Differential roles of Toll-like receptors 2 and 4 in in vitro responses of macrophages to Legionella pneumophila

Abstract

The role of Toll-like receptors (TLRs) in innate immunity to Legionella pneumophila, a gram-negative facultative intracellular bacterium, was studied by using bone marrow-derived macrophages and dendritic cells from TLR2-deficient (TLR2(-/-)), TLR4(-/-), and wild-type (WT) littermate (C57BL/6 x 129Sv) mice. Intracellular growth of L. pneumophila was enhanced within TLR2(-/-) macrophages compared to WT and TLR4(-/-) macrophages. There was no difference in the bacterial growth within dendritic cells from WT and TLR-deficient mice. Production of interleukin-12p40 (IL-12p40) and IL-10 after infection with L. pneumophila was attenuated in TLR2(-/-) macrophages compared to WT and TLR4(-/-) macrophages. Induction of IL-12p40, IL-10, and tumor necrosis factor alpha secretion from macrophages by the L. pneumophila dotO mutant, which cannot multiply within macrophages, and heat-killed bacteria, was similar to that caused by a viable virulent strain. There was no difference between the WT and its mutants in susceptibility to the cytopathic effect of bacteria. An L. pneumophila sonicated lysate induced IL-12p40 production by macrophages, but that of TLR2(-/-) macrophages was significantly lower than those of WT and TLR4(-/-) macrophages. Treatment of L. pneumophila sonicated lysate with proteinase K and heating did not abolish TLR2-dependent IL-12p40 production. Our results show that TLR2, but not TLR4, is involved in murine innate immunity against L. pneumophila, although other TLRs may also contribute to innate immunity against this organism.

FIG. 1.

(A) Intracellular growth of L. pneumophila in BMMs. BMMs were infected with L. pneumophila AA100jm at an MOI of 1 or 10. After a 2-h incubation, extracellular bacteria were washed out. The infected macrophages were cultured further. The number of viable bacteria in each well was determined by the CFU counting method. Symbols: ○, BMMs from WT mice; □, BMMs from TLR2^−/− mice; ▪, BMMs from TLR4^−/− mice. The data are means ± the SD of four wells. ✽, P < 0.05. Representative results of three independent experiments are shown. (B) Intracellular growth of L. pneumophila in BMDCs. BMDCs were infected with L. pneumophila AA100jm at an MOI of 10. After a 2-h incubation, extracellular bacteria were washed out. The infected macrophages were cultured further. The number of viable bacteria in each well was determined by the CFU counting method. Symbols: ○, BMMs from WT mice; □, BMMs from TLR2^−/− mice; ▪, BMDCs from TLR4^−/− mice. The data are means ± the SD of three wells. N.S, Not significantly different.

FIG. 2.

IL-12p40 production by macrophages infected with L. pneumophila. BMMs were infected with L. pneumophila AA100jm at an MOI of 1 or 10. After a 2-h incubation, extracellular bacteria were washed out. The infected macrophages were cultured further, and IL-12p40 levels in supernatant were determined by ELISA. Symbols: □, BMMs from WT mice; ▪, BMMs from TLR2^−/− mice; ▨, BMMs from TLR4^−/− mice. The data are means ± the SD of four wells. ✽, P < 0.05.

FIG. 3.

IL-10 production by macrophages infected with L. pneumophila. BMMs were infected with L. pneumophila AA100jm at an MOI of 1 or 10. After a 2-h incubation, extracellular bacteria were washed out. The infected macrophages were cultured further, and IL-10 levels in supernatant were determined by ELISA. Symbols: □, BMMs from WT mice; ▪, BMMs from TLR2^−/− mice; ▨, BMMs from TLR4^−/− mice. The data are means ± the SD of four wells. ✽, P < 0.05.

FIG. 4.

IL-12p40 production by macrophages stimulated with virulent strain, avirulent strain, or dead bacteria. BMMs were treated with L. pneumophila AA100jm (virulent strain), its dotO mutant (avirulent strain), or UV-killed bacteria (AA100jm strain) at an MOI of 10. After a 2-h incubation, extracellular bacteria were washed out. The infected macrophages were cultured for another 24 h, and the IL-12p40 levels in the supernatant were determined by ELISA. Symbols: □, BMMs from WT mice; ▪, BMMs from TLR2^−/− mice; ▨, BMMs from TLR4^−/− mice. The data are means ± the SD of four wells. ✽, P < 0.05.

FIG. 5.

Dose-response relationship of IL-12p40 production by macrophages stimulated with virulent strain, avirulent strain, or dead bacteria. BMMs were treated with L. pneumophila AA100jm (virulent strain), its dotO mutant (avirulent strain), or heat-killed bacteria (AA100jm strain) at various concentrations. The stimulated macrophages were further cultured for another 24 h. IL-12p40 levels in supernatant were determined by ELISA. Results for the wild-type virulent strain, the avirulent dotO mutant, and heat-killed bacteria are shown. Note the difference in the axis scale. Symbols: □, BMMs from WT mice; ▪, BMMs from TLR2^−/− mice; ▨, BMMs from TLR4^−/− mice. The data are means ± the SD of four wells. ✽, P < 0.05.

FIG. 6.

Dose-response relationship of IL-10 production by macrophages stimulated with virulent strain, avirulent strain, or dead bacteria. BMMs were treated with L. pneumophila AA100jm (virulent strain), its dotO mutant (avirulent strain), or heat-killed bacteria (AA100jm strain) at various concentrations. The stimulated macrophages were further cultured for another 24 h. IL-10 levels in supernatant were determined by ELISA. Results for the WT virulent strain, the avirulent dotO mutant, and heat-killed bacteria are shown. Note the difference in the axis scale. Symbols: □, BMMs from WT mice; ▪, BMMs from TLR2^−/− mice; ▨, BMMs from TLR4^−/− mice. The data are means ± the SD of four wells. ✽, P < 0.05.

FIG. 7.

Cytotoxic effects of virulent strain, avirulent strain, and dead bacteria against macrophages of WT, TLR2^−/−, and −/− mice. BMMs were treated with L. pneumophila AA100jm (virulent strain), its dotO mutant (avirulent strain), or heat-killed bacteria (AA100jm strain) at various concentrations. The stimulated macrophages were further cultured for another 24 h. Symbols: □, BMMs from WT mice; □, BMMs from TLR2^−/− mice; ▪, BMMs from TLR4^−/− mice. The data are means ± the SD of four wells.

FIG. 8.

IL-12p40 production by macrophages stimulated with various components of L. pneumophila lysate. BMMs were treated with L. pneumophila AA100jm sonic extract (50 μg/ml) or its fraction (>5 or <5 kDa). The sonicate and its fraction were treated with proteinase K and boiling where indicated. The stimulated macrophages were cultured for 24 h, and IL-12p40 levels in the supernatant were determined by ELISA. Symbols: □, BMMs from WT mice; ▪, BMMs from TLR2^−/− mice; ▨, BMMs from TLR4^−/− mice. The data are means ± the SD of four wells. ✽, P < 0.05.