Absence of TLR2 increases activated CD8⁺ T-cell recruitment in the lung during <i>Chlamydia</i> respiratory infection in early life.

<p>Flow cytometric analysis of single cell suspensions of lung tissue was used to determine the levels of total and activated CD4⁺ or CD8⁺ T-cells in sham inoculated and infected Wt and TLR^−/− pups at 7 and 14 dpi. T-cell populations were identified by selecting those cells, which were CD3⁺ and CD4⁺ or CD8⁺. These populations of (A) total CD4⁺ and (B) total CD8⁺ T-cells were then analysed for CD43 expression, as a marker of T-cell activation (C and D). Representative FACS scatter plots of these data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039460#pone.0039460.s002" target="_blank">Figure S2E and F</a>. dpi = days post-inoculation, n = 6–12 mice/group. Results are presented as means ± SEM. * denotes significant difference between sham inoculated and infected groups of the same strain at the same time point, # denotes significant difference between infected WT and TLR^−/− groups at the same time point. #p<0.05, **/##p<0.01, ***/###p<0.001.</p

Absence of TLR2 increases NK and neutrophil influx, and IL-17 transcript levels in the lung during <i>Chlamydia</i> respiratory infection in early life.

<p>Flow cytometric analysis of single cell suspensions of lung tissue was used to determine the levels of NK cells and neutrophils in sham inoculated and infected Wt and TLR^−/− pups at 3, 7 and 14 dpi. (A) NK cells and (B) neutrophils were identified as cells expressing CD3⁻ and CD49⁺ and CD11b⁺ and GR-1⁺, respectively. Representative FACS plots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039460#pone.0039460.s002" target="_blank">Figure S2A and B</a>. Total numbers of NK cells and neutrophils in the lung were calculated by multiplying total cell counts by the percentage of NK cells or neutrophils. Quantitative real-time-PCR was used to determine the transcript levels of (C) KC, (D) MIP-2 and (E) IL-17 in lung tissue at the same time points. dpi = days post-inoculation, n = 5–12 mice/group. Results are presented as means ± SEM. * denotes significant difference between sham inoculated and infected groups of the same strain at the same time point, # denotes significant difference between infected WT and TLR^−/− groups at the same time point. */#p<0.05, **p<0.01, ***/###p<0.001.</p

Absence of TLR2 reduces weight gain and increases clinical score during <i>Chlamydia</i> respiratory infection in early life.

<p>Wild type (Wt) and TLR^−/− pups were sham inoculated or infected. (A) Mice were weighed individually each day and the percentage weight gain calculated daily relative to the average weight of the litter prior to inoculation. n = 10–12 pups/group (5–6 pups/litter). (B) Clinical scores were also determined based on symptoms observed in litters daily as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039460#pone.0039460.s004" target="_blank">Table S1</a>. n = 2–4 litters. Results are presented as means ± SEM. # denotes significant difference between infected Wt and TLR^−/− groups. # p<0.05, ### p<0.001 for the whole curves.</p

Absence of TLR2 and 4 reduces IFNγ release and proliferation of <i>ex-</i>vivo stimulated T-cells during <i>Chlamydia</i> respiratory infection in early life.

<p>Single cell suspensions of mediastinal lymph nodes were prepared from sham inoculated and infected Wt and TLR^−/− pups at 14 dpi. (A) Total cell numbers in lung-draining mediatinal lymph nodes were determined by trypan blue exclusion. (B) Other mediatinal lymph node cells were stimulated with media only or MOMP and IFNγ levels in culture supernatants were determined by ELISA. (C) IFNγ protein levels in lung homogenates were also quantitated by ELISA. (D) Lymph node cell proliferation was assessed following stimulation with MOMP (2 µg/ml). Percentage proliferation was calculated relative to cells stimulated with media only. n = 6–12 mice/group. Results are presented as means ± SEM. * denotes significant difference between sham inoculated and infected groups of the same strain, # denotes significant difference between infected WT and TLR^−/− groups. */#p<0.05, **p<0.01, ***/###p<0.001.</p

Absence of TLR2 reduces phagocytosis of <i>Chlamydia</i> by neutrophils.

<p>Bone marrow was collected from Wild type (Wt), and TLR^−/− mice, grown, and non- and semi-adherent cells cultured with fluorescently (FITC) labelled latex beads. Neutrophils were identified as expressing CD11b⁺ and GR1⁺. The percentage of neutrophils that were FITC low, high, negative or positivewere determined. FACS scatter plots of these data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039460#pone.0039460.s003" target="_blank">Figure S3</a>. n = 3 replicates, results are presented as means ± SEM. * denotes significant different between WT and TLR^−/− groups. ***p<0.001.</p

Absence of TLR2 increases late DC recruitment in the lung during <i>Chlamydia</i> respiratory infection in early life.

<p>Flow cytometric analysis of single cell suspensions of lung tissue was used to determine the levels of mDCs and pDCs in of sham inoculated and infected Wt and TLR^−/− pups at 3 and 7 dpi. (A) mDCs and (B) pDCs were identified as cells expressing CD11b⁺, CD11c⁺ and GR-1⁻, or CD11b⁻, B220⁺ and pDCA⁺, respectively. Representative FACS plots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039460#pone.0039460.s002" target="_blank">Figure S2C and D</a>. dpi = days post-inoculation, n = 5–12 mice/group. Results are presented as means ± SEM. * denotes significant difference between sham inoculated and infected groups of the same strain at the same time point, # denotes significant difference between infected WT and TLR^−/− groups at the same time point. */#p<0.05, **/##p<0.01, ***/###p<0.001.</p

Absence of TLR2 reduces the clearance of <i>Chlamydia</i> respiratory infection in early life.

<p>Whole lungs were homogenized and DNA extracted. Quantitative real-time-PCR was used to determine the level of <i>Chlamydia</i> DNA in each sample relative to known standards. dpi = days post-inoculation, n = 5–9 mice/group. Results are presented as means ± SEM. # denotes significant difference between infected Wt and TLR^−/− groups. # p<0.05, ##p<0.01, ### p<0.001.</p

<i>C. muridarum</i> lung infection in infants, but not neonates, alters hematopoietic cells to increase AHR during AAD.

<p>BALB/c mice were infected with <i>C. muridarum</i> or sham inoculated (vehicle) as neonates, infants or adults. Nine weeks after infection bone marrow was extracted and adoptively transferred into age-matched, irradiated naïve 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 (<b>A</b>) neonate, (<b>B</b>) infant and (<b>C</b>) adult bone marrow chimeras. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042588#s3" target="_blank">Results</a> are represented as mean ± SEM. n = 6–8, from two independent experiments of 3–4 mice. Age-matched controls were run in parallel with every experiment. *P&lt;0.05 compared to un-infected allergic (Vehicle Chimera + Ova) controls.</p

<i>C. muridarum</i> lung infection in infants, but not neonates, alters hematopoietic cells to increase IL-13 in the lung during AAD.

<p>BALB/c mice were infected with <i>C. muridarum</i> or sham inoculated (vehicle) as neonates, infants or adults. Nine weeks after infection bone marrow was extracted and adoptively transferred into age-matched, irradiated naïve 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 (<b>A</b>) neonate, (<b>B</b>) infant and (<b>C</b>) adult bone marrow chimeras. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042588#s3" target="_blank">Results</a> are represented as mean ± SEM. n = 6–8, from two independent experiments of 3–4 mice. Age-matched controls were run in parallel with every experiment. *P&lt;0.05 compared to un-infected allergic (Vehicle Chimera + Ova) controls.</p

<i>C. muridarum</i> lung infection in infants, but not neonates, alters hematopoietic cells to induce MSC hyperplasia.

<p>BALB/c mice were infected with <i>C. muridarum</i> or sham inoculated (vehicle) as neonates, infants or adults. Nine weeks after infection bone marrow was extracted and adoptively transferred into age-matched irradiated, naïve 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 µm of basement membrane (BM) in the airways of (<b>A</b>) neonate, (<b>B</b>) infant and (<b>C</b>) adult bone marrow chimeras. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042588#s3" target="_blank">Results</a> are represented as mean ± SEM. n = 6–8, from two independent experiments of 3–4 mice. Age-matched controls were run in parallel with every experiment. *P&lt;0.05 compared to un-infected allergic (Vehicle Chimera + Ova) controls.</p