WNV compared with the control through the TGF-b1/Smad6–signaling pathway. 3156 ROLE OF Sema7A IN WNV PATHOGENESIS Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 6. Sema7A facilitates WNV pathogenesis through TGF-b1/Smad6 signaling. (A) Q-PCR results showing expression of tgf-beta1 mRNA levels at 24, 48, and 72 h p.i. with WNV in cortical neurons isolated from wild-type mice. Levels of tgf-beta1 mRNA were normalized to the levels of actin mRNA. (B) Expression of tgf-beta1 mRNA levels at 24, 48, and 72 h p.i. with WNV in cortical neurons isolated from wild-type or sema7A2/2 mice. (C) Expression of tgf-beta1 mRNA at 24, 48, and 72 h p.i. with WNV in HEK 293 cells transfected with tgf-beta1 or control siRNA. (D) Kinetics of WNV infection in HEK 293 cells transfected with tgf-beta1 or control siRNA at 24, 48, and 72 h p.i. (E) Q-PCR results showing smad6 mRNA levels in cortical neurons isolated from wild-type or sema7A2/2 mice at 48 h p.i. with WNV. (F) Q-PCR results showing expression of smad6 mRNA levels at 72 h p.i. with WNV in HEK 293 cells transfected with tgf-beta1 or control siRNA. (G) Immunoblot showing levels of Smad6 and Smad2 in HEK 293 cells transfected with tgf-beta1 siRNA at 48 and 72 h p.i. with WNV. Actin levels served as loading control. Representative gel images from two independent experiments are shown. Q-PCR results showing expression of smad6 mRNA (H) or the kinetics of WNV infection (I) at 48 and 72 h p.i. in HEK 293 cells transfected with smad6 or control siRNA. All data are representative of results obtained from two independent experiments, performed in triplicate. Error bars represent + SD from the mean value. Statistical signiﬁcance was calculated using the Student t test.

Discussion Sema7A upon WNV infection in blood and brain tissues suggests Viruses, such as vaccinia and alcelaphine herpesvirus, encode its role both in the peripheral organs and CNS. Second, mice that semaphorin A39R and AHV-sema, which achieve immunomodu- lack Sema7A had a reduced viral burden in the periphery during latory functions by mimicking vertebrate Sema7A (16, 26, 27, 33). early infection and reduced levels in the brain during late infec- The role of vertebrate Sema7A in viral pathogenesis has not been tion, suggesting a role for Sema7A both in the early and late directly addressed. In this article, we describe a crucial role for stages of WNV pathogenesis. Third, the level of TNF-a, which is Sema7A in WNV pathogenesis. Our finding that abrogation of important for WNV crossing the BBB, was significantly reduced Sema7A provides resistance to WNV infection in mice is sup- in mice that lack Sema7A. Fourth, in the absence of Sema7A, IgG ported by several lines of evidence. First, the upregulation of leakage into the brain was reduced upon WNV infection, impli- The Journal of Immunology 3157 cating a role for Sema7A in protecting BBB permeability. Last, in the nucleus (40, 41, 46). However, we did not observe any changes the absence of Sema7A, viral replication in cortical neurons was in the mRNA expression of either of these Smads upon WNV significantly reduced, suggesting a role for Sema7A in brain cells. infection in murine cortical neurons (Supplemental Fig. 3E) or in Sema7A is a potent stimulator of monocytes and neutrophils WNV-infected mice lacking Sema7A (data not shown). There is (25). Studies demonstrated that concentrations as low as 1 pM of increasing evidence for Smad2/3-independent pathways that me- Sema7A can stimulate monocyte cytokine production and che- diate the effects of TGF-b1 (34, 40, 41, 46). Overexpression motaxis. Indeed, the addition of Sema7A influenced monocyte studies in mammalian cells elucidated that Smad6 associates with chemotaxis 1000 times more efficiently than did MCP-1, and it TGF-b1 type I receptor and inhibits Smad2 and Smad1 phos- influenced neutrophil chemotaxis 100 times better than did IL-8 phorylation (46). This regulatory function of Smad6 may allow stimulation (25). Sema7A is abundantly expressed in monocytes, more precise regulation of TGF-b1–mediated Smad signaling neutrophils, and other lymphoid cells (25, 28, 29). A recent study (46). Based on our findings that WNV induces Smad6 expression showed a biphasic response of PMNs to WNV infection; they and that the absence of Sema7A or TGF-b1 affected this induc- serve as a reservoir for WNV replication and dissemination in the tion, we hypothesize that Sema7A functions via TGF-b1/Smad6 early stages of infection and later contribute to WNV clearance signaling to contribute to WNV pathogenesis. The reduced WNV (42). The reduced viremia in the early stage of WNV infection in infection in Smad6-silenced cells further supports this hypothesis. Sema7A-deficient mice (Fig. 2) suggests that Sema7A may par- Collectively, we propose that Sema7A deficiency may affect the ticipate in the replication of WNV at early stages in these cells and ability of the TGF-b1/Smad6 pathway to influence cytokine pro- at later stages in the dissemination of WNV to the peripheral duction, antiproteases, transcription factors, and receptor compo- tissues and brain. It is possible that the absence of Sema7A results nents that contribute to WNV pathogenesis. Downloaded from in slower recruitment of neutrophils to the site of WNV infection, Several GPI-anchored proteins perform diverse roles in immu- which may affect PMN-dependent viral replication and dissemi- noregulation (47). GPI-anchored proteins occur in microdomains nation. Peripheral blood monocytes are relatively immature pre- or “rafts” on the cell surface that can also be cleaved off from the cursor cells that can differentiate into macrophage or dendritic cell surface by proteolytic cleavage, resulting in a secreted form of cells based on environmental stimuli (43). Sema7A induces the the proteins (48). Sema7A is a GPI-anchored protein that is re-

maturation of monocytes toward dendritic cell morphology (43). leased from the cell surface by proteolysis, suggesting its role in http://www.jimmunol.org/ Studies show that TLR-3 binding to double-stranded viral RNA autocrine function (30, 49). Sema7A functions both as a cell inside infected dendritic cells leads to the production of inflam- surface and secreted molecule. Also, lipid rafts have been impli- matory cytokines, such as TNF-a (7). Our findings that Sema7A- cated in flavivirus entry into cells by the activation of PI3K/Akt deficient mice infected with WNV have lower production of signaling during the early stages of Japanese encephalitis virus TNF-a and less BBB permeability (Fig. 3) support our hypothesis infection in neural stem/progenitor cells (50). Sema7A plays a that, in the absence of Sema7A, there might be one possible an- central role in a PI3K/PKB/AKT-dependent pathway that con- tiviral mechanism that reduces maturation of monocytes to den- tributes to TGF-b1–induced fibrosis and modeling (34, 50). It is dritic cells. The reduction in dendritic cell maturation may lead possible that initiation and propagation of the signaling events to decreased TLR-3–mediated recognition of viral RNA and pro- taking place during WNV pathogenesis occur in these specialized by guest on September 24, 2021 duction of TNF-a that subsequently leads to less WNV crossing the Sema7A-containing membrane regions. We postulate that, in its BBB. We propose that Sema7A acts upstream of the host inflam- GPI-anchored form, Sema7A may facilitate viral entry on the cell matory reaction that results from WNV invasion. surface (when it is linked to lipid rafts) either by directly binding TGF-b1 is synthesized as precursor that is complexed with la- to the virus or indirectly by forming a complex with other cell tent TGF-b–binding proteins and is activated by a variety of surface receptors. The fact that WNV infection in vitro is inhibited mechanisms that include integrin binding (41, 44). Activated TGF- by the blockage of Sema7A further supports that it may be a re- b1 binds to heterodimeric receptors containing types I and II receptor or coreceptor for WNV. In its secreted form, Sema7A may ceptor components that have tyrosine kinase activity (41, 44). Our trigger downstream signaling in modulating the expression of studies demonstrate that mice lacking Sema7A exhibited a signifi- TGF-b1/Smad6 and other inflammatory cytokines that contribute cant reduction in TGF-b1 mRNA levels. The reduced TGF-b1 to WNV pathogenesis. With any of these models, the finding that mRNA levels correlated with reduced viral infection in cortical abrogation of Sema7A results in increased resistance to WNV neurons isolated from Sema7A-deficient animals (Fig. 6). Kang infection supports the role of Sema7A as a potential therapeutic et al. (34) showed that TGF-b1 induces the expression of Sema7A target for disease caused by WNV. in a pulmonary fibrosis model. A more conservative interpretation Collectively, these data provide direct evidence for the in- of the possible mechanisms is warranted, because our study pro- volvement of Sema7A in WNV pathogenesis. Our results em- poses that Sema7A contributes to WNV infection through TGF-b1/ phasize molecular mechanisms that WNV uses to establish and Smad–signaling pathways. It is possible that Sema7A induces invade the brain, leading to the cause of lethal encephalitis. Because TGF-b1, which, in turn, may induce Sema7A expression during treatment with Sema7A Ab is efficacious in mice and provides WNV infection. A feedback mechanism was identified for sem- protection in microvascular endothelial cells, murine cortical aphorin 3E signaling (45). We found that sema7A and tgf-b1 neurons, and human macrophages, our study opens up a promising mRNA levels are coordinately induced in mouse cortical neurons area for the development of Sema7A-blocking therapies for WNV during WNV infection (Figs. 4A, 6A). Although, we found that and possibly other flaviviral infections. Sema7A deficiency abrogates tgf-b1 mRNA levels, we did not b observe any changes in sema7A mRNA expression upon TGF- 1 Acknowledgments silencing during WNV infection (data not shown). This role of We thank Drs. Jack Elias and Bing Ma for providing sema7A2/2 mice. Sema7A in TGF-b1 signaling and in virus infection suggests We also thank Deborah Beck and Lida Yuan for technical assistance. that these are two mechanisms that can both contribute to WNV pathogenesis. TGF-b1 signals via Smads, where Smad2 and Smad3 are phosphorylated by TGF-bR1 kinase. The phosphory- Disclosures lated Smad2/3 complex binds to Smad4 and translocates Smad4 to The authors have no financial conflicts of interest. 3158 ROLE OF Sema7A IN WNV PATHOGENESIS

References poxvirus-encoded semaphorin induces cytokine production from monocytes and binds to a novel cellular semaphorin receptor, VESPR. Immunity 8: 473–482. 1. Klee, A. L., B. Maidin, B. Edwin, I. Poshni, F. Mostashari, A. Fine, M. Layton, 27. Xu, X., S. Ng, Z. L. Wu, D. Nguyen, S. Homburger, C. Seidel-Dugan, A. Ebens, and D. Nash. 2004. Long-term prognosis for clinical West Nile virus infection. and Y. Luo. 1998. Human semaphorin K1 is glycosylphosphatidylinositol-linked Emerg. Infect. Dis. 10: 1405–1411. and defines a new subfamily of viral-related semaphorins. J. Biol. Chem. 273: 2. Petersen, L. R., and A. A. Marfin. 2002. West Nile virus: a primer for the cli- 22428–22434. nician. Ann. Intern. Med. 137: 173–179. 28. Sato, Y., and H. Takahashi. 1998. Molecular cloning and expression of murine 3. Diamond, M. S., and R. S. Klein. 2004. West Nile virus: crossing the blood-brain homologue of semaphorin K1 gene. Biochim. Biophys. Acta 1443: 419–422. barrier. Nat. Med. 10: 1294–1295. 29. Mine, T., K. Harada, T. Matsumoto, H. Yamana, K. Shirouzu, K. Itoh, and 4. Lustig, S., U. Olshevsky, D. Ben-Nathan, B. E. Lachmi, M. Malkinson, A. Yamada. 2000. CDw108 expression during T-cell development. Tissue D. Kobiler, and M. Halevy. 2000. A live attenuated West Nile virus strain as Antigens 55: 429–436. a potential veterinary vaccine. Viral Immunol. 13: 401–410. 30. Angelisova´, P., K. Drbal, J. Cerny, I. Hilgert, and V. Horejsı´. 1999. Character- 5. Samuel, M. A., and M. S. Diamond. 2006. Pathogenesis of West Nile Virus ization of the human leukocyte GPI-anchored glycoprotein CDw108 and its infection: a balance between virulence, innate and adaptive immunity, and viral relation to other similar molecules. Immunobiology 200: 234–245. evasion. J. Virol. 80: 9349–9360. 31. Pendergast, A. M. 2002. The Abl family kinases: mechanisms of regulation and 6. Shrestha, B., D. Gottlieb, and M. S. Diamond. 2003. Infection and injury of signaling. Adv. Cancer Res. 85: 51–100. neurons by West Nile encephalitis virus. J. Virol. 77: 13203–13213. 32. Tamagnone, L., S. Artigiani, H. Chen, Z. He, G. I. Ming, H. Song, A. Chedotal, 7. Wang, T., T. Town, L. Alexopoulou, J. F. Anderson, E. Fikrig, and R. A. Flavell. M. L. Winberg, C. S. Goodman, M. Poo, et al. 1999. Plexins are a large family of 2004. Toll-like receptor 3 mediates West Nile virus entry into the brain causing receptors for transmembrane, secreted, and GPI-anchored semaphorins in ver- lethal encephalitis. Nat. Med. 10: 1366–1373. Cell 8. Arjona, A., H. G. Foellmer, T. Town, L. Leng, C. McDonald, T. Wang, tebrates. 99: 71–80. ´ S. J. Wong, R. R. Montgomery, E. Fikrig, and R. Bucala. 2007. Abrogation of 33. Smith, G. L., J. A. Symons, A. Khanna, A. Vanderplasschen, and A. Alcamı. macrophage migration inhibitory factor decreases West Nile virus lethality by 1997. Vaccinia virus immune evasion. Immunol. Rev. 159: 137–154. limiting viral neuroinvasion. J. Clin. Invest. 117: 3059–3066. 34. Kang, H. R., C. G. Lee, R. J. Homer, and J. A. Elias. 2007. Semaphorin 7A plays 9. Wang, P., J. Dai, F. Bai, K. F. Kong, S. J. Wong, R. R. Montgomery, J. A. Madri, a critical role in TGF-beta1-induced pulmonary fibrosis. J. Exp. Med. 204: 1083– 1093. and E. Fikrig. 2008. Matrix metalloproteinase 9 facilitates West Nile virus entry Downloaded from into the brain. J. Virol. 82: 8978–8985. 35. Sultana, H., H. G. Foellmer, G. Neelakanta, T. Oliphant, M. Engle, M. Ledizet, 10. Brinton, M. A. 2001. Host factors involved in West Nile virus replication. Ann. M. N. Krishnan, N. Bonafe´, K. G. Anthony, W. A. Marasco, et al. 2009. Fusion N. Y. Acad. Sci. 951: 207–219. loop peptide of the West Nile virus envelope protein is essential for pathogenesis 11. Mashimo, T., M. Lucas, D. Simon-Chazottes, M. P. Frenkiel, X. Montagutelli, and is recognized by a therapeutic cross-reactive human monoclonal antibody. J. P. E. Ceccaldi, V. Deubel, J. L. Guenet, and P. Despres. 2002. A nonsense Immunol. 183: 650–660. mutation in the gene encoding 29-59-oligoadenylate synthetase/L1 isoform is 36. Qian, F., X. Wang, L. Zhang, A. Lin, H. Zhao, E. Fikrig, and R. R. Montgomery. associated with West Nile virus susceptibility in laboratory mice. Proc. Natl. 2011. Impaired interferon signaling in dendritic cells from older donors infected Acad. Sci. USA 99: 11311–11316. in vitro with West Nile virus. J. Infect. Dis. 203: 1415–1424. 12. Dai, J., P. Wang, F. Bai, T. Town, and E. Fikrig. 2008. Icam-1 participates in the 37. Sultana, H., G. Neelakanta, F. S. Kantor, S. E. Malawista, D. Fish, http://www.jimmunol.org/ entry of west nile virus into the central nervous system. J. Virol. 82: 4164–4168. R. R. Montgomery, and E. Fikrig. 2010. Anaplasma phagocytophilum induces 13. Bai, F., T. Town, F. Qian, P. Wang, M. Kamanaka, T. M. Connolly, D. Gate, actin phosphorylation to selectively regulate gene transcription in Ixodes scap- R. R. Montgomery, R. A. Flavell, and E. Fikrig. 2009. IL-10 signaling blockade ularis ticks. J. Exp. Med. 207: 1727–1743. controls murine West Nile virus infection. PLoS Pathog. 5: e1000610. 38. Town, T., F. Bai, T. Wang, A. T. Kaplan, F. Qian, R. R. Montgomery, 14. Kolodkin, A. L., D. J. Matthes, T. P. O’Connor, N. H. Patel, A. Admon, D. Bentley, J. F. Anderson, R. A. Flavell, and E. Fikrig. 2009. Toll-like receptor 7 mitigates and C. S. Goodman. 1992. Fasciclin IV: sequence, expression, and function during lethal West Nile encephalitis via interleukin 23-dependent immune cell infil- growth cone guidance in the grasshopper embryo. Neuron 9: 831–845. tration and homing. Immunity 30: 242–253. 15. Hall, K. T., L. Boumsell, J. L. Schultze, V. A. Boussiotis, D. M. Dorfman, 39. Samuel, M. A., H. Wang, V. Siddharthan, J. D. Morrey, and M. S. Diamond. A. A. Cardoso, A. Bensussan, L. M. Nadler, and G. J. Freeman. 1996. Human 2007. Axonal transport mediates West Nile virus entry into the central nervous CD100, a novel leukocyte semaphorin that promotes B-cell aggregation and system and induces acute flaccid paralysis. Proc. Natl. Acad. Sci. USA 104: differentiation. Proc. Natl. Acad. Sci. USA 93: 11780–11785. 17140–17145. 16. Lange, C., T. Liehr, M. Goen, E. Gebhart, B. Fleckenstein, and A. Ensser. 1998. 40. Moustakas, A., S. Souchelnytskyi, and C. H. Heldin. 2001. Smad regulation in by guest on September 24, 2021 New eukaryotic semaphorins with close homology to semaphorins of DNA TGF-beta signal transduction. J. Cell Sci. 114: 4359–4369. viruses. Genomics 51: 340–350. 41. Leask, A., and D. J. Abraham. 2004. TGF-beta signaling and the fibrotic re- 17. Suzuki, K., T. Okuno, M. Yamamoto, R. J. Pasterkamp, N. Takegahara, sponse. FASEB J. 18: 816–827. H. Takamatsu, T. Kitao, J. Takagi, P. D. Rennert, A. L. Kolodkin, et al. 2007. 42. Bai, F., K. F. Kong, J. Dai, F. Qian, L. Zhang, C. R. Brown, E. Fikrig, and Semaphorin 7A initiates T-cell-mediated inflammatory responses through R. R. Montgomery. 2010. A paradoxical role for neutrophils in the pathogenesis alpha1beta1 integrin. Nature 446: 680–684. of West Nile virus. J. Infect. Dis. 202: 1804–1812. 18. Spriggs, M. K. 1999. Shared resources between the neural and immune systems: 43. Peters. J. H., R. Giessler, B. Thiele, and F. Steinbach. 1996. Dendritic cells: from semaphorins join the ranks. Curr. Opin. Immunol. 11: 387–391. ontogenic orphans to myelomonocytic descendants. Immunol. Today 17: 273– 19. Pasterkamp, R. J., and A. L. Kolodkin. 2003. Semaphorin junction: making 278. tracks toward neural connectivity. Curr. Opin. Neurobiol. 13: 79–89. 44. Annes, J. P., J. S. Munger, and D. B. Rifkin. 2003. Making sense of latent 20. Pasterkamp, R. J., J. J. Peschon, M. K. Spriggs, and A. L. Kolodkin. 2003. TGFbeta activation. J. Cell Sci. 116: 217–224. Semaphorin 7A promotes axon outgrowth through integrins and MAPKs. Nature 45. Kim, J., W. J. Oh, N. Gaiano, Y. Yoshida, and C. Gu. 2011. Semaphorin 3E- 424: 398–405. Plexin-D1 signaling regulates VEGF function in developmental angiogenesis via 21. Kolodkin, A. L., D. J. Matthes, and C. S. Goodman. 1993. The semaphorin genes a feedback mechanism. Genes Dev. 25: 1399–1411. encode a family of transmembrane and secreted growth cone guidance mole- 46. Kretzschmar, M., and J. Massague´. 1998. SMADs: mediators and regulators of cules. Cell 75: 1389–1399. TGF-beta signaling. Curr. Opin. Genet. Dev. 8: 103–111. 22. Luo, Y., D. Raible, and J. A. Raper. 1993. Collapsin: a protein in brain that 47. Horejsı´, V., K. Drbal, M. Cebecauer, J. Cerny, T. Brdicka, P. Angelisova´,and induces the collapse and paralysis of neuronal growth cones. Cell 75: 217–227. H. Stockinger. 1999. GPI-microdomains: a role in signalling via immunor- 23. Czopik, A. K., M. S. Bynoe, N. Palm, C. S. Raine, and R. Medzhitov. 2006. eceptors. Immunol. Today 20: 356–361. Semaphorin 7A is a negative regulator of T cell responses. Immunity 24: 591–600. 48. Friedrichson, T., and T. V. Kurzchalia. 1998. Microdomains of GPI-anchored 24. Delorme, G., F. Saltel, E. Bonnelye, P. Jurdic, and I. Machuca-Gayet. 2005. proteins in living cells revealed by crosslinking. Nature 394: 802–805. Expression and function of semaphorin 7A in bone cells. Biol. Cell 97: 589–597. 49. Suzuki, K., A. Kumanogoh, and H. Kikutani. 2008. Semaphorins and their 25. Holmes, S., A. M. Downs, A. Fosberry, P. D. Hayes, D. Michalovich, receptors in immune cell interactions. Nat. Immunol. 9: 17–23. P. Murdoch, K. Moores, J. Fox, K. Deen, G. Pettman, et al. 2002. Sema7A is 50. Das, S., S. Chakraborty, and A. Basu. 2010. Critical role of lipid rafts in virus a potent monocyte stimulator. Scand. J. Immunol. 56: 270–275. entry and activation of phosphoinositide 39 kinase/Akt signaling during early 26. Comeau, M. R., R. Johnson, R. F. DuBose, M. Petersen, P. Gearing, stages of Japanese encephalitis virus infection in neural stem/progenitor cells. J. T. VandenBos, L. Park, T. Farrah, R. M. Buller, J. I. Cohen, et al. 1998. A Neurochem. 115: 537–549.