protect or predispose to asthma. Numerous studies show an inverse association between Th1-inducing infections and the development of asthma [9,10,11]. However, infection with the intracellular bacterium Chlamydia pneumoniae is a notable exception and is increasingly linked with the development of asthma in both children and adults [4,5,7,8,12]. Respiratory infections with C. pneumoniae are common and usually asymptomatic but are responsible for up to 22% of all cases of community-acquired pneumonia requiring hospitalization [13,14]. Significantly, 50–80% of young adults have anti-C. pneumoniae antibodies [15,16], indicating the high prevalence of chlamydial respiratory tract infections in the community during the earlier stages of life. Resolution of infection is mediated by Th1 and interferon (IFN)-c-driven responses [17,18]. However, how Th1-inducing chlamydial lung infections are associated with increasing the severity of Th2-mediated asthma remain poorly understood. We have previously shown that Chlamydia can infect dendritic cells (DCs) and subvert their function to induce Th2 responses and AHR [19,20]. We have also shown that the Th2 cytokine IL-13, which is increased in the airways of asthmatics, enhances susceptibility to chlamydial infections in mice [21]. Furthermore we have recently demonstrated that chlamydial lung infection in early-life increases the severity of allergic airway disease (AAD) in later-life [7]. Infection of both neonatal and infant, but not adult, BALB/c mice increased the expression of IL- 13 in the lungs, the numbers of mucus secreting cells (MSC) around the airways and AHR during AAD in later-life [7]. We have begun to elucidate the mechanisms involved. Neonatal infection suppressed eosinophilic and Th2-mediated allergic inflammation, but increased systemic DC:T cell IL-13 release and altered lung structure by increasing the size of alveoli [7]. By contrast, infant infection enhanced eosinophilic and Th2-mediated allergic inflammation, but did not alter lung structure [7]. These results suggest that hematopoietic cells may have differential contributions to the mechanisms through which neonatal and infant infections increase the severity of AAD. Recent studies suggest that hematopoietic cells can respond directly to infections and inflammatory signals [22]. These cells give rise to myeloid and lymphoid immune cell lineages and can proliferate, and differentiate, to replace immune cells lost to cell death following infection. Hematopoietic cells have been shown to sense pathogen components directly via toll-like receptors (TLRs) [23]. Infection-induced, pro-inflammatory cytokine release may also activate hematopoietic cells [24,25] and aberrant cytokine- induced signalling may have negative effects on the function of these cells [24,25]. This may have long-term effects on the programming of the immune system and the nature of subsequent responses to antigens. The effects of chlamydial lung infection on hematopoietic cell function and subsequent AAD have not been investigated. In this study, we demonstrate that reconstitution of bone marrow from mice infected with C. muridarum as infants, but not neonates, increases the severity of AAD in later-life. Therefore, early-life infection-induced alterations in hematopoietic cells may play a previously unrecognised role in predisposing to severe AAD. Materials and Methods Ethics Statement All experiments were performed with approval from the animal ethics committees of The University of Newcastle and Garvan Institute/St. Vincent’s Hospital, NSW. Infection Alters Bone Marrow to Promote Asthma Animals Specific pathogen-free pregnant and non-pregnant BALB/c mice (6, 9, 12 or 15 week old) were obtained from the central animal house, The University of Newcastle or from Australian BioResources (Moss Vale, Australia). C. muridarum lung infection Neonatal (,24 hour old), infant (3 weeks old) or adult (6 weeks old) BALB/c mice were infected intranasally with C. muridarum (400 [neonate] or 100 [infant and adult] inclusion-forming units, ATCC VR-123, in 5 ml (neonate) or 30 ml (infant and adult) sucrose phosphate glutamate buffer [vehicle]) [6,7,26]. Controls were sham inoculated with equivalent volumes of vehicle intranasally. Generation of bone marrow chimeras and induction of AAD Nine weeks after neonatal, infant or adult infection, or sham inoculation, bone marrow was extracted from the hind limbs of donor mice and 16107 cells were intravenously transferred to recipient age-matched irradiated na¨ıve BALB/c mice. Recipient mice were irradiated twice (four hours between each irradiation) with 450RAD (4.5 Gy) prior to adoptive transfer of bone marrow [27]. The mice were left for a period of 8 weeks to allow for reconstitution of bone marrow and hematopoietic cells. It has been shown previously [27], that this method is sufficient for bone marrow reconstitution. AAD was then induced as previously described [6,7,28,29]. Mice were sensitized intraperitoneally to the model allergen Ovalbumin (Ova, 50 mg, Sigma, Missouri, USA) in Rehydrogel (1 mg, Reheis, Berkeley Heights, USA) in sterile saline (200 ml) and subsequently challenged 12 days later by intranasal administration of Ova (10 mg in 50 ml sterile saline) for 4 consecutive days. Mice were sacrificed 24 hours after the final Ova challenge and features of AAD were assessed (Figure 1). Determination of mediastinal lymph node cell cytokine release Mediastinal lymph node (MLN) cells were excised, single cell suspensions prepared, erythrocytes lysed and 16106 MLN cells plated out in 96 well culture plates in 200 ml of RPMI 1640 culture medium containing 10% FCS, 2 mM L-glutamine, 2 mM sodium pyruvate, 100 mg/ml penicillin, 100 mg/ml streptomycin, 50 mM 2-mercaptoethanol (GIBCO, Invitrogen Mount Waverly, Austra- lia). Cells were re-stimulated with Ova (200 mg/mL; Sigma) for 4 days and the concentration of IL-5, IL-13 and IFN-c in supernatants were analyzed by ELISA using paired antibodies (R&D systems, Gymea, Australia) as we have previously described [6,7,28,29,30,31,32,33]. Quantification of airway MSCs Lungs were formalin-fixed, embedded, and sectioned (4–6 mm). Sections were stained with periodic acid–schiff for enumeration of MSCs in the airways as we have previously described [32,34]. Assessment of lung function Lung function, in terms of AHR, was measured in anesthetized mice using whole-body plethysmography by determination of transpulmonary resistance and dynamic compliance in response to increasing doses of nebulized methacholine (Sigma) as we have previously described [6]. PLoS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e42588