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The unique permissiveness of A/J mouse macrophages for replication of Legionella pneumophila is caused by a deficiency in the Nod-like receptor (NLR) protein and intracellular sensor for L. pneumophila flagellin (Naip5). The signaling pathways and proteins activated by Naip5 sensing in macrophages were investigated. Transcript profiling of macrophages from susceptible A/J mice and from resistant A/J mice harboring a transgenic wild-type copy of Naip5 at 4 h following L. pneumophila infection suggested that two members of the Irf transcriptional regulator family, Irf1 and Irf8, are regulated in response to Naip5 sensing of L. pneumophila. We show that macrophages having defective alleles of either Irf1 (Irf1−/−) or its heterodimerization partner gene Irf8 (Irf8R294C) become permissive for L. pneumophila replication, indicating that both the Irf1 and Irf8 proteins are essential for macrophage defense against L. pneumophila. Moreover, macrophages doubly heterozygous (Naip5AJ/WT Irf8R294C/WT or Nlrc4−/+ Irf8R294C/WT) for combined loss-of-function mutations in Irf8 and in either Naip5 or Nlrc4 are highly susceptible to L. pneumophila, indicating that there is a strong genetic interaction between Irf8 and the NLR protein family in the macrophage response to L. pneumophila. Legionella-containing phagosomes (LCPs) formed in permissive Irf1−/− or Irf8R294C macrophages behave like LCPs formed in Naip5-insufficient and Nlrc4-deficient macrophages which fail to acidify. These results suggest that, in addition to Naip5 and Nlrc4, Irf1 and Irf8 play a critical role in the early response of macrophages to infection with L. pneumophila, including antagonizing the ability of L. pneumophila to block phagosome acidification. They also suggest that flagellin sensing by the NLR proteins Naip5 and Nlrc4 may be coupled to Irf1-Irf8-mediated transcriptional activation of key effector genes essential for macrophage resistance to L. pneumophila infection.
Legionella pneumophila is an intracellular gram-negative bacterium that is ubiquitous in aquatic environment. Following inhalation of L. pneumophila-contaminated water droplets, Legionella may replicate inside human alveolar macrophages and cause a severe form of pneumonia called Legionnaires' disease (14, 29) or a less severe flu-like disease, Pontiac fever (20). In permissive macrophages, Legionella interferes with normal phagosome maturation and survives in a phagosome that neither acidifies nor fuses with lysosomes for several hours (4, 19, 39, 42). On the other hand, a Legionella-containing phagosome (LCP) rapidly acquires markers of the endoplasmic reticulum, such as calnexin and glucose-6-phosphatase, and becomes studded with ribosomes (1, 9, 38, 46). This remodeling requires the Dot/Icm type IV secretion system that injects over 30 bacterial protein effectors into the cytosol of host cells during infection (2, 33, 34, 40, 53). These effectors are thought to manipulate host cell functions, enabling establishment of the replicative organelle and promoting intracellular survival of the pathogen.
In contrast to human macrophages, mouse macrophages are generally nonpermissive for intracellular replication of L. pneumophila, with the notable exception of the A/J mouse strain (55, 56). Susceptibility of murine macrophages to L. pneumophila ex vivo is caused by a single locus on chromosome 13, Lgn1 (7, 16). In vivo complementation studies with A/J mice transgenic for the Lgn1 region (8), in vitro gene-silencing experiments with macrophages (54), and recent studies with a null mutant (22) have established that Naip5 is the gene underlying the L. pneumophila susceptibility effect at Lgn1. Naip5 belongs to the Nod-like receptor (NLR) family, a group of cytoplasmic proteins that act as intracellular sensors of pathogen-associated molecular patterns (PAMPs) and that represent a first line of defense in innate immunity (10, 27, 47). NLR proteins (over 20 NLR proteins have been described for humans) have a modular structure with (i) a conserved leucine-rich repeat (LRR) responsible for ligand recognition, (ii) a nucleotide-binding domain (NBD) mediating protein oligomerization, and (iii) a signaling-interaction domain that is specific for each NLR subfamily and that includes a caspase-recruitment domain (CARD), a pyrin domain, or a baculovirus inhibitor of apoptosis repeat domain. Activation of NLR proteins by PAMPs causes inflammatory, microbicidal, and cell death responses in macrophages that are mediated by NF-κB, mitogen-activated protein kinase, or caspase-1 (41). Stimulation of Nlrc4, Nlrp1b, and Nlrp3 by their ligand causes assembly of an inflammasome, an intracellular protein complex that leads to activation of proinflammatory caspase-1 and caspase-5 and secretion of proinflammatory cytokines (26, 35, 48).
Genetic and biochemical data have shown that both Naip5 (baculovirus inhibitor of apoptosis repeat domain, NBD, and LRR) and Nlrc4 (CARD, NBD, and LRR) are essential for innate defense against L. pneumophila and Salmonella, acting as sensors for flagellin species produced by these bacteria (1, 11, 24, 30, 37). Likewise, the human orthologs hNAIP and hNLRC4 were recently shown to be required for inhibition of intracellular Legionella replication (52). Flagellin-induced activation of caspase-1 is abolished in Salmonella- and Pseudomonas-infected macrophages lacking either Nlrc4 (1, 11, 13, 30, 31) or the NLR adaptor protein ASC (CARD and pyrin domain) (13, 24, 44). In addition, ASC has been shown to associate with Nlrc4 via CARD-CARD domain interaction in vitro (15). This suggests a model in which flagellin engagement of the LRR domain of Nlrc4 induces formation of an inflammasome consisting of Nlrc4, ASC, and caspase-1, leading to activation of the latter, interleukin-1β (IL-1β) processing, and pyroptosis (13, 31, 43).
Parallel study of Naip5 activation of caspase-1 in response to L. pneumophila flagellin has painted a more complex picture. First, L. pneumophila-induced cell death is impaired in Naip5-insufficient macrophages (37). Second, Naip5-insufficient macrophages show reduced caspase-1 activation in response to L. pneumophila infection, as measured by cleavage of synthetic substrates and IL-1β production (37, 57). Third, caspase-1-deficient macrophages show some increase in permissiveness for L. pneumophila replication (1, 57). Finally, cytosolic flagellin appears to activate caspase-1 in a Naip5-dependent fashion (32, 37), and 35 amino acids of the carboxyl terminus of flagellin is sufficient to trigger Naip5-dependent inflammasome activation (22). These observations suggest that Naip5 is required for restriction of L. pneumophila replication in macrophages through inflammasome-mediated activation of caspase-1 in response to flagellin. On the other hand, processing of caspase-1 in response to L. pneumophila infection has been demonstrated in macrophages carrying the A/J hypomorphic allele of Naip5 (B6. Chr13-A/J) but not in Nlrc4-deficient macrophages (21, 22) or in macrophages from Naip5-null mice (knockout allele) (22). These studies indicate that mutations in the A/J allele of Naip5 do not abolish its caspase-1 activation function, while they do impair its capacity to restrict L. pneumophila replication, suggesting that there is partitioning between these two Naip5-associated functions. In addition, ASC is necessary for L. pneumophila-induced caspase-1 activation but is dispensable for bacterial restriction (3, 57). Finally, studies of the maturation of LCPs in Naip5-deficient macrophages (6, 9) and in Nlrc4-deficient macrophages (1) have shown that Naip5 and Nlrc4 sensing of L. pneumophila products occurs very rapidly following phagocytosis (within 1 h).
These results suggest that Naip5 may participate in some signaling pathways (in addition to caspase-1) that play important roles in the early macrophage response and overall defense against L. pneumophila. In the present study, we used transcript profiling of Naip5-insufficient and Naip5-sufficient macrophages to obtain insight into the genes and pathways that are regulated in a Naip5-dependent fashion in response to L. pneumophila infection. Our results show that the transcriptional regulators Irf1 and Irf8 are rapidly induced in macrophages following infection. In addition, inactivation mutations in either Irf1 or Irf8 abrogate resistance to L. pneumophila. Finally, studies with macrophages doubly heterozygous for mutations in Irf8 and Naip5 or Nlrc4 demonstrated that there is a strong genetic interaction between members of the Irf and NLR families in the anti-Legionella defense of macrophages. This suggests that Naip5-Nlrc4 signaling in response to Legionella flagellin is closely linked to Irf-dependent transcription of genes encoding proteins essential for restriction of L. pneumophila growth in macrophages.
A/J, C57BL/6J (B6), and Irf1−/− mutant mice (with a B6 background) were purchased from Jackson ImmunoResearch Laboratories. Transgenic mice that express a Naip5 resistance allele from the B6 strain with the genetically susceptible A/J background have been described previously (8). These A/J mice correspond to an N8 generation intercross carrying (BAC+) or not carrying (BAC−) a B6-derived BAC clone harboring a wild-type Naip5 resistance allele. BXH-2 mice were obtained from N. Copeland and N. Jenkins (National Cancer Institute, Frederick, MD) and were maintained as a breeding colony at McGill University. The IL-12−/− mutant mice were provided by M. M Stevenson (McGill University, Montreal, Canada). The Nlrc4−/− mutant mice were kindly provided by Millennium Pharmaceuticals, Inc., and R. A. Flavell (Yale University). All mice were maintained and handled according to guidelines of the Canadian Council on Animal Care.
Bone marrow-derived macrophages (BMDMs) were isolated from femurs of 12- to 16-week-old mice and were cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma) containing 10% heat-inactivated fetal bovine serum (HI-FBS), 20% L-cell-conditioned medium (LCCM), 100 U/ml penicillin, and 100 μg/ml streptomycin in bacteriological grade dishes (Fischer) at 37°C in a humidified atmosphere containing 5% CO2. Seven days later, cells were harvested by gentle washing of the monolayer with phosphate-buffered saline containing citrate. Cells were plated in 150-mm tissue culture-grade plastic plates (15 × 106 cells per plate; Corning) in DMEM containing 10% HI-FBS, 10% LCCM, and 100 μg/ml of thymidine (Sigma) without antibiotics. Macrophages were cultured for an additional 24 h prior to use.
L. pneumophila Philadelphia-1 strain Lp02, a thymidine auxotroph derivative of strain Lp01, was a kind gift from Craig Roy (Yale University School of Medicine, New Haven, CT). The dotA mutant was a kind gift from Howard Shuman (Columbia University, New York, NY). An flaA deletion (corresponding to nucleotides 1478105 to 1479574 of the Lp01 genome) was generated in strain Lp02 by use of the allelic exchange vector pSR47S kindly provided by Russell Vance (Harvard Medical School, Boston, MA). The Lp02, dotA, and ΔflaA strains were cultured to stationary phase in N-(2-acetamido)-2-aminoethanesulfonic acid (ACES) (Sigma)-buffered yeast extract broth supplemented with 100 μg/ml of thymidine and used to infect BMDMs. BMDMs were exposed to L. pneumophila at a multiplicity of infection of 10:1 for 1 h at 37°C, and then cells were washed with warm DMEM and incubated for the period of time indicated below in DMEM supplemented with 10% HI-FBS, 10% LCCM, and 100 μg/ml of thymidine. Bacterial replication is expressed as the log increase in the number of CFU determined by lysis of macrophages with distilled water and plating of the cell lysates onto BCYE agar plates.
Total cellular RNA was extracted from BMDMs using a commercial reagent (TRIzol; Invitrogen) according to the manufacturer's recommendations. Macrophages were harvested in 2 ml (final volume) of TRIzol reagent. The samples were incubated for 5 min at 20°C, which was followed by chloroform extraction. The aqueous phase was removed, and nucleic acids were precipitated with isopropanol. Pellets were washed with 75% ethanol and dissolved in RNase-free water treated with 0.1% diethlypyrocarbamate. The integrity of each of the RNA preparations was verified by electrophoresis on 1% formaldehyde-containing agarose gels. In some instances, further RNA purification was performed using RNeasy columns (Qiagen) and 100 μg of total RNA according to the manufacturer's recommendations.
Mouse 15k v.4 cDNA spotted arrays were generated and hybridized by the UHN Microarray Facility (Toronto, Ontario, Canada) and analyzed as previously described. Individual spots from 16-bit TIFF digitized images were quantified using the QuantArray software (Perkin Elmer). Raw data generated by QuantArray were normalized using the GeneSpring software package (Silicon Genetics) and the Lowess scatter smoothing algorithm. We analyzed six hybridizations consisting of dye swap hybridizations of three biological replicates for BAC− versus BAC+. Genes with reproducible changes in transcript abundance were identified using the “one-class” algorithm in Significance Analysis of Microarrays (SAM). SAM assigns a score to each gene on the basis of the change in expression relative to the standard deviation of repeated measurements for that gene. Genes showing significant differences in expression were chosen using a false discovery rate of <0.05% and were further culled by keeping only the genes with a relative variation of >1.5. Results were visualized using the GeneSpring software.
For semiquantitative reverse transcription (RT)-PCR, independent RNA samples (n = 3) from the same experimental group were pooled, and 3 μg was converted to cDNA with reverse transcriptase (Moloney murine leukemia virus reverse transcriptase; Invitrogen) in a 20-μl RT reaction mixture, as previously described (25). One microliter of the RT reaction mixture was used for Taq DNA polymerase (Invitrogen)-mediated PCR amplification; the cycling parameters included an initial denaturation step (3 min at 94°C), followed by 16 to 20 cycles of 30 s at 94°C, 30 s at 55°C, and 1 min at 72°C and a final elongation step consisting of 7 min at 72°C. Amplicons were resolved in 1% agarose gels analyzed under UV light and were transferred to GeneScreen Plus membranes (Dupont, NEN Research Products). PCR primers were designed using the reported gene sequences. After transfer, DNA was UV cross-linked and prehybridized for at least 4 h at 65°C in a solution containing 10% dextran sulfate, 1% sodium dodecyl sulfate (SDS), and 1 M NaCl with 200 μg ml−1 of salmon sperm DNA. Hybridization was then performed overnight at 65°C with an [α-32P]dATP-labeled specific DNA fragment (100,000 cpm/ml of buffer) corresponding to each target gene. After incubation, the membrane was washed twice with 2× SSC-0.1% SDS (15 min per wash, 42°C), once with 2× SSC-0.5% SDS (30 min, 65°C), and once with 0.5× SSC-0.5% SDS (30 min, 65°C) (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate).
Total RNA (15 μg) was separated by electrophoresis on a 1% agarose gel containing 0.66 M formaldehyde and blotted onto a GeneScreen membrane (Perkin Elmer). Irf1 expression was monitored by hybridization to a gene-specific probe labeled with [α-32P]dATP using Klenow fragment DNA polymerase. The membrane was hybridized at 65°C in a solution containing 10% dextran sulfate, 2× SSC, 1% SDS, and 200 μg/ml salmon sperm DNA for 16 h. The membrane was washed twice at 65°C for 15 min in 2× SSC-0.1% SDS and then for 30 min with 2× SSC-0.5% SDS and for 30 min with 0.5× SSC-0.5% SDS. The membrane was stripped by boiling it in 10 mM Tris-HCl, 1 mM EDTA, 1% SDS before rehybridization. The signal was quantified using a phosphorimager.
Bacteria were grown as described above. Before infection, 2 × 108 bacteria were transferred to a microcentrifuge tube, pelleted, and suspended in 1 ml of a fluorescein isothiocyanate solution (FITC) (0.5 mg/ml; Sigma) in 100 mM NaHCO3 (pH 8). Bacteria were incubated for 20 min at room temperature, washed three times in 1 ml of NaHCO3 (pH 8), and resuspended in DMEM.
Measurements of phagosomal pH were obtained by fluorescence ratio imaging. BMDMs were plated on a coverslip and were infected the next day with fluorescein-treated L. pneumophila. The labeled bacteria were added to cells at a multiplicity of infection of 10:1, which was followed by centrifugation for 5 min at 4°C and incubation for 15 min at 37°C. Extracellular bacteria were removed by three washes with DMEM. The remaining nonphagocytosed bacteria were labeled with rat anti-Legionella antibody (1:100) for 5 min, followed by Cy3-conjugated anti-rat immunoglobulin G (1:100) for 5 min on ice. Phagosomes were allowed to mature for 1 h at 37°C, and then cells were placed in a chamber on the stage of a Zeiss microscope (Carl Zeiss Inc., Thornwood, NY) equipped with a ×100 oil immersion objective. A Sutter filter wheel was used to alternately position the two excitation filters (440 and 490 nm) in front of a xenon lamp. Image acquisition was controlled by MetaMorph software (Universal Imaging Corp., West Chester, PA). Calibration of the ratio of fluorescence to pH was performed in situ for each experiment by equilibrating the cells in isotonic K+-rich medium buffered to various pH values (between 4.5 and 7.5) in the presence of the K+/H+ ionophore nigericin (5 μM) as described previously (17). Calibration curves were constructed by plotting the extracellular pH, which is assumed to be identical to the internal pH, versus the corresponding fluorescence ratio.
In the present study, we set out to identify genes, proteins, and biochemical pathways that are important for preventing intracellular replication of L. pneumophila in macrophages and whose activity may be under the control of early signaling by the Naip5 protein. In these experiments, we used BMDMs obtained from A/J (Lgn1s) transgenic mice harboring (BAC+, resistant) or not harboring (BAC−, permissive) a recombinant BAC clone expressing the resistant Naip5 allele from strain B6 (Lgn1r). During the first 4 h following infection, both macrophage populations can restrict Legionella replication. However, by 8 h, the effect of the Naip5 gene can be detected, as there is active L. pneumophila replication in BAC− macrophages, while BAC+ macrophages can suppress bacterial replication. The difference in the number of CFU continues to increase over the next 72 h (Fig. (Fig.1A1A).
To look for genes and pathways regulated by L. pneumophila infection in a Naip5-dependent or -independent fashion, we carried out transcriptional profiling with cDNA microarrays, comparing RNA populations of infected BMDMs from BAC+ and BAC− mice. In this experiment, BAC− macrophages allowed 3.2-fold replication of L. pneumophila over 24 h, whereas for BAC+ transgenic macrophages there was a 1.8-fold decrease in the number of CFU over the same period (Fig. (Fig.1B).1B). At 4 h, the numbers of CFU recovered from the BAC+ and BAC− groups were similar (Fig. (Fig.1B),1B), and therefore differences in gene expression profiles detected at that time point were more likely to reflect the direct effect of the Naip5 gene and not secondary effects associated with differences in the intracellular bacterial loads. Transcript profiles were obtained for BAC− and BAC+ BMDMs either prior to infection or 4 h following infection. Using the “one-class” algorithm in the SAM application (P < 0.05, as determined by a t test; cutoff, 1.5-fold change; false discovery rate, 0.5%) (Fig. (Fig.1C),1C), we detected 138 transcripts significantly regulated by infection in BMDMs from both the BAC+ and BAC− groups following infection with L. pneumophila (Table (Table1;1; see Table S1 in the supplemental material for the complete list). In addition, we detected 17 transcripts that were differentially expressed only in BAC+ cells in response to infection and 49 transcripts specific to BAC− cells (Table (Table1).1). For the genes significantly regulated at 4 h postinfection only in BAC− cells, only in BAC+ cells, or in both BAC− and BAC+ cells, gene ontology analysis identified transcripts associated with signal transduction, immune response, and transcription (Table (Table1).1). As the cDNA microarrays used here contained only 15,000 transcripts, the number of genes identified in each class is likely to be an underestimate.
A semiquantitative RT-PCR approach was used to validate the differential expression of several genes detected by transcript profiling. In these experiments, we prioritized genes potentially implicated in immune functions. The Gapdh and β-Actin mRNA levels were not modulated in response to infection and were used as an internal control for normalization. Robust upregulation of Gro1, NFκBiα, Mlp, and Irf1 in response to L. pneumophila infection was verified (Fig. (Fig.2A).2A). In a time course experiment, maximal Irf1 induction was observed as early as 1 h postinfection (data not shown). We further confirmed infection-dependent induction of Irf1 and of its heterodimerization partner, Irf8, by Northern blot analysis (Fig. (Fig.2B).2B). Interestingly, we noted that induction of Irf1 and Irf8 mRNA expression in macrophages infected with L. pneumophila was somewhat greater in Naip5-sufficient (BAC+) macrophages than in Naip5-insufficient (BAC−) macrophages (Fig. (Fig.2B).2B). These results suggest that the transcriptional regulators Irf1 and Irf8 may be part of the signaling program downstream of Naip5.
Irf1 is a transcriptional regulator and a member of the interferon response factor (IRF) family, a group of nine proteins that play a critical role in cellular responses to bacterial and viral pathogens by activating transcription of certain gene families in response to early signaling by different classes of interferon. Notably, Irf3 is required for L. pneumophila-induced beta interferon (IFN-β) expression and for control of intracellular replication of L. pneumophila in lung epithelial cells (36). However, the role of other IRF family members in macrophage defense against L. pneumophila infection has not been studied previously. Irf1 heterodimerizes with Irf8 to stimulate transcription of IFN-γ-responsive genes that have an interferon-stimulated response element sequence in their regulatory regions, including IL-12p40 and iNOS. In vivo, this leads to activation of CD4+ T cells and NK cells for production of IFN-γ, thereby amplifying the initial signal and contributing to Th1 polarization of the early T-cell response (45).
To address the role of Irf1 and Irf8 in macrophage defense against L. pneumophila, we monitored replication of L. pneumophila in macrophages from mice having a nonfunctional allele at each locus. The Irf1−/− mutant used was generated using an otherwise Legionella-resistant B6 background (Lgn1r). Likewise, BXH-2 is a recombinant inbred mouse strain derived from C3H/HeJ (Lgn1r) and B6 (Lgn1r) that has a loss-of-function mutation (R294C) in Irf8 that we have reported previously (50). BMDMs from Irf1- and Irf8-deficient mice were infected in vitro with L. pneumophila Lp02, and 72 h later the total numbers of CFU were determined. Control BMDMs from permissive A/J and BAC− controls supported a 1.5-log increase in the number of CFU, while resistant controls (B6, BAC+) could completely suppress L. pneumophila replication over the same period (Fig. (Fig.3).3). Irf1−/− macrophages seemed to be partially impaired in the ability to control L. pneumophila replication (0.5-log increase), while Irf8R294C mutant macrophages were completely susceptible to infection (Fig. (Fig.3).3). These results demonstrate that Irf1 and Irf8 are required for macrophage defense against L. pneumophila.
Macrophages infected with intracellular parasites, including L. pneumophila, respond by producing IL-12. An intact IFN-γ-IL-12 cytokine loop is required for expression of a number of protective responses, including expression of effector molecules and bactericidal enzymes synthesized by macrophages. In addition, IL-12 is a major transcriptional target of Irf1 and Irf8 (28). Therefore, we assessed the possibility that Irf1−/− and Irf8R294C macrophages have increased susceptibility due to a deficiency in IL-12 production by examining the intracellular replication of L. pneumophila in macrophages from B6 mice lacking an IL-12p40 gene (IL-12p40−/−). Macrophages from IL-12p40−/− mice were as resistant to L. pneumophila infection as macrophages from B6 controls at 72 h postinfection (Fig. (Fig.4A).4A). The specificity of the increased permissiveness of macrophages lacking Irf1 or Irf8 for intracellular replication of L. pneumophila was validated with two additional controls. Irf1−/− and Irf8R294C macrophages did not suffer from general impairment of bacteriostatic and bactericidal functions, as both populations could block replication of an avirulent dotA mutant defective in the type IV secretion system (Fig. (Fig.4B).4B). On the other hand, complete (B6, BAC+) or partial (Irf1−/−) inhibition of L. pneumophila replication was dependent on recognition of bacterial flagellin since macrophages with wild-type and Irf-deficient genotypes are equally susceptible to infection with an L. pneumophila flagellin (ΔflaA) mutant (Fig. (Fig.4C).4C). Finally, pretreatment of macrophages from mice with all genotypes with IFN-γ completely abolished intracellular replication of L. pneumophila (Fig. (Fig.4D).4D). Together, these results indicate that Irf1 and Irf8 are required for the intrinsic resistance of macrophages to intracellular replication of L. pneumophila. However, the protective effect of Irf1 and Irf8 in macrophages ex vivo is IL-12p40 independent and can be corrected by exposure to IFN-γ.
We further investigated a possible link between flagellin-dependent Naip5 signaling, inflammasome activation, and stimulation of Irf-dependent gene transcription. It has been proposed that the response to L. pneumophila flagellin involves activation of a Naip5-Nlrc4-containing inflammasome, and loss-of-function mutations in Naip5 or Nlrc4 abrogate macrophage resistance to L. pneumophila infection (1, 32, 37). Therefore, we set out to test possible genetic interactions between Irf8 and either Naip5 or Nlrc4 by creating macrophages partially deficient in both pathways and testing the effects of the partial impairment on resistance to L. pneumophila infection. For this, we isolated BMDMs from (i) (A/J × BXH-2)F1 mice that were doubly heterozygous for loss of function at Naip5 and Irf8 (Naip5S/R Irf8R294C/wt) and (ii) (Nlrc4−/− × BXH-2)F1 mice that were doubly heterozygous for loss of function at Nlrc4 and Irf8 (Nlrc4−/+ Irf8R294C/wt). The Nlrc4 mutation was created in an otherwise L. pneumophila-resistant B6 background (11). BMDMs from these mice were infected in vitro with L. pneumophila Lp02, and bacterial replication was monitored 72 h postinfection (Fig. (Fig.5).5). In these experiments, we also tested BMDMs from additional control groups of singly heterozygous animals (Irf8R294C/wt and Nlrc4−/+) which were found to be resistant to infection (data not shown). In addition, we assayed BMDMs from (BXH-2 × Irf1−/−)F1 mice that were doubly heterozygous for Irf1 and Irf8 mutations (Irf8R294C/wt Irf1−/+). These macrophages were found to be nonpermissive for L. pneumophila replication (Fig. (Fig.5),5), indicating that a combined partial loss of function at Irf1 and Irf8 is not sufficient to cause susceptibility. By contrast, BMDMs doubly heterozygous (Naip5S/R Irf8R294C/wt or Nlrc4−/+ Irf8R294C/wt) for combined loss-of-function mutations in Irf8 and in either Naip5 or Nlrc4 were found to be highly susceptible to L. pneumophila (Fig. (Fig.5).5). The degree of susceptibility of these double heterozygotes was as great as that of BMDMs from mice homozygous for either Irf8 (BXH-2), Naip5 (A/J), or Nlrc4 mutations (Fig. (Fig.55 and data not shown). These results indicate that there is strong genetic interaction between Irf8 and Naip5 or Nlrc4 that restricts L. pneumophila replication in macrophages. They suggest a model in which sensing of flagellin and activation of the inflammasome via Naip5 and Nlrc4 either are Irf8 dependent or cause downstream Irf8-dependent transcriptional activation of additional genes and pathways critical for defense against L pneumophila.
We previously showed that in wild-type B6 macrophages, Naip5 antagonizes the ability of L. pneumophila to modulate phagosome maturation toward an endoplasmic reticulum-derived replicative organelle, causing instead phagosome maturation of a fully acidified phagolysosome within 1 h following infection (9). A similar effect of Nlrc4 on maturation of an LCP was also demonstrated (within 2 h after infection) and was shown to be flagellin dependent and cell death independent (1). We investigated whether the permissiveness for L. pneumophila replication detected in Irf1−/− and in Irf8R294C mutant macrophages had effects on phagosome maturation similar to those observed in Naip5-insufficient (A/J) and Nlrc4-deficient macrophages. For this, we used live-cell microscopy to monitor in real time acidification of LCPs formed in macrophages isolated from BAC−, BAC+, B6, Nlrc4−/−, Irf1−/−, and Irf8R294C mice. Macrophages were infected with L. pneumophila Lp02 labeled by covalent attachment of fluorescein, a pH-sensitive ratiometric dye (pKa 6.4), using a protocol that we have described previously (17). In these experiments, L. pneumophila infection was synchronized using a 5-min centrifugation step, followed by 15 min of incubation at 37°C to allow phagocytosis. Nonphagocytosed bacteria were removed by washing. In addition, to ascertain that only internalized bacteria were quantified, possible residual extracellular bacteria were labeled with an anti-Legionella antibody, followed by a Cy3-conjugated secondary antibody. Phagosome maturation was allowed to take place for 1 h following phagocytosis.
Figure Figure6A6A shows the results of a representative experiment performed with B6 wild-type macrophages. The upper left panel shows a phase-contrast microscopy image (differential interfering contrast) of macrophages interacting with two live L. pneumophila cells (as shown by the FITC image), one of which is intracellular and the other of which is extracellular (positive for Cy3 dye staining). As shown in the fluorescence ratio image in the bottom right panel, the intracellular bacterium is in an acidic environment (determined using the pseudocolor calibration scale on the right), while the other bacterium is exposed to extracellular medium which has a near-neutral pH. Calibration of the ratio of fluorescence to pH was performed in situ for each experiment by equilibrating the cells in isotonic K+-rich medium buffered to various pH values (between pH 4.5 and 7.5) in the presence of the K+/H+ ionophore nigericin (5 μM) as described previously (17).
The pH of phagosomes containing L. pneumophila Lp02 formed in macrophages from wild-type B6 and Naip5-sufficient BAC+ mice was significantly more acidic than that of L. pneumophila phagosomes formed in Naip5-insufficient BAC−, Nlrc4−/−, Irf1−/−, and Irf8R294C mutant macrophages (pH 5.4 ± 0.1 and pH 6.2 ± 0.2, respectively) (Fig. (Fig.6B).6B). The phagosomal pH in all cell types was indistinguishable after addition of the vacuolar H+ ATPase inhibitor nigericin (not shown). Importantly, the attenuated phagosomal acidification seen in Naip5-insufficient, Nlrc4- or Irf1-deficient, or Irf8R294C mutant macrophages was specific for L. pneumophila wild-type strain Lp02, as phagosomes containing avirulent dotA bacteria acidified normally (Fig. (Fig.6B);6B); i.e., they acidified to an extent comparable to that observed in phagosomes formed in B6 and BAC+ macrophages. This confirms that the phagosomal acidification mechanism is fully competent in all cell types and that the cells differ only in their responsiveness to virulent wild-type L. pneumophila. In addition, this response is flagellin dependent, as shown by the lack of acidification of flagellin-deficient mutant (Δfla) LCPs (Fig. (Fig.6B).6B). In conclusion, the pattern of the phagosomal acidification defect caused by nonfunctional Irf8 is similar to and concomitant with the pattern caused by Naip5 insufficiency (Fig. (Fig.6C).6C). These results suggest that Irf8 expression, either constitutive or inducible following rapid flagellin- and Naip5-dependent signaling, is required to antagonize the ability of L. pneumophila to modulate phagosome maturation.
In vivo complementation data, silencing studies, and recent experiments with a knockout mouse mutant have shown that permissiveness of A/J macrophages for infection with L. pneumophila is caused by a partial loss of function (hypomorphic allele) of the Naip5 gene (8, 22, 54). Naip5 is a member of the NLR family of intracellular sensors of PAMPs. More recently, it was observed that loss of function for another NLR protein, Nlrc4, also causes susceptibility to infection with L. pneumophila (1, 57). Upon engagement with their ligands, namely bacterial flagellin, anthrax lethal toxin, and ATP-uric acid crystals, Nlrc4, Nlrp1b, and Nlrp3, respectively, have been shown to assemble to form a so-called inflammasome (12), an inflammatory caspase-activator complex composed of an NLR protein platform (Nlrp1b, Nlrp3, or Nlrc4), an inflammatory caspase (caspase-1 or caspase-5), and an adaptor molecule (ASC). A model has been proposed (32, 37, 57) in which recognition of flagellin by Naip5 and Nlrc4 results in inflammasome assembly, caspase-1 activation, and ultimately inhibition of intracellular L. pneumophila replication. This model is based on the following observations: (i) macrophages deficient in Naip5 or Nlrc4 cannot restrict intracellular replication of L. pneumophila (1, 8, 22, 54); (ii) gene silencing of hNAIP or hNLRC4 in human cells leads to enhanced bacterial growth, and overexpression of both molecules strongly reduces Legionella replication (52); (iii) in L. pneumophila-infected macrophages, caspase-1 activation, IL-1β secretion, and cell death are abolished in the absence of Naip5 or Nlrc4 (11, 22, 37); (iv) flagellin gene-deficient L. pneumophila (ΔflaA) can replicate in otherwise resistant macrophages (32, 37); and (v) Naip5 physically interacts in vitro with Nlrc4 (57). Thus, flagellin derived from intracellular L. pneumophila would be detected by Naip5 and/or Nlrc4, which would heterodimerize through NBD interaction. Nlrc4 would recruit and activate caspase-1 through CARD-CARD interaction. Then activated caspase-1 may cleave proinflammatory cytokines (IL-1β and IL-18) into their active forms (Fig. (Fig.7)7) and also induce apoptotic cell death in infected macrophages.
On the other hand, parallel studies have suggested that Naip5 may play supplementary roles in very early events following phagocytosis of L. pneumophila by macrophages (in addition to caspase-1 activation). Indeed, the Naip5 and Nlrc4 status (either wild type or mutant) has dramatic consequences for the early maturation of L. pneumophila-containing phagosomes in macrophages, with differential recruitment of the lysosomal markers cathepsin D and Lamp1 and the endoplasmic reticulum markers BAP31 and calnexin being noticed as early as 1 to 2 h postinfection (1, 6, 9). With this in mind, we set out to identify genes, proteins, and biochemical pathways that are important for preventing intracellular replication of L. pneumophila in macrophages and whose activity may be influenced by early Naip5 signaling. Transcript profiling was used to identify genes differentially expressed in Naip5-insufficient and Naip5-sufficient macrophages 4 h following infection with L. pneumophila. Irf1 was found to be upregulated by L. pneumophila infection in macrophages, and the degree of induction was further modulated by the Naip5 status (Fig. (Fig.11 and and2).2). Further, we demonstrated that loss-of-function mutations in either Irf1 or its coactivator and heterodimerization partner Irf8 cause increased susceptibility to L. pneumophila infection (Fig. (Fig.3).3). This susceptibility occurs in the context of functional Naip5 signaling of otherwise resistant B6 macrophages. To ascertain that Legionella susceptibility in BXH-2 mice is specifically linked to the R294C mutant Irf8 variant and not to additional effects of the mixed C3H/HeJ-B6 genetic background of the strain, we first verified the resistance phenotype of both parental strains (see Fig. S3 in the supplemental material) and then we produced a (BXH-2 × A/J)F2 offspring, and F2 mice homozygous for the wild-type or mutant Irf8 allele were identified by genotyping. Scrambling of genetic background effects in these F2 mice allowed us to verify the effect of Irf8 alleles on the response to Legionella. Macrophages from F2 mice homozygous for the Irf8R294C mutant allele were found to be significantly more permissive for Legionella replication than macrophages from F2 mice carrying the wild-type Irf8 allele (see Fig. S1 in the supplemental material). Together, these results demonstrate that sensing and signaling by the NLR protein Naip5 and transcriptional activation by Irf1-Irf8 are both absolutely required to prevent intracellular replication of L. pneumophila in macrophages.
Irf1 and Irf8 are members of the IRF family of transcriptional regulators that play a critical role in innate immunity. Irf8 acts as a coactivator with Irf1 to stimulate transcription of IFN-γ-responsive genes that have an interferon-stimulated response element sequence in their regulatory regions, including IL-12p40, but it can also act as a corepressor with Irf2 to antagonize Irf1-dependent transcriptional activation. Furthermore, Irf8 can further heterodimerize with PU.1 and other Ets proteins to activate transcription of genes containing IFN-γ activation site or Ets/IRF composite element promoter elements, including Igκ, p67phox, p91phox, CD20, IL-1, Tlr4, and genes encoding members of the macrophage scavenger receptor family (45). Therefore, (i) the rapid induction of Irf1 and Irf8 mRNA expression in response to L. pneumophila infection and (ii) the increased susceptibility of Irf1−/− and Irf8R294C mutant macrophages suggest a model in which immediate early activation of Irf1-Irf8-dependent transcription in response to phagocytosis of L. pneumophila by macrophages (including flagellin sensing by Naip5) is absolutely essential to restrict intracellular replication of this bacterium. Although this represents our favored model, one must also consider the fact that Irf1 and Irf8 are known to play critical roles in maturation of several myeloid lineages, including NK cells, dendritic cells, and macrophages (45). However, several lines of evidence argue against a generalized defect in the antimicrobial defenses of Irf1−/− and Irf8R294C macrophages to account for increased susceptibility of these cells to L. pneumophila infection, for the following reasons: (i) BXH-2 mice (Irf8R294C) have numbers of F4/80-positive macrophages in their spleens comparable to the numbers in B6 controls (data not shown), (ii) Irf1−/− and Irf8R294C macrophages can efficiently kill avirulent dotA L. pneumophila mutants (Fig. (Fig.4),4), (iii) Irf8R294C mutant BXH-2 mice can control early replication of Mycobacterium bovis BCG in vivo more efficiently that the Nramp1G169D mutant mouse strain (49), and (iv) mice having a null mutation in Irf8 become susceptible to infection with certain viruses while they remain resistant to infection with other viruses (18). Nevertheless, it is still possible that Irf1 and Irf8 are required for expression of a macrophage protein(s) that may be required for Naip5 signaling and/or for restriction of intracellular of Legionella replication.
What is the functional relationship between Naip5-Nlrc4 sensing of L. pneumophila leading to inflammasome activation, Irf1-Irf8 transcriptional activation, and macrophage defenses against L. pneumophila? One possibility is that both pathways are essential and function in parallel, possibly responding to distinct subsets of L. pneumophila stimuli, where Irf1 and Irf8 are required for the transcription of key components of these pathways (Fig. (Fig.7A).7A). This may involve flagellin-independent signaling by Toll-like receptor family members or other receptors to activate IRF family members (as observed in Naip5-insufficient mice) and flagellin-dependent activation of the Naip5-Nlrc4 inflammasome to activate the caspase-1 cascade. Irf1, Irf5, and Irf7 have been identified as key effectors in Toll-like receptor signaling. Irf3 and Irf7 are also involved in cascade events from RIG-I/MDA5, a cytosolic pathogen receptor (45). We have observed that induction of caspase-1 mRNA expression in response to Legionella infection in macrophages is not dependent on either Naip5 or Irf1 and Irf8 (see Fig. S2 in the supplemental material). In addition, we observed similar expression of caspase-1 at the protein level in Irf1−/−, Irf8R294C, and B6 BMDMs (see Fig. S4A in the supplemental material), and caspase-1 activation following infection by L. pneumophila seems not to be impaired in Irf1−/− or Irf8R294C BMDMs (see Fig. S4B in the supplemental material). Although caspase-1−/− macrophages have been shown to be more permissive for Legionella replication than wild-type macrophages (57), caspase-1 activation observed in A/J and ASC−/− macrophages does not seem to be sufficient for Legionella restriction (3, 21, 22, 57). An additional and exciting possibility is that Irf1 and Irf8 represent immediate-early downstream mediators of Naip5-Nlrc4 inflammasome signaling, acting to amplify the transcriptional response of macrophages to L. pneumophila infection (Fig. (Fig.7B).7B). This scenario is supported by different key observations. First, macrophages that are doubly heterozygous (Naip5S/R Irf8R294C/wt or Nlrc4−/+ Irf8R294C/wt) for combined loss-of-function mutations in Irf8 and in either Naip5 or Nlrc4 are highly susceptible to L. pneumophila. Although this is not absolute proof, such a strong genetic interaction between Irf8 and NLR family members is compatible with the hypothesis that both proteins are part of the same signaling pathway in macrophages following phagocytosis of L. pneumophila (Fig. (Fig.6).6). Second, we observed that homozygosity for loss of function of Irf1, Irf8, Naip5, and Nlrc4 is phenotypically expressed as a rapid defect in acidification of the L. pneumophila-containing phagosome, taking place within 1 h following phagocytosis. This concordance in the temporal and subcellular expression of the defect is also in agreement with the hypothesis that both sets of proteins are parts of the same pathways (Fig. (Fig.5).5). Finally, we observed that activation of Irf1 and Irf8 transcriptional targets in macrophages (including IL-12p40 and iNOS) following L. pneumophila infection is dependent on flagellin, the key ligand of Naip5 and Nlrc4, and is not detected when macrophages are infected with flagellin-deficient L. pneumophila mutants (data not shown).
Together, these results suggest that there is a link between L. pneumophila sensing by the NLR proteins Naip5 and Nlrc4 and Irf1-Irf8 transcriptional activation of a number of effector genes that play a critical role in the macrophage response to L. pneumophila (Fig. (Fig.7).7). These results are in agreement with a recent study showing that there must be cooperation between cytokine signaling (tumor necrosis factor alpha and type I interferon) and Naip5-Nlrc4 signaling for L. pneumophila restriction by macrophages (5). It is noteworthy that both tumor necrosis factor alpha and type I interferon signaling have been found to be regulated by Irf1 and/or Irf8 (45, 51). Although there are many potential transcriptional targets of Irf1 and Irf8 that are known to be critical for the macrophage antimicrobial arsenal, an obvious and attractive set of targets to explain the Naip5-dependent effect on modulation of L. pneumophila phagosome maturation in macrophages is the family of p47 GTPases (23). The genes encoding these proteins, which include IRG-47, LRG-47, TGTP, IGTP, IIGP1, and GTP 1, are interferon-responsive genes that are recruited to the nascent phagosomes. IRG-47 and IGTP are regulated by Irf1 very rapidly (1 to 4 h) and are known to play an important role in maturation of phagosomes into phagolysosomes, a process impaired in L. pneumophila phagosomes formed in Naip5- and Nlrc4-deficient macrophages (1, 6, 9).
P.G. is a distinguished scientist of the Canadian Institutes of Health Research and a James McGill Professor of Biochemistry. A.F. was supported by a fellowship from the Canadian Institutes of Health Research.
Editor: J. L. Flynn
Published ahead of print on 31 August 2009.
†Supplemental material for this article may be found at http://iai.asm.org/.