CR3-dependent negative regulation of human eosinophils by Mycobacterium bovis BCG lipoarabinomannan

Immunology Letters 143 (2012) 202–207

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CR3-dependent negative regulation of human eosinophils by Mycobacterium bovis BCG lipoarabinomannan Virginie Driss a,e , Emmanuel Hermann a,f,g , Fanny Legrand a,h , Sylvie Loiseau a,h , Marie Delbeke a,h , Laurent Kremer b,c , Yann Guerardel d , David Dombrowicz a,i , Monique Capron a,h,∗ a

Inserm, U547, Lille, France; Université Lille 2, Lille, France; Institut Pasteur de Lille, 59019 Lille, France Laboratoire de Dynamique des Interactions Membranaires Normales ET Pathologiques, Université de Montpellier II et I, CNRS; UMR 5235, case 107, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France c Inserm, DIMNP, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France d CNRS UMR 8576, Lille, France; Université des Sciences ET Technologies de Lille, 59655 Villeneuve d’Ascq Cedex, France e Inserm U837, Institut pour la Recherche sur le Cancer de Lille, Université Lille 2, Lille, France f Université Lille2, INSERM U1019,CNRS UMR 8204, IPL, F-59000 Lille, France g UDSL, Faculté des Sciences Pharmaceutiques et Biologiques, F-59006 Lille, France h Inserm U995, Faculté de Médecine, Université Lille 2, Lille France i Inserm U1011, Université Lille 2, Institut Pasteur de Lille, Lille, France b

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Article history: Received 5 December 2011 Received in revised form 16 February 2012 Accepted 16 February 2012 Available online 25 February 2012 Keywords: Eosinophil Complement receptor Mycobacterium Lipoarabinomannan Innate immunity

a b s t r a c t Eosinophils have recently been shown to participate in innate immune responses against mycobacteria. We have investigated whether Mycobacterium bovis BCG regulate the human eosinophil immune response. A negative correlation between mycobacteria internalization and eosinophil activation was observed. In addition, mannose-capped lipoarabinomannan from M. bovis BCG (ManLAM) failed to induce a significant release of eosinophil peroxidase and TNF-␣. Noteworthy, ManLAM exhibited a potent inhibitory effect on eosinophil peroxidase release by TLR2-activated eosinophils involving the complement receptor-3 molecule and the phosphatidylinositol-3 kinase pathway. ManLAM, generally present in pathogenic mycobacteria, plays an important role in modulating eosinophil-dependent immune response. © 2012 Elsevier B.V. All rights reserved.

1. Introduction The mycobacterial cell wall comprises a vast array of proteins, lipids and lipoglycans, including lipoarabinomannan (LAM) and its related biosynthetic precursor lipomannan (LM), known to modulate the host immune system [1,2]. Both Mycobacterium tuberculosis and Mycobacterium bovis BCG express mannose-capped LAM (ManLAM), inhibiting IL-12 production in LPS-stimulated human mononuclear phagocytes and in dendritic cells (DCs) through binding to mannose receptor (MR) [3]. LAM from M. tuberculosis also recognizes dendritic cell-specific ICAM 3-grabbing non-integrin (DC-SIGN) molecule [4] and prevents mycobacteria- or LPS-induced DC maturation [5]. Moreover, other immune receptors, such as complement receptors (CR), are also involved in the interactions with mycobacteria [6]. Taken together, these studies illustrate close

relationships between innate receptors and cell wall-associated lipoglycans in regulating innate immune responses. Most studies have focused on macrophages and DCs to study receptor/ligand interactions influencing host resistance or susceptibility to mycobacterial infection. However, little is known regarding the eosinophil surface receptors potentially involved in eosinophil-mycobacteria interactions, despite the fact that eosinophil recruitment within mycobacterial granuloma has been observed in different animal models [7]. We have recently provided evidence for a direct TLR2-dependent interaction of M. bovis BCG with human eosinophils [8]. Since eosinophils have the potential to express CR, especially CR3 (CD11b/CD18) [9], this study aims to investigate whether M. bovis BCG can interact with eosinophil CR and may influence the subsequent CR-mediated activation process. 2. Materials and methods

∗ Corresponding author at: Inserm U995. Faculté de Médecine. Pôle Recherche. 1 place Verdun 59045 Lille Cedex. France. Tel.: +33 0 6 62 03 27 13; fax: +33 0 3 20 96 86 62. E-mail address: [email protected] (M. Capron). 0165-2478/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2012.02.011

2.1. Eosinophil and lipoglycan purification Eosinophils were isolated from peripheral blood of donors following informed consent. Eosinophils were separated from

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peripheral-blood mononuclear cells (PBMCs) by Percoll centrifugation as previously described [10]. Eosinophil purity checked by cytocentrifugated preparations after RAL555 coloration was found to be >98%. Lipomannan and lipoarabinomannan from M. bovis BCG were purified as previously described [11]. 2.2. Mycobacterial cultures M. bovis BCG Pasteur strain was obtained from Dr. C. Locht (Inserm U1019, Pasteur Institute, Lille, France) and maintained at 37 ◦ C in Sauton’s liquid medium. 2.3. Eosinophil degranulation (2 × 105

cells in 100 ␮L) were incubated for 2 h in Eosinophils RPMI 1640 without phenol red, with stimuli at 37 ◦ C in 5% CO2 . EPO activity in eosinophil supernatants was measured by oxidation of H2 O2 by luminol as previously described [10]. Chemiluminescence was measured with a luminometer (VictorTM Wallac). For inhibition experiments, eosinophils were preincubated with blocking anti-CR3 monoclonal antibody (mAb) (Bear-1; Santa Cruz, California, USA), blocking anti-CR4 mAb (BU15; Santa Cruz Biotechnology, California, USA), total anti-mouse (mIgG) isotype control (Jackson ImmunoResearch Laboratories, West Grove, PA), PI3K inhibitor (LY294002; Promega, Wisconsin, USA) for 30 min at 37 ◦ C before addition of the stimulus without washing. Cell viability was tested using the trypan blue test after treatment. 2.4. Cytokine stimulation assay After 18 h of eosinophil stimulation, the levels of TNF-␣ in the supernatants were determined by performing an ELISA (Diaclone) according to the manufacturer’s recommendations. 2.5. Internalization assays 2.5.1. Carboxyfluorescein diacetate-succinimidyl ester (CFSE)-labeled eosinophils Cells were resuspended at a concentration of 4 × 106 cells/mL in PBS. CFSE (Invitrogen, California) was added to a final concentration of 20 ␮M and incubated at room temperature for 10 min in the darkness. The cells were washed three times with DPBS. 2.5.2. PKH26-labeled mycobacteria Mycobacteria were washed and resuspended at a concentration of 108 bacteria/mL in diluent C. Mycobacteria were incubated with 20 ␮M PKH26 (Sigma–Aldrich) for 15 min at room temperature. Reaction was blocked by decomplemented FCS for 1 min. The mycobacteria were washed three times with DPBS. On the day of each experiment, CFSE-labeled eosinophils were incubated with PKH26-labeled mycobacteria at different mycobacteria:eosinophils ratios (5:1, 10:1, 20:1) in RPMI 1640 without phenol red and without serum at 37 ◦ C in 5% CO2 . After selected incubation times, eosinophils were washed in cold RPMI to stop internalization and remove non-adherent mycobacteria. Trypan blue (5 mg/mL) was added on slides to quench adherent mycobacteria and those remaining in the extracellular medium [12]. The trypan blue-treated cells were washed three times with DPBS before fixation in 1% paraformaldehyde in PBS, pH 7.4, 30 min at room temperature and stored at 4 ◦ C in the dark until analyze. Cells containing fluorescent mycobacteria were counted by alternately viewing them by phase contrast and fluorescence microscopy. For each condition, at least 100 cells were counted. Inhibitory experiments were performed as described above (eosinophils were

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preincubated with blocking anti-CR3 monoclonal antibody mAb or PI3K inhibitor before mycobacteria infection). 2.6. Flow cytometry analysis Human eosinophils (2 × 106 /mL) were stimulated for 2 h in RPMI 1640 with or without stimulus as indicated in figure legends. 2 × 105 cells were incubated in PBS containing 1% BSA with detection antibody or isotype control at 4 ◦ C for 40 min. Cell surface staining was performed on cells with PE-anti-CD69 (BD Pharmingen), or the matched isotype control PE-mIgG1 (BD Pharmingen). The cells were then washed and were immediately analyzed on a FACSCaliburTM . 2.7. Statistical analysis All data were expressed as mean ± S.E.M. All statistical analyses were performed using SPSS software. Normality of data samples was assessed with the Normality test of Shapiro and Wilk. Parametric Student’s t-test for paired experiments was employed to compare two variables. ANOVA followed by Dunnett’s post-test were employed for comparisons of more than two data sets. A P value of less than 0.05 was used to indicate statistical significance. *P < 0.05 and **P < 0.01 are presented on the figures. 3. Results 3.1. Human eosinophils internalize M. bovis BCG Eosinophils are characterized by several functional properties, including bacterial internalization [13]. First, the ability of eosinophils to ingest live M. bovis BCG was studied. Purified human eosinophils were incubated for different times (0, 5, 10, 20 and 40 min) in the presence of mycobacterial suspensions at a multiplicity of infection (MOI) of 10:1 (Fig. 1A). In order to discriminate between extracellular adherent M. bovis BCG and those that had been internalized, we used the trypan blue quenching technique (see material and methods). Adjusting the focus at different depths, we show that Human eosinophils contained one to four red fluorescent mycobacteria, which appeared clearly intracellular, by fluorescence microscopy (Fig. 1B). Interestingly, these data demonstrate the efficient internalization of M. bovis BCG by human eosinophils. 3.2. CR3 modulates eosinophil activation in response to M. bovis BCG Macrophage phagocytosis of M. tuberculosis can be mediated by CR3 and CR4 [14]. Similarly, in this study, we investigated if M. bovis BCG internalization could be mediated by CR3 and CR4. We showed that M. bovis BCG internalization by eosinophils was significantly inhibited by the anti-CR3 antibody (73.3 ± 3.4% inhibition). No effect was observed with either the anti-CR4 antibody or the isotype control antibody (Fig. 2A). As we could not exclude the possibility that eosinophil activation might also be affected by the anti-CR3 antibody, we studied cytotoxic specific granule proteins, such as EPO or cytokine release, such as TNF␣ (Fig. 2B and C). We demonstrated EPO and TNF␣ releases by eosinophils in the presence of M. bovis BCG at a ratio 10:1. There was also a significant increase of EPO (by 33.3 ± 10.4%) and TNF␣ (51.13 ± 6.5%) when cells were pre-treated with the blocking anti-CR3 antibody. No increase was observed when the anti-CR4 or the isotype control was added. To further understand the involvement of bacteria internalization in eosinophil activation, eosinophils were pre-treated with 5 ␮g/mL cytochalasin D (Fig. 2D). Indeed, cytochalasin D blocked

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the entry of M. bovis BCG by the disruption of actin cytoskeleton. After having verified that cytochalasin D inhibits mycobacteria internalization, we have measured the level of EPO release after addition of mycobacteria on cytochalasin D-treated eosinophils. Results shown in Fig. 2D indicate that eosinophil pretreatment with cytochalasin D caused a slight but significant increase of EPO released by eosinophils in the presence of M. bovis BCG. To explore the relationship between the activation status of eosinophils and mycobacteria ingestion, we performed experiments with double staining: (CFSE-labeled mycobacteria and APC-anti-CD69), analyzed by flow cytometry (Fig. 2E). After the trypan blue quenching, no surface CD69 expression was observed on eosinophils ingesting CFSE-labeled mycobacteria (CFSE+ eosinophils). CFSE− eosinophils correspond to eosinophils binding extracellular mycobacteria or without mycobacteria. Only eosinophils without internalized mycobacteria were CD69+ . Interestingly, when mycobacteria internalization was inhibited by anti-CR3, 90.9% of eosinophils expressed CD69. Taken together, these data suggest that, M. bovis BCG internalization could modulate eosinophil activation regarding EPO release.

3.3. CR3-dependent M. bovis BCG internalization and CR3-dependent eosinophil activation involves PI3K

Fig. 1. M. bovis-BCG internalization by human eosinophils. (A) CFSE-eosinophils were incubated with or without PKH26-mycobacteria (mycobacteria: eosinophils = 10:1) at 37 ◦ C at different times (0, 5, 10, 20 and 40 min). Data are shown as the mean of 3 independent experiments ± SEM, expressed in percentage of eosinophils with fluorescent mycobacteria. (B) The percentage of eosinophils ingesting 1, 2, 3 and 4 fluorescent mycobacteria was determined by fluorescence microscopy after 40 min of incubation. Data are shown as the mean of 4 independent experiments ± SEM.

Previous data have shown that phosphoinositol 3 kinase(PI3K) is rapidly activated following CR3 engagement [15]. We then focused our attention on the PI3K pathway and determined whether this signaling molecule plays a role in CR3-dependent internalization of M. bovis BCG and in EPO degranulation or TNF␣ release (Fig. 3A–C). Pre-incubation of eosinophils with LY294002 inhibited M. bovis BCG uptake in a dose-dependent manner. Degranulation of EPO and release of TNF␣ in response to M. bovis BCG was found to be significantly higher when LY294002 was added (30.7 ± 15.8% and 44.6 ± 3.2%). These data suggest that CR3 associated with the PI3K signaling pathway in eosinophils plays a role in the M. bovis BCG internalization and negatively affects eosinophil activation.

Fig. 2. Involvement of CR3 in M. bovis BCG internalization and eosinophil activation. (A) Regulation of mycobacterial internalization (mycobacteria:eosinophils = 10:1 or 0:1), (B) EPO and (C) TNF␣ release by anti-CR3 and anti-CR4 blocking antibodies (20 ␮g/mL) or an isotype control antibody was measured. (D) After 30 min of incubation with cytochalasin D (5 ␮g/mL), eosinophils were stimulated with mycobacteria (10:1) and EPO degranulation was measured. (E) Purified human eosinophils were incubated with CFSE-labeled mycobacteria at 10:1 ratio (40 min, 37 ◦ C). For internalization inhibition experiment, eosinophils were pretreated with anti-CR3 antibody. Data are shown as the mean of 3 independent experiments ± SEM.

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Fig. 3. Role of PI3 kinase pathways in M. bovis BCG internalization and eosinophil activation. Purified eosinophils were pre-treated with or without LY294002, a PI3 kinase inhibitor (0.2, 2 or 20 ␮M) at 37 ◦ C for 30 min and were incubated with M. bovis BCG (10:1). Mycobacterial internalization (A), EPO (B) and TNF␣ (C) releases were determined (n = 3).

3.4. ManLAM fails to induce EPO release by eosinophils The mycobacterial cell wall composition and more particularly LM/LAM ratio might be a crucial factor in determining the virulence of a mycobacterial species and the outcome of the infection. For instance, an approximate LM/ManLAM ratio (w/w) of 1:2.5 was found in M. bovis BCG [11,16]. In order to unravel mycobacterial lipoglycans possibly interacting with CR3, we investigated the potential of lipoarabinomannan and lipomannan purified from M. bovis BCG to activate or downregulate eosinophil degranulation (Fig. 4). Eosinophil degranulation by ManLAM was negligible compared to eosinophil activation induced by LM (Fig. 4A). Thus ManLAM fails to induce eosinophil activation whereas LM activates eosinophils. We, then, investigated if ManLAM could down-regulate the eosinophil activation induced by LM. Because in our previous work we have shown that TLR2 is a functional innate receptor involved in the response of eosinophils to LM from M. bovis BCG [8], we tested the possibility that ManLAM may control this pathway of eosinophil activation. We have

chosen to use Pam3CSK4, a TLR2 specific agonist, rather than LM which has other potential receptors on eosinophils as EPO response to LM was reduced by 66.1% (±20%) when eosinophils were pretreated with a blocking anti-TLR2 antibody. Eosinophils were pre-incubated with different concentrations of ManLAM, and activated with Pam3CSK4 (Fig. 4B). ManLAM significantly inhibited EPO release from Pam3CSK4-activated eosinophils. 3.5. ManLAM modulates eosinophil activation mediated by TLR2 We next investigated the involvement of CR3 and PI3K in ManLAM inhibition of Pam3CSK4-induced eosinophil activation using the anti-CR3 antibody and LY294002 (Fig. 5A.) In the presence of ManLAM, eosinophil response to the TLR2 agonist was restored when the anti-CR3 antibody (76.8 ± 5.3% increase) or LY294002 (72.2 ± 17.9% increase) were added. To note, anti-CR3 antibody and LY294002 had no effect on Pam3CSK4-induced eosinophil activation. CD69 is a cell surface activation marker for human eosinophils [17]. Pam3CSK4-induced CD69 expression on human eosinophils as

Fig. 4. Stimulatory effects of LAM and LM, from M. bovis BCG on EPO and TNF␣ release by eosinophils. Human eosinophils were incubated with or without LM/LAM from M. bovis BCG at serially diluted concentrations. (A) After eosinophil stimulation with mycobacterial components from M. bovis BCG, EPO or TNF␣ release was measured in supernatants (n = 3). (B) Measurement of EPO release by eosinophils pre-incubated with or without ManLAM (0.1 and 1 ␮g/mL) at 37 ◦ C for 30 min, further activated with Pam3CSK4 (0.5 ␮g/mL (n = 4).

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Fig. 5. Impairment of TLR2-mediated eosinophil activation by ManLAM. (A) After pre-treatment with or without CR3 blocking antibodies (20 ␮g/mL) or LY294002 (20 ␮M), EPO release by eosinophils following stimulation for 2 h with PAM (0.5 ␮g/mL) in the presence or absence of ManLAM (1 ␮g/mL) was measured (n = 3). (B) CD69 expression was analyzed by flow cytometry and MFI are indicated (n = 3).

shown in Fig. 5. The addition of ManLAM reduced this expression to 42.6% (25.1 versus 10.1) confirming the inhibitory role of ManLAM on TLR2-activated eosinophils. Overall, these results support the finding that ManLAM inhibits TLR2-mediated eosinophil activation in a direct or indirect way involving the CR3 molecule ligation and the PI3K-dependent pathway.

4. Discussion The recognition of mycobacteria by eosinophils is also a multireceptor-mediated event. Although we failed to detect MR and DC-SIGN on purified human eosinophils by RT-PCR experiments or using flow cytometry (data not shown), TLR2 and ␥␦ TCR are expressed on human eosinophils and could interact respectively with LM [8] and TUBag [18], a non-peptidic phosphorylated antigen purified from M. bovis BCG. Thereby, mycobacteria act on alternative pathways in eosinophils by utilizing a specific combination of receptors to modulate immune responses. In this study, our results provide evidence that human eosinophils can interact and internalize M. bovis BCG. Consistent with the literature that considers CR3 as a phagocytic receptor for mycobacteria on macrophages [19], we have shown that CR3 was involved in mycobacteria internalization by eosinophils. In addition, our data support the fact that internalization of mycobacteria contributes to the inhibition of eosinophils’ activation. However, the mycobacterial ligand(s) involved remained unknown and an important question raised by this study was the mechanism mediating this inhibition. Because M. bovis BCG show extensive mannose capping of the arabinan termini of LAM [20] and because the lectin domain of CR3 interacts with mannose, we hypothesized that cell surface exposed CR3 could allow eosinophils to recognize ManLAM, and as

a consequence mediates bacterial uptake. Indeed, in macrophagelike J774 cells, ManLAM enhances LPS-induced TNF-␣ production but inhibits NO secretion by engaging scavenger receptors [21]. Moreover, ManLAM from M. tuberculosis can down-regulate IL12 production by LPS-stimulated macrophages and DCs through MR and DC-SIGN ligation [5]. On the other hand, CR3 ligation has recently been reported to inhibit the cytokine production induced by TLR4 triggering on DCs [22]. Here, we showed that ManLAM can inhibit Pam3CSK4-induced EPO and TNF␣ release by eosinophils, suggesting that ManLAM can interfere with TLR2-mediated signals. Our results suggest that ManLAM exert an anti-inflammatory role, leading to suppression of eosinophil activation in a CR3-dependent manner. Thus, ManLAM exerted the same anti-inflammatory CR3dependent effect on eosinophils’ activation that internalization. However, we cannot exclude the possibility that the internalization effect observed could be independent of ManLAM and related to other uncharacterized mycobacterial factors. Further investigation should be conducted to determine if ManLAM can directly interact with the CR3 molecule as previously hypothesized. Indeed, others have reported a cooperation between CR3 and CD14 molecules in ManLAM recognition during mycobacteria phagocytosis [23]. In macrophages, CR3/MAC-1-mediated myelin phagocytosis pathway uses the PI3K signaling pathway [24]. Two distinct and specific PI3K inhibitors, wortmannin (data not shown) and LY294002, allowed us to establish that immunomodulatory activity of ManLAM was mediated by the PI3K signal transduction pathway. In our previous work, we have shown that LY294002 had no effect on ROS release by M. bovis BCG-activated eosinophils [8]. In addition, the same result was obtained with anti-CR3 blocking antibodies (data not shown). ROS and EPO release are two mechanisms with distinct molecular pathways. Indeed, ROS are neosynthesized by NADPH oxidase complex while eosinophil peroxidase is stored in the matrix of the secondary crystalloid granules and released

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following degranulation [25]. Our findings are in agreement with a study conducted by Mishra et al., who confirmed the ability of wortmannin and LY294002 to inhibit selectively paraformaldehyde (PFA) induced respiratory burst but not PFA-induced LTC4 secretion by eosinophils [26]. The ability to modulate eosinophil activation through interactions between CR3 and ManLAM is encouraging for the development of future therapeutic approaches. Notably, the use of ManLAM may be relevant, in conditions involving clinical eosinophilic inflammation, such as allergy or hypereosinophilic syndromes, diseases under which eosinophils have deleterious functions. In this regard, these data indicate that ManLAM could contribute to increase the therapeutic arsenal for the treatment of allergic airway inflammation [27]. Conflict of interest The authors declare no financial or commercial conflict of interest. Acknowledgments LK was supported by a grant from the Centre National de la Recherche Scientifique (CNRS) (Action Thématique Incitative sur Programme « Microbiologie Fondamentale »). YG was supported by ANR005N/FEDER028F. We thank Dr. C. Locht and C. Rouanet (Inserm U629, Institut de Biologie de Lille, Lille, FRANCE) for providing M. bovis BCG. We thank Dr. C. Brinster for her assistance. References [1] Briken V, Porcelli SA, Besra GS, Kremer L. Mycobacterial lipoarabinomannan and related lipoglycans: from biogenesis to modulation of the immune response. Mol Microbiol 2004;53:391–403. [2] Welin A, Winberg ME, Abdalla H, Sarndahl E, Rasmusson B, Stendahl O, et al. Incorporation of Mycobacterium tuberculosis lipoarabinomannan into macrophage membrane rafts is a prerequisite for the phagosomal maturation block. Infect Immun 2008;76:2882–7. [3] Nigou J, Zelle-Rieser C, Gilleron M, Thurnher M, Puzo G. Mannosylated lipoarabinomannans inhibit IL-12 production by human dendritic cells: evidence for a negative signal delivered through the mannose receptor. J Immunol 2001;166:7477–85. [4] Maeda N, Nigou J, Herrmann JL, Jackson M, Amara A, Lagrange PH, et al. The cell surface receptor DC-SIGN discriminates between Mycobacterium species through selective recognition of the mannose caps on lipoarabinomannan. J Biol Chem 2003;278:5513–6. [5] Geijtenbeek TB, Van Vliet SJ, Koppel EA, Sanchez-Hernandez M, Vandenbroucke-Grauls CM, Appelmelk B, et al. Mycobacteria target DC-SIGN to suppress dendritic cell function. J Exp Med 2003;197:7–17. [6] Velasco-Velazquez MA, Barrera D, Gonzalez-Arenas A, Rosales C, AgramonteHevia J. Macrophage—Mycobacterium tuberculosis interactions: role of complement receptor 3. Microb Pathog 2003;35:125–31. [7] Lasco TM, Turner OC, Cassone L, Sugawara I, Yamada H, McMurray DN, et al. Rapid accumulation of eosinophils in lung lesions in guinea pigs infected with Mycobacterium tuberculosis. Infect Immun 2004;72:1147–9.

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