Multiple MyD88-dependent responses contribute to pulmonary clearance of Legionella pneumophila

Abstract

MyD88-dependent signalling is important for secretion of early inflammatory cytokines and host protection in response to Legionella pneumophila infection. Although toll-like receptor (TLR)2 contributes to MyD88-dependent clearance of L. pneumophila, TLR-independent functions of MyD88 could also be important. To determine why MyD88 is critical for host protection to L. pneumophila, the contribution of multiple TLRs and IL-18 receptor (IL-18R)-dependent interferon-gamma (IFN-gamma) production in a mouse was examined. Mice deficient for TLR5 or TLR9, or deficient for TLR2 along with either TLR5 or TLR9, were competent for controlling bacterial replication and had no apparent defects in cytokine production compared with control mice. MyD88-dependent production of IFN-gamma in the lung was mediated primarily by natural killer cells and required IL-18R signalling. Reducing IFN-gamma levels did not greatly affect the kinetics of L. pneumophila replication or clearance in infected mice. Additionally, IFN-gamma-deficient mice did not have a susceptibility phenotype as severe as the MyD88-deficient mice and were able to control a pulmonary infection by L. pneumophila. Thus, MyD88-dependent innate immune responses induced by L. pneumophila involve both TLR-dependent responses and IL-18R-dependent production of IFN-gamma by natural killer cells, and these MyD88-dependent pathways can function independently to provide host protection against an intracellular pathogen.

Fig. 1

L. pneumophila infection of TLR9-deficient mice. A. TLR9-deficient mice (TLR9^−/−) and heterozygous littermates (TLR9^+/−) were infected with 10⁶ L. pneumophila intranasally. Three mice were sacrificed at the indicated days post infection and average number of CFU ± S.D. detected in TLR9-deficient (dotted line) and TLR9-sufficient mice (solid line) are indicated. B. BALF from infected mice was assayed for the indicated cytokines at day 2 and day 3 post infection by ELISA. Each point represents data from a single TLR9-deficient (open circles) or a TLR9-sufficient mouse (solid circles). The lines indicate the means calculated from the data for the two groups of mice. There was no statistical significance between the two groups of mice (P > 0.05). C. TLR2-deficient mice that were either deficient in TLR9 (TLR2^−/− TLR9^−/−) or sufficient for TLR9 (TLR2^−/− TLR9^+/−) were infected with L. pneumophila intranasally. Three mice were sacrificed at the indicated days post infection and average number of CFU ± S.D. detected in TLR9-deficient (dotted line) and TLR9-sufficient mice (solid line) are indicated. D. BALF from infected mice was assayed for the indicated cytokines at day 2 and day 3 post infection by ELISA. Each point represents data from a single TLR2^−/− TLR9^−/− (open circles) or a TLR2^−/− TLR9^+/− mouse (solid circles). The lines indicate the means calculated from the data for the two groups of mice. There was no statistical significance between the two groups of mice (P > 0.05). E. TLR2^−/− TLR9^+/−(solid line) and TLR2^−/− TLR9^−/− mice (dotted line) were infected intranasally with 10⁷ L. pneumophila and survival was monitored (n = 10 animals/group). The graph represents the percentage of mice surviving at the time points indicated. There was no statistical difference between the two groups of mice (P > 0.05).

Fig. 2

L. pneumophila infection of TLR5-deficient mice. A. TLR5-deficient mice (TLR5^−/−) and heterozygous littermates (TLR5^+/−) were infected with 10⁶ L. pneumophila intranasally. Three mice were sacrificed at the indicated days post infection and average number of CFU ± S.D. detected in TLR5-deficient (dotted line) and TLR5-sufficient mice (solid line) are indicated. B. BALF from infected mice was assayed for the indicated cytokines at day 2 and day 3 post infection by ELISA. Each point represents data from a single TLR5-deficient (open circles) or a TLR5-sufficient mouse (solid circles). The lines indicate the means calculated from the data for the two groups of mice. There was no statistical significance between the two groups of mice (P > 0.05) except for IFN-γ at day 2 post infection (*P < 0.05). C. TLR2-deficient mice that were either deficient in TLR5 (TLR2^−/− TLR5^−/−) or sufficient for TLR5 (TLR2^−/− TLR5^+/−) were infected with L. pneumophila intranasally. Three mice were sacrificed at the indicated days post infection and average number of CFU ± S.D. detected in TLR5-deficient (dotted line) and TLR5-sufficient mice (solid line) are indicated. D. BALF from infected mice was assayed for the indicated cytokines at day 2 and day 3 post infection by ELISA. Each point represents data from a single TLR2^−/− TLR5^−/− (open circles) or a TLR2^−/− TLR5^+/− mouse (solid circles). The lines indicate the means calculated from the data for the two groups of mice. There was no statistical significance between the two groups of mice (P > 0.05). E. Peritoneal macrophages from TLR5^+/− (Control), TLR2^−/−, and TLR2^−/−TLR5^−/− mice were incubated with either WT (solid circles) or ΔflaA (open circles) L. pneumophila. Supernatants were collected 24 h post infection, and TNF-α levels were measured by ELISA. Each data point represents the cytokine content of one well from cells pooled from six mice.

Fig. 3

A protective innate immune response to L. pneumophila is induced in mice deficient in TLR2, TLR5 and TLR9 signaling. A. TLR2-deficient mice that were either TLR9-deficient (TLR2^−/−TLR9^−/−) or TLR9-sufficient (TLR2^−/− TLR9^+/−) were infected with 10⁶ CFU of the L. pneumophila ΔflaA strain intranasally. Three mice were sacrificed at the indicated days post infection and average number of CFU ± S.D. detected in TLR9-deficient (dotted line) and TLR9-sufficient mice (solid line) are indicated. B. TLR2^−/− TLR9^−/− mice were infected with either WT or ΔflaA L. pneumophila. Mice were sacrificed at day 2 and day 3 post infection and bacteria were measured in the lung. The number of WT (closed circles) and ΔflaA (open circles) L. pneumophila detected in the lung are plotted for individual mice sacrificed at the times indicated. The lines indicate the average number of CFUs for each strain at each time point (** P < 0.005). C. BALF from infected TLR2^−/−TLR9^−/− mice was assayed for the indicated cytokines at day 2 and day 3 post infection by ELISA. Each point represents data from a single mouse infected with either WT (solid circles) or ΔflaA L. pneumophila (open circles) (*P < 0.01; **P < 0.005). D and E. BMMs from TLR2^−/−TLR9^−/− and TLR2^−/−TLR9^+/− mice (D) or alveolar macrophages from TLR2^−/−TLR9^−/− mice (E) were incubated with either WT (solid circles) or ΔflaA (open circles) L. pneumophila. Supernatants were collected 24 h post infection, and IL-12 p40 (D) or TNF-α(E) levels were determined by ELISA. For BMMs, all data points represent the average cytokine concentration determined from three wells infected independently.

Fig. 4

IL-18R signaling is important of NK cell production of IFN-γ. A. MyD88^+/− (left panels) and MyD88^−/− mice (right panels) were either infected intranasally with 10⁶ L. pneumophila (solid line) or administered PBS intranasally (dotted line). At day 2 post infection, cell suspensions from isolated lungs were stained for DX5, NKG2ACE, and intracellular IFN-γ and examined by flow cytometry. Top panels are DX5⁺ NKG2ACE⁺ lymphocytes. Bottom panels are DX5⁻ lymphocytes. Results are representative of independent data from three mice. B. MyD88^+/− and MyD88^−/− mice were either infected intranasally with 10⁶ L. pneumophila or administered PBS intranasally. IL-18 levels in BALF were measured at day 2 and day 3 post infection by ELISA. Each point represents data from a single MyD88^−/− (open circles) or a MyD88^+/− mouse (solid circles) and the mean value for each group is indicated by the line (*P < 0.05, **P < 0.005).

Fig. 5

L. pneumophila clearance from the lung can occur independent of IL-18 signaling. A. IL-18R1-deficient (IL-18R1^−/−) and C57BL/6 wild-type (WT) mice were infected intranasally with 10⁶ CFU of the L. pneumophila ΔflaA strain and mice were sacrificed at day 2 post infection. IFN-γ and IL-12 p40 in BALF was measured by ELISA (left and middle panel, respectively) and bacterial CFU in lung lysates were determined (right panel). Each point represents data from a single IL-18R1^−/− (open circles) or WT (solid circles) mouse. The lines indicate the means calculated from the data for the two groups of mice (*P < 0.05; **P < 0.0005). B. IL-18R1^−/−, MyD88^−/−, and WT mice on the C57BL/6 background were infected intranasally with 1×10⁶ CFU of the L. pneumophila ΔflaA strain. Mice were sacrificed in groups of 3 and bacterial numbers were determined from lung lysates at the indicated time points. The average number of CFUs detected for the IL-18R1^−/− (line with open circles), MyD88^−/− (dotted line), and WT (solid line) mice are indicated. C. WT (left panel), IL-18R1^−/− (middle panel), and MyD88^−/− (right panel) mice in a C57BL/6 background were infected intranasally with 10⁶ L. pneumophilaΔflaA. At day 2 post infection, cell suspensions from isolated lungs were stained for DX5, NKG2ACE, and intracellular IFN-γ and examined by flow cytometry. Results are representative of independent data from three mice.

Fig. 6

L. pneumophila clearance from the lung can occur independent of NK cells. A. MyD88^+/− mice were injected with α-asialo GM-1 or PBS one day prior to intranasal infection with 10⁶ L. pneumophila. IFN-γ in BALF was measured at day 2 post infection by ELISA. The reduction in IFN-γ levels in NK cell-depleted mice compared to non NK-cell depleted mice is significant (P < 0.05). B. MyD88^+/− mice were injected with α-asialo GM-1 (dashed line) or PBS (solid line) one day prior to intranasal infection with 10⁶ L. pneumophila. CFUs in lung lysates were determined for at indicated time points. There was no statistical significance between the two groups of mice at day 2 and day 4 post infection (P > 0.05).

Fig. 7

IFN-γ^−/− mice are more resistant to L. pneumophila infection than MyD88^−/− mice. A. IFN-γ^−/−, MyD88^−/−, and WT mice on the C57BL/6 background were infected intranasally with 10⁶ CFU of the L. pneumophilaΔflaA strain. Mice were sacrificed in groups of 3 and bacterial numbers were determined from lung lysates at the indicated time points. The average number of CFUs detected for the IFN-γ^−/− (line with open circles), MyD88^−/− (dotted line), and WT (solid line) mice are indicated. B. CFUs were measured from the spleens (left panel) and livers (right panel) of infected mice. The average number of L. pneumophila detected for IFN-γ^−/− (shaded bars), MyD88^−/− (open bars), and WT (solid bars) are indicated (ND = not detectable). C. Survival was monitored in infected mice (n = 10 animals/group). The graph represents the percent of surviving mice. The difference between the WT and IFN-γ^−/− curves versus the MyD88^−/− curve was significant (P < 0.0001).