Enhanced growth restriction of Legionella pneumophila in endotoxin-treated macrophages.

Macrophages from A/J mice are permissive for growth of Legionella pneumophila, an intracellular opportunistic pathogen that grows preferentially in macrophages. Macrophages from other mouse strains are highly resistant to growth of Legionella. In the present study, it was found that macrophages from A/J mice are readily activated by pretreatment with lipopolysaccharide (LPS), so that the cells do not permit Legionella to replicate in vitro, as occurs when untreated macrophages from A/J mice are cultured with these organisms for 48 hr. The augmentation of Legionella growth inhibition by LPS-activated macrophages from nonpermissive BDF1 mice also occurred. After in vitro infection, there was a 1000-fold increase in the number of Legionella in A/J macrophages and approximately a 10-fold increase in BDF1 macrophages, but LPS treatment of macrophages from either strain resulted in marked growth restrictions. This suppression was both dose dependent as well as dependent upon the time of addition of the LPS to the macrophages. Furthermore, the lipid A component of LPS was found to be as effective as the intact LPS in activating macrophages to inhibit the intracellular growth of Legionella. Further studies concerning the mechanisms involved are clearly warranted and in progress.     

Type I IFN protects permissive macrophages from Legionella pneumophila infection through an IFN-gamma-independent pathway

Legionella pneumophila is an intracellular pathogen whose replication in macrophages is mainly controlled by IFN-gamma. Freshly isolated peritoneal macrophages elicited in vivo with thioglycolate (TG) from A/J mice are highly permissive to L. pneumophila growth in vitro, while TG-elicited macrophages from CD1 mice are resistant. In this study, we show that when CD1 TG-macrophages are cultured for 7 days, they become permissive to Legionella infection. We demonstrate that treatment with type I IFN (IFN-alphabeta) totally inhibits the growth of L. pneumophila in both freshly isolated A/J and in vitro-aged CD1 TG-macrophages. IFN-alphabeta protective effect on permissive macrophages was comparable to that induced by IFN-gamma. Even low doses of either IFN-alpha or IFN-beta alone were effective in inhibiting L. pneumophila multiplication in macrophage cultures. Notably, treatment of resistant, freshly isolated CD1 TG-macrophages with Ab to mouse IFN-alphabeta significantly enhanced their susceptibility to Legionella infection in vitro, thus implying a role of endogenous IFN-alphabeta in mediating the natural resistance of macrophages to L. pneumophila infection. Finally, addition of anti-IFN-gamma-neutralizing Ab did not restore Legionella growth in IFN-alpha- or IFN-beta-treated A/J or CD1 permissive macrophages, indicating that IFN-alphabeta effect was not mediated by IFN-gamma. This observation was further confirmed by the finding that IFN-alphabeta was effective in inhibiting L. pneumophila replication in macrophages from IFN-gamma receptor-deficient mice. Taken together, our results provide the first evidence for a role of IFN-alphabeta in the control of L. pneumophila infection in mouse models of susceptible macrophages and suggest the existence of different pathways for the control of intracellular bacteria in macrophages.     

Legionella pneumophila growth in macrophages from susceptible mice is genetically controlled

Growth of the intracellular opportunistic bacterium Legionella pneumophila in macrophages from A/J mice is a vigorous as growth in macrophages from susceptible guinea pigs and human monocytes, whereas growth is inhibited in macrophages from other mouse strains, such as nonpermissive BALB/c mice. Permissiveness versus nonpermissiveness of macrophages from A/J versus BALB/c mice appeared to be controlled by a genetic mechanism dependent upon a single gene or a closely clustered family of genes. Susceptibility versus resistance of macrophages from F1 offspring of these two strains of mice and macrophages from backcrossed mice prepared from F1 hybrids and the original parental strain showed a segregation of permissiveness for growth of Legionella in vitro, consistent with genetic control.     

Genetic control of macrophage susceptibility to infection by Legionella pneumophila

Abstract Legionella pneumophila readily grows in cultures of thioglycollate (TGC)-induced macrophages (MPs) from A/J mice, but not in MPs from BALB/c mice or other mouse strains. In the present study, the growth of Legionella pneumophila in MPs from A/J and BALB/c mice, as well as hybrids of the two strains and back-crossed mice, was investigated to determine whether the permissiveness of growth of these bacteria was due to an inherited trait of the MPs. The MPs from all A/J mice supported the growth of Legionella , regardless of whether they were obtained from TGC or casein injected donors, but the cells from the mice given TGC supported growth of L. pneumophila much better than cells from mice injected with casein. Furthermore, MPs obtained from all BALB/c mice treated with either TGC or casein were nonpermissive for the growth of L. pneumophila . MPs from approximately 46% of the back-crossed ACF1 to A/J mice were permissive for L. pneumophila growth, while MPs from all ACF1 to back-crossed BALB/c mice were found to be nonpermissive. MPs from approximately 19% of ACF2 mice were permissive for L. pneumophila . Killing activities of MPs using temperature sensitive mutants of Salmonella typhimurium were variable and did not correlate with permissiveness or nonpermissiveness for growth of L. pneumophila . In addition, the number of inflammatory cells in the peritoneal cavity induced in response to TGC did not correlate with the permissiveness or nonpermissiveness of the MPs from various mouse strains to Legionella , indicating the permissive nature of the cells is controlled by genetic mechanisms involving a recessive phenotype but differs from resistance genes such as Ity important for replication of S. typhimurium .     

Grouping of 20 reference strains of Legionella species by the growth ability within mouse and guinea pig macrophages

20 Reference strains of Legionella species, isolated from human, were classified according to their ability to grow within thioglycolate-induced peritoneal macrophages of mice and guinea pigs. Inbred and congenic mice were used to study the effect of the natural resistance genes Lgn1 and Bcg that are expressed phenotypically in the mouse macrophages. The Lgn1 gene controlled the intracellular growth of Legionella pneumophila Philadelphia-1 and Legionella jordanis GIFU 12657, but the Bcg gene did not affect the intracellular growth of any organism examined. Based on these results and the growth ability in guinea pig macrophages, the 20 reference strains were divided into four groups. This grouping will help us to understand a variety of modes of interaction between Legionella species and macrophages.     

Enhanced growth of Legionella pneumophila in tetrahydrocannabinol-treated macrophages.

Legionella pneumophila is an opportunistic intracellular pathogen that infects macrophages, both in vivo and in vitro. Tetrahydrocannabinol is a major psychoactive component of marijuana and can affect the functional activity of macrophages. In the present study, it was found that the treatment of macrophage cultures from permissive A/J mice with THC enhanced the growth of Legionella in these cells. Legionella grew much better in macrophages treated with low doses of THC, which caused no alteration in the number or viability of macrophages, as compared with growth in untreated cells. Furthermore, lipopolysaccharide-treated A/J mouse macrophages restricted the growth of Legionella, but this growth restriction was overcome by the addition of THC to LPS-treated macrophage cultures after infection. Thus, it is apparent that THC has the ability to enhance the growth of the intracellular opportunistic pathogen Legionella that grows in A/J mouse macrophages.     

Tetrahydrocannabinol treatment suppresses growth restriction of Legionella pneumophila in murine macrophage cultures.

Legionella pneumophila is a facultative intracellular pathogen which readily grows in human and guinea pig macrophages and in peritoneal exudate macrophages from A/J mice. Macrophage cultures capable of supporting the growth of Legionella can be used to test the potency of biologically active substances suspected of modulating host mechanisms of resistance to infection. Accordingly, this model was used to evaluate the influence of delta-9-tetrahydro-cannabinol (THC) on macrophage resistance to infection with an intracellular pathogen. Pretreatment of the macrophages with THC in the concentration range of 2.5 micrograms/ml (8 microM) to 5.0 micrograms/ml (16 microM) had little if any effect on the ability of the macrophages to either ingest or support the replication of Legionella. However, THC treatment of cells following Legionella infection resulted in increased numbers of bacteria recoverable from the macrophage cultures. Stimulation of the macrophage cultures with the activating agent lipopolysaccharide (LPS) was effective in reducing the ability of Legionella to grow in the cells. However, treatment of the LPS activated macrophages with THC resulted in greater growth of the Legionella in the cultures, indicating that the drug abolished the LPS induced enhanced resistance. These results demonstrate that THC treatment of macrophages following infection rather than before infection with Legionella promotes the replication of the bacteria within the macrophages. In addition, drug treatment suppresses the growth restricting potential of macrophages activated by LPS.     

Tumor necrosis factor induces resistance of macrophages to Legionella pneumophila infection

Legionella pneumophila is an ubiquitous opportunistic intracellular pathogen that replicates readily in thioglycollate-elicited peritoneal macrophages from genetically susceptible A/J mice. Treatment of macrophage cultures in vitro with tumor necrosis factor-alpha (TNF-alpha) induced resistance of the macrophages to infection by Legionella as compared with control macrophages treated with medium alone. Addition of small amounts of monoclonal antibody to TNF-alpha restored susceptibility of the macrophages. Furthermore, antibody to the proinflammatory cytokine interleukin-1 (IL-1) alpha/beta increased resistance, but recombinant IL-1 had little effect. Such decreased susceptibility to Legionella growth in anti-IL-1 antibody-treated cultures corresponded with enhanced levels of TNF-alpha in the supernatants of the treated cells. An antibody to another proinflammatory cytokine with known immunoregulatory properties (i.e., IL-6) had little or no effect on the ability of the macrophages to be infected by Legionella and, furthermore, treatment with recombinant IL-6, similar to recombinant IL-1, did not modify the ability of the cells to be infected in vitro. These results indicate that TNF-alpha is important in controlling L. pneumophila replication, and IL-1 can regulate TNF-alpha levels, affecting susceptibility of macrophages to infection with an intracellular opportunistic pathogen like Legionella.     

Restriction of Legionella pneumophila growth in macrophages requires the concerted action of cytokine and Naip5/Ipaf signalling pathways

Macrophages from the C57BL/6 (B6) mouse strain restrict intracellular growth of Legionella pneumophila, whereas A/J macrophages are highly permissive. The mechanism by which B6 macrophages restrict Legionella growth remains poorly understood, but is known to require the cytosolic microbe sensors Naip5 (Birc1e) and Ipaf. We hypothesized that Naip5 and Ipaf may act in partnership with other antimicrobial signalling pathways in macrophages. Indeed, we found that macrophages lacking either tumour necrosis factor (TNF)-alpha or type I interferon (IFN) signalling are permissive for growth of L. pneumophila, even in the presence of functional Naip5 and Ipaf alleles. Similarly, macrophages lacking Naip5 and/or Ipaf signalling were permissive even though we found that Naip5 or Ipaf were not required for induction of TNF-alpha and type I IFN. Therefore, our data suggest that the mechanism by which B6 macrophages restrict intracellular replication of L. pneumophila is more complex than previously appreciated, and involves the concerted action of cytokine and intracellular microbe sensor signalling pathways.     

Rapid reduction of Legionella pneumophila on stainless steel with zeolite coatings containing silver and zinc ions

AIMS: To determine the rate of reduction of Legionella pneumophila by stainless steel surfaces with zeolite ceramic coatings containing 2.5% (w/w) silver (Ag) and 14% zinc (Zn) ions. METHODS AND RESULTS: Stainless steel pans with and without Ag/Zn coatings were inoculated with solutions of Leg. pneumophila ATCC 33155 and incubated at 37 degrees C. Survival was monitored using the spread-plate technique on selective buffered charcoal yeast extract agar. Significant reductions of Leg. pneumophila were effected by the Ag/Zn zeolite coatings within 2 h of exposure. CONCLUSIONS, Significance and Impact of the Study: Zeolite ceramic Ag/Zn coatings impart significant anti-Legionella properties to stainless steel surfaces. Coated stainless steel could be used in the manufacture of air ducts, condensation pans and intake and exhaust vents. These products have the potential to reduce numbers of Legionella in air-handling systems.     

Fate of Legionella pneumophila in macrophages of C57BL/6 chronic granulomatous disease mice

We compared the intracellular survival and growth of Legionella pneumophila Philadelphia-1 in peritoneal macrophages obtained from A/J, C57BL/6, and X-linked chronic granulomatous disease (CGD) mice produced from C57BL/6 strain. The initial killing was observed in A/J and C57BL/6 macrophages at 2, 4 and 6 hr after in vitro phagocytosis, but not in the CGD macrophages. Thereafter, there was a 10-fold increase of CFU in A/J macrophages. The bacteria, however, did not proliferate in C57BL/6 and CGD macrophages at 24 or 48 hr after in vitro phagocytosis. These results suggest that effector molecules for the initial killing are a superoxide anion and its metabolites, and Lgn1 gene product inhibits the intracellular growth of L. pneumophila independently of NADPH oxidase.     

Interferon-gamma reverses the evasion of Birc1e/Naip5 gene mediated murine macrophage immunity by Legionella pneumophila mutant lacking flagellin

Legionella pneumophila is the etiologic agent of Legionnaires' disease. This bacterium contains a single monopolar flagellum, of which the FlaA subunit is a major protein constituent. The murine macrophage resistance against this bacterium is controlled by the Birc1e/Naip5 gene, which belongs to the NOD family. We evaluated the intracellular growth of the flaA mutant bacteria as well as another aflagellated fliA mutant, within bone marrow-derived macrophages from mice with an intact (C57BL/6, BALB/c) or mutated (A/J) Birc1e/Naip5 gene. The flaA mutant L. pneumophila multiplied within C57BL/6 and BALB/c macrophages while the wild-type strain did not. Cell viability was not impaired until 3 days after infection when the flaA mutant bacteria replicated 10(2-3)-fold in macrophages, implying that L. pneumophila inhibited host cell death during the early phase of intracellular replication. The addition of recombinant interferon-gamma (IFN-gamma) to the infected macrophages restricted replication of the flaA mutant within macrophages; these treated cells also showed enhanced nitric oxide production, although inhibition of nitric oxide production did not affect the IFN-gamma induced inhibition of Legionella replication. These findings suggested that IFN-gamma activated macrophages to restrict the intracellular growth of the L. pneumophila flaA mutant by a NO independent pathway.     

Birc1e/Naip5 rapidly antagonizes modulation of phagosome maturation by Legionella pneumophila

Legionella survives intracellularly by preventing fusion with lysosomes, due to phagosome escape from the endocytic pathway at an early stage of phagosome maturation, and by creating a replicative organelle that acquires endoplasmic reticulum (ER) characteristics through sustained interactions and fusion with the ER. Intracellular replication of Legionella pneumophila in mouse macrophages is controlled by the Lgn1 locus. Functional complementation in vivo has identified the Birc1e/Naip5 gene as being responsible for the Lgn1 effect. To understand the function and temporal site of action of Birc1e/Naip5 in susceptibility to L. pneumophila, we examined the biogenesis of Legionella-containing vacuoles (LCVs) formed in permissive A/J macrophages and in their Birc1e/Naip5 transgenic non-permissive counterpart. Birc1e/Naip5 effects on acquisition of lysosomal and ER markers were evident within 1-2 h following infection. A significantly higher proportion of LCVs formed in Birc1e/Naip5 transgenic macrophages had acquired the lysosomal markers cathepsin D and Lamp1 by 2 h post infection, whereas a significantly higher proportion of LCVs formed in permissive macrophages were positively stained for the ER markers BAP31 and calnexin, 6 h post infection. Likewise, studies by electron microscopy showed acquisition of lysosomal contents (horseradish peroxidase), within the first hour following phagocytic uptake, by LCVs formed in Birc1e/Naip5 transgenic macrophages and delivery of the ER marker glucose 6-phosphatase (G6Pase) only to the lumen of LCVs formed in A/J macrophages. Finally, a larger proportion of LCVs formed in A/J macrophages were studded with ribosomes 24 h post infection, compared with LCVs formed in Birc1e/Naip5 transgenic macrophages. These results suggest that sensing of L. pneumophila products by Birc1e/Naip5 in macrophages occurs rapidly following phagocytosis, a process that antagonizes the ability of L. pneumophila to remodel its phagosome into a specialized vacuole with ER characteristics.     

The Nod-like receptor family member Naip5/Birc1e restricts Legionella pneumophila growth independently of caspase-1 activation

Similar to Ipaf and caspase-1, the Nod-like receptor protein Naip5 restricts intracellular proliferation of Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaires' disease. Thus, Naip5 has been suggested to regulate Legionella replication inside macrophages through the activation of caspase-1. In this study, we show that cytosolic delivery of recombinant flagellin activated caspase-1 in A/J macrophages carrying a mutant Naip5 allele, and in C57BL/6 (B6) macrophages congenic for the mutant Naip5 allele (B6-Naip5(A/J)), but not in Ipaf(-/-) cells. In line with these results, A/J and B6-Naip5(A/J) macrophages induced high levels of caspase-1 activation and IL-1beta secretion when infected with Legionella. In addition, transgenic expression of a functional Naip5 allele in A/J macrophages did not alter Legionella-induced caspase-1 activation and IL-1beta secretion. Notably, defective Naip5 signaling renders B6-Naip5(A/J) macrophages permissive for Legionella proliferation despite normal caspase-1 activation. These results indicate that the restriction of intracellular Legionella replication is more complex than previously appreciated and requires both Ipaf-dependent caspase-1 activation as well as functional Naip5 signaling.     

Multiplication of Legionella pneumophila Philadelphia-1 in cultured peritoneal macrophages and its correlation to susceptibility of animals

Intracellular growth of Legionella pneumophila Philadelphia-1 strain in peritoneal macrophages (PMP) from various rodents was measured and its correlation to the level of susceptibility of the animal was examined. In guinea pig PMP, the organism grew well and the guinea pig was very susceptible to it (50% lethal dose, LD50 = 7.6 X 10(4)). On the other hand, the bacteria hardly multiplied in mouse PMP and the animal was resistant to infection (LD50 = 6.7 X 10(7)). Intracellular growth rate correlated well with susceptibility in these animals. In golden hamsters, a discrepancy between intracellular growth and susceptibility was found. The organism grew intracellularly as rapid as in guinea pig PMP, but the golden hamster was very resistant to infection (LD50 = 2.2 X 10(8)). In rat PMP, the organism did not grow intracellularly during a 24-h period of infection, but started to grow after that and the growth rate thereafter was as rapid as in guinea pig PMP. WKA rats were resistant and the LD50 in the animal was 1.9 X 10(7). In vivo natural resistance of rats and golden hamsters to the organism was considered to be a result of other factors than macrophages.     

Autophagy is an immediate macrophage response to Legionella pneumophila

After ingestion by macrophages, Legionella pneumophila enter spacious vacuoles that are quickly enveloped by endoplasmic reticulum (ER), then slowly transferred to lysosomes. Here we demonstrate that the macrophage autophagy machinery recognizes the pathogen phagosome as cargo for lysosome delivery. The autophagy conjugation enzyme Atg7 immediately translocated to phagosomes harbouring virulent Legionella. Subsequently, Atg8, a second autophagy enzyme, and monodansyl-cadaverine (MDC), a dye that accumulates in acidic autophagosomes, decorated the pathogen vacuoles. The autophagy machinery responded to 10-30 kDa species released into culture supernatants by Type IV secretion-competent Legionella, as judged by the macrophages' processing of Atg8 and formation of vacuoles that sequentially acquired Atg7, Atg8 and MDC. When compared with autophagosomes stimulated by rapamycin, Legionella vacuoles acquired Atg7, Atg8 and MDC more slowly, and Atg8 processing was also delayed. Moreover, compared with autophagosomes of Legionella-permissive naip5 mutant A/J macrophages, those of resistant C57BL/6 J macrophages matured quickly, preventing efficient Legionella replication. Accordingly, we discuss a model in which macrophages elevate autophagy as a barrier to infection, a decision influenced by regulatory interactions between Naip proteins and caspases.     

Association of Lps gene with natural resistance of mouse macrophages against Legionella pneumophila.

Peritoneal macrophages obtained from lipopolysaccharide (LPS)-low responder C3H/HeJ mice (J) permitted the intracellular growth of the bacterium in macrophages of (J x N) F1 progeny was between the parent strains, showing that the traits were co-dominantly expressed. Correlation between intracellular bacterial growth in macrophages and LPS response of spleen cells was examined. Negative correlation was found between the two factors in F2, (J x F1) backcross and (N x F1) backcross progeny. This result implies that Lps gene controls the innate resistance of murine macrophages against the bacteria. Although macrophages of A/J strain also permit intracellular growth of L. pneumophila, gene complementation analysis of A/J and C3H/HeJ mice made clear that the gene control in C3H/HeJ differs from that of A/J strain. Macrophages of C57BL/10ScN, which is LPS-low responder line obtained from C57BL/10, were also defective in controlling the bacterial growth when compared to C57BL/10 mice. We suggest that the Lps gene also controls the natural resistance of murine macrophages against L. pneumophila.     

Role of protein kinase C-alpha in the control of infection by intracellular pathogens in macrophages.

The protein kinase C (PKC) family regulates macrophage function involved in host defense against infection. In this study, we investigated the role of macrophage PKC-alpha in the uptake and subsequent fate of Leishmania donovani promastigotes and Legionella pneumophila infections. To this end, we used clones of the murine macrophage cell line RAW 264.7 overexpressing a dominant-negative (DN) mutant of PKC-alpha. While phagocytosis of L. donovani promastigotes was not affected by DN PKC-alpha overexpression, their intracellular survival was enhanced by 10- to 20-fold at 48 h postinfection. Intracellular survival of a L. donovani mutant defective in lipophosphoglycan repeating units synthesis, which normally is rapidly degraded in phagolysosomes, was enhanced by 100-fold at 48 h postinfection. However, IFN-gamma-induced leishmanicidal activity was not affected by DN PKC-alpha overexpression. Similar to macrophages from genetically resistant C57BL/6 mice, control RAW 264.7 cells were not permissive for the intracellular replication of Legionella pneumophila. In contrast, DN PKC-alpha-overexpressing RAW 264.7 clones were phenotypically similar to macrophages from genetically susceptible A/J mice, as they allowed intracellular replication of L. pneumophila. Permissiveness to L. pneumophila was not the consequence of a general defect in the microbicidal capacities because killing of a temperature-sensitive mutant of Pseudomonas aeruginosa was normal in DN PKC-alpha-overexpressing RAW 264.7 clones. Collectively, these results support a role for PKC-alpha in the regulation of innate macrophage functions involved in the control of infection by intracellular parasites.     

Legionella pneumophila EnhC is required for efficient replication in tumour necrosis factor alpha-stimulated macrophages

Legionella pneumophila enhC ^- mutants were originally identified as being defective for uptake into host cells. In this work, we found that the absence of EnhC resulted in defective intracellular growth when dissemination of intracellular bacteria to neighbouring cells was expected to occur. No such defect was observed during growth within the amoeba Dictyostelium discoideum. Culture supernatants containing the secreted products of infected macrophages added to host cells restricted the growth of the Δ enhC strain, while tumour necrosis factor α (TNF-α), at concentrations similar to those found in macrophage culture supernatants, could reproduce the growth restriction exerted by culture supernatants on L. pneumophila Δ enhC . The absence of EnhC also caused defective trafficking of the Legionella- containing vacuole in TNF-α-treated macrophages. EnhC was shown to be an envelope-associated protein largely localized to the periplasm, with its expression induced in post-exponential phase, as is true for many virulence-associated proteins. Furthermore, the absence of EnhC appeared to affect survival under stress conditions, as the Δ enhC mutant was more susceptible to H₂O₂ treatment than the wild-type strain. EnhC therefore is a unique virulence factor that is required for growth specifically when macrophages have heightened potential to restrict microbial replication.