Microbes and Infection 16 (2014) 985e990 www.elsevier.com/locate/micinf
Recognition of Legionella pneumophila nucleic acids by innate immune receptors Larissa D. Cunha, Dario S. Zamboni* Department of Cell Biology, Ribeir~ao Preto Medical School, University of S~ao Paulo (FMRP/USP), Ribeir~ao Preto, SP 14049-900, Brazil Received 10 July 2014; accepted 18 August 2014 Available online 27 August 2014
Abstract Innate immune receptors evolved to sense conserved molecules that are present in microbes or are released during non-physiological conditions. Activation of these receptors is essential for early restriction of microbial infections and generation of adaptive immunity. Among the conserved molecules sensed by innate immune receptors are the nucleic acids, which are abundantly contained in all infectious organisms including virus, bacteria, fungi and parasites. In this review we focus in the innate immune proteins that function to sense nucleic acids from the intracellular bacterial pathogen Legionella pneumophila and the importance of these processes to the outcome of the infection. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Legionella; Nucleic acid sensing; Innate immune receptors
1. Introduction Legionella pneumophila is the etiological agent of Legionnaire's disease, an acute pneumonia that affects mostly immunosuppressed patients, with an elevated mortality risk [1]. L. pneumophila is a facultative intracellular Gramnegative bacterium that parasite free-living protozoa of fresh water reservoirs, thus human contamination occurs by inhalation of aerosols containing with infected protozoan host [2]. In humans, L. pneumophila thrives in endosomal-like vacuoles (LCV, Legionella-containing vacuole), which are formed in alveolar macrophages and monocytes, eventually invading non-professional phagocytes such as epithelial cells [3,4]. In order to establish and maintain the LCV, the bacteria modulate host cell vesicular traffic and endosomal maturation pathway, inducing recruitment of vesicles derived from endoplasmic * Corresponding author. Department of Cell Biology, Ribeir~ao Preto Medical School, University of S~ao Paulo (FMRP/USP), Av. Bandeirantes 3900, Ribeir~ao Preto, SP 14049-900, Brazil. Tel.: þ55 (16) 3602 3265; fax: þ55 (16) 3633 1786. E-mail address: [email protected] (D.S. Zamboni).
reticulum while avoiding LCV fusion with lysosomes [5]. Legionella express a unique type IVB secretion system denominated Dot/Icm, through which more than 300 bacterial effector proteins are secreted into host cytosol to subvert host functions Contributing to LCV maintenance [6,7]. Consequently, L. pneumophila mutants that express defective Dot/ Icm machinery fail to establish a functional LCV and are rapidly controlled by the host cells [8,9]. Murine models of infection with L. pneumophila have effectively contributed to unravel the mechanisms that lead to infection control by a competent host. Effective innate immune responses are essential to control L. pneumophila infection [10]. Susceptibility to infection by L. pneumophila in A/J mice has been mapped to a gene called Naip5 or Birc1e, encoded within the chromosome 13 locus Lgn1 [11e14]. Birc1e (NAIP5) is a cytosolic receptor that belongs to the Nod-like family. Recognition of bacterial flagellin that escapes into macrophages cytosol through the Dot/Icm system interacts with NAIP5, forming a complex with CARDcontaining NLRC4 (also a member of Nod-like receptor family), and triggering caspase-1 recruitment and activation in a molecular platform generally denominated inflammasome
http://dx.doi.org/10.1016/j.micinf.2014.08.008 1286-4579/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
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[15e18]. The NAIP5/NLRC4 pathway of inflammasome activation in response to flagellin is considered the main mechanism involved with generation of responses that culminate in infection control [19e21]. In accordance, restrictive C57BL/6 mice are susceptible to early pulmonary bacterial multiplication in the absence of Naip5, Nlrc4 or caspase-1, as well as by infection with flagellin deficient L. pneumophila ( flaA) [15,22]. Neutrophil recruitment to the lungs of infected mice is required for successful eradication of L. pneumophila. Deficiency on Myd88, an adaptor of Toll-like (TLR) receptors, IL1 and IL-18 receptors, is well known to impact neutrophil recruitment and infection control and different players have already been shown to collaborate to this process [23e25]. In addition, the Nod-like receptor Nod2 and the adaptor molecule Rip2, which are known to trigger NF-kB signaling pathway, also contribute to recruitment of neutrophils to the lungs of infected mice [26]. Type I interferon (IFN) production, induced by pathways that trigger activation of transcription factor IRF3, is another important player in the immune response against L. pneumophila. Mice lacking type I interferon (IFN) signaling are defective in bacterial clearance by mechanisms not completely understood [27e29]. Finally, other components of the innate immune response, such as the Nod-like receptor NLRP3, also participate in the recognition of L. pneumophila [30,31]. In this review, we discuss the role of nucleic acid recognition in immune responses to L. pneumophila. So far, different signaling pathways dependent on nucleic acid sensing have already been shown to recognize L. pneumophila and contribute to infection control. Among them are the recognition of bacterial DNA by TLR9 and induction of type I interferon mediated by IRF3 activation. Moreover, modulation by L. pneumophila Dot/Icm effectors of host cell responses driven by acid nucleic sensing is also discussed. 2. TLRs activation and DNA-mediated TLR9 activation in response to L. pneumophila As previously mentioned, innate immune responses mediated by the adaptor protein Myd88 are pivotal to clearance of L. pneumophila [23e25]. Recognition of L. pneumophila peptidoglycans and lipopeptides by TLR2 contributes to bacterial clearance in the lungs of infected mice [24,25,32,33]. Moreover, alveolar macrophages deficient in TLR5 produce less TNF-a as compared to cells from wild-type animals [34]. Although Tlr5/ mice restrict L. pneumophila infection normally, they show lung pathology consistent with development of pneumonia in the late stages of infection [34]. IL-18 signaling, mediated by Myd88, was also shown to be important for IFN-g induction by NK cells [35]. In agreement, IFN-g deficiency moderately impairs L. pneumophila clearance [23,35]. Recently, it was shown that the IL-1a production in the lungs of infected mice was dependent on the expression of a functional Dot/Icm dependent system that mediates neutrophil recruitment to the infection site [36]. Consequently, IL-1a deficient mice failed to control infection by virulent L. pneumophila [36].
The initial studies supporting a role of TLR9 for L. pneumophila recognition demonstrated that infection with L. pneumophila or stimulation with heat-killed bacteria induced IL-12p40 secretion in macrophages [37]. Of note, the cytokine production was reduced by treatment of cells with chloroquine, an inhibitor of endosome acidification, which is essential for TLR9 activation [37]. Further studies performed with macrophages derived from Tlr2//Tlr9/ indicated that these cells produced similar levels of IL-12 compared Tlr2/ littermates controls, suggesting that in macrophages TLR9 is not essential for cytokine production in response to L. pneumophila infection [35]. Of note, these studies were performed using cells encoding the Naip5 mutant allele derived from the A/J mice background. Therefore, the TLR9-mediated restriction of L. pneumophila replication is not affected by Naip5mediated flagellin recognition. In the case of dendritic cells (DC), inhibition of Tlr9 activation by either chloroquine or by an inhibitory ligand of TLR9 (ODN2088) reduced IL-12p40 secretion in cells derived from both susceptible A/J and restrictive BALB/c mice (that express a functional Naip5) [37]. Importantly, Tlr9/ mice in the restrictive BALB/c genetic background are more susceptible to pulmonary infection with L. pneumophila, with increased mortality and bacterial burden in comparison to wild-type mice in an intratracheal route of infection [38]. Susceptibility of mice deficient for Tlr9 could be associated with reduced levels of myeloid DC and CD4þ lymphocytes in the lung of infected mice [38]. This impairment observed in Tlr9/ mice was associated with isolation from infected mice of alveolar macrophages displaying alternative polarization, with reduced phagocytosis of pulmonary bacteria and expression of nitric oxide, which is important to bacterial clearance. As Tlr9/ macrophages infected in vitro does not display impaired bacterial phagocytosis, it is suggestive that the cytokine milieu in the lungs is important for macrophage response to Legionella. Indeed, Tlr9/ mice presented reduced production of chemokines and type I cytokines, such as MCP-1, TNF-a, IL-12 and IFN-g. IL-12, for instance, stimulates IFN-g production by NK cells, which is important to host resistance to L. pneumophila infection. Importantly, Tlr9-dependent chemokine and cytokine profile in the lungs and bacterial clearance could be restored by adoptive transfer of wild-type dendritic cells directly in the lungs of Tlr9/ mice. These data unequivocally supported a role for TLR9-mediated DNA sensing for host immunity against L. pneumophila. Therefore, recognition of L. pneumophila by TLR9 is important for bacterial sensing by dendritic cells, collaborating to the development of a chemokine and cytokine microenvironment that sustain effective activation of macrophages in the lungs. Finally, is important to emphasize that so far, mice deficient to each component that signal through Myd88, as well as doubleknockouts such as Tlr2//Tlr5/ and Tlr2//Tlr9/, fail to recapitulate the severe defect in L. pneumophila clearance present by Myd88/ mice [35]. Therefore, despite the important role of TLR2, -5 and -9 for innate immune recognition of L. pneumophila, the severe deficiency in MyD88 suggest the
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existence of a redundant function of TLRs and/or additional receptors operate in the MyD88-mediated innate immunity to L. pneumophila. 3. Induction of type I IFN in response to L. pneumophila Induction of antimicrobial genes through type I interferon (e.g., IFN-a and IFN-b) signaling is a classic antiviral response. During viral infections, type I IFN transcription is mediated by activation of transcription factors IRF3 and IRF7, upon sensing of viral dsRNA by cytosolic helicases retinoic acid-inducible gene I (RIG-I) or melanoma differentiationassociated gene 5 (MDA5), both requiring the common adaptor protein mitochondrial antiviral signaling protein (MAVS/IPS-1) [39]. However, it is now well accepted that type I interferon is also relevant in immune responses to intracellular bacterial pathogens such as Listeria monocytogenes, Mycobacterium tuberculosis, Franciella tularensis and Brucella abortus [40e43]. Production of IFN-b in response to bacteria has been suggested to occur by recognition of cytosolic dsDNA, supported by the fact that purified bacterial DNA can induce type I IFN production by BMM [44]. Recently, different sensors leading to IRF3 activation and type I IFN expression by recognition of cytosolic DNA have emerged, such as RNA polymerase III (PolIII) [45,46], DNAdependent activator of IFN- regulatory factors (DAI)/Z-DNAbinding protein (ZBP)1 [47], STING [48] and cGAS [49]. The Dot/Icm machinery diverged from ancestral bacterial DNA conjugation system, while retaining the capacity to translocate plasmidial DNA [50]. Therefore, recognition of cytosolic bacterial DNA during the infection with L. pneumophila is likely to occur. In fact, infection of bone marrowderived macrophages and epithelial-like A549 human cells revealed that the bacteria trigger IRF3-dependent IFN-b expression dependently on a functional Dot/Icm secretion system but independently of bacterial flagellin [44,51]. In macrophages, IFN induction is independent of TLR receptors or de novo protein synthesis by the host cell [44]. Accordingly, epithelial cells silenced for IRF3 and macrophages Irf3/ are more susceptible to infection with L. pneumophila [27,51]. These phenotypes could be reverted by treatment with recombinant IFN-b, thus suggesting that type I IFNs contributes to the restriction of L. pneumophila replication in macrophages. Interestingly, induction of Irgm1, an immunity-related GTPase induced by type I IFN response, is partially required for resistance to L. pneumophila in macrophages [27]. Chiu et al. (2009) demonstrated that AT-rich cytosolic DNA triggers IFN production through a mechanism involving Polymerase III (Pol III)-dependent synthesis of poly (A-U) and recognition of newly synthetized RNA by RIG-I. Pol III inhibition was found to significantly reduce IFN-b production in response to L. pneumophila, also impairing infection control. Macrophages treated with Pol III inhibitor ML60218 were susceptible to bacterial infection, and resistance was restored by treatment with IFN-b. Results by other authors argued that, although MDA5 and RIG1 partially contribute to induction of IFN-b in response to L. pneumophila in BMM, this response
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occurs by recognition of bacterial RNA, not DNA [52]. This latter work also argued that inhibition of Pol III activity by ML60218 does not impair resistance to L. pneumophila. Although the adaptor IPS-1 is required downstream for IFN-b, both in BMM and in vivo, Ips-1/ mice do not fail to control bacterial multiplication [45,52]. Interestingly, in human epithelial cells, the use of RNAi was used to demonstrate that MDA5, RIG1 and other CARD-containing receptors (such as ASC and Nod2) are not necessary for IFN induction, suggesting that in humans other receptors may contribute to IFN production in response to bacteria [51]. Finally, differently than L. pneumophila, mice deficient in IFN-b signaling are more resistant to infections with Listeria monocytogenes and with Brucella abortus [53,54]. This contrasting role of type I IFN in host resistance suggests that type I IFN might be a pathway subverted by certain bacterial pathogens thus influencing infection outcome in a pathogen specific fashion. Most recently, induction of a unique eukaryotic cyclic dinucleotide, denominated cyclic guanosine monophosphateadenosine monophosphate (cyclic GMP-AMP, or cGAMP), has been recognized as a major pathway of type IFN expression in response to cytosolic DNA in innate immune cells [55]. Cyclic dinucleotides are conserved secondary messengers in bacterial signaling cascades, and had been previously suggested as immunomodulatory molecules in bacterial infection of murine cells [56]. Nonetheless cGAMP is produced in response to DNA recognition by a cGAMP synthase (cGAS), belonging to the nucleotidyltransferase family [49]. Of note, cGAMP intermediates activation of STING, also important for IRF3-dependent induction of type I IFN [55,57,58]. The axis cGAS/cGAMP/STING contributes to IFN-b production by human macrophages in response to Listeria monocytogenes [59]. Interestingly, silencing of STING by RNAi reduces IFNb in BMM infected with L. pneumophila [27]. In this context, it will be important to verify whether cGAS and cGAMP synthesis are also key players in the generation of type I IFN in response to L. pneumophila. 4. Inflammasome activation in response to recognition of L. pneumophila nucleic acid Induction of inflammasome and caspase-1 activation by the Nod-like receptors NAIP5/NLRC4 is pivotal for recognition of L. pneumophila by murine macrophages and infection control [18e21]. However, additional cytosolic sensors that trigger inflammasome activation also participate in a flagellinindependent pathway for caspase-1 activation in response to L. pneumophila. Of note, the central adaptor protein of inflammasome receptors ASC, is required for cleavage of caspase-1 dependent and independent of expression of flagellin by L. pneumophila [60]. In addition, non-canonical inflammasome activation mediated by caspase-11, most likely in response to cytosolic L. pneumophila LPS, triggers flagellin-independent pore formation in the macrophages membranes and regulates cleavage of caspase-1 by NLRP3 [30]. Similarly to ASC, the NLRP3 receptor is essential to caspase-1 cleavage in response to flaA
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L. pneumophila, as caspase-1 cleavage and IL-1b is abrogated in Nlrp3/ macrophages [30]. In this context, it is important to emphasize that L. pneumophila RNA has been shown to activate NLRP3-dependent inflammasome activation [31]. The role of NLRP3 in the recognition of RNA:DNA hybrid complexes has been recently demonstrated, corroborating the importance of this sensor for recognition and signaling against the presence of foreign nucleic acid inside the cell [61]. In this context, it was reported that NLRP3 participate in the recognition of prokaryotic mRNA thus favoring the recognition of microbial viability, a feature that would account for differential discrimination of live versus dead microbes. It is still unclear if this pathway operates for recognition of L. pneumophila. The observations that live L. pneumophila that is deficient in functional Dot/Icm fail to trigger NLRP3 activation do not favor this hypothesis. An important DNA-sensing inflammasome was revealed in 2009, this complex is involved in recognition of cytosolic DNA and is composed by the cytosolic helicase AIM2 and uses the adaptor protein ASC to trigger caspase-1 activation, the so-called AIM2 inflammasome [62e65]. AIM2 directly interacts with DNA, and have already been shown to participate in macrophage response to DNA virus, such as vaccinia virus and mouse cytomegalovirus, and bacterial pathogens, such as Francisella tularensis and L. monocytogenes [66e70]. It is unclear if AIM2 participate in L. pneumophila recognition. Certainly the L. pneumophila DNA trigger AIM2 activation, but it has been reported that a Dot/Icm effector SdhA, which was known to inhibit host cell death and stabilize the vacuole containing bacteria, prevent the access of bacterial DNA to the cytosol, thus inhibiting the AIM2 recognition of L. pneumophila by human macrophage-like cells and murine macrophages [71e73]. Thus, determination of the role of AIM2 in bacterial recognition and host resistance during L. pneumophila infections may be a subject of additional investigation. 5. Conclusion The resistance to infectious organisms, including L. pneumophila, relies on the innate immune recognition of invading organisms. Understanding of this process is essential to unravel the mechanisms underlying host resistance. Although the recognition of L. pneumophila flagellin by cytosolic receptors NAIP5 and NLRC4 represent a pivotal mechanism for host resistance, recent studies identified additional mechanisms that operate independent on the axis flagellin/NAIP5/NLRC4. In this context, the recognition of L. pneumophila DNA and RNA may effectively contribute to early innate immune detection of the pathogen. Different innate immune receptors have already been shown to be involved in the recognition of the L. pneumophila nucleic acids, including TLR9, Pol III, NLRP3 and AIM2 (Fig. 1). These receptors trigger different signaling pathways, such as NF-kB pathway, signaling through type I interferon and cleavage of caspase-1, that result in expression, activation and secretion of diverse chemokines and cytokines. Activation of these pathways may favor the
Fig. 1. Overview of innate immune responses pathways triggered by sensing of L. pneumophila DNA. LCV indicate the Legionella-containing vacuole. DNA releases from death (dotted square in blue) bacteria trigger TLR9 activation in the LCV membrane leading to NF-kB activation. Nucleic acid from live L. pneumophila (square in blue) translocate into the host cell cytoplasm in a process dependent of the bacterial Dot/Icm. This process enables activation of RIG1/MDA5, STING and AIM2 pathways, which culminated in caspase-1 activation (via AIM2 and ASC) and IFN-b production. The Dot/Icm effector protein SdhA was shown to stabilize the vacuole containing bacteria preventing the access of bacterial DNA to the cytosol, thus reducing AIM2 activation in response to L. pneumophila infection.
boosting an effective innate immune response for host resistance during infections with Legionella. We envisage that in the near future additional pathways for DNA and RNA recognition will be identified as relevant for host response during L. pneumophila infections. Moreover, further investigation using L. pneumophila as a model of infection may facilitate identification of novel nucleic acid recognition pathways that operate in response to infections with pathogenic microbes. Conflict of interest There is no conflict of interest. Acknowledgments We apologize to our colleagues whose papers we were unable to cite due to space limitations. Work in our laboratory is supported by grants from the Fundaç~ao de Amparo a Pesquisa do Estado de S~ao Paulo (FAPESP, Grants 2012/09363-6, 2013/08216-2, and 2014/04684-4) and Instituto Nacional de Ci^encia e Tecnologia em Vacinas (INCTV/CNPq). D.S.Z. is a research fellow from CNPq. References [1] Phin N, Parry-Ford F, Harrison T, Stagg HR, Zhang N, Kumar K, et al. Epidemiology and clinical management of Legionnaires' disease. Lancet Infect Dis 2014;14:70713e3.
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Recognition of Legionella pneumophila nucleic acids by innate immune receptors Larissa D. Cunha, Dario S. Zamboni* Department of Cell Biology, Ribeir~ao Preto Medical School, University of S~ao Paulo (FMRP/USP), Ribeir~ao Preto, SP 14049-900, Brazil Received 10 July 2014; accepted 18 August 2014 Available online 27 August 2014
Abstract Innate immune receptors evolved to sense conserved molecules that are present in microbes or are released during non-physiological conditions. Activation of these receptors is essential for early restriction of microbial infections and generation of adaptive immunity. Among the conserved molecules sensed by innate immune receptors are the nucleic acids, which are abundantly contained in all infectious organisms including virus, bacteria, fungi and parasites. In this review we focus in the innate immune proteins that function to sense nucleic acids from the intracellular bacterial pathogen Legionella pneumophila and the importance of these processes to the outcome of the infection. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
Keywords: Legionella; Nucleic acid sensing; Innate immune receptors
1. Introduction Legionella pneumophila is the etiological agent of Legionnaire's disease, an acute pneumonia that affects mostly immunosuppressed patients, with an elevated mortality risk [1]. L. pneumophila is a facultative intracellular Gramnegative bacterium that parasite free-living protozoa of fresh water reservoirs, thus human contamination occurs by inhalation of aerosols containing with infected protozoan host [2]. In humans, L. pneumophila thrives in endosomal-like vacuoles (LCV, Legionella-containing vacuole), which are formed in alveolar macrophages and monocytes, eventually invading non-professional phagocytes such as epithelial cells [3,4]. In order to establish and maintain the LCV, the bacteria modulate host cell vesicular traffic and endosomal maturation pathway, inducing recruitment of vesicles derived from endoplasmic * Corresponding author. Department of Cell Biology, Ribeir~ao Preto Medical School, University of S~ao Paulo (FMRP/USP), Av. Bandeirantes 3900, Ribeir~ao Preto, SP 14049-900, Brazil. Tel.: þ55 (16) 3602 3265; fax: þ55 (16) 3633 1786. E-mail address: [email protected] (D.S. Zamboni).
reticulum while avoiding LCV fusion with lysosomes [5]. Legionella express a unique type IVB secretion system denominated Dot/Icm, through which more than 300 bacterial effector proteins are secreted into host cytosol to subvert host functions Contributing to LCV maintenance [6,7]. Consequently, L. pneumophila mutants that express defective Dot/ Icm machinery fail to establish a functional LCV and are rapidly controlled by the host cells [8,9]. Murine models of infection with L. pneumophila have effectively contributed to unravel the mechanisms that lead to infection control by a competent host. Effective innate immune responses are essential to control L. pneumophila infection [10]. Susceptibility to infection by L. pneumophila in A/J mice has been mapped to a gene called Naip5 or Birc1e, encoded within the chromosome 13 locus Lgn1 [11e14]. Birc1e (NAIP5) is a cytosolic receptor that belongs to the Nod-like family. Recognition of bacterial flagellin that escapes into macrophages cytosol through the Dot/Icm system interacts with NAIP5, forming a complex with CARDcontaining NLRC4 (also a member of Nod-like receptor family), and triggering caspase-1 recruitment and activation in a molecular platform generally denominated inflammasome
http://dx.doi.org/10.1016/j.micinf.2014.08.008 1286-4579/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.
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[15e18]. The NAIP5/NLRC4 pathway of inflammasome activation in response to flagellin is considered the main mechanism involved with generation of responses that culminate in infection control [19e21]. In accordance, restrictive C57BL/6 mice are susceptible to early pulmonary bacterial multiplication in the absence of Naip5, Nlrc4 or caspase-1, as well as by infection with flagellin deficient L. pneumophila ( flaA) [15,22]. Neutrophil recruitment to the lungs of infected mice is required for successful eradication of L. pneumophila. Deficiency on Myd88, an adaptor of Toll-like (TLR) receptors, IL1 and IL-18 receptors, is well known to impact neutrophil recruitment and infection control and different players have already been shown to collaborate to this process [23e25]. In addition, the Nod-like receptor Nod2 and the adaptor molecule Rip2, which are known to trigger NF-kB signaling pathway, also contribute to recruitment of neutrophils to the lungs of infected mice [26]. Type I interferon (IFN) production, induced by pathways that trigger activation of transcription factor IRF3, is another important player in the immune response against L. pneumophila. Mice lacking type I interferon (IFN) signaling are defective in bacterial clearance by mechanisms not completely understood [27e29]. Finally, other components of the innate immune response, such as the Nod-like receptor NLRP3, also participate in the recognition of L. pneumophila [30,31]. In this review, we discuss the role of nucleic acid recognition in immune responses to L. pneumophila. So far, different signaling pathways dependent on nucleic acid sensing have already been shown to recognize L. pneumophila and contribute to infection control. Among them are the recognition of bacterial DNA by TLR9 and induction of type I interferon mediated by IRF3 activation. Moreover, modulation by L. pneumophila Dot/Icm effectors of host cell responses driven by acid nucleic sensing is also discussed. 2. TLRs activation and DNA-mediated TLR9 activation in response to L. pneumophila As previously mentioned, innate immune responses mediated by the adaptor protein Myd88 are pivotal to clearance of L. pneumophila [23e25]. Recognition of L. pneumophila peptidoglycans and lipopeptides by TLR2 contributes to bacterial clearance in the lungs of infected mice [24,25,32,33]. Moreover, alveolar macrophages deficient in TLR5 produce less TNF-a as compared to cells from wild-type animals [34]. Although Tlr5/ mice restrict L. pneumophila infection normally, they show lung pathology consistent with development of pneumonia in the late stages of infection [34]. IL-18 signaling, mediated by Myd88, was also shown to be important for IFN-g induction by NK cells [35]. In agreement, IFN-g deficiency moderately impairs L. pneumophila clearance [23,35]. Recently, it was shown that the IL-1a production in the lungs of infected mice was dependent on the expression of a functional Dot/Icm dependent system that mediates neutrophil recruitment to the infection site [36]. Consequently, IL-1a deficient mice failed to control infection by virulent L. pneumophila [36].
The initial studies supporting a role of TLR9 for L. pneumophila recognition demonstrated that infection with L. pneumophila or stimulation with heat-killed bacteria induced IL-12p40 secretion in macrophages [37]. Of note, the cytokine production was reduced by treatment of cells with chloroquine, an inhibitor of endosome acidification, which is essential for TLR9 activation [37]. Further studies performed with macrophages derived from Tlr2//Tlr9/ indicated that these cells produced similar levels of IL-12 compared Tlr2/ littermates controls, suggesting that in macrophages TLR9 is not essential for cytokine production in response to L. pneumophila infection [35]. Of note, these studies were performed using cells encoding the Naip5 mutant allele derived from the A/J mice background. Therefore, the TLR9-mediated restriction of L. pneumophila replication is not affected by Naip5mediated flagellin recognition. In the case of dendritic cells (DC), inhibition of Tlr9 activation by either chloroquine or by an inhibitory ligand of TLR9 (ODN2088) reduced IL-12p40 secretion in cells derived from both susceptible A/J and restrictive BALB/c mice (that express a functional Naip5) [37]. Importantly, Tlr9/ mice in the restrictive BALB/c genetic background are more susceptible to pulmonary infection with L. pneumophila, with increased mortality and bacterial burden in comparison to wild-type mice in an intratracheal route of infection [38]. Susceptibility of mice deficient for Tlr9 could be associated with reduced levels of myeloid DC and CD4þ lymphocytes in the lung of infected mice [38]. This impairment observed in Tlr9/ mice was associated with isolation from infected mice of alveolar macrophages displaying alternative polarization, with reduced phagocytosis of pulmonary bacteria and expression of nitric oxide, which is important to bacterial clearance. As Tlr9/ macrophages infected in vitro does not display impaired bacterial phagocytosis, it is suggestive that the cytokine milieu in the lungs is important for macrophage response to Legionella. Indeed, Tlr9/ mice presented reduced production of chemokines and type I cytokines, such as MCP-1, TNF-a, IL-12 and IFN-g. IL-12, for instance, stimulates IFN-g production by NK cells, which is important to host resistance to L. pneumophila infection. Importantly, Tlr9-dependent chemokine and cytokine profile in the lungs and bacterial clearance could be restored by adoptive transfer of wild-type dendritic cells directly in the lungs of Tlr9/ mice. These data unequivocally supported a role for TLR9-mediated DNA sensing for host immunity against L. pneumophila. Therefore, recognition of L. pneumophila by TLR9 is important for bacterial sensing by dendritic cells, collaborating to the development of a chemokine and cytokine microenvironment that sustain effective activation of macrophages in the lungs. Finally, is important to emphasize that so far, mice deficient to each component that signal through Myd88, as well as doubleknockouts such as Tlr2//Tlr5/ and Tlr2//Tlr9/, fail to recapitulate the severe defect in L. pneumophila clearance present by Myd88/ mice [35]. Therefore, despite the important role of TLR2, -5 and -9 for innate immune recognition of L. pneumophila, the severe deficiency in MyD88 suggest the
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existence of a redundant function of TLRs and/or additional receptors operate in the MyD88-mediated innate immunity to L. pneumophila. 3. Induction of type I IFN in response to L. pneumophila Induction of antimicrobial genes through type I interferon (e.g., IFN-a and IFN-b) signaling is a classic antiviral response. During viral infections, type I IFN transcription is mediated by activation of transcription factors IRF3 and IRF7, upon sensing of viral dsRNA by cytosolic helicases retinoic acid-inducible gene I (RIG-I) or melanoma differentiationassociated gene 5 (MDA5), both requiring the common adaptor protein mitochondrial antiviral signaling protein (MAVS/IPS-1) [39]. However, it is now well accepted that type I interferon is also relevant in immune responses to intracellular bacterial pathogens such as Listeria monocytogenes, Mycobacterium tuberculosis, Franciella tularensis and Brucella abortus [40e43]. Production of IFN-b in response to bacteria has been suggested to occur by recognition of cytosolic dsDNA, supported by the fact that purified bacterial DNA can induce type I IFN production by BMM [44]. Recently, different sensors leading to IRF3 activation and type I IFN expression by recognition of cytosolic DNA have emerged, such as RNA polymerase III (PolIII) [45,46], DNAdependent activator of IFN- regulatory factors (DAI)/Z-DNAbinding protein (ZBP)1 [47], STING [48] and cGAS [49]. The Dot/Icm machinery diverged from ancestral bacterial DNA conjugation system, while retaining the capacity to translocate plasmidial DNA [50]. Therefore, recognition of cytosolic bacterial DNA during the infection with L. pneumophila is likely to occur. In fact, infection of bone marrowderived macrophages and epithelial-like A549 human cells revealed that the bacteria trigger IRF3-dependent IFN-b expression dependently on a functional Dot/Icm secretion system but independently of bacterial flagellin [44,51]. In macrophages, IFN induction is independent of TLR receptors or de novo protein synthesis by the host cell [44]. Accordingly, epithelial cells silenced for IRF3 and macrophages Irf3/ are more susceptible to infection with L. pneumophila [27,51]. These phenotypes could be reverted by treatment with recombinant IFN-b, thus suggesting that type I IFNs contributes to the restriction of L. pneumophila replication in macrophages. Interestingly, induction of Irgm1, an immunity-related GTPase induced by type I IFN response, is partially required for resistance to L. pneumophila in macrophages [27]. Chiu et al. (2009) demonstrated that AT-rich cytosolic DNA triggers IFN production through a mechanism involving Polymerase III (Pol III)-dependent synthesis of poly (A-U) and recognition of newly synthetized RNA by RIG-I. Pol III inhibition was found to significantly reduce IFN-b production in response to L. pneumophila, also impairing infection control. Macrophages treated with Pol III inhibitor ML60218 were susceptible to bacterial infection, and resistance was restored by treatment with IFN-b. Results by other authors argued that, although MDA5 and RIG1 partially contribute to induction of IFN-b in response to L. pneumophila in BMM, this response
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occurs by recognition of bacterial RNA, not DNA [52]. This latter work also argued that inhibition of Pol III activity by ML60218 does not impair resistance to L. pneumophila. Although the adaptor IPS-1 is required downstream for IFN-b, both in BMM and in vivo, Ips-1/ mice do not fail to control bacterial multiplication [45,52]. Interestingly, in human epithelial cells, the use of RNAi was used to demonstrate that MDA5, RIG1 and other CARD-containing receptors (such as ASC and Nod2) are not necessary for IFN induction, suggesting that in humans other receptors may contribute to IFN production in response to bacteria [51]. Finally, differently than L. pneumophila, mice deficient in IFN-b signaling are more resistant to infections with Listeria monocytogenes and with Brucella abortus [53,54]. This contrasting role of type I IFN in host resistance suggests that type I IFN might be a pathway subverted by certain bacterial pathogens thus influencing infection outcome in a pathogen specific fashion. Most recently, induction of a unique eukaryotic cyclic dinucleotide, denominated cyclic guanosine monophosphateadenosine monophosphate (cyclic GMP-AMP, or cGAMP), has been recognized as a major pathway of type IFN expression in response to cytosolic DNA in innate immune cells [55]. Cyclic dinucleotides are conserved secondary messengers in bacterial signaling cascades, and had been previously suggested as immunomodulatory molecules in bacterial infection of murine cells [56]. Nonetheless cGAMP is produced in response to DNA recognition by a cGAMP synthase (cGAS), belonging to the nucleotidyltransferase family [49]. Of note, cGAMP intermediates activation of STING, also important for IRF3-dependent induction of type I IFN [55,57,58]. The axis cGAS/cGAMP/STING contributes to IFN-b production by human macrophages in response to Listeria monocytogenes [59]. Interestingly, silencing of STING by RNAi reduces IFNb in BMM infected with L. pneumophila [27]. In this context, it will be important to verify whether cGAS and cGAMP synthesis are also key players in the generation of type I IFN in response to L. pneumophila. 4. Inflammasome activation in response to recognition of L. pneumophila nucleic acid Induction of inflammasome and caspase-1 activation by the Nod-like receptors NAIP5/NLRC4 is pivotal for recognition of L. pneumophila by murine macrophages and infection control [18e21]. However, additional cytosolic sensors that trigger inflammasome activation also participate in a flagellinindependent pathway for caspase-1 activation in response to L. pneumophila. Of note, the central adaptor protein of inflammasome receptors ASC, is required for cleavage of caspase-1 dependent and independent of expression of flagellin by L. pneumophila [60]. In addition, non-canonical inflammasome activation mediated by caspase-11, most likely in response to cytosolic L. pneumophila LPS, triggers flagellin-independent pore formation in the macrophages membranes and regulates cleavage of caspase-1 by NLRP3 [30]. Similarly to ASC, the NLRP3 receptor is essential to caspase-1 cleavage in response to flaA
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L. pneumophila, as caspase-1 cleavage and IL-1b is abrogated in Nlrp3/ macrophages [30]. In this context, it is important to emphasize that L. pneumophila RNA has been shown to activate NLRP3-dependent inflammasome activation [31]. The role of NLRP3 in the recognition of RNA:DNA hybrid complexes has been recently demonstrated, corroborating the importance of this sensor for recognition and signaling against the presence of foreign nucleic acid inside the cell [61]. In this context, it was reported that NLRP3 participate in the recognition of prokaryotic mRNA thus favoring the recognition of microbial viability, a feature that would account for differential discrimination of live versus dead microbes. It is still unclear if this pathway operates for recognition of L. pneumophila. The observations that live L. pneumophila that is deficient in functional Dot/Icm fail to trigger NLRP3 activation do not favor this hypothesis. An important DNA-sensing inflammasome was revealed in 2009, this complex is involved in recognition of cytosolic DNA and is composed by the cytosolic helicase AIM2 and uses the adaptor protein ASC to trigger caspase-1 activation, the so-called AIM2 inflammasome [62e65]. AIM2 directly interacts with DNA, and have already been shown to participate in macrophage response to DNA virus, such as vaccinia virus and mouse cytomegalovirus, and bacterial pathogens, such as Francisella tularensis and L. monocytogenes [66e70]. It is unclear if AIM2 participate in L. pneumophila recognition. Certainly the L. pneumophila DNA trigger AIM2 activation, but it has been reported that a Dot/Icm effector SdhA, which was known to inhibit host cell death and stabilize the vacuole containing bacteria, prevent the access of bacterial DNA to the cytosol, thus inhibiting the AIM2 recognition of L. pneumophila by human macrophage-like cells and murine macrophages [71e73]. Thus, determination of the role of AIM2 in bacterial recognition and host resistance during L. pneumophila infections may be a subject of additional investigation. 5. Conclusion The resistance to infectious organisms, including L. pneumophila, relies on the innate immune recognition of invading organisms. Understanding of this process is essential to unravel the mechanisms underlying host resistance. Although the recognition of L. pneumophila flagellin by cytosolic receptors NAIP5 and NLRC4 represent a pivotal mechanism for host resistance, recent studies identified additional mechanisms that operate independent on the axis flagellin/NAIP5/NLRC4. In this context, the recognition of L. pneumophila DNA and RNA may effectively contribute to early innate immune detection of the pathogen. Different innate immune receptors have already been shown to be involved in the recognition of the L. pneumophila nucleic acids, including TLR9, Pol III, NLRP3 and AIM2 (Fig. 1). These receptors trigger different signaling pathways, such as NF-kB pathway, signaling through type I interferon and cleavage of caspase-1, that result in expression, activation and secretion of diverse chemokines and cytokines. Activation of these pathways may favor the
Fig. 1. Overview of innate immune responses pathways triggered by sensing of L. pneumophila DNA. LCV indicate the Legionella-containing vacuole. DNA releases from death (dotted square in blue) bacteria trigger TLR9 activation in the LCV membrane leading to NF-kB activation. Nucleic acid from live L. pneumophila (square in blue) translocate into the host cell cytoplasm in a process dependent of the bacterial Dot/Icm. This process enables activation of RIG1/MDA5, STING and AIM2 pathways, which culminated in caspase-1 activation (via AIM2 and ASC) and IFN-b production. The Dot/Icm effector protein SdhA was shown to stabilize the vacuole containing bacteria preventing the access of bacterial DNA to the cytosol, thus reducing AIM2 activation in response to L. pneumophila infection.
boosting an effective innate immune response for host resistance during infections with Legionella. We envisage that in the near future additional pathways for DNA and RNA recognition will be identified as relevant for host response during L. pneumophila infections. Moreover, further investigation using L. pneumophila as a model of infection may facilitate identification of novel nucleic acid recognition pathways that operate in response to infections with pathogenic microbes. Conflict of interest There is no conflict of interest. Acknowledgments We apologize to our colleagues whose papers we were unable to cite due to space limitations. Work in our laboratory is supported by grants from the Fundaç~ao de Amparo a Pesquisa do Estado de S~ao Paulo (FAPESP, Grants 2012/09363-6, 2013/08216-2, and 2014/04684-4) and Instituto Nacional de Ci^encia e Tecnologia em Vacinas (INCTV/CNPq). D.S.Z. is a research fellow from CNPq. References [1] Phin N, Parry-Ford F, Harrison T, Stagg HR, Zhang N, Kumar K, et al. Epidemiology and clinical management of Legionnaires' disease. Lancet Infect Dis 2014;14:70713e3.
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