Lung CD200 Receptor Activation Abrogates Airway Hyperresponsiveness in Experimental Asthma.

AJRCMB Articles in Press. Published on 08-January-2015 as 10.1165/rcmb.2014-0229OC

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Lung CD200R activation abrogates airway hyperresponsiveness in experimental asthma Jean-François Lauzon-Joset; Anick Langlois; Laetitia JA Lai; Kim Santerre; Audrey LeeGosselin; Ynuk Bossé; David Marsolais#; Elyse Y Bissonnette#. #

: both senior authors contributed equally to the work.

Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Québec (CRIUCPQ), Department of Medicine, Laval University, QC, Canada.

Running title: CD200 inhibits asthma

Corresponding author:

Dr. David Marsolais 2725, chemin Ste-Foy, CRIUCPQ Quebec, QC Canada G1V 4G5 Phone: 418-656-4760 FAX: 418-656-4509

Jean-François Lauzon-Joset: [email protected] Anick Langlois: [email protected] Laetitia JA Lai: [email protected] Kim Santerre: [email protected] Audrey Lee-Gosselin: [email protected] Ynuk Bossé: [email protected] David Marsolais: [email protected] Elyse Y Bissonnette: [email protected]

This work was supported by Fondation J.D. Bégin. JFLJ was supported by Fonds de Recherche du Québec - Santé. DM is a FRQ-S Junior 1 Scholar. Conception and design: AL, DM, EB; Experimentation: JFLJ, AL, LL, KS, ALG; Analysis and interpretation: JFLJ, AL, DM, EB; Drafting and revision of the manuscript: JFLJ, YB, DM, EB.

1 Copyright © 2015 by the American Thoracic Society


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Abstract In allergic asthma, homeostatic pathways are dysregulated, which leads to an immune response towards normally innocuous antigens. The CD200-CD200R pathway is a central regulator of inflammation and CD200 expression was recently found to be downregulated in circulating leukocytes of asthmatic patients. Given the antiinflammatory properties of CD200, we investigated whether local delivery of recombinant CD200 (rCD200) could reinstate lung homeostasis in an experimental model of asthma. Brown Norway rats were sensitized with ovalbumin (OVA) and Alum. rCD200 was intratracheally administered 24 h before OVA challenge and airway responsiveness to methacholine was measured 24 h after the allergen challenge. Inflammation was also assessed by measuring cell recruitment and cytokine levels in bronchoalveolar lavages, as well as lung and draining lymph node accumulation of dendritic cells (DC) and T cells. In sensitized rats, rCD200 abolished airway hyperresponsiveness (AHR), while the sham treatment had no effect. In addition, rCD200 strongly reduced OVA-induced lung accumulation of myeloid DC (mDC), CD4+ T cells, and Th2 cells. This was associated with a strong reduction of OVA-induced IL-13 level and with an increase of IL-10 in supernatants of bronchoalveolar lavages. Lung eosinophilia and draining lymph nodes accumulation of mDC and T cells were not affected by rCD200. Overall, these data reveal that rCD200 can inhibit AHR in a model of asthma by a multi-step mechanism associated with local alterations of the T cell response and the cytokine milieu. Key Words: Allergic asthma, CD200, Airway hyperresponsiveness, Th2 cells, Lung cytokine milieu.



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Introduction Allergic asthma is an inflammatory lung disorder that originates from excessive immune activation toward antigens. Although many cell types are involved in asthma pathogenesis, the activation of dendritic cells (DC), mainly myeloid DC (mDC), is sufficient to trigger the asthmatic cascade (1, 2). Indeed, mDC sample allergens in the airways and can promote T cells to develop an antigen-specific Th2 response. In turn, cytokines, such as IL-13, are upregulated in a Th2 environment and promote the development of airway inflammation and hyperresponsiveness (AHR) (3, 4). Inflammation and AHR are two typical features of asthma and the root causes of asthma symptoms (5, 6). Treating either one of these features is thus conducive to salutary effects. The asthmatic inflammatory cascade can be enabled, at least partially, by dysregulation of homeostatic pathways that normally limit airway inflammation (7, 8). In that regard, lung expresses high level of anti-inflammatory molecules, including the CD200 (9). Yet, few homeostatic mechanisms have been shown to be dysregulated in asthma pathogenesis. Interaction between CD200 and its receptor (CD200R) plays an important role in the regulation of immune responses (10). CD200 is a highly conserved membrane molecule present on many cell types, including epithelial cells, lymphocytes, and some myeloid cells. CD200 cannot transduce signals directly inside the cells because it is devoid of intracellular domain. Alternatively, it fosters anti-inflammatory cascades by activating its cognate receptor, the CD200R (11). Low level of CD200 is associated with autoimmune disease progression, such as arthritis and neurodegenerative disorders (12).



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Conversely, high CD200 expression promotes immune ignorance, which can enable cancer progression (12). As for the role of CD200 in lungs, the main focus has been on virus-induced inflammation, showing that CD200 limits both the amplitude and the duration of the inflammatory response (13). In asthma, CD200 expression is reduced on peripheral blood cells of asthmatic patients during exacerbation (14). Together, these data suggest that the dysregulation of CD200 in asthma may be involved in allergic airway inflammation and asthma pathogenesis. Our hypothesis is that pulmonary delivery of CD200 can protect against asthma pathogenesis. Using a rat model of allergic airway inflammation, we show for the first time that local delivery of recombinant CD200 (rCD200) completely abrogates AHR. The effect was independent of an alteration of airway smooth muscle contractility, but it was associated with a reduction of IL-13 level in bronchoalveolar lavage (BAL), concurrent with an increase of IL-10. Moreover, rCD200 alleviated mDC accumulation and reduced Th2 cell recruitment into the lungs. However, rCD200 did not reduce eosinophil numbers in BAL. Thus, our study reveals a local role for CD200 in controlling AHR and Th2 inflammation in experimental asthma.



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Materials and Methods Animals and allergen exposure Brown Norway SSN rats were bred and maintained at the CRIUCPQ. The protocol was approved by Laval University Animal Care Committee in accordance with the guidelines of the Canadian Council on Animal Care. Sensitization was performed on 8- to 12-weeks-old males by i.p. injection of 1 mg OVA (Sigma Chemical, St. Louis (MO), USA) with 10 mg aluminum hydroxide (Sigma Chemical). On day 20 after sensitization, animals received intratracheal (i.t.) administration of either 100 µmol of a recombinant mouse CD200 fused with a human IgG1 Fc (rCD200; R&D, Minneapolis (MN), USA) or recombinant human IgG1 Fc (Sham; R&D). On day 21, naïve and sensitized animals were challenged by intranasal instillation of 75 µg of OVAAlexaFluor647 (Invitrogen, Grand Island (NY) USA) and tissues were harvested 24 h later. Analysis of responsiveness to methacholine Respiratory mechanics were measured 24 h after OVA challenge as previously described (15). Briefly, rats were anaesthetized, tracheotomized and connected to a small animal ventilator (FX4; FlexiVent, SCIREQ, Inc., Montreal (QC), Canada). Escalating doses of methacholine (MCh) were nebulized and respiratory system resistance (RRS) was assessed. The maximal response obtained after each dose was compared between groups. Effect of CD200 on the contractility of isolated tracheas was also evaluated ex vivo based on the methods described in details in the online supplement. CD200 PCR



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The paries membranaceus was excised of the trachea of naïve and OVAsensitized/challenged rats. The epithelium was then removed by scraping to isolate the smooth muscle tissue. Total RNA was extracted with TRIzol reagent, according to the manufacturer's instructions (Invitrogen Canada). Both cDNA synthesis and PCR amplification were performed using the SuperScript III One-Step RT-PCR system with a Platinum Taq DNA polymerase (Invitrogen Canada) according to the manufacturer's instructions. After 30 amplification cycles, PCR products were visualized by gel electrophoresis and densitometry was performed with the National Institutes of Health Image software. Cell isolation and preparation BAL were performed as previously described (16). Cell types were identified using cytospins stained with Diff-Quick (Gibco BRL, Burlington (ON), Canada). Lung tissue and draining lymph nodes (dLN) were prepared as previously reported (17). Identification of cell populations was performed using monoclonal antibodies (Biolegend, San Diego (CA), USA; BD Pharmingen, San Diego (CA), USA; R&D) directed against cell-surface antigens and intracellular markers. mDC are CD11b+/MHC IIhigh, pDC are CD11b–/MHC II+/CD4+ and Th2 cells are CD3+/CD4+/IL-4+. Data were acquired on a BD FACS Aria II (Becton Dickinson, Franklin Lakes, (NJ) USA) and analyzed using Flowjo software (Tree star Inc, Ashland (OR), USA). Cytokines in BAL



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Cytokine levels were measured in the first 5 mL of the BAL. ELISA were performed with DuoSet kit (R&D Systems) to measure IL-5, IL-13, TNF, IL-10, and TGF-β. Results are expressed per ml of BAL. Statistics Prism (GraphPad Software, La Jolla (CA), USA) was used for all statistical analyses, which includes one-way and two-way ANOVAs with Bonferroni post hoc test. Data are presented as mean ± SEM and p values < 0.05 were considered significant.



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Results Local administration of CD200 prevents AHR development, without directly altering tracheal contractility We evaluated whether local delivery of a recombinant CD200 (rCD200) can inhibit key features of asthma in a validated model of allergic airway inflammation in Brown Norway rats (18, 19). Naïve and sensitized rats, pre-treated or not with either the rCD200 or the isotype-matched recombinant Fc (sham), were challenged with OVA. 24 h later, respiratory system resistance (RRS) was evaluated in response to incremental doses of MCh. Sensitized animals that were either sham-treated or left untreated showed AHR to incremental doses of MCh compared with naïve animals (Figure 1A). In contrast, sensitized animals treated with rCD200 demonstrated a degree of responsiveness similar to naïve animals (Figure 1A). rCD200 neither altered MCh responsiveness in naïve animals (data not shown) nor affected baseline resistance in sensitized rats (0.13 ± 0.04 versus 0.19 ± 0.05 cmH2O · s/ml in sensitized animals treated with sham versus rCD200, respectively). To determine if rCD200 prevented AHR by directly attenuating the responsiveness of airway smooth muscle (ASM) to MCh, we measured the effect of rCD200 on isolated trachea contractility (Figure 1B-C). Neither the resting tension nor the contractile response to MCh was affected by rCD200 (Figure 1C). To further investigate the responsiveness of ASM to rCD200, we assessed CD200R mRNA expression in ASM of naïve and sensitized rats with/without allergen challenge. CD200R was not expressed in ASM in any of the conditions studied (Figure 1D). Thus, rCD200 inhibited AHR, without directly altering ASM contractility. 8 Copyright © 2015 by the American Thoracic Society

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rCD200 does not alter eosinophil recruitment, but modulates the expression of cytokines in BAL Given that the degree of airway responsiveness is influenced by many facets of inflammation, we harvested BAL 24 h after OVA challenge to investigate whether rCD200 interferes with allergen-induced inflammation. Total cell count in the BAL was increased in sensitized animals compared with the naïve group (Figure 2A), mainly because of eosinophil recruitment (Figure 2B). No modulation was observed between sensitized animals treated or not with either sham or rCD200, for both total cell count and eosinophil number (Figure 2). Furthermore, naïve animals treated with rCD200 showed similar BAL cell counts to untreated naïve controls (data not shown). Levels of the pro-inflammatory cytokines IL-5, IL-13 and TNF; and of the antiinflammatory cytokines IL-10 and TGF-β were also measured in BAL 24 h following allergen challenge. Whereas IL-13 level was increased in BAL of sensitized animals (sham or left untreated) compared with the naïve group, CD200 treatment prevented IL13 increase (Figure 3A). Furthermore, IL-10 level was increased in BAL of rCD200treated rats compared to all other groups (Figure 3B). IL-5, TNF, and TGF- β levels remained the same in all groups studied (Table 1). Therefore, rCD200 administration increased IL-10 production and prevented the increase of IL-13 induced by sensitization and challenge with OVA. DC and Th2 cell recruitment in the lung is inhibited by rCD200 Another factor that contributes to AHR development is the DC/Th2 axis (20). We first measured whether rCD200 reduced the accumulation of lung DC subsets. DC (MHC



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IIhigh/autofluorescencelow) were divided into myeloid DC (mDC; CD11b+) and plasmacytoid DC (pDC; CD11b-/CD4+) (Figure 4A). As previously reported (7, 21), mDC is the major lung DC subset in both naïve and sensitized rats following allergen challenge (Figure 4B). Sensitized animals that were sham-treated or left untreated had significantly more lung mDC compared with naïve rats. rCD200 treatment abolished mDC accumulation in sensitized rats (Figure 4B). rCD200-treated and -untreated naïve animals had similar numbers of lung mDC (5.9 ± 1.0 x 105 and 4.0 ± 0.8 x 105 cells per g of lung, respectively). In contrast to mDC, no variation in the number of pDC was observed between groups (Figure 4B). We then determined if the lack of DC accumulation in rCD200-treated animals translated into alterations in lung lymphocyte populations following OVA exposure (Figure 5A-C), given that the latter are involved in AHR development (22). Sensitized animals had more CD4+ T cells and Th2 cells compared with naïve rats (Figure 5B-D). Sensitized animals treated with rCD200 had a reduced number of CD4+ T cells (decreased by 42%) and Th2 cells (decreased by 49%) compared with the sham-treated group (Figure 5). In the absence of allergic inflammation, rCD200 had no effect on T cell populations (data not shown). Thus, rCD200 inhibited allergen-induced mDC recruitment, as well as Th2 cell accumulation in the lungs of allergen-sensitized animals. rCD200 does not alter allergen-induced Th2 accumulation in draining lymph nodes of asthmatic animals mDC migrate from lungs to draining lymph nodes (dLN) to induce the expansion of antigen-specific T cells. Given that rCD200 reduced lung accumulation of


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inflammatory Th2 cells, we investigated whether rCD200 interfered with the migration of allergen-loaded DC to dLN. In sensitized animals, there was an increased number of OVA+ mDC compared with naïve rats, whereas OVA+ pDC number was not significantly increased (Figure 6A). No modulation of allergen-loaded DC in dLN was observed in sensitized animals treated with rCD200 compared with sensitized animals that were either left untreated or treated with sham. Similarly, dLN CD4+ T cells (Figure 6B) and Th2 cells (Figure 6C) were both increased by sensitization and challenge, and these increases were also observed in both sham and rCD200 groups. Therefore, rCD200 did not alter DC and T cells in dLN.



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Discussion CD200 belongs to a novel family of cell-surface receptors that modulate the immune response. Indeed, the interaction of CD200 with CD200R is central in immunity, as it controls the activation of myeloid cell subsets (23) and it was proven effective in the treatment of several experimental immune diseases (24, 25). Although the inception of allergic asthma involved the dysregulation of myeloid cells and that CD200 acts on myeloid cells, no report has heretofore addressed the local role of CD200 in the context of asthma. Herein, we demonstrate that local administration of rCD200 completely inhibits AHR development in an experimental model of asthma. We also show that the decrease in airway responsiveness in asthmatic animals treated with rCD200 is not related to a direct inhibitory effect of rCD200 on ASM contractility. Instead, the normal level of airway responsiveness in sensitized animals treated with rCD200 occurs in conjunction with a complete prevention of mDC and Th2 cell accumulations within the lungs. In agreement with dampened T cell responses, rCD200 increases the production of the anti-inflammatory cytokine IL-10 and prevents the increase of IL-13. Together, these results indicate that local delivery of rCD200 is sufficient to inhibit AHR and that the mechanisms potentially arise from alleviation of mDC and Th2 cell accumulations. We show for the first time that airway delivery of rCD200 inhibits AHR in response to an allergen challenge. AHR is recognized as a distinguished feature of asthma (26) and it is used in clinic to confirm the diagnosis of asthma (27, 28). It is also accepted that the degree of airway responsiveness is a proper surrogate of the airway response that is expected to occur during the natural course of asthma when inflammation-derived spasmogens, such as leukotrienes, prostaglandin D2 and others, are endogenously


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released. Thus, AHR is thought to contribute importantly to asthma symptoms. Strategies that aim at preventing AHR development are thus conducive to salutary effects in the control of asthma symptoms. Our results demonstrated that, although rCD200 inhibits AHR, it does not act directly on ASM contractility. Our ex vivo study on isolated tracheas suggested that rCD200 does not affect the contractile capacity of ASM directly or through the epithelium (since the epithelium was intact in tracheal segments used to assess ASM contractility). We further demonstrated that CD200R mRNA is not expressed on ASM derived from either naïve or OVA sensitized/challenged rats. This provides a convincing explanation for the lack of direct effect of rCD200 on ASM. It is therefore likely that rCD200 prevented AHR in vivo by decreasing the contractile capacity of ASM via indirect mechanisms. It is well understood that the contractile capacity of ASM is not fixed, but rather plastic (adaptable) and that numerous inflammatory mediators that are overexpressed in the lung of asthmatics can modulate the contractile capacity of ASM (29). In that regard, we showed that airway delivery of rCD200 modulates soluble factors that can substantially influence many facets of the asthmatic responses, including Th2 immune activation, AHR, and ASM contractility. For example, the prevention of IL13 upregulation by rCD200 is of great interest. In asthmatic models, blockade of the IL13 pathway inhibits AHR development (5, 30) and IL-13 was repeatedly shown to increase the contractile capacity of ASM (reviewed in (31)). Thus, it is tempting to speculate that the ability of rCD200 to prevent IL-13 upregulation may concomitantly prevent the increased contractile capacity of ASM and, as a result, prevent the development of AHR. Several cell types in the lung can generate IL-13, but its major



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sources are Th2 cells (32).The lack of IL-13 upregulation following allergen challenge in sensitized rats treated with rCD200 may thus be a consequence of the failure to recruit or amplify Th2 cells into the lung. Accordingly, blockade of the IL-13 pathway (3-5, 30) or of CD4+ T cells (33, 34) were proven effective to inhibit the development of AHR. We also observed a concomitant increase of IL-10 in rCD200-treated rats. Conflicting literature exists on the induction of regulatory T cells by the CD200 pathway. For instance, a neutralizing anti-CD200 antibody favors regulatory T cell polarization (35), while upregulation of CD200 is associated with increased regulatory T cell levels in vivo (36), which agrees with the ability of the soluble form of CD200 to favor regulatory T cell development (37). Our results showing rCD200 to increase IL-10 levels, for which regulatory T cells are major producers, support that rCD200 could act similarly to endogenous soluble CD200 by favoring regulatory T cell development or activity. Alternatively, rCD200 could act on other cell types documented to release IL-10, including DC. The blockade of Th2 cell recruitment by rCD200 may thus occur through the increased expression of IL-10. In line with this conjecture, the overexpression of IL10 reduces allergen-induced AHR (38). Together, these results suggest that rCD200 may alleviate AHR by altering the inflammatory environment, mainly by switching the cytokine balance (IL-10 vs IL-13) towards homeostasis. rCD200 also reduced the accumulation of mDC and Th2 cells in the lungs in our study. This is consistent with several other studies. Indeed, Snelgroove et al (13) observed that CD200–/– mice demonstrated an enhanced accumulation of DC and CD4+ T cells in the lungs in their influenza model (13). Also, Li et al (39) showed that rCD200 reduces DC migration and T cell activation in vitro. Taken together, these results suggest


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that the prevention of AHR and the alteration of the cytokine environment by rCD200 may occur by affecting the functions or the mobilization of mDC/Th2 cells into the lungs. This is further supported by the ability of the CD200-CD200R pathway to shift the balance of cytokines released by DC towards a regulatory phenotype (36). Interestingly, in our setting, rCD200 did not alter the accumulation of mDC nor the Th2 polarization of T cells in the dLN. The possibility that rCD200 could eventually interfere with dLN biology in a chronic setting remains. Our results thus suggest that in the context of an acute response, rCD200 potently interferes with local immunopathogenic mechanisms, most likely through the activation of CD200R. Indeed, in human and rats, the CD200R is the only receptor that has been described to bind CD200 (23), even though other receptors closely related to CD200R have been described in mice (CD200R2-4), their affinity for CD200 is still questioned (40-42). Thus, rCD200 could alter mDC recruitment to the lungs either via the binding of CD200R on mDC or via the activation of CD200R on adjacent cells in the lungs (e.g. alveolar macrophages). While effective at preventing AHR and Th2 cell accumulation, rCD200 administration did not alter eosinophil accumulation. This is reminiscent of the effect of IL-13 inhibition, which abrogated AHR without affecting eosinophilia (5). Although there is still a debate on the source of eosinophil chemotactic mediators, many studies report that the epithelial cells are major producers of eotaxins (43). Given that CD200R is not expressed by the epithelium (10), rCD200 administration cannot directly modulate epithelial cell functions. Therefore, epithelium production of eotaxins or other eosinophilic chemokines may rest unaltered in the presence of rCD200 and eosinophilia



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proceeds as in the absence of CD200. Notwithstanding the potentially detrimental effect of prolong eosinophilic inflammation (44), our results clearly indicate that resolving eosinophilia is not required for rCD200 to interfere with the acute development of AHR induced by allergen sensitization and challenge. Instead, our results are in agreement with new evidence supporting that T cells could be sufficient to modulate contractile properties of ASM (45). Our results also support the idea that the CD200 pathway might apply to clinical cases where AHR is uncoupled from eosinophilic inflammation (46, 47). Overall, this study is the first to demonstrate that administration of rCD200 abrogates the development of one of the root causes of asthma, namely AHR. The finding is important as it potentially carries major clinical implication. Yet, we need to stress out the limitation of the prophylactic course of rCD200 treatment in our study. The possibility remains that the response to rCD200 can be modified in chronically and extensively remodeled airways. We also partially unveiled the operational mechanisms by which rCD200 inhibits AHR development. CD200 seems to act by stopping the recruitment of mDC and T cells into the lungs, either directly or by increasing IL-10 production. In turn, the absence of Th2 cell recruitment into the lungs is likely linked with the decreased IL-13 level, a cytokine that fosters AHR by increasing ASM contractile capacity. Since rCD200 could act on many targets, including mDC, mast cells and alveolar macrophages, we are currently extending the mechanism of action of rCD200 to these cell types. Collectively, our results represent the foundational work that paved the way to further studies that will determine whether the modulation of the CD200 pathway is a viable strategy to interfere with AHR in asthma.


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Acknowledgements We thank Emilie Bernatchez and Marc Veillette for technical help, and Dr Yvon Cormier for critical review of the manuscript.



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References 1.

Lewkowich IP, Lajoie S, Clark JR, Herman NS, Sproles AA, Wills-Karp M.

Allergen uptake, activation, and IL-23 production by pulmonary myeloid DCs drives airway hyperresponsiveness in asthma-susceptible mice. PLoS One 2008;3:e3879. 2.

Plantinga M, Guilliams M, Vanheerswynghels M, Deswarte K, Branco-Madeira

F, Toussaint W, Vanhoutte L, Neyt K, Killeen N, Malissen B, Hammad H, Lambrecht BN. Conventional and Monocyte-Derived CD11b(+) Dendritic Cells Initiate and Maintain T Helper 2 Cell-Mediated Immunity to House Dust Mite Allergen. Immunity 2013;38:322-335. 3.

Grunig G, Warnock M, Wakil AE, Venkayya R, Brombacher F, Rennick DM,

Sheppard D, Mohrs M, Donaldson DD, Locksley RM, Corry DB. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 1998;282:2261-2263. 4.

Wills-Karp M, Luyimbazi J, Xu X, Schofield B, Neben TY, Karp CL, Donaldson

DD. Interleukin-13: central mediator of allergic asthma. Science 1998;282:2258-2261. 5.

Leigh R, Ellis R, Wattie J, Donaldson DD, Inman MD. Is interleukin-13 critical in

maintaining airway hyperresponsiveness in allergen-challenged mice? American journal of respiratory and critical care medicine 2004;170:851-856. 6.

Busse WW. The relationship of airway hyperresponsiveness and airway

inflammation: Airway hyperresponsiveness in asthma: its measurement and clinical significance. Chest 2010;138:4S-10S. 7.

Umetsu DT, Dekruyff RH. Immune dysregulation in asthma. Curr Opin Immunol

2006;18:727-732.



Page 19 of 35

8.

Lipscomb MF, Wilder JA. Immune dysregulation as a cause for allergic asthma.

Curr Opin Pulm Med 1999;5:10-20. 9.

Wissinger E, Goulding J, Hussell T. Immune homeostasis in the respiratory tract

and its impact on heterologous infection. Semin Immunol 2009;21:147-155. 10.

Holt PG, Strickland DH. The CD200-CD200R axis in local control of lung

inflammation. Nature immunology 2008;9:1011-1013. 11.

Gorczynski RM. CD200:CD200R-Mediated Regulation of Immunity. ISRN

Immunology 2012;2012:18. 12.

Holmannova D, Kolackova M, Kondelkova K, Kunes P, Krejsek J, Ctirad A.

CD200/CD200R paired potent inhibitory molecules regulating immune and inflammatory responses; Part II: CD200/CD200R potential clinical applications. Acta medica 2012;55:59-65. 13.

Snelgrove RJ, Goulding J, Didierlaurent AM, Lyonga D, Vekaria S, Edwards L,

Gwyer E, Sedgwick JD, Barclay AN, Hussell T. A critical function for CD200 in lung immune homeostasis and the severity of influenza infection. Nature immunology 2008;9:1074-1083. 14.

Aoki T, Matsumoto Y, Hirata K, Ochiai K, Okada M, Ichikawa K, Shibasaki M,

Arinami T, Sumazaki R, Noguchi E. Expression profiling of genes related to asthma exacerbations. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology 2009;39:213-221. 15.

Labonte I, Hassan M, Risse PA, Tsuchiya K, Laviolette M, Lauzon AM, Martin

JG. The effects of repeated allergen challenge on airway smooth muscle structural and



Page 20 of 35

molecular remodeling in a rat model of allergic asthma. American journal of physiology Lung cellular and molecular physiology 2009;297:L698-705. 16.

Careau E, Turmel V, Lauzon-Joset JF, Bissonnette EY. Alveolar macrophages

reduce airway hyperresponsiveness and modulate cytokine levels. Experimental lung research 2010;36:255-261. 17.

Strickland DH, Thomas JA, Mok D, Blank F, McKenna KL, Larcombe AN, Sly

PD, Holt PG. Defective aeroallergen surveillance by airway mucosal dendritic cells as a determinant of risk for persistent airways hyper-responsiveness in experimental asthma. Mucosal immunology 2012;5:332-341. 18.

Tschernig T, Neumann D, Pich A, Dorsch M, Pabst R. Experimental bronchial

asthma - the strength of the species rat. Current drug targets 2008;9:466-469. 19.

Martin JG, Tamaoka M. Rat models of asthma and chronic obstructive lung

disease. Pulmonary pharmacology & therapeutics 2006;19:377-385. 20.

Koya T, Matsuda H, Matsubara S, Miyahara N, Dakhama A, Takeda K, Gelfand

EW. Differential effects of dendritic cell transfer on airway hyperresponsiveness and inflammation. American journal of respiratory cell and molecular biology 2009;41:271280. 21.

Lauzon-Joset JF, Marsolais D, Langlois A, Bissonnette EY. Dysregulation of

alveolar macrophages unleashes dendritic cell-mediated mechanisms of allergic airway inflammation. Mucosal immunology 2014;7:155-164. 22.

Venkayya R, Lam M, Willkom M, Grunig G, Corry DB, Erle DJ. The Th2

lymphocyte products IL-4 and IL-13 rapidly induce airway hyperresponsiveness through



Page 21 of 35

direct effects on resident airway cells. American journal of respiratory cell and molecular biology 2002;26:202-208. 23.

Minas K, Liversidge J. Is the CD200/CD200 receptor interaction more than just a

myeloid cell inhibitory signal? Crit Rev Immunol 2006;26:213-230. 24.

Liu Y, Bando Y, Vargas-Lowy D, Elyaman W, Khoury SJ, Huang T, Reif K,

Chitnis T. CD200R1 agonist attenuates mechanisms of chronic disease in a murine model of multiple sclerosis. The Journal of neuroscience : the official journal of the Society for Neuroscience 2010;30:2025-2038. 25.

Gorczynski RM, Chen Z, Yu K, Hu J. CD200 immunoadhesin suppresses

collagen-induced arthritis in mice. Clinical immunology 2001;101:328-334. 26.

Woolcock AJ, Salome CM, Yan K. The shape of the dose-response curve to

histamine in asthmatic and normal subjects. Am Rev Respir Dis 1984;130:71-75. 27.

Crapo RO, Casaburi R, Coates AL, Enright PL, Hankinson JL, Irvin CG,

MacIntyre NR, McKay RT, Wanger JS, Anderson SD, Cockcroft DW, Fish JE, Sterk PJ. Guidelines for methacholine and exercise challenge testing-1999. This official statement of the American Thoracic Society was adopted by the ATS Board of Directors, July 1999. American journal of respiratory and critical care medicine 2000;161:309-329. 28.

Bateman ED, Hurd SS, Barnes PJ, Bousquet J, Drazen JM, FitzGerald M, Gibson

P, Ohta K, O'Byrne P, Pedersen SE, Pizzichini E, Sullivan SD, Wenzel SE, Zar HJ. Global strategy for asthma management and prevention: GINA executive summary. Eur Respir J 2008;31:143-178. 29.

Bosse Y. Asthmatic airway hyperresponsiveness: the ants in the tree. Trends in

molecular medicine 2012;18:627-633.



30.

Page 22 of 35

Yang G, Volk A, Petley T, Emmell E, Giles-Komar J, Shang X, Li J, Das AM,

Shealy D, Griswold DE, Li L. Anti-IL-13 monoclonal antibody inhibits airway hyperresponsiveness, inflammation and airway remodeling. Cytokine 2004;28:224-232. 31.

Bossé Y, Paré PD. The contractile properties of airway smooth muscle: How their

defects can be linked to asthmatic airway hyperresponsiveness? Curr Respir Med Rev 2013;9:42-68. 32.

Hershey GK. IL-13 receptors and signaling pathways: an evolving web. The

Journal of allergy and clinical immunology 2003;111:677-690; quiz 691. 33.

Gavett SH, Chen X, Finkelman F, Wills-Karp M. Depletion of murine CD4+ T

lymphocytes

prevents

antigen-induced

airway

hyperreactivity

and

pulmonary

eosinophilia. American journal of respiratory cell and molecular biology 1994;10:587593. 34.

Mishima H, Hojo M, Watanabe A, Hamid QA, Martin JG. CD4+ T cells can

induce airway hyperresponsiveness to allergen challenge in the brown norway rat. American journal of respiratory and critical care medicine 1998;158:1863-1870. 35.

Rygiel TP, Luijk B, Meyaard L. Use of an anti-CD200 antibody for prolonging

the survival of allografts: a patent evaluation of WO2012106634A1. Expert opinion on therapeutic patents 2013;23:389-392. 36.

Memarian A, Nourizadeh M, Masoumi F, Tabrizi M, Emami AH, Alimoghaddam

K, Hadjati J, Mirahmadian M, Jeddi-Tehrani M. Upregulation of CD200 is associated with Foxp3+ regulatory T cell expansion and disease progression in acute myeloid leukemia.

Tumour

biology

:

the

journal

of

the

International

Society

for

Oncodevelopmental Biology and Medicine 2013;34:531-542.



Page 23 of 35

37.

Gorczynski R, Chen Z, Khatri I, Yu K. sCD200 present in mice receiving cardiac

and skin allografts causes immunosuppression in vitro and induces Tregs. Transplantation 2013;95:442-447. 38.

Oh JW, Seroogy CM, Meyer EH, Akbari O, Berry G, Fathman CG, Dekruyff RH,

Umetsu DT. CD4 T-helper cells engineered to produce IL-10 prevent allergen-induced airway hyperreactivity and inflammation. The Journal of allergy and clinical immunology 2002;110:460-468. 39.

Li Y, Zhao LD, Tong LS, Qian SN, Ren Y, Zhang L, Ding X, Chen Y, Wang YX,

Zhang W, Zeng XF, Zhang FC, Tang FL, Zhang X, Ba DN, He W, Cao XT, Lipsky PE. Aberrant CD200/CD200R1 expression and function in systemic lupus erythematosus contributes to abnormal T-cell responsiveness and dendritic cell activity. Arthritis research & therapy 2012;14:R123. 40.

Wright GJ, Cherwinski H, Foster-Cuevas M, Brooke G, Puklavec MJ, Bigler M,

Song Y, Jenmalm M, Gorman D, McClanahan T, Liu MR, Brown MH, Sedgwick JD, Phillips JH, Barclay AN. Characterization of the CD200 receptor family in mice and humans and their interactions with CD200. Journal of immunology 2003;171:3034-3046. 41.

Hatherley D, Cherwinski HM, Moshref M, Barclay AN. Recombinant CD200

protein does not bind activating proteins closely related to CD200 receptor. Journal of immunology 2005;175:2469-2474. 42.

Gorczynski R, Chen Z, Kai Y, Lee L, Wong S, Marsden PA. CD200 is a ligand

for all members of the CD200R family of immunoregulatory molecules. Journal of immunology 2004;172:7744-7749.



43.

Page 24 of 35

Ying S, Robinson DS, Meng Q, Rottman J, Kennedy R, Ringler DJ, Mackay CR,

Daugherty BL, Springer MS, Durham SR, Williams TJ, Kay AB. Enhanced expression of eotaxin and CCR3 mRNA and protein in atopic asthma. Association with airway hyperresponsiveness and predominant co-localization of eotaxin mRNA to bronchial epithelial and endothelial cells. European journal of immunology 1997;27:3507-3516. 44.

Humbles AA, Lloyd CM, McMillan SJ, Friend DS, Xanthou G, McKenna EE,

Ghiran S, Gerard NP, Yu C, Orkin SH, Gerard C. A critical role for eosinophils in allergic airways remodeling. Science 2004;305:1776-1779. 45.

Matusovsky OS, Nakada EM, Kachmar L, Fixman ED, Lauzon AM. CD4+ T

cells enhance the unloaded shortening velocity of airway smooth muscle by altering the contractile protein expression. The Journal of physiology 2014;592:2999-3012. 46.

Alvarez MJ, Olaguibel JM, Garcia BE, Rodriquez A, Tabar AI, Urbiola E.

Airway inflammation in asthma and perennial allergic rhinitis. Relationship with nonspecific bronchial responsiveness and maximal airway narrowing. Allergy 2000;55:355-362. 47.

Crimi E, Spanevello A, Neri M, Ind PW, Rossi GA, Brusasco V. Dissociation

between airway inflammation and airway hyperresponsiveness in allergic asthma. American journal of respiratory and critical care medicine 1998;157:4-9.


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Table 1: Cytokine levels in BAL supernatant Groups of rats were sensitized i.p. with ovalbumin (OVA)/Alum and received recombinant IgG (Sham) or CD200 chimera (rCD200) i.t. 24 h before allergen challenge. Naïve and sensitized animals were challenged with OVA and BAL fluids were harvested after 24 h to measure cytokine levels. TNF, IL-5, and TGF were measured by ELISA. No difference was observed between the groups. n = 4-7.

Naïve Sensitized Sham rCD200

TNF IL-5 TGF (pg/mL) (pg/mL) (pg/mL) 21 ± 4 12 ± 2 39 ± 9 25 ± 3 13 ± 4 42 ± 6 27 ± 5 12 ± 3 41 ± 6 19 ± 3 14 ± 6 36 ± 9



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Figure legends Figure 1: rCD200 reduces airway hyperresponsiveness, but does not affect smooth muscle contractility Rats were sensitized i.p. with ovalbumin (OVA)/Alum and received recombinant IgG (Sham) or CD200 chimera (rCD200) i.t. 24 h before allergen challenge. Naïve and sensitized animals were challenged with OVA and the respiratory system resistance (RRS) was analyzed after 24 h. A) RRS was assessed after nebulisation with incremental doses of MCh. n = 4-7. B-C) The contractile capacity of trachea was assessed with MCh stimulations (see supplemental methods for details). D) ASM from naïve, OVAsensitized, and OVA-sensitized/challenged rats were isolated and CD200R expression was assessed by PCR. Positive controls are a macrophage cell line (NR-8383) and a mast cell line (RBL-2H3). This is a representative experiment out of 3. Asterisk indicates significant differences (P < 0.05) compared with the naïve group. Figure 2: rCD200 does not modulate cell recruitment in BAL Rats were sensitized i.p. with ovalbumin (OVA)/Alum and received recombinant IgG (Sham) or CD200 chimera (rCD200) i.t. 24 h before allergen challenge. Naïve and sensitized animals were challenged with OVA and BAL were analyzed after 24 h. A) Total cell counts and B) cellularity were measured after Trypan blue and DiffQuick stainings, respectively. AM: alveolar macrophages; Eos: eosinophils; Neutro: neutrophils. n = 4-7. Asterisk indicates significant differences (P < 0.05) compared with the naïve group. Figure 3: rCD200 treatment reduces IL-13 and increases IL-10 levels in BAL



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Rats were sensitized i.p. with ovalbumin (OVA)/Alum and received recombinant IgG (Sham) or CD200 chimera (rCD200) i.t. 24 h before allergen challenge. Naïve and sensitized animals were challenged with OVA and BAL fluids were harvested after 24 h to measure cytokine levels. A) IL-13 and B) IL-10 were measured by ELISA. n = 4-7. Asterisk indicates significant differences (P < 0.05) compared with the naïve group. Figure 4: mDC recruitment to the lung of asthmatic rats is inhibited by rCD200 treatment Rats were sensitized i.p. with ovalbumin (OVA)/Alum and received recombinant IgG (Sham) or CD200 chimera (rCD200) i.t. 24 h before allergen challenge. Naïve and sensitized animals were challenged with OVA and after 24 h, single cell suspensions were prepared from lungs and analysed by flow cytometry. Myeloid DC (mDC) were gated on non-autofluorescent cells and identified as CD11b+ and major histocompatibility complex (MHC) II+ cells, whereas plasmacytoid DC (pDC) were gated on nonautofluorescent CD4+ cells and identified as CD11b–, CD4+ and MHC II+ cells. mDC and pDC A) frequencies of total lung cells and B) number per g of lung were measured. n = 4-7. Asterisk indicates significant differences (P < 0.05). Figure 5: rCD200 reduces the accumulation of Th2 cells in the lung of asthmatic animals Rats were sensitized i.p. with ovalbumin (OVA)/Alum and received recombinant IgG (Sham) or CD200 chimera (rCD200) i.t. 24 h before allergen challenge. Naïve and sensitized animals were challenged with OVA and after 24 h, single cell suspensions were prepared from lungs and analysed by flow cytometry. CD4+ T cell (CD3+) and IL-4+



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Th2 cell frequencies of total lung cells (A;C, respectively) and number per g of lung (B;D, respectively) were measured. n = 4-7. Asterisk indicates significant differences (P < 0.05). Figure 6: rCD200 does not alter dLN DC and T cell numbers Rats were sensitized i.p. with ovalbumin (OVA)/Alum and received recombinant IgG (Sham) or CD200 chimera (rCD200) i.t. 24 h before allergen challenge. Naïve and sensitized animals were challenged with OVA-AlexaFluor647 and after 24 h, single cell suspensions were prepared from draining lymph nodes (dLN). OVA+ DC was assessed by measuring the frequency of AlexaFluor647+ DC using flow cytometry. A) OVA+ mDC and pDC number in dLN were measured. CD4+ T cell and IL-4+ Th2 numbers of total dLN cells (B;C, respectively) were measured. n = 4-7. Asterisk indicates significant differences (P < 0.05) compared with the naïve group.


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Online data supplement

Material and methods

Contractility of isolated trachea Tracheas were excised from naïve Brown Norway rats and mounted within a 37°C–water jacketed organ bath filled with Krebs solution. The resting tension was set to 0.5 g (Unstimulated; Uns). The contractility was measured with an isometric force transducer (Harvard Apparatus, Holliston (MA), USA) and recorded by a data acquisition system (MP 150 of Biopac Systems and AcqKnowledge 3.7.3 software). The tracheas were stimulated with MCh at 10-5 M during 5 min until 3 sequential contractions displayed similar values (Ctrl). Each trachea was then treated with a single dose (100 µmol) of either sham or rCD200. After 60 min of incubation, a final stimulation with MCh at 10-5 M was performed. The results presented are the total force (i.e. resting tension + the force generated by MCh) and are expressed relative to their own control obtained prior to the incubation with either molecule.


Asthma and airway hyperresponsiveness.

Beta-agonists, airway hyperresponsiveness, and asthma.

Small airway impairment and bronchial hyperresponsiveness in asthma onset.

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Association of Specific Immunoglobulin E to Staphylococcal Enterotoxin with Airway Hyperresponsiveness in Asthma Patients.

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Exhaled Nitric Oxide and Airway Hyperresponsiveness to Adenosine 5'-monophosphate and Methacholine in Children with Asthma.

Separate and combined effects of corticosteroids and bronchodilators on airflow obstruction and airway hyperresponsiveness in asthma.

P2Y12 antagonist attenuates eosinophilic inflammation and airway hyperresponsiveness in a mouse model of asthma.

Interleukin 33 exacerbates antigen driven airway hyperresponsiveness, inflammation and remodeling in a mouse model of asthma.

Fenretinide prevents inflammation and airway hyperresponsiveness in a mouse model of allergic asthma.

Concomitant exposure to ovalbumin and endotoxin augments airway inflammation but not airway hyperresponsiveness in a murine model of asthma.

Commentaries on Viewpoint: Airway smooth muscle and airway hyperresponsiveness in human asthma: have we chased the wrong horse?

Lymphokine-induced airway hyperresponsiveness in the rat.

Chimeric Antigen Receptor-Redirected Regulatory T Cells Suppress Experimental Allergic Airway Inflammation, a Model of Asthma.

Changes of airway smooth muscle in experimental asthma.

Airway smooth muscle mechanics and biochemistry in experimental asthma.