Toll-like receptor 4-dependent early elicited tumor necrosis factor alpha expression is critical for innate host defense against Bordetella bronchiseptica

Abstract

Toll-like receptor 4 (TLR4) mediates the response to lipopolysaccharide, and its activation induces the expression of a large number of inflammatory genes, many of which are also induced by other pathogen-associated molecular patterns. Interestingly, the subset of genes that are dependent on TLR4 for optimal expression during gram-negative bacterial infection has not been determined. We have previously shown that TLR4-deficient mice rapidly develop acute pneumonia after inoculation with Bordetella bronchiseptica, suggesting that TLR4 is required for expression of early elicited gene products in this model. Microarray analysis with macrophages derived from wild-type and TLR4-deficient mice was used to identify genes whose expression, within 1 h of bacterial exposure, is dependent on TLR4. The results of this investigation suggest that TLR4 is not required for the majority of the transcriptional response to B. bronchiseptica. However, early tumor necrosis factor alpha (TNF-alpha) mRNA expression is primarily dependent on TLR4 and in vitro and in vivo protein levels substantiate this finding. TLR4-deficient mice and TNF-alpha-/- mice are similarly susceptible to infection with relatively low doses of B. bronchiseptica and in vivo neutralization studies indicate that it is the TLR4-dependent early elicited TNF-alpha response that is critical for preventing severe pneumonia and limiting bacterial growth. These results suggest that one critical role for TLR4 is the generation of a robust but transient TNF-alpha response that is critical to innate host defense during acute gram-negative respiratory infection.

FIG. 1.

Display of the global transcriptional response of WT and TLR4^d BMMΦ exposed to 10 ng of purified B. bronchiseptica LPS/ml for 1 h (A) and live B. bronchiseptica at an MOI of 10 for 1 h or 30 min (B). Red indicates an upregulated gene, whereas green indicates a downregulated gene. A complete set of array data is available at http://www.vetsci.psu.edu/personnel/faculty/harvill.cfm.

FIG. 2.

Fold induction of genes whose mean fold induction was 4 or greater in WT but not in TLR4-deficient BMMφ after 1 h of exposure to B. bronchiseptica. Bars: □, WT; ▪, TLR4 deficient.

FIG. 3.

In vitro TNF-α response from BMMΦ at 24 h after exposure to the indicated concentrations (in nanograms/milliliter) of purified B. bronchiseptica LPS or 10 μg of Staphylococcus aureus PGN/ml (M = medium control) (A), at 24 h after exposure to the indicated MOIs of heat-killed B. bronchiseptica (Bb) (C), and at 2 h after exposed to live B. bronchiseptica at an MOI of ca. 5 (B). Bars: □, WT; ▪, TLR4 deficient (n = 3 to 4). The data are representative of three different experiments.

FIG. 4.

(A) Lung TNF-α levels at 2 h postinoculation with 5 × 10⁵ CFU of B. bronchiseptica (Inf) or a PBS control (sham) mice. (B) Lung TNF-α levels at 0.5, 1, 2, 6, 12, 24, 48, and 72 h following inoculation with 5 × 10⁵ CFU of B. bronchiseptica. Symbols: □, WT; ▪, TLR4 deficient. n = 3 to 4 per group. The data are representative of 3 different experiments.

FIG. 5.

Survival curve of WT C3H and TLR4-deficient mice (A) or WT C57BL/6 and TNF-α^−/− mice (B) inoculated with the indicated CFU of B. bronchiseptica. Symbols: ⋄, WT; ♦, TLR4 deficient and TNF-α^−/−. n = 8 to 10 per group.

FIG. 6.

(A) Lung pathology scores of H&E-stained lung sections on day 3 postinoculation with 5 × 10⁵ CFU of B. bronchiseptica; (B) representative field of H&E-stained lung sections; (C) CFU of B. bronchiseptica recovered from the lungs of mice on day 3 postinoculation. n = 3 to 4 per group. The data are representative of three different experiments.