Measles virus hemagglutinin triggers intracellular signaling in ... - Nature

Cellular & Molecular Immunology (2016) 13, 828–838 ß 2016 CSI and USTC. All rights reserved 1672-7681/16 $32.00 www.nature.com/cmi

RESEARCH ARTICLE

Measles virus hemagglutinin triggers intracellular signaling in CD150-expressing dendritic cells and inhibits immune response Olga Romanets-Korbut1,6, Larysa M. Kovalevska6, Tsukasa Seya7, Svetlana P. Sidorenko6 and Branka Horvat1–5 Measles virus (MV) is highly contagious pathogen, which causes a profound immunosuppression, resulting in high infant mortality. This virus infects dendritic cells (DCs) following the binding of MV hemagglutinin (MV-H) to CD150 receptor and alters DC functions by a mechanism that is not completely understood. We have analyzed the effect of MV-H interaction with CD150-expressing DCs on the DC signaling pathways and consequent phenotypic and functional changes in the absence of infectious context. We demonstrated that contact between CD150 on human DCs and MV-H expressed on membrane of transfected CHO cells was sufficient to modulate the activity of two major regulatory pathways of DC differentiation and function: to stimulate Akt and inhibit p38 MAPK phosphorylation, without concomitant ERK1/2 activation. Furthermore, interaction with MV-H decreased the expression level of DC activation markers CD80, CD83, CD86, and HLA-DR and strongly downregulated IL-12 production but did not modulate IL-10 secretion. Moreover, contact with MV-H suppressed DC-mediated T-cell alloproliferation, demonstrating profound alteration of DC maturation and functions. Finally, engagement of CD150 by MV-H in mice transgenic for human CD150 decreased inflammatory responses, showing the immunosuppressive effect of CD150–MV-H interaction in vivo. Altogether, these results uncover novel mechanism of MV-induced immunosuppression, implicating modulation of cell signaling pathways following MV-H interaction with CD150-expressing DCs and reveal anti-inflammatory effects of CD150 stimulation. Cellular & Molecular Immunology (2016) 13, 828–838;doi:10.1038/cmi.2015.55;published online 15 June 2015 Keywords: dendritic cells; measles virus; signaling pathways; SLAM/CD150

INTRODUCTION Measles remains one of the most deadly vaccine-preventable diseases, with recent outbreaks in numerous industrialized countries1. Although vaccination has controlled measles in many countries, the disease still represents a significant cause of child mortality. Measles virus (MV) is transmitted by a respiratory way and causes a systemic infection, which may be followed with a profound suppression of the immune system. Immune responses induced by MV infection are paradoxically associated with depressed responses to non-measles virus antigens for several weeks to months after resolution of the acute illness. This MV-induced immunosuppression renders individuals more susceptible to secondary bacterial and viral

infections and is responsible for most of measles-related morbidity and mortality2. Myeloid dendritic cells (DCs) and lymphocytes were shown to be principal targets of MV infection in peripheral tissues3. Different subsets of DCs, including skin Langerhans cells4, peripheral blood DCs5, CD341-derived DCs4, and monocyte-derived DCs6 are permissive to MV infection and seem to have subset-specific interferon-inducing systems7. Viral infection induces formation of DC syncytia, followed by the loss of the DC’s capacity to stimulate naive CD41 T cells resulting in inhibition of CD41 T-cell proliferation4–6. MV-induced inhibition of T-cell functions was suggested to be mediated through either transmission of infectious virus to T cells,

1 CIRI, International Center for Infectiology Research, IbIV team, Universite´ de Lyon, Lyon, France; 2Inserm, U1111, Lyon, France; 3CNRS, UMR5308, Lyon, France; 4Universite´ Lyon 1, Lyon, France; 5Ecole Normale Supe´rieure de Lyon, Lyon, France; 6R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NASU, Kyiv, Ukraine and 7Department of Microbiology and Immunology, Graduate School of Medicine, Hokkaido University, Kita-ku, Sapporo, Japan Correspondence: B Horvat CIRI 1111, 21 Avenue Tony Garnier, 69365 Lyon cedex 07, France E-mail: [email protected] Received: 2 March 2015 Revised: 17 May 2015 Accepted: 17 May 2015

Measles virus-CD150 interaction and immunosuppression O Romanets-Korbut et al

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leading to a cell cycle block8,9 and/or delivery of inhibitory signals via infected DCs4–6. In addition, MV binds DC-SIGN10, which could lead to the inhibition of type I IFN responses in DCs via blocking of RIG-I and MDA5 dephosphorylation11. Due to a crucial role of DCs in the immunoregulation, those studies indicate that MV infection interferes with DC functions and may represent the critical mechanism of immunosuppression. However, the molecular mechanism of MV-mediated perturbation of DC functions and particularly the contribution of direct interaction between viral proteins and DC membrane receptors remains unclear. Three different cell surface receptors have been identified to interact with MV envelope glycoprotein hemagglutinin (H), permitting viral entry into cells: CD46 for laboratory MV strains12,13, CD150 for both wild-type (wt) and laboratory MV strains14, and nectin-4 (PVRL4), recently shown to mediate the viral egress from the respiratory tract15,16. CD150 (IPO3 or SLAM, for Signaling Lymphocytic Activation Molecule) is a transmembrane protein which in humans is encoded by the SLAMF1 gene and is expressed on activated T and B cells, DCs, and monocytes17,18. CD150 functions as a co-receptor molecule that modulates signaling via antigen receptors19. The cytoplasmic tail of CD150 can bind the key SH2-containing components of signal transduction pathways, such as SHP-1, SHP-2, and SHIP, adaptor molecules SH2D1A/SAP and EAT2, as well as Src-family kinases, including Fyn, FynT, Lyn, and Fgr, and also the p85 regulatory subunit of phosphatidylinositol-3 kinase20–22. CD150 was shown to regulate Akt (v-Akt murine thymoma viral oncogen)/PKB (protein kinase B) and MAPK (mitogen-activated protein kinase) signaling pathways in human B cells23,24 and to induce Akt phosphorylation in CD41 T lymphocytes20. By binding to the CD150 cytoplasmic tail, the adaptor protein SH2D1A/SAP works as a molecular switch that regulates CD150-mediated signaling pathways25. CD150 engagement increases T-cell antigen receptor-mediated protein kinase C h (PKCh) recruitment, nuclear p50 NF-kB levels, NF-kB1 activation, and IL-4 production in the SH2D1A-dependent but Fyn-independent fashion25. However, CD150-mediated signal transduction pathways in DCs are currently unknown. Production of different viral proteins during MV infection profoundly modulates biology of an infected cell26 and makes the functional study of a particular virus-host cell protein interaction difficult to implement. To better understand the cellular and molecular basis of MV-induced regulation of DC functions and role of CD150, we thus generated a model that allowed focusing the study to the interaction of MV-H with human DCs, in the absence of the infectious context. We examined in cellulo the effect of wt MV-H on DC signaling pathways, including Akt and MAPK (p38 MAPK, ERK1/2), as well as DC phenotype and functions. In addition, we studied the effect of CD150 engagement by MV-H in vivo on the generation of the inflammatory responses in mice transgenic for human CD150. Our results demonstrate the important changes in signaling pathways, phenotype and function of

DCs, triggered after interaction with MV-H and suggest a new mechanism of MV-induced immunosuppression revealing thus novel aspects of CD150-mediated regulation in the immunobiology of DCs and inflammatory responses. MATERIALS AND METHODS Cell culture The B lymphoblastoid cell line MP-1 (kindly provided by Dr E. Clark, University of Washington, Seattle, WA, USA) was maintained in RPMI 1640 medium containing 10% FCS, 2 mM Lglutamine, 10 mM HEPES, and antibiotics. CHO (Chinese hamster ovary) cells (from ATCC) and CHO transfectants were cultured in DMEM medium supplemented as above. Human peripheral blood was obtained from healthy donors from the Blood Transfusion Centre (Lyon, France). PBMCs were isolated by density Ficoll/Hypaque gradient centrifugation and then centrifuged through a 50% Percoll gradient (Pharmacia Fine Chemicals, Sweeden) for 20 min at 400g. Peripheral blood lymphocytes (PBLs) were recovered from the high-density fraction and monocytes from the light-density fraction at the interface. CD31 T lymphocytes were isolated from high-density fraction, using microbeads (Miltenyi Biotech, France) and magnetic cell separation with MACS Separator. DCs were generated in vitro from the adherent fraction of purified monocytes, treated for 6 days at 5 3 105 monocytes/mL with IL-4 (250 U/mL, Peprotech, USA) and GM-CSF (500 U/mL, Peprotec). T cells and CD1d1 DCs were further cultured in RPMI 1640 medium containing 10% FCS, 2 mM L-glutamine, 10 mM HEPES, and antibiotics. All cell lines were tested to be negative for mycoplasma infection. Virus The wild-type MV strain, G954 (genotype B3.2)27 was produced on Vero-SLAM cells. Recombinant vesicular stomatitis virus (VSV) expressing the MV hemagglutinin (Edmonston strain)28, and the recombinant VSV control strain (kindly provided by Dr J.K. Rose, USA) were propagated on Vero cells, and harvested when a strong cytopathic effect was observed. Virus titers were determined by PFU assay on Vero-SLAM/Vero cell monolayers. For the infection-free injection, all viruses were inactivated by 30 min exposure at 4 uC to 254 nm UV irradiation. Viral inactivation was confirmed by the plaque assay on Vero cells. 5 3 106 PFU of UV-inactivated viruses were injected in mice intraperitoneally (i.p.) in all in vivo experiments. Human PBLs were infected by wt MV (G954) at MOI of 1 and further analyzed for MV-H expression. Production of stably transfected CHO-H cell line, cell stimulation, and cytokine detection by ELISA The plasmid pCXN2-KA-H containing H protein from wt MV strain KA (kindly provided by Dr Y. Yanagi, Kyshu Univer, Japan) was used to transfect CHO cells using Lipofectamine Transfection Reagent (Life Technologies, France) according to the manufacturer’s protocol and selective antibiotic G418. Cellular & Molecular Immunology


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Single-cell colonies were amplified and analyzed by flow cytometry using anti-MV-H mAb cl55, to select CHO-H cell line. CHO-H and CHO cells were used to stimulate MP-1 and DC in co-cultures with 1:5 (DC/MP-1:CHO/CHO-H) cell ratio. The production of cytokines by DCs was measured in culture supernatants using human IL-12 (p70) ELISA kit (BD Biosciences, Belgium) and human IL-10 ELISA kit (RayBiotech, USA) according to the manufacturer’s instructions. In some experiments, IL-12 production by DCs was stimulated by 10 ng/mL LPS for 12 h followed or not by CHO/ CHO-H co-culture. Cell stimulation and western blot analysis Cells were pelleted, resuspended at 107/mL in supplemented RPMI 1640 medium, equilibrated at 37 uC for 10–15 min and stimulated using 10 mg/mL anti-CD150 mAb (IgG1, clone IPO3, IEPOR NASU, Kiev, Ukraine), or co-cultured with either CHO or CHO-H cells as described above. To block TLR-2 and DC-SIGN signaling, DCs were incubated with 200 mg/ mL anti-TLR2 (InvivoGen, USA) or 20 mg/mL anti-DCSIGN AZN-D1 antibodies (kindly provided by Dr T.B.H. Geijtenbeek from the University of Amsterdam) for 1 h before further stimulation with CHO/CHO-H. The ligand for TLR2, FSL-1 (20 ng/mL) (InvivoGen, USA) was used as positive control for cell stimulation for 30 min. Stimulations with mAbs were stopped at various time points by diluting the cell suspensions into more than 10 V of ice-cold PBS with 0.1% NaN3. Cells were pelleted and washed with ice-cold PBS before lysis in 1% Triton lysis buffer, supplemented with inhibitors of proteases and phosphatases. Then the samples were boiled for 5 min in Laemmly buffer with 2-ME and resolved in standard 10% SDS-PAGE, followed by western blotting, using rabbit antisera against phospho-Akt (Ser473), phospho-p38 MAPK (T180/ Y182), and phospho-ERK1/2 (Thr202/Tyr204) (Cell Signaling Technology, USA) and mouse anti-CD45 (IEPOR NASU, Kiev, Ukraine). Protein expression was detected by HRP-conjugated secondary Abs, either anti-mouse (Amersham, UK) or anti-rabbit (Promega, USA), visualized using Chemiluminescent Reagent Kit Covalight (Covalab, France). CD45 was used as a loading control for DC and MP-1 cells on the same membrane. Flow cytometric analysis DCs were stimulated with CHO/CHO-H cells as described above and then stained with anti-CD80, CD1d, and HLA-DR (FITC-labeled), CD11c (PE-labeled) CD83 (PeCy5-labeled), and CD86 (PECy7-labeled) (all from BD-Biosciences), antiCD46 (MCI 20.6)29 and anti-MV-H Cl5530 mAbs, followed by goat anti-mouse IgG-PE or FITC-labeled (BD Biosciences, Belgium). Viable cells were acquired on FACSCalibur 3C cytometer (BD Biosciences, Belgium) and FACS analysis was performed using CellQuestPro software followed by FlowJo (Tree Star Inc, USA) software analysis. The DC subpopulation used for the analysis of surface markers expression was gated from the entire live cell population as CD11c-positive. Cellular & Molecular Immunology

Allogeneic MLR and T-cell proliferation DCs, differentiated from peripheral blood monocytes, were stimulated with CHO/CHO-H cells as described above, removed from the co-culture and then UV-irradiated at 0.25 J/cm2 for 30 min. Freshly generated or CHO/CHO-H co-cultured DCs, removed as a non-adherent fraction from the coculture, were added at 104 cells/well in round-bottomed 96-well plates to 2 3 104 CD31 T cells, isolated from allogeneic PBLs. After 6 days, cells were pulsed with 1 mCi of [3H]thymidine and harvested after 16 h of cultivation. Thymidine incorporation was determined using MicroBeta2 plate counter (Perkin Elmer, USA). Results were expressed as mean cpm 6 SD of quadruplicate wells. Murine DC isolation and intracellular IL-12 detection Murine DCs were purified from inguinal, axillary, and popliteal lymph nodes (LNs) (4–5 mice/group, treated with DNFB as described above). LNs were homogenized, incubated in RPMI containing 2% FCS, 1 mg/mL Collagenase 1A and 0.1 mg/mL DNAseI at 137 uC for 25 min, then filtered through 100 mm membrane to obtain single-cell suspension. Cell suspension was centrifuged on OptiPrep gradient for 20 min at 600g. Cells at the interface were collected, washed once, and analyzed. For the detection of intracellular IL-12, cell suspensions containing enriched DC (106 cells/mL) were first incubated with 1 ml/mL GolgiPlug (BD Biosciences, USA) for 8 h to block cytokine secretion (37 uC, 5% CO2). FccR was blocked with rat mAb 2.4G2, and cells were stained for surface marker using anti-CD11c-FITC (BD Pharmingen, USA) Abs. Intracellular cytokines were stained using the Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer’s instructions and rat anti-IL-12 p40/p70 Abs C15.6-PE or rat IgG1 isotype control R3-34-PE (BD Pharmingen). Flow cytometric analysis was carried out on BD Accuri C6 cytometer (Becton Dickinson, USA). Contact hypersensitivity assay Mice transgenic for the human CD150 with the expression pattern in tissues closely correlating with humans, were generated as described previously31, crossed into C57BL/6 background more than 10 generations and maintained in PBES (Plateau de Biologie Experimentale de la Souris) of ENSLyon. About 8- to 12-week-old CD150tg mice or C57Bl/6 mice (Charles River, France) were injected i.p. with mock preparation, VSV or VSV-H UV-irradiated viruses at 5 3 106 PFU, and contact hypersensitivity to dinitrofluorobenzene (DNFB) was assayed as described32. The protocol was approved by the Regional Animal Care Committee (CREEA). Statistical analysis All statistical analyses were performed with GraphPad Prism 5 software, using Mann–Whitney U test and unpaired two-tailed Student’s t-test for the comparison between two groups and one-way ANOVA for three groups. Differences were considered to be statistically significant when P , 0.05.


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RESULTS MV-H interaction with CD150 increases Akt phosphorylation in human DCs MV was shown to disrupt Akt kinase activation in T cells and this modulation of signal transduction was suggested to play the crucial role in MV-induced immunosuppression33. As PI3K/ Akt pathway is one of the signaling pathways linked to CD150 engagement in lymphocytes20,22, we have assumed that MV may also trigger Akt in monocyte-derived human DCs. Our previous studies demonstrated that both infectious and UV-inactivated MV particles, in addition to the presence of viral envelope glycoproteins, contain an important quantity of free MV nucleoprotein, which can stimulate Fc receptor expressed on numerous cells, including DCs34,35. To be able to analyze DC-MV-H interaction in the absence of infectious context, we have generated CHO cell line stably expressing MV-H (CHO-H cells). This line allowed us to focus our study to the role of MV-H protein in the absence of other MV proteins, including fusion protein and nucleoprotein, both known to have biological activity33–35. Obtained CHO-H cell line stably expresses MV-H from the wt MV strain at the level comparable to MV-H expression on PBLs, infected with wt MV (Figure 1). Co-culture of DCs with CHO induced a low level of Akt phosphorylation (Figure 2a), probably due to the heterologous cell membrane interaction. However, the incubation of DCs with CHO-H cells strongly increased the level of Akt phosphorylation at Ser473 starting from 2 h and up to 24 h of co-culture (Figure 2a and e). Analogously to DCs, in B-cell line MP-1 the incubation with CHO-H cells-activated Akt, but at later time points, after 12–24 h (Figure 3c). Similar stimulation of Akt was also observed after ligation with anti-CD150 mAb IPO3 in both DCs (Figure 2b) and MP-1 (Figure 3a)23, although at earlier time points, probably because in this experimental conditions an establishment of cell–cell contact was not required to trigger signaling. Expression of CD45 and Akt phosphorylation was not observed in CHO cells (data not shown), allowing us thus to exclude potential CHO contamination in analyzed DCs lysates. In addition to CD150, MV-H may also interact with two other surface molecules expressed on DCs, TLR236, and DCSIGN10. To analyze whether these molecules could participate in MV-H induced signaling, since CD150 blocking antibodies are not available, we tested if specific blocking mAbs, antiTLR2 mAb (Figure 2c and f) and anti-DC-SIGN, AZN-D1 (Figure 2d and f)37 could affect the Akt phosphorylation. The results are additionally presented in relative units obtained after normalization against the level of CD45 (Figure 2f). Presence of anti-TLR2 and anti-DC-SIGN blocking antibodies did not interfere with CHO-H-induced Akt phosphorylation, suggesting that in DCs MV-H activates Akt-mediated signaling pathway via CD150 receptor.

human DCs38. We have thus explored the effect of MV-H on the phosphorylation of p38 MAPK at T180/Y182 and ERK1/2 at T202/Y204 residues in CD150 expressing DCs. While p38 MAPK was already activated in DCs, the co-culture of DCs with CHO cells did not further modulate the phosphorylation of p38 MAPK (pp38 MAPK). In contrast, the level of pp38 MAPK was decreased already after 5 min of incubation with CHO-H, and this effect was still observed after 24 h of co-culture (Figure 4a, b, d, and e). Reduced levels of pp38 MAPK were regularly observed with DCs from different donors and when pooled results from several experiments were analyzed, the statistical difference was obtained at 6 h of coculture. Interestingly, stimulation with anti-CD150 mAb resulted in bimodal modulation of p38 MAPK phosphorylation, starting with downregulation, followed by elevation of pp38 MAPK level 30 min later (Figure 4c). These data suggest that CD150 ligation on DCs with two different ligands, mAb and MV-H, may have diverse effects on p38 MAPK pathway, reflecting possibly different valence of the interaction, mAb being bivalent and MV-H polyvalent, as expressed in numerous copies on the cell surface, in addition to their recognition of potentially different epitopes at CD150. The downregulation of p38 MAPK phosphorylation was still observed after MV-H stimulation in the presence of anti-TLR2 blocking mAb (Figure 4d and f), indicating that in human DCs TLR2 does not mediate pp38 MAPK inhibition in human DCs. Stimulation of DC-SIGN was shown not to lead to the modulation of p38 MAPK in DCs39. CD150 engagement either with mAb or MV-H did not affect the level of pp38 MAPK in B cells (Figure 4g), indicating thus DC-specific modulation of this signaling pathway. We did not detect any p38 MAPK phosphorylation in CHO cells (data not shown), excluding thus the contamination from CHO cells. In contrast to p38 MAPK, we did not observe any changes in the activation of ERK1/2 MAPK in any tested conditions (data not shown). Altogether, these results suggest that CD150 ligation with MV-H modulates the p38 MAPK signaling in human DCs.

MV-H interaction with CD150 decreases p38 MAPK phosphorylation in DCs MAPK, particularly p38 MAPK and extracellular signal-regulated kinase (ERK), play an important role in the maturation of

MV-H-induced signaling triggers phenotypic and functional changes in human DCs Since intracellular signaling pathways regulate phenotypic and functional maturation of DCs, we then analyzed the effect of

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Figure 2 CD150 modulates Akt signaling pathway in human DCs. DCs were co-cultured with CHO/CHO-H cells (a, c–d) at 1:5 ratio or stimulated with 10 mg/mL of anti-CD150 mAbs (clone IPO3) (b) for the different time. Activation of signaling pathways via TLR2 and DC-SIGN receptors was blocked with specific anti-TLR2 (c) and anti-DC-SIGN (d) mAbs. FSL-1 was used as specific ligands for TLR2 stimulation. Cell lysates were analyzed for pAkt (S473) expression by western blot with CD45 as loading control. The level of pAkt was normalized against the level of CD45 using TotalLab program (e, f). (e) The results are expressed as mean (6SD) from 3–6 independent experiments, and differences observed at 6, 12, and 24 h were statistically significant (*P , 0.05, **P , 0.001).

MV-H on phenotype and functions of CD150-expressing DCs generated from human monocytes (Figure 5a). As expected, co-culture with CHO-H cells induced downregulation of CD150 on DCs, but not CD46, due to the selective utilization of CD150 receptor by wt MV (Figure 5a). Co-culture of DCs with CHO cells induced certain DC maturation, as demonstrated by the increase of DC activation markers: HLA-DR, CD80, CD83, and CD86 (Figure 5a). However, the incubation of DCs with CHO-H cells in the same experimental conditions induced the decreased expression level of all tested DC costimulatory molecules important for the DC function in the activation of the immune response. We did not observe any MV-H effect on phenotype of CD40 ligand-activated DCs (three independent experiments, data not shown), suggesting that MV-H-induced phenotypic changes may depend on the differentiation stage of DCs, at the moment when MV-H signaling is triggered. We further analyzed whether DC-MV-H interaction may influence the production of IL-12, a critical cytokine for DCmediated regulation of cellular immune response40. The LPS stimulation induced the high level of IL-12 secretion by DCs, Cellular & Molecular Immunology

which was to a lower extent seen when DCs were co-cultured with CHO cells (Figure 5b), in accord with the CHO-induced commencement of DC phenotypic maturation (Figure 5a). However, the incubation of DCs with CHO-H significantly reduced the level of IL-12 production, compared to co-culture with CHO (Figure 5b). The same effect was also observed with DCs stimulated by LPS either before CHO/CHO-H co-culture (Figure 5b), confirming the inhibitory effect of MV-H on IL-12 production in different experimental conditions. In contrast, IL-10 production by DCs was not affected (Figure 5c). Finally, to evaluate the effect of MV-H on DC-induced T-cell stimulation, we analyzed proliferation of CD31 T lymphocytes in response to allogeneic DCs. Monocyte-derived DCs were cultured for 24 h with either CHO or CHO-H cells, non-adherent cells were removed from the culture and used for the allostimulation of purified CD31 T lymphocytes. Although co-culture of DCs with CHO did not affect DC immnostimulatory properties, pre-incubation with CHO-H significantly decreased T-cell allostimulation (Figure 5d). Altogether, these data strongly imply that MV-H is able to trigger a profound alteration of DC maturation and function.


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(Figure 6b). Furthermore, VSV and VSV-H did not show any differential effect on the development of inflammatory response in C57BL/6 mice. These results suggest the anti-inflammatory effect of CD150 engagement in vivo and imply that MV-H-induced CD150 signaling may be involved in the inhibition of cellular immune responses and thus play an important role in pathogenesis of measles.

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Figure 3 MV-H modulates Akt signaling pathway in human B-lymphoblastoid cell line MP-1 in the absence of an infectious context. MP-1 cells were stimulated by anti-CD150 antibodies IPO3 (a) or co-cultured with CHO (b) or CHO-H (c) cells at 1:5 ratio for the different time. Cell lysates were analyzed for pAkt (S473) expression by western blot with CD45 as loading control. Data represent one of the three independent experiments.

MV-H-CD150 interaction inhibits inflammatory immune response in vivo We then analyzed the immunological consequence of MV-HCD150 interaction in vivo, using mice expressing transgenic human CD150 (CD150tg) in the similar pattern as humans31. CD150 was engaged by injecting UV-inactivated vesicular stomatitis virus (UV-VSV), expressing or not MV-H28. This approach has already been shown to stimulate efficiently immune cell membrane receptors in transgenic mice in vivo32. In accord to in vitro data, DCs isolated from lymph nodes of five VSV-H-injected CD150tg mice produced lower levels of IL-12 comparing to VSV-injected CD150tg mice, while IL-12 production in C57BL/6 mice was not changed (Figure 6a). Interestingly, the decrease in IL-12 producing cells was seen in both CD11c1 and CD11c- cell populations. To further confirm whether the observed effect may be relevant for the effect of MV-H in vivo, we analyzed contact hypersensitivity response, the type of cellular immunity known to depend on DC function41. MV infection results with a suppression of delayed-type hypersensitivity responses to recall antigens, such as tuberculin42. Therefore, we analyzed whether interaction between MV-H and human CD150 may be involved in the inhibition of inflammatory response such as hypersensitivity reaction to the hapten DNFB. While CD150 transgenic mice injected with VSV developed good inflammatory response, the reaction was significantly reduced when they were injected with VSV-H

DISCUSSION MV infection implicates complex process of the production of several viral proteins and RNA, which could all interfere with cell functions2,26. DCs are one of the first targets of MV and play a critical role in viral dissemination but also help viral invasion by inhibiting the host immune response. Although some of MV immunosuppressive strategies are well described, the role of the direct interaction between wt MV-H and CD150 receptor on DCs and consecutive signaling events with potential impact on MV-caused inhibition of immune reaction is still unknown. Our results have demonstrated that MV-H interaction with both DCs and B cells, activates Akt (Figures 2 and 3). Although CD150-induced activation of Akt phosphorylation was observed in T and B lymphocytes20,22, UV-inactivated vaccine strain of MV was shown to inhibit IL-2 dependent activation of Akt pathway in T cells33. These disparities may be linked to differences in the cell types and viral strains, distinct mode of cell activation and finally surface interaction of lymphocytes with the MV glycoprotein complex independently of CD150. Our results suggest that that MV-H may have the F-independent effects on the cells of the immune system, in addition to their possible conjoint action on T lymphocytes. Among downstream targets of CD150-activated Akt kinase in B cells are GSK-3b protein kinase and transcription factor FoxO123. Which upstream and downstream elements of Akt signaling machinery are linked to CD150–MV-H engagement in DCs, remains to be determined. In accordance with some previous data43,44, DCs generated from human monocytes expressed CD150 (Figure 5a). This expression was slightly increased after 24 h co-culture with CHO cells, presumably due to further DC maturation, as reflected by elevation of CD83 and HLA-DR expression and p38MAPK activation. In contrary, 24 h co-culture of DCs with CHO-H cells diminished the level of cell surface expression of CD150, HLA-DR, CD80, CD83, and CD86 (Figure 5a), similarly to the effect of UV-inactivated MV, previously observed43. MV infection was shown to downregulate co-stimulatory molecules and MHC proteins on murine DCs expressing human CD150 receptor45. Moreover, CD150 self-ligation on human DCs also leads to surface downregulation of CD83, CD86, and HLA-DR46, so it is likely that the phenotypic changes upon MV-H-DC interaction benefit from MV-Hinduced CD150 signaling. Interestingly, infection of canine DCs with canine distemper virus, a close relative of MV also using CD150 as the entry receptor, equally decreases the surface levels of HLA-DR, CD80, and CD8647, suggesting that viral Cellular & Molecular Immunology


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Figure 4 CD150-mediated p38MAPK phosphorylation is decreased upon MV-H-CD150 interaction in DCs. DCs were either co-cultured with CHO/ CHO-H cells at 1:5 ratio (a, b) or stimulated with 10 mg/mL of anti-CD150 IPO-3 antibody at different time points (c). Activation of signaling pathways via TLR2 receptor was blocked with specific anti-TLR2 antibodies (d). Cell lysates were analyzed for pp38 MAPK (T180/Y182) expression by western blot with CD45 as a loading control. The level of ppMAPK38 was normalized against the level of CD45 using TotalLab program (e, f). (e) The results are expressed as mean (6SD) from at least three independent experiments and differences observed at 6 h were statistically significant (*P , 0.05, Student’s t-test). (g) MP-1 cells were either stimulated with 10 mg/mL of anti-CD150 IPO-3 antibody or co-cultured with CHO/CHO-H cells at 1:5 ratio at different time points.

interaction with CD150 may have similar consequences for different Moribilliviruses in their natural hosts. Another hallmark of MV-induced immunosuppression, inhibition of IL-12 production, was reported in MV-infected children48, rhesus macaques during measles49, wt MV-infected mice expressing human SLAM50 and primary human monocytes infected by MV51. The production of IL-12 was also impaired after CD150/CD150 self-ligation in human DCs46 and an activation of Akt was shown to be followed by a decrease in IL-12 production52,53. For the first time, our results demonstrate that interaction between MV-H and human DCs, in the absence of an infectious context, may also lead to the inhibition of IL-12 production (Figure 5b) and thus participate in the generation of the immunosuppressive state, observed in measles. Cellular & Molecular Immunology

Our results imply that MV-H triggering could inhibit p38 MAPK phosphorylation in DCs (Figure 4). Since p38 MAPK is considered as the major positive regulator of DC phenotypic maturation and IL-12 cytokine production54, it is possible that this signaling pathway is involved in the phenotypic and functional changes in DCs, following MV-H contact. Several studies have shown decreased lymphocyte proliferation in measles patients55,56, or hSLAM1 transgenic mice57, as well as abrogation of DC allostimulatory capacities upon interaction with both wt MV4,45 and vaccine MV strains4,6. Our results indicate that MV-H interaction with CD150 expressing DC could play an important role in these immunosuppressive consequences of MV infection. Aberrant maturation of DCs could lead not only to the insufficient stimulation of effector cells, but also favor induction of T regulatory cells, increased in both, measles


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Figure 5 Interaction of human DCs with MV-H causes changes in DC maturation and function. (a) Downregulation of several surface markers expression in DCs. DCs, co-cultured with CHO (solid line) or CHO-H cells (dashed line) at 1:5 ratio for 24 h, were stained for CD150, CD46, CD80, HLA-DR, CD83, and CD86 (light-gray histogram: nonstained DCs; dark-gray histogram: DCs before CHO/CHO-H co-cultures). Data represent one of the four independent experiments with different donors. IL-12 (b) and IL-10 (c) production by DCs was measured by ELISA in supernatants of either freshly generated DCs (‘‘medium’’), or DCs, co-cultured with CHO or CHO-H cells at 1:5 ratio for 24 h (‘‘CHO/CHO-H’’). Alternatively, DCs were stimulated by 10 ng/mL LPS for 12 h followed or not by CHO/CHO-H co-culture (‘‘LPS, CHO/CHO-H’’ or ‘‘LPS’’ respectively). Data represent one of the four independent experiments with different donors. (d) Human peripheral blood CD3-positive T cells (2 3 104) were cultured in quadruplicates for 6 days with culture medium, 104 of UV-irradiated DCs, either freshly generated (DC) or previously co-cultured 24 h with either CHO (DC CHO) or CHO-H (DC CHO-H) at 1:5 ratio. T-cell proliferation was measured on day 6 by [3H] thymidine uptake during last 16 h of culture. Results, expressed as mean cpm (6SD), are from one of 6 representative experiments. The statistical significance was determined by unpaired two-tailed Student’s t-test (*P , 0.05, ***P , 0.0001).

patients58 and MV-infected CD150tg mice59. It is possible that MV-H–CD150 interaction may be involved in the modulation of other cell types function, like memory T lymphocytes, which are particularly affected by MV infection in macaque model60. Finally, we showed the inhibition of inflammatory responses in human CD150 transgenic mice, after engagement of CD150 in vivo (Figure 6). It was reported that only MV-H from wt, but not from Edmonston MV strain, activates TLR2 signaling36. The utilization of VSV expressing Edmonston MV-H in our study excludes TLR2 implication from the observed antiinflammatory effect in mice. Furthermore, the absence of VSV-H effect on contact hypersensitivity reactions in

non-transgenic mice, implying that anti-inflammatory effect of MV-H passes via the interaction with CD150, rather than murine DC-SIGN or TLR2. This was supported by the absence of anti-TLR2 and anti-DC-SIGN blocking mAbs effect on signaling events in human DCs in vitro. Our data strongly suggest that MV-H–CD150 interaction may play an important role in the inhibition of cellular immune response that are characteristic for measles42. These results also elucidate the anti-inflammatory capacity of CD150 signaling, which may have therapeutic potential. Various stimuli mediate DC responses via differential regulation of Akt and MAPKs52,53. The inhibition of the immune Cellular & Molecular Immunology


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response in measles may be attributed, at least in part, to the modulation of CD150-mediated signaling pathways by MV-H. Therefore, our results present an evidence for the novel mechanism of MV-induced immunosuppression, where direct contact between MV-H and CD150 expressing DCs is sufficient to transmit intracellular signals via MAPK and Akt pathway to alter DC functions (Figure 7). In addition to free viral particles, 1

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Figure 7 Schematic presentation of mechanisms possibly involved in MV-H-induced modulation of the immunobiology of DCs and inhibition of inflammatory immune responses. MV-H interaction with CD150expressing DCs modifies signaling pathways Akt and p38MAPK (1) and affects DC maturation and function (decreased expression of DC maturation markers, inhibition of IL-12 production and T-cell proliferation) (2). CD150 engagement with MV-H in vivo results in the inhibition of inflammatory responses in humanized CD150tg mice (3). Modulation of cell transduction pathways in DCs and inhibition of their maturation and function are likely to be linked to the suppression of inflammatory responses, seen in measles patients (dashed arrows). Cellular & Molecular Immunology

MV-H is expressed on all infected cells and therefore can easily come in contact with circulating DCs. This interaction could amplify the effect of viral replication in the inhibition of the immune response and thus play an important role in measles immunosuppression. Obtained results reveal the immunosuppressive capacity of CD150 when it is engaged with a viral ligand, expanding the immunomodulatory properties of this receptor. COMPETING INTERESTS The authors declare no financial or commercial conflict of interest. ACKNOWLEDGEMENTS The work was supported by INSERM, Ligue contre le Cancer, Ministere des affaires Etrangeres, Partenariat Curien FrancoUkrainien ‘‘Dnipro’’ and The State Fund for Fundamental Research of Ukraine, National Academy of Sciences of Ukraine. Olga RomanetsKorbut was supported by FEBS Summer and Collaborative Fellowships, Bourse de Gouvernement Francais and AccueilDoc from Region Rhone Alpes. Authors thank to Dr T.B.H. Geijtenbeek (University of Amsterdam) for generous gift of AZN-D1 mAb and Dr D. Gerlier and members of the CIRI-INSERM-U1111 group ‘‘Immunobiology of viral infections’’ for their help in the achievement of this study.

1 Perry RT, Gacic-Dobo M, Dabbagh A et al. Global control and regional elimination of measles, 2000-2012. MMWR Morb Mortal Wkly Rep 2014; 63: 103–107.


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