Cooperation between multiple microbial pattern recognition systems is important for host protection against the intracellular pathogen Legionella pneumophila

Abstract

Multiple pattern recognition systems have been shown to initiate innate immune responses to microbial pathogens. The degree to which these detection systems cooperate with each other to provide host protection is unknown. Here, we investigated the importance of several immune surveillance pathways in protecting mice against lethal infection by the intracellular pathogen Legionella pneumophila, the causative agent of a severe pneumonia called Legionnaires' disease. Rip2 and Naip5/NLRC4 signaling was found to contribute to the innate immune response generated against L. pneumophila in the lung. Elimination of Rip2 or Naip5/NLRC4 signaling in MyD88-deficient mice resulted in increased replication and dissemination of L. pneumophila and higher rates of mortality. Irradiated wild-type mice receiving bone marrow cells from pattern recognition receptor-deficient mice displayed L. pneumophila infection phenotypes similar to those of donor mice. Rip2 and Naip5/NLRC4 signaling provided additive effects in protecting MyD88-deficient mice from lethal infection by L. pneumophila, with the contribution of Naip5/NLRC4 being slightly greater than that of Rip2. Thus, activation of the Rip2, MyD88, and Naip5/NLRC4 signaling pathways triggers a coordinated and synergistic response that protects the host against lethal infection by L. pneumophila. These data provide new insight into how different pattern recognition systems interact functionally to generate innate immune responses that protect the host from lethal infection by activating cellular pathways that restrict intracellular replication of L. pneumophila and by recruiting to the site of infection additional phagocytes that eliminate extracellular bacteria.

FIG. 1.

L. pneumophila activates Rip2-dependent pathways in vivo. WT and Rip2-deficient (Rip2^−/−) mice in a C57BL/6 background were given a high intranasal dose (1 × 10⁶ CFU) of L. pneumophila ΔflaA. For each group, five mice were sacrificed at days 2, 3, and 5 postinfection as indicated below each graph. (A) BALF from infected mice was assayed for the indicated cytokines by ELISA. Each circle represents data obtained from a single mouse. The lines indicate the mean values calculated from the data for the two groups of mice. *, P < 0.05. (B) Bacterial numbers were determined from lung lysates at the indicated time points. There was no statistical significance between the two groups of mice (P > 0.05).

FIG. 2.

Rip2 is important for suppression of L. pneumophila replication in MyD88-deficient mice. (A) MyD88-deficient (MyD88^−/−) and Rip2/MyD88-deficient (Rip2^−/−MyD88^−/−) mice in a C57BL/6 background were given a low intranasal dose (4 × 10⁴ CFU) of L. pneumophila ΔflaA. At day 2 and day 5 postinfection, seven mice from each group were sacrificed, and bacterial numbers were determined from the lung, blood, liver, and spleen. Each point represents data from a single mouse. The lines indicate the mean values calculated from the data for the two groups of mice. The difference between the two groups of mice was statistically significant at day 5. *, P < 0.005. (B) BMMs from WT (filled circles), Rip2^−/− (open circles), MyD88^−/− (filled squares), and Rip2^−/− MyD88^−/− (open squares) mice were infected with L. pneumophila ΔflaA. The numbers of bacterial CFU were determined at 1 h and 48 h postinfection. Intracellular growth is expressed as the fold increase in the number of CFU detected over this period. Each point represents data for BMMs derived from a single mouse. All data points represent the average increase in the number of bacterial CFU determined from three wells infected independently. Each line indicates the mean calculated from the data for the two different mice.

FIG. 3.

Cellular recruitment defects in Rip2/MyD88-deficient mice after L. pneumophila infection. MyD88^−/− and Rip2^−/− MyD88^−/− mice in a C57BL/6 background were given a low intranasal dose (4 × 10⁴ CFU) of L. pneumophila ΔflaA. Seven mice from each group were sacrificed at days 2 and 5 postinfection. (A) BALF from infected mice was assayed for the indicated cytokines by ELISA. Each point represents data from a single mouse. The horizontal lines indicate the mean values calculated from the data for the two groups of mice. (B and C) Cell suspensions from isolated lungs were stained for CD11b, Gr-1, and F4/80 and examined by flow cytometry. (B) Representative plots indicating the frequency of neutrophils (CD11b⁺, Gr-1⁺, and F4/80⁻; upper-left quadrant) and inflammatory monocytes (CD11b⁺, Gr-1⁺, and F4/80⁺; upper-right quadrant) from an individual MyD88^−/− or Rip2^−/− MyD88^−/− mouse sacrificed at day 2 or day 5. (C) Average percentage of neutrophils or inflammatory monocytes from individual MyD88^−/− or Rip2^−/− MyD88^−/− mice sacrificed at day 2 or day 5. Plots for mouse groups are arranged as in panel A. *, P < 0.05; **, P < 0.005.

FIG. 4.

Mice deficient for both Rip2 and MyD88 are highly susceptible to lethal infection by L. pneumophila. MyD88^−/− and Rip2^−/− MyD88^−/− mice in a C57BL/6 background were given a low intranasal dose (4 × 10⁴ CFU) of L. pneumophila ΔflaA. (A) The graph represents the percentage of MyD88^−/− (n = 19) and Rip2^−/− MyD88^−/− (n = 18) mice surviving at the time points indicated. The difference between the two groups of mice was significant (P < 0.0001). (B) Surviving MyD88^−/− mice were sacrificed at day 30 postinfection, and the average number of CFU in the lung was measured. There were no detectible bacteria in either the liver or the spleen of these mice, and there were no surviving Rip2/MyD88-deficient mice to analyze. These data are in contrast to immune-sufficient mice, which did not have detectible bacteria in the lung at day 30 postinfection. Each point represents data from a single MyD88^−/− mouse, and the line represents the average from all mice.

FIG. 5.

Host protection from L. pneumophila infection requires activation of innate signaling pathways in bone marrow-derived cells. Bone marrow-chimeric mice were generated by irradiating wild-type (WT) mice and reconstituting them with donor bone marrow from either WT (WT>WT), MyD88-deficient (MyD88^−/−>WT), or Rip2/MyD88-deficient (Rip2^−/− MyD88^−/−>WT) mice. Mice were given a low intranasal dose (4 × 10⁴ CFU) of L. pneumophila ΔflaA. (A) Bacterial CFU in the lung and spleen was measured at 5 days postinfection for irradiated mice (open circles) receiving bone marrow from wild-type, MyD88^−/−, or Rip2^−/− MyD88^−/− donor mice and for mice with the same genetic background as the donors (filled circles). (B) BALF from infected mice was assayed for IL-6. Each point represents data from a single mouse. The lines indicate the means calculated from data for each group of mice. *, P < 0.05; **, P < 0.01.

FIG. 6.

Cooperative signaling between multiple pattern recognition systems is important for a protective immune response against L. pneumophila. Rip2^−/−, MyD88^−/−, and Rip2^−/− MyD88^−/− mice in a C57BL/6 background were given a high intranasal dose (1 × 10⁶ CFU) of either wild-type L. pneumophila or L. pneumophila ΔflaA. (A) At day 5 postinfection bacterial CFU were measured in the lung. Data in the left panel (day 0) represent CFU measured in the lungs of control animals sacrificed 4 h after intranasal infection. Each point represents data from a single mouse. The lines indicate the means calculated from the data for the two groups of mice. The dashed line indicates the lower limit of detection. *, P < 0.05 (B) The graph on the left indicates the survival of Rip2^−/−, MyD88^−/−, and Rip2^−/− MyD88^−/− mice after a high intranasal dose (1 × 10⁶ CFU) of wild-type L. pneumophila (n = 9 mice for each group). The difference between the MyD88^−/− and Rip2^−/− MyD88^−/− mice was significant (P < 0.01). The graph on the right shows the number of L. pneumophila bacteria in the lungs of mice surviving until day 21. (C) Survival of Rip2^−/−, MyD88^−/−, and Rip2^−/− MyD88^−/− mice after a high intranasal dose (1 × 10⁶ CFU) of L. pneumophila ΔflaA (n = 3 mice for each group). The difference between the MyD88^−/− and Rip2^−/− MyD88^−/− mice was significant (P < 0.05).