Inflammasome Activation in Legionella-Infected Macrophages.

Legionella pneumophila is a gram-negative bacterium that infects many species of unicellular protozoa in freshwater environments. The human infection is accidental, and the bacteria may not have evolved strategies to bypass innate immune signaling in mammalian macrophages. Thus, L. pneumophila triggers many innate immune pathways including inflammasome activation. The inflammasomes are multimolecular platforms assembled in the host cell cytoplasm and lead to activation of inflammatory caspases. Inflammasome activation leads to secretion of inflammatory cytokines, such as IL-1ß and IL-18, and an inflammatory form of cell death called pyroptosis, which initiates with the induction of a pore in the macrophage membranes. In this chapter we provide detailed protocols to evaluate Legionella-induced inflammasome activation in macrophages, including real-time pore formation assay, western blotting to detect activation of inflammatory caspases (cleavage and pulldown), and the measurement of inflammatory cytokines.

Legionella longbeachae Is Immunologically Silent and Highly Virulent In Vivo.

BACKGROUND: Legionella longbeachae (Llo) and Legionella pneumophila (Lpn) are the most common pneumonia-causing agents of the genus. Although both species can be lethal to humans and are highly prevalent, little is known about the molecular pathogenesis of Llo infections. In murine models of infection, Lpn infection is self-limited, whereas Llo infection is lethal. METHODS: We used mouse macrophages, human macrophages, human epithelial cells, and mouse infections in vivo to evaluate multiple parameters of the infection. RESULTS: We determined that the Llo Dot/Icm secretion system is critical for virulence. Different than Lpn, Llo disseminates and the animals develop a severe pulmonary failure, as demonstrated by lung mechanics and blood oxygenation assays. As compared to Lpn, Llo is immunologically silent and fails to trigger the production of cytokines in human pulmonary epithelial cells and in mouse and human macrophages. Infections in Tnfr1-/-, Ifng-/-, and Il12p40-/- mice supported the participation of cytokines for the resistance phenotype. CONCLUSIONS: Both Lpn and Llo require the Dot/Icm system for pathogenesis, but the infection outcome is strikingly different. Llo is immunologically silent, highly virulent, and lethal. The differences reported herein may reflect unappreciated clinical differences in patients infected with Lpn or Llo.

Different STAT Transcription Complexes Drive Early and Delayed Responses to Type I IFNs.

IFNs, which transduce pivotal signals through Stat1 and Stat2, effectively suppress the replication of Legionella pneumophila in primary murine macrophages. Although the ability of IFN-γ to impede L. pneumophila growth is fully dependent on Stat1, IFN-αß unexpectedly suppresses L. pneumophila growth in both Stat1- and Stat2-deficient macrophages. New studies demonstrating that the robust response to IFN-αß is lost in Stat1-Stat2 double-knockout macrophages suggest that Stat1 and Stat2 are functionally redundant in their ability to direct an innate response toward L. pneumophila. Because the ability of IFN-αß to signal through Stat1-dependent complexes (i.e., Stat1-Stat1 and Stat1-Stat2 dimers) has been well characterized, the current studies focus on how Stat2 is able to direct a potent response to IFN-αß in the absence of Stat1. These studies reveal that IFN-αß is able to drive the formation of a Stat2 and IFN regulatory factor 9 complex that drives the expression of a subset of IFN-stimulated genes, but with substantially delayed kinetics. These observations raise the possibility that this pathway evolved in response to microbes that have devised strategies to subvert Stat1-dependent responses.

In vitro derivation of macrophage from guinea pig bone marrow with human M-CSF.

The guinea pig has a storied history as a model in the study of infectious disease and immunology. Because of reproducibility of data and availability of various reagents, inbred mice have since supplanted the guinea pig as the animal model-of-choice in these fields. However, several clinically-significant microorganisms do not cause the same pathology in mice, or mice may not be susceptible to these infections. These demonstrate the utility of other animal models - either as the primary method to study a particular infection, or to confirm or refute findings in the mouse before translating basic science into clinical practice. The mononuclear phagocyte, or macrophage (Mφ), plays a key role in antigen presentation and the pathogenesis of intracellular bacteria, such as Mycobacterium tuberculosis and Legionella pneumophila. Because of variable yield and difficult extraction from tissue, the preferred method of producing Mφ for in vitro studies is to expand murine bone marrow (BM) precursors with mouse macrophage colony-stimulating factor (M-CSF). This has not been shown in the guinea pig. Here, we report the empiric observation that human M-CSF - but not mouse M-CSF, nor human granulocyte/macrophage colony-stimulating factor - can be used to induce BM precursor differentiation into bonafide Mφ. The differentiated cells appeared as enlarged adherent cells, capable of both pinocytosis and large particle phagocytosis. Furthermore, we showed that these guinea pig BM-derived Mφ, similar to human monocyte/Mφ lines but unlike most murine BM Mφ, support growth of wild type L. pneumophila. This method may prove useful for in vitro studies of Mφ in the guinea pig, as well as in the translation of results found using mouse BM-derived Mφ towards studies in human immunology and infectious disease.

Legionella jordanis in hematopoietic SCT patients radiographically mimicking invasive mold infection.

Opportunistic pulmonary infections are a major cause of post-transplant morbidity and mortality. Among these infections, Aspergillus is a common cause of fatal pneumonia. Owing to the precarious clinical condition of many patients who acquire invasive mold infections, clinicians often treat them on the basis of radiographic findings, such as the halo sign. However, in patients who do not respond to treatment or who have uncommon presentations, bronchoscopy or lung biopsy looking for other pathogens should be considered. This study describes two cases in which the radiographic halo signs characteristic of Aspergillus were in fact due to Legionella jordanis, a pathogen that has been culture proven only in two patients previously (both of whom had underlying lung pathology) and diagnosed by serologic evidence in several other patients. In immunocompromised patients, Legionella can present as a cavitary lesion. Thus, presumptive treatment for this organism should be considered in post-transplant patients who do not have a classic presentation for invasive fungal infection and/or who fail to respond to conventional treatment. These cases illustrate the importance of obtaining tissue cultures to differentiate among the wide variety of pathogens present in this patient population.

Differential requirements for NAIP5 in activation of the NLRC4 inflammasome.

Inflammasomes are cytosolic multiprotein complexes that assemble in response to infectious or noxious stimuli and activate the CASPASE-1 protease. The inflammasome containing the nucleotide binding domain-leucine-rich repeat (NBD-LRR) protein NLRC4 (interleukin-converting enzyme protease-activating factor [IPAF]) responds to the cytosolic presence of bacterial proteins such as flagellin or the inner rod component of bacterial type III secretion systems (e.g., Salmonella PrgJ). In some instances, such as infection with Legionella pneumophila, the activation of the NLRC4 inflammasome requires the presence of a second NBD-LRR protein, NAIP5. NAIP5 also is required for NLRC4 activation by the minimal C-terminal flagellin peptide, which is sufficient to activate NLRC4. However, NLRC4 activation is not always dependent upon NAIP5. In this report, we define the molecular requirements for NAIP5 in the activation of the NLRC4 inflammasome. We demonstrate that the N terminus of flagellin can relieve the requirement for NAIP5 during the activation of the NLRC4 inflammasome. We also demonstrate that NLRC4 responds to the Salmonella protein PrgJ independently of NAIP5. Our results indicate that NAIP5 regulates the apparent specificity of the NLRC4 inflammasome for distinct bacterial ligands.

Legionnaires disease in cannabis smokers.

Tobacco smoking is a well-recognized risk factor for Legionnaires disease. However, it may be potentiated by cannabis use, as there is strong evidence that Δ(9)-tetrahydrocannabinol impairs immune functions in vitro and in vivo. We report herein two out of three cases of severe Legionnaires disease in three men with no overt comorbid illnesses, aged 38, 28, and 48 years, respectively. All of them were heavy cigarette and cannabis smokers.

Genetic susceptibility and caspase activation in mouse and human macrophages are distinct for Legionella longbeachae and L. pneumophila.

Legionella pneumophila is the predominant cause of Legionnaires' disease in the United States and Europe, while Legionella longbeachae is the common cause of the disease in Western Australia. Although clinical manifestations by both intracellular pathogens are very similar, recent studies have shown that phagosome biogeneses of both species within human macrophages are distinct (R. Asare and Y. Abu Kwaik, Cell. Microbiol., in press). Most inbred mouse strains are resistant to infection by L. pneumophila, with the exception of the A/J mouse strain, and this genetic susceptibility is associated with polymorphism in the naip5 allele and flagellin-mediated early activation of caspase 1 and pyropoptosis in nonpermissive mouse macrophages. Here, we show that genetic susceptibility of mice to infection by L. longbeachae is independent of allelic polymorphism of naip5. L. longbeachae replicates within bone marrow-derived macrophages and in the lungs of A/J, C57BL/6, and BALB/c mice, while L. pneumophila replicates in macrophages in vitro and in the lungs of the A/J mouse strain only. Quantitative real-time PCR studies on infected A/J and C57BL/6 mouse bone marrow-derived macrophages show that both L. longbeachae and L. pneumophila trigger similar levels of naip5 expression, but the levels are higher in infected C57BL/6 mouse macrophages. In contrast to L. pneumophila, L. longbeachae has no detectable pore-forming activity and does not activate caspase 1 in A/J and C57BL/6 mouse or human macrophages, despite flagellation. Unlike L. pneumophila, L. longbeachae triggers only a modest activation of caspase 3 and low levels of apoptosis in human and murine macrophages in vitro and in the lungs of infected mice at late stages of infection. We conclude that despite flagellation, infection by L. longbeachae is independent of polymorphism in the naip5 allele and L. longbeachae does not trigger the activation of caspase 1, caspase 3, or late-stage apoptosis in mouse and human macrophages. Neither species triggers caspase 1 activation in human macrophages.

Activated macrophages infected with Legionella inhibit T cells by means of MyD88-dependent production of prostaglandins.

To understand how macrophages (Mphi) activated with IFN-gamma modulate the adaptive immune response to intracellular pathogens, the interaction of IFN-gamma-treated bone marrow-derived murine Mphi (BMphi) with Legionella pneumophila was investigated. Although Legionella was able to evade phagosome lysosome fusion initially, and was capable of de novo protein synthesis within IFN-gamma-treated BMphi, intracellular growth of Legionella was restricted. It was determined that activated BMphi infected with Legionella suppressed IFN-gamma production by Ag-specific CD4 and CD8 T cells. A factor sufficient for suppression of T cell responses was present in culture supernatants isolated from activated BMphi following Legionella infection. Signaling pathways requiring MyD88 and TLR2 were important for production of a factor produced by IFN-gamma-treated BMphi that interfered with effector T cell functions. Cyclooxygenase-2-dependent production of PGs by IFN-gamma-treated BMphi infected with Legionella was required for inhibition of effector T cell responses. From these data we conclude that activated Mphi can down-modulate Ag-specific T cell responses after they encounter bacterial pathogens through production of PGs, which may be important in preventing unnecessary immune-mediated damage to host tissues.

[Increased risk of infection with biological immunomodifying antirheumatic agents. Clear guidelines are necessary as shown by case reports]. / Okad risk för infektion med biologiska immunmodifierande antireumatika.

Several potent immunosuppressive drugs have become available in the new millennium for patients with rheumatologic diseases, Crohn's disease and other autoimmune disorders. Five patient cases from Växjö central hospital (uptake area 178 000 individuals) with Listeria meningitis, Pneumocystis jiroveci and tuberculosis pneumonia, Listeria sepsis, Legionella pneumonia and E coli sepsis are described. A doubled risk for infections has previously been observed for RA patients, as compared to healthy individuals. There is clearly an increased risk of tuberculosis (depending on the actual and historic environmental prevalence) for patients on TNF antagonists, and therefore tuberculosis screening is now mandatory before start of therapy. Since TNF has a central role in the immune defence, an increased risk of opportunistic infections like listeriosis. mycobacteriosis, and invasive fungal infections has been established. Eight hospitals in southern Sweden participate in a register for the use of TNF blockers in rheumatologic diseases (South Swedish Arthritis Treatment Group, SSATG). Guidelines for screening and treatment of latent and active tuberculosis, possible prophylactic antibiotic treatment for endocarditis and vaccination programs for patients on TNF antagonists are discussed.

V gamma 9V delta 2 T cells in human legionellosis.

In humans, expansion of circulating Vgamma9Vdelta2 T cells seems to be a pathophysiological denominator shared by protozoan and intracellular bacterial diseases. The assumption was tested here on legionellosis, a condition conforming to the category but not yet described with respect to gammadelta T cells. Levels of Vgamma9Vdelta2 T cells in peripheral blood were measured at various intervals in 14 subjects undergoing a Pontiac fever-like disease, shown by serological investigation to be caused by Legionella micdadei. In samples obtained 4 to 6 days after the onset of the disease, the mean percentage (+/- the standard deviation) of Vgamma9Vdelta2+ T cells among CD3+ cells was 1.0% +/- 0.5%, compared to 5.0% +/- 3.9% in healthy control subjects (P < 0.001). Thereafter, a pronounced increase occurred and at 2 to 7 weeks after onset, mean peak levels were as high as approximately equal to 15%. During the next 6 months, values slowly declined, although without reaching the normal range. Percentages of gammadelta+ T cells expressing tumor necrosis factor alpha or gamma interferon in response to phorbol myristate acetate were assayed in vitro. At 14 to 16 days after the onset of disease, the expression of both cytokines was increased (P < 0.01), whereas at 5 to 7 weeks, the expression of tumor necrosis factor alpha was decreased (P < 0.05), possibly reflecting modulation of an inflammatory response. In conclusion, Pontiac fever was found to be associated with a pronounced and long-lasting expansion of Vgamma9Vdelta2 T cells, implying that the subset may also be pathophysiologically important in a mild and transient form of intracellular bacterial diseases. Surprisingly, the expansion was preceded by a depletion of circulatory Vgamma9Vdelta2 T cells. Possibly, Vgamma9Vdelta2 T cells are initially recruited to a site of infection before they expand in response to antigen and occur in high numbers in blood.

Serum cytokines in patients with Legionella pneumonia: relative predominance of Th1-type cytokines.

Serum samples from 14 patients with Legionella pneumonia were examined for the presence of cytokines. In spite of high levels of serum C-reactive protein in all patients during the acute phase in only four cases (one involving interleukin-1beta [IL-1beta], three involving IL-6, and none involving tumor necrosis factor alpha) was the concentration of cytokines more than 100 pg/ml. Th2 cytokines IL-4 and IL-10 were detected in only one patient each. In contrast, significant increases of serum gamma interferon (IFN-gamma) and IL-12 levels were observed during the acute phase in 6 and 11 cases, respectively. Interestingly, although serum IFN-gamma levels diminished thereafter, in seven cases IL-12 levels remained high or increased further during the convalescent phase. In an additional 22 cases clinically suspected to be but not diagnosed as Legionella pneumonia, increases of serum IL-12 levels were observed in 16 cases, whereas the remaining 6 cases showed no detectable IL-12. Our results demonstrate the relative predominance of Th1 cytokine production in Legionella pneumonia. Although the role and significance of prolonged increases in IL-12 levels in Legionella disease are unknown, our results should prompt further investigation of the host immune response in terms of Th1 and Th2 balance in legionellosis.

Western blot analysis of immune response to Legionella bozemanii antigens.

The immune response to individual major antigens of Legionella bozemanii was studied in 67 sera from 26 inpatients with febrile disease using immunoblotting techniques. All the patients had fever of unknown origin and showed strong serological reactions to L bozemanii that cross-reacted with Rickettsia typhi, as determined by a standard indirect microimmunofluorescence assay. Sera analysed by western blotting reacted with 12-14 molecular components of L bozemanii with apparent molecular weights ranging from 14,000 to 94,000 daltons. These reactions compared well with a reference system using antisera of rabbits immunised with the same strain of Legionella. The three major cross-reactive components of R typhi were polypeptide antigens of 94,000, 67,000 and 43,000 daltons. It is concluded that western blotting can help in the differential diagnosis of patients with fever of unknown origin whose sera cross-react to L bozemanii and R typhi.

Alveolar macrophage activation in experimental legionellosis.

Legionella pneumophila is a facultative intracellular parasite of alveolar macrophages. In vitro studies have shown that lymphokine-activated mononuclear phagocytes inhibit intracellular replication of L. pneumophila. To determine if recovery from legionellosis is associated with activation of alveolar macrophages in vivo to resist L. pneumophila, we studied an animal model of Legionnaires' disease. Rats were exposed to aerosolized L. pneumophila and alveolar macrophages were harvested during the recovery phase of infection. We compared these alveolar exudate macrophages with normal resident alveolar macrophages for the capacity to support or inhibit the intracellular growth of L. pneumophila. We also measured Ia expression as a marker of immunologic activation, and studied binding of bacteria, superoxide release, and the expression of transferrin receptors as potential mechanisms of resistance to L. pneumophila. For perspective on the specificity of these responses, we also studied alveolar exudate cells elicited by inhalation of heat-killed L. pneumophila, live Listeria monocytogenes, and live Escherichia coli. We found that alveolar exudate macrophages elicited by live L. pneumophila, but not heat-killed L. pneumophila, resisted the intracellular growth of L. pneumophila. Exudate macrophages in resolving legionellosis exhibited increased Ia expression, diminished superoxide production, and downregulation of transferrin receptors. Binding of L. pneumophila to exudate macrophages was indistinguishable from that to resident macrophages in the presence of normal serum, and augmented in the presence of immune serum. Alveolar exudate macrophages elicited by E. coli also inhibited growth of L. pneumophila, and exhibited a modest increase in Ia expression without change in transferrin receptors. Exudate cells induced by L. monocytogenes exhibited up-regulation of Ia without diminution of superoxide release. Alveolar cells harvested after inhalation of heat-killed L. pneumophila did not differ from resident alveolar macrophages in the expression of surface markers. These findings suggest that alveolar macrophages are immunologically activated in vivo to serve as effector cells in resolving legionellosis, and that live bacteria are required to induce this expression of immunity. The mechanism of resistance to parasitism by L. pneumophila may entail restriction of the intracellular availability of iron, but does not involve diminished bacterial binding or an augmented respiratory burst.

Genetic control of natural resistance in mouse macrophages regulating intracellular Legionella pneumophila multiplication in vitro.

It is known that Legionella pneumophila proliferates in peritoneal macrophage cultures derived from A/J mice but not in macrophage cultures derived from many other strains, including C57BL/6 mice. To analyze the genetic control of this trait and the location of the Legionella resistance-susceptibility gene, we prepared segregating progeny of A/J and C57BL/6 mice and determined the levels of susceptibility of individual mice. Peritoneal macrophages were collected by injecting thioglycolate medium, and macrophage monolayers were infected in vitro with L. pneumophila Philadelphia-1. Counting of colonies on buffered charcoal yeast extract agar plates and Gimenez staining of macrophage monolayers were carried out daily. There was a 10-fold increase in bacterial burden 1 day after infection and a 100-fold increase after 2 days in A/J (susceptible) macrophages. The increase in bacterial burden was always less than 10-fold in macrophages from C57BL/6 (resistant) progenitors, A/J x C57BL/6 F1 hybrids, and C57BL/6 x F1 backcross progeny. The ratios of resistant individuals to susceptible individuals were 22:6 for F2 progeny and 20:22 for A/J x F1 backcross progeny. The fact that the organism did not proliferate in macrophages from B10.A mice demonstrated that major histocompatibility antigens did not regulate the macrophage resistance of C57BL/6-derived mice. The sex and coat color genes of mice were not linked to the resistance-susceptibility gene. We suggest that resistance and susceptibility are controlled by a single gene or closely linked genes which are autosomal and that the resistance allele is dominant. The results of a comparison of the strain distribution pattern of this trait with the distribution pattern of 185 allelic markers in A/J x C57BL/6 and C57BL/6 x A/J recombinant inbred strains suggest that this susceptibility-resistance gene is located in the proximal part of chromosome 15.

Role of gamma interferon in induction of natural killer activity by Legionella pneumophila in vitro and in an experimental murine infection model.

Legionella pneumophila has been shown to induce gamma interferon (IFN-gamma) both in vitro and in vivo during experimental infections of mice. With complement-mediated serologic depletion of murine splenocytes, the cellular sources of IFN-gamma following in vitro stimulation with L. pneumophila antigens were Thy-1.2+, Lyt-2-, L3T4-, and asialo-GM1+, which is consistent with the natural killer (NK) cell phenotype. Additionally, Percoll density discontinuous centrifugation demonstrated that maximal production of IFN coincided with high NK activity in fractions which were enriched for large granular lymphocytes. Furthermore, 18- to 24-h incubation of splenocytes with L. pneumophila whole-cell vaccine resulted in augmented NK cytotoxic activity against YAC-1 tumor target cells in a 51Cr release assay. The addition of macrophages to purified large granular lymphocyte populations augmented both IFN-gamma production and NK activity, suggesting that antigen is required for optimal responses. In an experimental infection model using an intratracheal inoculation route, NK activity was enhanced in the spleen, peripheral blood, and lung cells of infected mice, with maximal stimulation in the lung leukocytes at the site of infection. The results of the present study indicate that NK cells respond in vivo and in vitro to stimulation by L. pneumophila by producing IFN-gamma and by increased cytolytic activity.

Legionella pneumophila immunity and immunomodulation: nature and mechanisms.

L. pneumophila is a facultative intracellular opportunistic pathogen ubiquitously present in the environment. Much is now known concerning the ecological niche of this organism as well as many other characteristics of these bacteria, including physiology and biochemistry. However, much less is known about immune mechanisms responsible for host resistance vs susceptibility. Not only outer membrane protein rich fractions but also LPS-rich components are potent immunogens, both in experimental animals such as susceptible guinea pigs and more resistant rodent species like rats and mice. Immunity to these organisms can be readily observed by a variety of serologic techniques. Antibody titers increase rapidly after exposure of individuals to these bacteria either by infection or immunization. However, such antibody does not appear to play an important role in host resistance. Serum antibody plus complement is not lytic for the bacteria in vitro. Furthermore, antibody appears to promote the phagocytosis of the bacteria by monocytes and/or macrophages in culture but such phagocytosis does not result in killing of the bacteria, merely an enhanced uptake and subsequent replication of the organisms. Studies on cellular immunity have focused attention on the role of T lymphocytes, monocytes and macrophages. In addition, cutaneous hypersensitivity is readily induced by infection or immunization of experimental animals with Legionella or antigenic components. In vitro correlates of hypersensitivity is also readily evident after infection or immunization. Although lymphoid cells from guinea pigs only show evidence of responsiveness to Legionella antigens by the lymphocyte blastogenic reaction after animals have been sensitized, peripheral blood monocytes from man as well as splenocytes from mice show evidence of responsiveness to Legionella even before known infection or sensitization. However, higher blastogenic responses become evident after sensitization or infection. In addition, interleukins, such as interleukin 1 and 2, as well as interferon and tumor necrotizing factor, appear in response to Legionella antigens and seem to play a role in resistance mechanisms. Cellular replication of Legionella in monocytes from man as well as macrophages from susceptible animals seems related to susceptibility or resistance to these organisms. Further analyses of the nature and mechanism of humoral vs cellular immune responses to Legionella antigens will provide valuable information about immunity and resistance to these intracellular pathogens in susceptible individuals.

Role of the alveolar macrophage in host defense and immunity to Legionella micdadei pneumonia in the guinea pig.

Guinea pigs develop a lethal pneumonia after intratracheal infection with Legionella micdadei, and the lung displays pathological changes similar to those observed in humans. To investigate the role of the resident alveolar macrophage in the pathogenesis of L. micdadei pneumonia, guinea pig alveolar macrophages obtained by bronchoalveolar lavage were cultured in vitro and infected with L. micdadei. In the absence of opsonins L. micdadei was phagocytized by, and multiplied within, alveolar macrophages with greater than a 100-fold increase in cell-associated colony forming units over 20 h. L. micdadei opsonized with complement or antibody multiplied within alveolar macrophages at the same rate as unopsonized bacteria. Guinea pigs which were treated with antimicrobials after infection with L. micdadei and recovered from the pneumonia were immune to challenge with an otherwise lethal inoculum of L. micdadei. However, the growth curve of both unopsonized and opsonized L. micdadei in the alveolar macrophages from immune animals was essentially identical to that in macrophages from susceptible animals. Thus, the resident alveolar macrophage is not capable of limiting the growth of Legionella. Rather, the alveolar macrophages appear to be the primary site of Legionella multiplication within the lung. Although alveolar macrophages may participate in other aspects of pulmonary immunity to the legionellae, these data indicate that the alveolar macrophage alone does not act as an effector cell in cell-mediated immunity to Legionella.