Infection Alters Bone Marrow to Promote Asthma Figure 6. C. muridarum lung infection in infants, but not neonates, alters hematopoietic cells to increase IL-13 in the lung during AAD. BALB/c mice were infected with C. muridarum or sham inoculated (vehicle) as neonates, infants or adults. Nine weeks after infection bone marrow was extracted and adoptively transferred into age-matched, irradiated na¨ıve mice. Eight weeks after bone marrow reconstitution, chimeras were subjected to Ova-induced AAD and the development of AAD was assessed. IL-13 protein in lung homgenates (A) neonate, (B) infant and (C) adult bone marrow chimeras. Results are represented as mean 6 SEM. n = 6–8, from two independent experiments of 3–4 mice. Age-matched controls were run in parallel with every experiment. *P,0.05 compared to un-infected allergic (Vehicle Chimera + Ova) controls. doi:10.1371/journal.pone.0042588.g006 In contrast to the affects on transpulmonary resistance, dynamic compliance was unaffected. The large airways are the major contributors to transpulmonary resistance, whereas dynamic compliance is a measure of the elasticity of the small airways and parenchyma [35]. It is likely therefore that infection-induced changes to hematopoietic cells predominantly affected the larger airways in this study. This is supported by the observations that MSC numbers around the large airways were also altered. There were no differences in all except one of the features of AAD in chimeras generated from sham-inoculated mice. There were no differences between the allergic controls from the three different age groups (Vehicle+Chimera) in terms of MSC numbers, transpulmonary resistance or IL-13 protein in the lung during AAD. This demonstrates that the bone marrow of the chimeras was reconstituted to the same level. The only difference was in the magnitude of MLN cytokine release into culture supernatants. The MLN culture experiments were performed at different times for the different groups. This assay is an ex vivo 6 day cell culture assay that is subject to day to day variation and results are compared between groups performed at the same time and not between different experiments carried out at different times. It is not as robust as MSC enumeration, lung function and IL-13 protein in the lung, all of which are direct representations of what occurred in vivo. Notably all mice were adults at the time bone marrow was extracted and reconstituted. Mice infected or sham-inoculated as neonates, infants or adults were 9, 12 and 15 weeks old at time of bone marrow extraction and reconstitution, respectively. Bone marrow was reconstituted into age-matched controls (9, 12 and 15 week old mice for neonate, infant and adult groups, respectively). Each infected group was compared to its relevant age-matched sham-inoculated control, which was run in parallel with each experiment. For this study we have used an Ova-induced model of AAD, which involves systemic sensitization and airway challenge. Asthma does have a systemic component although it manifests in the respiratory system. We have previously demonstrated that early-life Chlamydia lung infection drives more severe Ova-induced AAD in later-life [7]. Thus, the use of this model allowed us to investigate the effects of infection-induced changes in the bone barrow compartment on infection-enhanced AAD in later-life. Our study could be extended by using a house dust mite model of AAD in which sensitization occurs through the respiratory mucosa [34]. Such a study would answer different but important questions that relate to how infection-induced changes in the bone marrow affect mucosal sensitization. Our results which show that neonatal infection alters hemato- poietic cells to suppress, rather than promote the development of AAD in later-life both support and contrast with what we observed in intact (i.e. wild type) mice in our previous studies [6,7]. In the previous studies neonatal infection also suppressed Th2 and inflammatory responses. However, infection at this age increased MSC and AHR and thus the severity of AAD by inducing systemic IL-13 responses and altering lung structure. The enhanced IL-13 responses involved increased expression in the lung and systemic DC:T-cell release, which correlated with increased MSC numbers and AHR [7]. This contrasts with our current study, which shows that IL-13 protein levels were decreased in the lungs. This indicates that this component of the effects of a neonatal infection does depend on hematopoietic cells. However, we did not investigate the effects of infection on systemic DC:T-cell release in this study. The alterations in lung structure involved alveolar enlargement, which may significantly contribute to changes in lung function [7]. Increased alveolar diameter results in the reduction of alveolar attachments to the airway wall, which decreases airway support and elastic recoil and may lead to enhanced transpulmonary resistance and reduced tissue compli- ance [36]. The effects on IL-13 responses and lung structure are unlikely to be controlled by the hematopoietic system and in the current study it is not surprising that neonatal infection does not increase the severity of AAD through these processes. Neonatal infection did not alter the release of IL-5 or IL-13 from Ova-stimulated MLN cells but reduced MSC numbers and AHR, which was associated with attenuated IL-13 production in the lung. It is likely that reductions in IL-13 production in the lung contribute to the reductions in MSC numbers and AHR. Potential explanations for these differential effects in MLNs and lungs are that infection may have differential effects on different compart- ments so that T-cells in the MLNs can still release sufficient amounts of IL-5 and IL-13, but they may not be able to migrate to the lung to induce MSC hyperplasia and AHR. Another explanation could be that systemic DC and/or T-cell function could be altered. Neonatal infection-induced changes in lung structure in intact wild-type mice is also likely to contribute to infection-induced severe AAD [7]. In contrast to what occurred in neonates, infection of infants altered hematopoietic cells to increase the severity of AAD in later- life by increasing Th2 (IL-5 and IL-13) cytokine release from MLN cells, MSC numbers, transpulmonary resistance and IL-13 production in the lung. This is consistent with what occurs in intact mice, where infection of infants increased the release of the pro-asthmatic Th2 cytokines IL-5 and IL-13, which was associated PLoS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e42588