Resident CD11c+ Lung Cells Are Impaired by Anthrax Toxins after Spore Infection

MAJOR ARTICLE Resident CD11c+ Lung Cells Are Impaired by Anthrax Toxins after Spore Infection Aurélie Cleret,1 Anne Quesnel-Hellmann,1 Jacques Mathieu,2 Dominique Vidal,1 and Jean-Nicolas Tournier1 1 Unité d’Immunobiologie, Département de Biologie des Agents Transmissibles, and 2Unité de Radiobiologie et Inflammation, Département de Radiobiologie et de Radiopathologie, Centre de Recherche du Service de Santé des Armées, La Tronche, France Bacillus anthracis is a gram-positive, spore-forming bacterium and is the causal agent of zoonotic anthrax disease. It has recently become a major concern, after its use as a bioweapon in terrorist attacks in 2001. B. anthracis has 2 major virulence factors: (1) the capsule encoded by the plasmid pXO2 and (2) the 2 toxins produced by the association of 3 factors encoded by the plasmid pXO1. These 3 factors are protective antigen (PA), lethal factor (LF), and edema factor (EF). PA associates with LF to form lethal toxin (LT) and associates with EF to form edema toxin (ET). PA binds to at least 2 cellular receptors and, after being cleaved by a furin-like protease, self-assembles to form a hep- Received 18 November 2005; accepted 4 February 2006; electronically published 26 May 2006. Presented in part: 12th International Congress of Mucosal Immunology, Boston, MA, 25–30 June 2005 (abstract 53928). Potential conflicts of interest: none reported. Financial support: Délégation Générale pour l’Armement (grant CO 010808); Service de Santé des Armées (135OP3B LFR EMA). Reprints or correspondence: Dr. Jean-Nicolas Tournier, Unité d’Immunobiologie, Département de Biologie des Agents Transmissibles, CRSSA, 24 ave. des maquis du Grésivaudan, 38702 La Tronche, France (). The Journal of Infectious Diseases 2006; 194:86–94  2006 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2006/19401-0013$15.00 86 • JID 2006:194 (1 July) • Cleret et al. tameric prepore that facilitates the entry of LF/EF moieties into the cytosol, where they exert their toxic activities [1]. LF is a zinc-dependent metalloprotease that cleaves most of the mitogen-activated protein kinase kinases [2], whereas EF is a calcium- and calmodulindependent adenylate cyclase that increases intracellular cAMP concentration [3, 4]. The pathogenesis of inhalational anthrax disease, especially the crucial step of crossing the alveolocapillar space, is not well understood [5, 6]. Inhaled spores are phagocytosed in the alveolar spaces by antigen-presenting cells (APCs). Phagocytes then carry the spores from the alveolus to the mediastinal lymph nodes [7, 8]. The spores germinate within their intracellular niche and release vegetative bacteria extracellularly, which causes septicemia associated with a release of the toxins and, ultimately, causes host death. For decades, research focused on alveolar macrophages (AMs), which were thought to be the “Trojan horse” for the pathogen [9]. However, AMs are not thought to play a role in antigen traffic to lymph nodes [10]. Also, macrophage-depleted mice are still susceptible to aerosol infection, demonstrating the crucial role of another, as-yet-unidentified population of cells [11]. Resident lung dendritic cells Bacillus anthracis secretes 2 toxins: lethal toxin (LT) and edema toxin (ET). We investigated their role in the physiopathologic mechanisms of inhalational anthrax by evaluating murine lung dendritic cell (LDC) functions after infection with B. anthracis strains secreting LT, ET, or both or with a nontoxinogenic strain. Three lung cell populations gated on CD11c/CD11b expression were obtained after lung digestion: (1) CD11chigh/CD11blow (alveolar macrophages), (2) CD11cintermediate (int)/CD11bint (LDCs), and (3) CD11clow/CD11bhigh (interstitial macrophages or monocytes). After infection with LT-secreting strains, a decrease in costimulatory molecule expression on LDCs was observed. All CD11c+ cells infected with a nontoxinogenic strain secreted tumor necrosis factor (TNF)–a, interleukin (IL)–10, and IL-6. LT-secreting strains inhibited overall cytokine secretion, whereas the ET-secreting strain inhibited only TNF-a secretion and increased IL-6 secretion. Similar results were obtained after preincubation with purified toxins. Our results suggest that anthrax toxins secreted during infection impair LDC function and suppress the innate immune response. MATERIALS AND METHODS Mice. Male 6–12-week-old BALB/c (H-2d) mice (Janvier) were housed in clean, standard conditions at our animal-care facility. The local ethics committee approved our animal experiments. B. anthracis strains. We used the following B. anthracis strains: the parental Sterne strain 7702 (pXO1+); single-mutant derivatives RP10 Dlef and RP9 Dcya, which secrete PA-EF and PA-LF, respectively [23]; and double-mutant RP42 Dlef/Dcya, which secretes PA only [24] (all from M. Mock, Institut Pasteur, Paris, France). Isolation of CD11c+ cells from lung and generation of BMDCs. BALB/c mice were killed, and bronchoalveolar lavage (BAL) was performed 4 times, by intratracheal instillation of 1 mL of PBS (Gibco; Invitrogen) supplemented with 2 mmol/ L EDTA (Sigma-Aldrich). The thoracic cavity was opened, and lungs were perfused with 5 mL of PBS–2 mmol/L EDTA via the right ventricular cavity of the heart. The lungs were aseptically removed, cut into small pieces, and digested twice, in RPMI 1640 medium (Sigma-Aldrich) containing collagenase type I (1 mg/mL; Worthington Biochemical) and DNAse I (50 U; Sigma-Aldrich), by incubation for 30 min in a humidified incubator at 37C in a 5% CO2 atmosphere. Enzyme activity was stopped by washing the digested tissues in PBS–10 mmol/ L EDTA for 10 min on ice. The digested lungs were further disrupted by gently forcing the tissue through 2 nylon screens (pore size, 70 mm and 40 mm; BD Biosciences). After red-bloodcell lysis (in NH4Cl solution), cells were separated using CD11c microbeads (Miltenyi Biotec). We collected the positive fraction, and cell suspensions were counted and viability assessed by trypan-blue exclusion. All reagents used for isolation and culture of cells were certified as being endotoxin free. BMDCs were generated as described elsewhere [15, 25]. In vitro infection and toxin-exposure conditions. CD11c+ cells were seeded at 1.5 ⫻ 10 6 cells/mL in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS; Invitrogen) and 2 mmol/L l-glutamine (Sigma-Aldrich) for 1 h in a humidified incubator at 37C in a 5% CO2 atmosphere. Spores of B. anthracis were heat activated (70C for 10 min), and cells were infected with the spores for 1 h at an MOI of 20 [15, 26, 27]. The cells were washed twice in RPMI supplemented with 2.5 mg/mL gentamycin (Invitrogen) to kill any remaining extracellular bacteria [26] and were resuspended in RPMI–5% FBS–gentamycin. After 17 h of culture, followed by centrifugation, supernatants were sampled, and cells were harvested. Noninfected CD11c+ lung cells were us (...truncated)