Infection Alters Bone Marrow to Promote Asthma Figure 1. Experimental protocol. BALB/c mice were infected intranasally with C. muridarum as neonates (,24 hours old), infants (3 weeks old) or adults (6 weeks old). Controls were inoculated with vehicle (sucrose phosphate glutamate buffer). Nine weeks (63 days) after inoculation, bone marrow was extracted and reconstituted into age-matched irradiated, naive mice. The mice were left for a period of 8 weeks to allow for reconstitution of hematopoietic cells. Mice were then sensitized intraperitoneally (Day 120) to Ovalbumin (Ova) and subsequently challenged 12 days later by intranasal administration of Ova for 4 consecutive days (Day 132–135). Mice were sacrificed 24 hours after the final Ova challenge (Day 136) and features of AAD were assessed. doi:10.1371/journal.pone.0042588.g001 Quantification of IL-13 protein in lung homogenates Whole lungs were homogenized in 1 mL of RIPA buffer (Sigma) and total protein was extracted according to manufactur- ers recommendations. Extracted total protein from lung homog- enates was quantified using BCA Protein Assay Kits (Pierce, Scorsby, Australia). IL-13 protein in lung homogenates was analyzed by ELISA (R&D systems). Data analysis Results are presented as means 6 SEM with each experimental group consisting of 6–8 mice, from two independent experiments (3– 4 mice per experiment). Each experimental age group was run in parallel with its age-matched control. Between group comparisons of transpulmonary resistance and dynamic compliance (whole curve analysis) were performed using One-way repeated-measures AN- OVA. The Mann-Whitney non-parametric test was used for all other comparisons. Analyses were conducted using GraphPad Prism 5 (GraphPad Software, La Jolla, USA). Analysis of statistical outliers was performed using the Grubb’s outlier test for GraphPad Prism 5. Results Establishment of AAD in bone marrow chimeras To establish the chimera system, bone marrow was extracted from na¨ıve (i.e. un-infected) BALB/c mice and transferred to age- matched irradiated na¨ıve mice. After reconstitution of hematopoi- etic cells (8 weeks later), AAD was induced and assessed. Allergic mice (Chimera+Ova groups) had significantly increased AHR (increased transpulmonary resistance (Figure 2A) and decreased dynamic compliance (Figure 2B)) compared to non-allergic (Chimera+Sham) controls. They also had increased MSC numbers around the airways (Figure 2C) and Ova-specific Th2 (IL-5 and IL- 13), but not Th1 (IFN-c [data not shown]) cytokine release from MLN cells restimulated with Ova (Figure 2D–E). Allergic mice also had significantly increased IL-13 in the lung (Figure 2F) compared to non-allergic (Chimera+Sham) controls. Notably, the develop- ment of AAD in chimeras did not involve the influx of eosinophils into the lung (data not shown). These results demonstrate that AAD could be induced after reconstitution of bone marrow. C. muridarum lung infection in infants, but not neonates, alters hematopoietic cells to increase Ova-specific Th2 cytokine release from MLN cells during AAD We have previously shown that neonatal infection suppressed, whereas infant infection increased Ova-specific Th2 cytokine (IL-5 and IL-13) release from MLN cell cultures, whilst an adult infection had no effect [7]. Here we show that reconstitution of na¨ıve mice with bone marrow from mice infected (C. muridarum Chimera+Ova) as neonates had no effect on Ova-specific IL-5 or IL-13 release in AAD compared to age-matched, un-infected allergic (Vehicle Chimera+Ova) controls (Figure 3A). In contrast, reconstitution with bone marrow from mice infected as infants increased IL-5 and IL-13 release from MLN cells in AAD (Figure 3B). Reconstitution with bone marrow from mice infected as adults increased IL-5 but had no effect on IL-13 release (Figure 3C). These results suggest that infant, but not neonatal or adult infection, alters hematopoietic cells within the bone marrow to promote increased Th2 cytokine release in AAD. C. muridarum lung infection in infants, but not neonates, alters hematopoietic cells to induce MSC hyperplasia during AAD We have previously shown that neonatal and infant, but not adult chlamydial lung infection results in increased MSC numbers in the airways during AAD in later-life [7]. Reconstitution of na¨ıve mice with bone marrow from mice infected (C. muridarum Chimera+Ova) as neonates resulted in a small but significant reduction, whilst bone marrow from mice infected as infants increased the number of MSCs in the airways in AAD, compared to age-matched, un-infected allergic (Vehicle Chimera+Ova) controls (Figure 4A and 4B, respectively). Reconstitution with bone marrow from mice infected as adults had no effect (Figure 4C). These results suggest that infant, but not neonatal or adult infection, alters hematopoietic cells within the bone marrow, leading to increased MSC numbers in AAD. C. muridarum lung infection in infants, but not neonates, alters hematopoietic cells to increase AHR during AAD We have previously shown that neonatal and infant, but not adult chlamydial lung infection increases AHR during AAD in later-life [7]. Thus, we investigated whether infection in early-life altered hematopoietic cells to increase AHR. Reconstitution of na¨ıve mice with bone marrow from mice infected (C. muridarum Chimera+Ova) as neonates suppressed transpulmonary resistance during AAD, compared to age-matched, un-infected allergic (Vehicle Chimer- a+Ova) controls (Figure 5A). In stark contrast, reconstitution with bone marrow from mice infected as infants increased transpulmonary resistance (Figure 5B). An adult infection had no effect (Figure 5C). Reconstitution with bone marrow from mice infected as neonates, infants or adults had no effect on dynamic compliance during AAD (data not shown). These results suggest that infant, but not neonatal or adult infection, alters hematopoietic cells within the bone marrow and promotes increased AHR in AAD. PLoS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e42588