Cooperative Interactions between TLR4 and TLR9 Regulate Interleukin 23 and 17 Production in a Murine Model of Gram Negative Bacterial Pneumonia

Toll like receptors play an important role in lung host defense against bacterial pathogens. In this study, we investigated independent and cooperative functions of TLR4 and TLR9 in microbial clearance and systemic dissemination during Gram-negative bacterial pneumonia. To access these responses, wildtype Balb/c mice, mice with defective TLR4 signaling (TLR4lps-d), mice deficient in TLR9 (TLR9−/−) and TLR4/9 double mutant mice (TLR4lps-d/TLR9−/−) were challenged with K. pneumoniae, then time-dependent lung bacterial clearance and systemic dissemination determined. We found impaired lung bacterial clearance in TLR4 and TLR9 single mutant mice, whereas the greatest impairment in clearance was observed in TLR4lps-d/TLR9−/− double mutant mice. Early lung expression of TNF-α, IL-12, and chemokines was TLR4 dependent, while IFN-γ production and the later expression of TNF-α and IL-12 was dependent on TLR9. Classical activation of lung macrophages and maximal induction of IL-23 and IL-17 required both TLR4 and TLR9. Finally, the i.t. instillation of IL-17 partially restored anti-bacterial immunity in TLR4lps-d/TLR9−/− double mutant mice. In conclusion, our studies indicate that TLR4 and TLR9 have both non-redundant and cooperative roles in lung innate responses during Gram-negative bacterial pneumonia and are both critical for IL-17 driven antibacterial host response

Expression of IL-23 in WT and mutant mice in-vivo and in-vitro.

<p>(A) Whole lung IL-23 production after i.t. <i>K. pneumonia</i> administration. n = 5 in each group. *p<0.05 as compared to WT post i.t. bacteria. (B) Secretion of IL-23 by BMDC incubated with heat killed <i>K. pneumoniae</i> for 18 hrs. n = 5 in each group. *p<0.01 as compared to WT BMDC; #p<0.05 as compared to single mutants.</p

Bacterial clearance (A) and, lung expression of chemokines (B), in <i>Klebsiella</i>-infected WT and TLR4^lps-d/TLR9^−/− mice post i.t. IL-17 treatment.

<p>WT or TLR4^lps-d/TLR9^−/− mice were challenged with 8×10² CFU <i>K. pneumoniae</i> followed immediately by intratracheal administration of 1 µg rm IL-17A or vehicle. (A) Blood and lungs were collected at 24 hours post challenge and CFU quantitated. (B) Whole lung KC/CXCL1 and MIP-2/CXCL2 levels in WT and TLR4^lps-d/TLR9^−/− mice 24 hours post treatment with <i>K. pneumoniae</i>. n = 3−5 per group. *p<0.01 and τ<0.001.</p

Lung and blood CFU from WT, TLR4^lps-d, TLR9^−/−, and TLR4^lps-d/TLR9^−/− mice 24 hrs and 48 hrs post i.t. <i>Klebsiella</i> challenge.

<p>Mice were given 5×10² CFU <i>Klebsiella</i> i.t., lungs and blood harvested at designated time points and CFU assessed. Panel A shows lung and blood CFU at 24 hrs and panel B is lung and blood CFU at 48 hrs post i.t. <i>Klebsiella</i> challenge. CFU are expressed in mean log₁₀ ± SEM. n = 6−8, * p<0.05 as compared to WT mice, # p<0.01 as compared to single mutants, †<0.05 as compared to TLR9^−/− mice.</p

IL-17 expression by lung γδ and CD4+ T cells after i.t. <i>K. pneumoniae</i> administration.

<p>Flow cytometric analysis showing the number and percentage of γδ (A) and CD4+ T cells (B) with intracellular IL-17, 24 hours after i.t. <i>K. pneumoniae</i> challenge. n = 4 in each group. *p<0.05 as compared to WT post i.t. <i>K. pneumoniae</i>.</p

Expression of iNOS and FIZZ by alveolar macrophages.

<p>WT, TLR4^lps-d, TLR9^−/−, and TLR4^lps-d/TLR9^−/− mice were challenged with 5×10² CFU <i>Klebsiella</i> i.t. and BAL performed at 24 hours. Alveolar macrophages were harvested by adherence purification and iNOS and Fizz expression measured by RT-PCR. n = 5 in each group. *p<0.01 as compared to WT, †<0.05 as compared to TLR9^−/− mice.</p

Survival after i.t. <i>Klebsiella</i> challenge in WT, TLR4^lps-d, TLR9^−/−, and TLR4^lps-d/TLR9^−/− mice.

<p>n = 12 in each group. *p<0.01 as compared to WT mice. #p<0.01 as compared to TLR9^−/− mice. Combined from 2 separate experiments.</p

Cytokine production in whole lung after i.t. bacterial challenge.

<p>WT, TLR4^lps-d, TLR9^−/−, and TLR4^lps-d/TLR9^−/− mice were challenged with 5×10² CFU <i>Klebsiella</i>, then lungs harvested 6 hrs and 24 hrs post bacterial challenge. Cytokine levels were measured by ELISA. n = 5 in each group. *p<0.01 as compared to WT, # p<0.01 as compared to single mutant mice.</p