Infection Alters Bone Marrow to Promote Asthma Figure 4. C. muridarum lung infection in infants, but not neonates, alters hematopoietic cells to induce MSC hyperplasia. 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. Numbers of mucus secreting cells (MSCs) within 100 mm of basement membrane (BM) in the airways of (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.g004 C. muridarum lung infection in infants alters hematopoietic cells to increase IL-13 in the lung during AAD Given that MLN IL-13 release did not correlate with decreased MSC numbers and transpulmonary resistance in the neonatal group, we assessed IL-13 protein in the lung. Reconstitution of na¨ıve mice with bone marrow from mice infected (C. muridarum Chimera+Ova) as neonates suppressed IL-13 production during AAD, compared to age-matched, un-infected allergic (Vehicle Chimera+Ova) controls (Figure 6A). In stark contrast, reconstitu- tion with bone marrow from mice infected as infants increased IL- 13 production in the lung (Figure 6B). An adult infection had no effect (Figure 6C). These results suggest that an infant infection alters hematopoietic cells within the bone marrow and promotes increased IL-13 in the lung during AAD. Discussion In this investigation we demonstrate that C. muridarum lung infection in infants alters cells in the bone marrow to increase the severity of AAD in later-life. It is most likely that hematopoietic cells are the cells that are affected. We first developed a model of Ova-induced AAD, which has mucus hypersecretion and AHR as hallmark features, in a chimeric system. We then used this model to show that chlamydial lung infection in infants had long lasting effects on bone marrow cells. When bone marrow was transferred into irradiated na¨ıve recipients nine weeks after infant infection, the release of pro-asthmatic Th2-associated cytokines (IL-5 and IL-13) from MLN cells, MSC numbers around the airways, transpulmonary resistance and IL-13 levels in the lung were all increased during Ova-induced AAD. Adoptive transfer of bone marrow from mice infected as neonates had no effect on Th2- cytokine release, and suppressed the number of MSCs around the airways, transpulmonary resistance and IL-13 in the lung. Infection of adult mice had minimal effects. Therefore, an infant, but not neonatal, chlamydial lung infection, increases AAD in later-life through mechanisms that involve alterations in either the number or phenotype of hematopoietic cells. This is the first study to show that the age of infection can have differential effects on hematopoietic cells resulting in either enhanced, or suppressed, AAD in later-life. Figure 5. C. muridarum lung infection in infants, but not neonates, alters hematopoietic cells to increase AHR 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. Transpulmonary resistance at the maximal dose of methacholine administered (10 mg/mL) in (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.g005 PLoS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e42588