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Brucella abortus DNA is a major bacterial agonist to activate the host innate immune system
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Microbes and Infection xx (2014) 1e6 www.elsevier.com/locate/micinf
Brucella abortus DNA is a major bacterial agonist to activate the host innate immune system Priscila Carneiro Campos, Marco Tulio Ribeiro Gomes, Gabriela Guimar~aes, Miriam Maria Silva Costa Franco, Fernanda Martins Marim, Sergio Costa Oliveira*
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Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Av. Antonio Carlos 6627, Pampulha, 31270-901 Belo Horizonte, MG, Brazil Received 30 June 2014; accepted 20 August 2014
Abstract Immunity against Brucella abortus depends on the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Signaling pathways triggered by Brucella DNA involves TLR9, AIM2 and possibly STING and MAVS. Herein, we review the advances in B. abortus DNA sensing by host innate immune receptors and the progress in this field. © 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur.
Keywords: Brucella abortus; Innate immunity; TLRs; Cytosolic sensors; Bacterial DNA
1. Introduction The innate immune system is the first line of defense against invading pathogens and other danger signals, acting through the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) [1]. These PRRs sense foreign molecules shared by a large group of microbes. For example, many Toll-like receptors (TLRs), NOD-like receptors (NLRs) or RIG-I receptors (RLRs) recognize lipopolysaccharide, peptidoglycan or RNA motifs that are expressed by different bacteria or viruses but not by the host [2]. Other receptors, however, are activated by common molecules expressed by microbes and/or the host if these molecules are located at unphysiological cellular compartments. The DNA, for instance, is a molecule present in the pathogen and it is highly abundant in the nucleus of host cells.
* Corresponding author. Tel./fax: þ55 31 34092666. E-mail addresses: [email protected], [email protected] (S.C. Oliveira).
Its presence in other cell compartments including endosomes and the cytosol activates DNA recognition systems to detect DNA derived from invading pathogens (DNA PAMPs) and disturbed self (DNA DAMPs) [3]. Unmethylated CpG DNA (abundant in many pathogen genomes) and classical B form double-stranded DNA (dsDNA) are examples of motifs which have the ability to stimulate immune responses through DNA sensors such as TLR9 on endosomal membranes [4]; and cGAS (Cyclic GMP-AMP synthase), AIM2 (absent in melanoma 2) and RNA polymerase III [5] in the cytosol, respectively. These sensors signal through specific adapter molecules including MyD88, STING (stimulator of interferon genes), MAVS (mitochondrial antiviral-signaling protein) and ASC (apoptosis-associated speck-like protein containing a CARD) to stimulate production of proinflammatory cytokines or type I IFN, or to activate inflammasomes [6e9]. Several groups have described DNA-sensing receptors involved in innate immune response to bacterial species, such as Mycobacterium tuberculosis [10], Listeria monocytogenes [11e13], Francisella tularensis [11,14] and Brucella abortus [15e16]. Our research group is currently identifying host
http://dx.doi.org/10.1016/j.micinf.2014.08.010 1286-4579/© 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur. Please cite this article in press as: Campos PC, et al., Brucella abortus DNA is a major bacterial agonist to activate the host innate immune system, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.010
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receptors that recognize B. abortus-derived DNA in a murine model. B. abortus is a Gram-negative facultative intracellular bacterium, that causes brucellosis, with pathological manifestations of arthritis, endocarditis, and meningitis in humans, and in cattle it leads to abortion and infertility resulting in serious economic losses to the livestock industry [17]. This pathogen resist killing by neutrophils, it replicates inside macrophages and dendritic cells (DCs), maintaining a long lasting interaction with host cells. Immunity against Brucella infection involves mainly Th1 cells and CD8þ T lymphocytes, gamma-interferon (IFN-g) and tumor necrosis factor alpha (TNF-a) production and activated macrophages and DCs [18e19]. We have identified Brucella DNA as a major bacterial component to induce type I IFN and our study revealed that this molecule operates through a mechanism dependent on RNA polymerase III to be sensed probably by an unknown receptor via the adapter molecule STING [20]. It is also possible that STING acts as a DNA sensor itself and the mitochondrial protein MAVS could be involved in the RNA pol III and/or STING-mediated responses. Besides, we determined that AIM2, which senses Brucella DNA in the cytosol, is partially required for caspase-1 activation and IL-1b secretion [16]. This review summarizes the current knowledge about host cell response to microbial DNA, with emphasis on recent advances on B. abortus-derived DNA sensing pathways and we discuss the new findings in this field. 1.1. The role of the endosomal receptor TLR9 in sensing B. abortus DNA TLRs were the first PRRs to be identified and since then the most well characterized. TLRs are type I integral membrane proteins with ectodomains containing leucine-rich repeats for PAMP recognition, a transmembrane domain, and a cytosolic Toll/IL-1 receptor (TIR) domain responsible for transducing signals to downstream adapters including TRIF and MyD88 [21]. There are 5 identified TLRs involved in nucleic acid sensing: TLR3, TLR7, TLR8, TLR9, and TLR13, although TLR8 is not functional in mice and TLR13 has not been found in humans yet [22e24]. These receptors are responsible for sensing a diversity of pathogen-derived molecules. The sensing of nucleic acids performed by these TLRs which reside within the endoplasmic reticulum induces its traffic to endosomal compartment in the presence of stimuli [25]. TLR3 senses dsRNA, TLR7/TLR8 sense ssRNA and TLR9 senses unmethylated cytosine-guanosine (CpG) DNA motifs that are common for bacterial but rare in mammalian genomes [26,27]. The adaptor protein MyD88 plays a pivotal role in the cell signaling initiated upon TLRs (except for TLR3) or IL-1R family activation. PAMPs generally induce TLR dimerization leading the recruitment of MyD88 followed by the interleukin-1 receptorassociated kinase (IRAK) family activation [28]. This process generates multiple cellular responses including induction of inflammatory cytokines secretion through activation of nuclear factor (NF)-kB and AP-1 transcription factors [29]. However, activation of TLR7 and TLR9 alternatively might elicit the production of type-I IFNs by activating the IFN regulatory
factor-7 (IRF-7) [27]. Since the adaptor MyD88 and IRAK proteins show this central role in cellular response to PAMPs, it is conceived that these molecules participate in the innate immune response against pathogens. Regarding B. abortus infection, it was shown that MyD88/ and IRAK4/ mice are inefficient to control infection and macrophages or DC derived from these mice produce low levels of proinflammatory cytokines in response to this pathogen [30e32]. The essential role attributed to MyD88 and IRAK-4 proteins argues to the participation of TLRs during the control of B. abortus infection. Initially, it was shown that TLR2 plays no role controlling infection in mice [30]. Subsequently, it was shown by our group that TLR6 is important for host resistance since knockout mice for this receptor are more susceptible to B. abortus infection [33]. Regarding TLR9, it was demonstrated that this receptor is implicated, at least at early phases of infection, in host resistance to B. abortus in mice [31]. Furthermore, it was shown that bacterial DNA extracted from heat killed B. abortus induces IL-12 production by dendritic cells dependent on TLR9. Moreover, DNA methylation abrogates the production of inflammatory cytokines by splenocytes, suggesting that stimulatory property of DNA resides in CpG motifs that are not methylated [15]. As mentioned before, the activation of TLR9 signaling cascades also induces type I IFN secretion which might trigger expression of the interferon-inducible resistance proteins at late time points. For instance, murine macrophages infected with B. abortus produce interferon-inducible resistance proteins such as the members of the IRG (immunity-related GTPases) family. Remarkably, albeit this induction requires the adaptor molecule MyD88 it does not involve TLR2/TLR4/ TLR5/TLR9 [34]. In addition, our group demonstrated that type I IFN secretion induced by B. abortus or its DNA is independent of TLR2/TLR4/TLR9 recognition but requires the adaptor molecule MyD88. This pathway leads to type-I IFN secretion and regulation of IRG family proteins expression independent on TLR signaling but engaging MyD88, which suggests the participation of an unknown receptor sensing B. abortus and its DNA [20]. Although it seems that TLR9 is not required for type-I IFN induction, the role of TLR9 to control B. abortus infection and inflammatory cytokines production was well documented as described before [31]. In addition, we suggest that inflammatory cytokine signaling pathway, MAPKs (ERK1/2, p38 and JNK) and p65 NF-kB phosphorylation are impaired in TLR9/ macrophages activated by B. abortus or its DNA (unpublished results). Taken together, these results suggest that B. abortus DNA contains motifs that act as an immune cell activator, and the sensing of this molecule by TLR9 orchestrates a robust immune response that helps to control B. abortus infection. 1.2. Cytosolic molecules involved in sensing B. abortus DNA In addition to the endosomal TLR-dependent nucleic acid sensing pathway, cytosolic nucleic acid sensors are also
Please cite this article in press as: Campos PC, et al., Brucella abortus DNA is a major bacterial agonist to activate the host innate immune system, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.010
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involved in the recognition of pathogen-derived DNA. One of these pathways involves AIM2 that leads to inflammasome activation which via caspase-1 controls the catalytic cleavage of the pro-forms of the cytokines IL-1b and IL-18 [11,35]. A second pathway involves other sensors like MAVS and STING and activation of the TANK-binding kinase (TBK1)-Interferon Regulatory Factor (IRF)-3 signaling pathway to trigger transcriptional induction of type I IFN genes and NF-kB activation to produce proinflammatory cytokines (Fig. 1) [6,9,12,36]. AIM2 is a cytosolic dsDNA sensor which belongs to the hematopoietic interferon-inducible nuclear HIN200 protein family (also called PYHIN protein family) characterized by Nterminal pyrin (PYD) domain and a C-terminal hematopoietic interferon-inducible nuclear antigen with 200 amino acid repeats (HIN200) domain [36,37]. Although all PYHIN family members can bind to DNA, only AIM2 interacts with ASC through homotypic PYDePYD domains and is primarily localized in the cytoplasm [11]. In this context, AIM2 binds to dsDNA via its HIN200 domain and oligomerizes with ASC to initiate the formation of a caspase-1-activating inflammasome, leading to the secretion of proinflammatory cytokines, including IL-1b and IL-18 [9,27,36,38].
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Some studies have demonstrated that AIM2 is required for the host defense against DNA viruses such as vaccinia virus and murine cytomegalovirus (MCMV) [9,11,36,39] and against intracellular bacteria such as F. tularensis [11,40e42], L. monocytogenes [11,43,44], M. tuberculosis [45], Mycobacterium bovis [46] and B. abortus [16]. AIM2 is critical in stimulating proinflammatory responses during F. tularensis infection since AIM2/ mice presented rapid mortality, decreased production of IL-18 and increased microbial load [11,41,47]. L. monocytogenes infection is sensed by multiple inflammasomes that cooperatively coordinate a strong caspase-1 activation and proinflammatory response. Consequently, AIM2 acts in a redundant manner with other inflammasomes (NLRP3 and NLRC4) in sensing L. monocytogenes, with a prevalent involvement of the AIM2inflammasome [11,43e44]. Moreover, another study showed that AIM2/ mice were highly susceptible to intratracheal infection with M. tuberculosis and infected AIM2/ macrophages showed severely impaired secretion of IL-1b and IL-18 as well as activation of the inflammasome, determined by caspase-1 cleavage [45]. A more recent study that virulent M. bovis induces AIM2-inflammasome activation in macrophages
Fig. 1. Overview of B. abortus DNA recognition pathways. B. abortus DNA is sensed by TLR9 through a MyD88-dependent endosomal pathway leading to induction of proinflammatory cytokines. Additionally, bacterial DNA induces type I interferon through an unknown receptor in a MyD88-dependent but TLRindependent pathway. Two different DNA motifs, AT-rich dsDNA and dsDNA, can be recognized by cytosolic receptors. RNA polymerase III (RNA pol III) detects AT-rich dsDNA and converts it into triphosphorylated RNA (pppRNA). The pppRNA is recognized by RIG-I which in turn interacts with the mitochondrial protein MAVS. This route culminates in the production of proinflammatory cytokines and type I interferon. The AT-rich dsDNA can also be recognized by cGAS/ cGAMP, interacting with STING and inducing type I interferon. Recent reports suggest a direct interaction between MAVS and STING, indicating an alternative pathway of cytosolic DNA sensing. Alternatively, bacterial dsDNA is sensed by AIM2-inflammasome in the cytosol leading to IL-1b secretion. Solid arrows are related to pathways involved in B. abortus DNA-mediated response already described (Huang et al., 2005; Macedo et al., 2008; de Almeida et al., 2011; Gomes et al., 2013). Dashed arrows represent B. abortus DNA-related pathways currently under investigation. Colored molecules correspond to proteins cited in this review. Please cite this article in press as: Campos PC, et al., Brucella abortus DNA is a major bacterial agonist to activate the host innate immune system, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.010
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further emphasizes the potential for AIM2-inflammasome in host defense against mycobacteria [46]. Recently, our research group showed that ASC inflammasomes, mainly AIM2 sensing DNA and NLRP3, are essential for the secretion of caspase-1-dependent IL-1b and for the resistance of mice to infection with B. abortus [16]. In vitro data showed that bacterial DNA induced caspase-1 activation and IL-1b secretion in WT cells but not in ASC and caspase-1 KO macrophages. Moreover, it was observed a significant reduction in IL-1b secretion and caspase-1 activation in AIM2/ macrophages transfected with B. abortus DNA, indicating an important role of AIM2 in recognizing B. abortus genomic DNA and further caspase-1 activation and IL-1b secretion. Additionally, consistent with the in vitro findings, AIM2, ASC, and caspase-1 deficient mice showed a reduced resistance to B. abortus at four weeks postinfection, determined by bacterial burden in the spleens. Therefore, these studies establish AIM2 as an important antimicrobial sensor and a key determinant of protective immunity to pathogens. AIM2 definitely acts as a sensor of B. abortus DNA and plays a critical role in caspase1-mediated cytokine processing during infection [16]. Another DNA sensing pathway involves the Stimulator of Interferon Genes (STING, also referred to as TMEM173, MPYS and MITA) that is a transmembrane-spanning protein located in the endoplasmic reticulum (ER) and upon exposure to cytosolic DNA it translocates to certain perinuclear sites. It is expressed in many cell types such as macrophages, dendritic cells, T cells, endothelial, epithelial cells and select fibroblasts [48,49]. STING functions as a dimer and it contains 4 transmembrane helices and a globular carboxy-terminal domain (CTD) that protrudes into the cytosol. The very C-terminal tail (CTT) of this domain maintains STING in an autoinhibited state [50,51]. STING is essential for IFN-b induction by exogenous DNA ligands and also by various DNA pathogens after infection [48]. Burdette et al. first demonstrated that STING acts as a direct molecule sensor for cyclic diguanylate monophosphate (c-di-GMP) and other cyclic dinucleotides (which function as conserved signaling molecules in bacteria) [52]. Sun et al. demonstrated that cGAMP synthase (cGAS) is a cytosolic DNA sensor and also reveal that cGAS generates the second messenger cyclic-GMP-AMP (cGAMP), which binds to active STING [53]. It was unclear whether STING is directly involved in the sensing of DNA species, or whether upstream molecules facilitate this purpose, however, recent evidence indicates that STING also associates with dsDNA directly [54]. These findings suggest that STING act not only as an adapter, but can also function as a receptor that participates in DNA sensing directly. STING activates and recruits TANK-binding kinase 1 (TBK1) to phosphorylate interferon regulatory factor 3 (IRF3) and it enables IKK to phosphorylate IkBa, leading to IkBa degradation. Subsequently, IRF3 dimer and NF-kB translocate into the nucleus to induce the production of type I IFNs and other proinflammatory cytokines [55,56]. Some studies have demonstrated that STING is required for effective innate immune signaling processes. Macrophages from mutant mouse strain showed that STING is essential for
the type I IFN response to both c-di-GMP and c-di-AMP and for response to L. monocytogenes in vivo [57]. It has additionally been shown that M. tuberculosis triggers the STING/ IRF3 pathway, since macrophages derived from STING/ mice were unable to activates IRF3 translocation when infected with M. tuberculosis [10]. Barker et al. showed that primary mouse lung fibroblast (MLFs) from STING/ mice were unable to mount a type I IFN response to Chlamydia trachomatis and produced more Chlamydia infectious units than wild-type MLFs. In addition, they also showed that Chlamydia synthesizes c-di-AMP during infection [58]. Previous studies reported by our research group showed that B. abortus-induced IFN-b is dependent on STING. Realtime RT-PCR analysis demonstrates that RAW 264.7 macrophages transfected with siRNA directed against STING exhibited a significant decrease in IFN-b mRNA levels during B. abortus infection (55%) or upon stimulation with bacterial DNA (93%) compared to siRNA non-transfected cells. This result demonstrates that IFN-b expression induced by B. abortus-derived DNA requires STING as an adapter [20]. Moreover, additional results performed by our research team corroborates that STING plays an important role in the response to infection with B. abortus in vitro. Macrophages derived from STING/ mice showed reduction in IL-12 cytokine production, when infected with the bacteria or stimulated with bacterial DNA (unpublished data). The role of STING in innate signaling pathways leading to type I IFN and proinflammatory cytokines production, as well as its contribution to control B. abortus infection is under investigation in our laboratory. The molecule STING has also been reported to be a MAVS-interacting protein, in a mitochondrial complex [48]. Although targeted to the endoplasmic reticulum, STING interacts with the adapter MAVS which, in turn, it may activates TRAF3/TBK1/IKKi and IRF3/IRF7, resulting in expression of type I IFN and also leading to activation of NF-kB pathway [59,60]. MAVS (mitochondrial antiviral-signaling receptor, also known as IPS -1, VISA or Cardif) is involved in DNA sensing by the retinoic acid-inducible gene-I (RIG-I) pathway through the RNA polymerase III (RNA pol III) activity. This enzyme converts AT-rich dsDNA into uncapped 50 triphosphate-bearing RNA, which serves as an agonist for RIG-I. The protein MAVS functions downstream of RIG-I and upstream of IkB and IRF3 phosphorylation [61]. MAVS was initially described as an intracellular molecule involved in viral double-stranded RNA sensing. It contains an N-terminal CARD-like domain that interacts with the CARD of RIG-I and melanoma differentiation-associated gene-5 (MDA-5) [62e65]. Since that, several investigators had investigated the role of this molecule in different viral infections [66,67]. Nevertheless, only few studies have been reported about its role in the recognition of bacterial nucleic acid and the results are controversial. Previous reports suggested that IFN induction by L. monocytogenes occurs in a MAVS-independent, and thereby cytosolic RIG-I independent way [68]. However in 2010, Buss et al. described a role of MAVS in mediated IFN-b response and control of
Please cite this article in press as: Campos PC, et al., Brucella abortus DNA is a major bacterial agonist to activate the host innate immune system, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.010
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Chlamydophila pneumonia replication [69]. Additionally, Hagmann et al. have shown that siRNA-mediated knock-down of MAVS extensively inhibited type I IFN production in A549 cells (a lung epithelial cell line) infected with L. monocytogenes but did not in THP-1 cells [70], indicating that the role of MAVS in inducing type I IFN production could be cell type-dependent. Since MAVS is a common adapter molecule to different cytosolic receptors from RLR families (RIG-I and MDA-5), it is an important target molecule to be studied during B. abortus infection. We are now carrying on experiments to define the role of MAVS on B. abortus nucleic acid sensing. Preliminary results from our group implicated the adapter molecule MAVS in Brucella nucleic acid recognition. However, further studies are required to define MAVS function during B. abortus infection. 2. Concluding remarks
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There is a considerable amount of evidence that indicates the capacity of Brucella sp to avoid or interfere with components of the host innate immune responses that plays a critical role in their virulence. It has been suggested that Brucella has developed a stealth strategy through PAMPs reduction, modification and hiding, to ensure low stimulatory activity and toxicity for cells. This strategy allows Brucella to reach its replication niche before activating antimicrobial mechanisms by host immune responses. However, inside the host cells, Brucella releases vital molecules for the bacteria such as its DNA. Then, Brucella DNA is sensed by endosomal TLR9 and by cytosolic AIM2-inflammasome to induce proinflammatory cytokine production that contributes to host control of infection. Additionally, in this review we discuss the potential role of the adapter molecules STING and MAVS in cytosolic Brucella DNA sensing. However, further studies are required to elucidate this complex circuit by which the host innate immune system recognizes Brucella-derived nucleic acids. Acknowledgments
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This work was supported by grants from CNPq, CNPq/ CONICET, FAPEMIG, FAPEMIG/CNPq (PRONEX), CAPES/PNPD, CNPq/MAPA, CNPq/REPENSA and INCTVacinas. References [1] Vilaysane A, Muruve DA. The innate immune response to DNA. Semin Immunol 2009;21:208e14. [2] Medzhitov R, Janeway Jr CA. Innate immunity: the virtues of a nonclonal system of recognition. Cell 1997;91:295e8. [3] Ablasser A, Hertrich C, Wassermann R, Hornung V. Nucleic acid driven sterile inflammation. Clin Immunol 2013;147:207e15. [4] Wagner H. Toll meets bacterial CpG-DNA. Immunity 2001;14:499e502. [5] Chiu YH, Macmillan JB, Chen ZJ. RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway. Cell 2009;138:576e91. [6] Ishii KJ, Coban C, Kato H, Takahashi K, Torii Y, Takeshita F, et al. A toll-like receptor-independent antiviral response induced by doublestranded B-form DNA. Nat Immunol 2006;7:40e8.
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MODEL
Microbes and Infection xx (2014) 1e6 www.elsevier.com/locate/micinf
Brucella abortus DNA is a major bacterial agonist to activate the host innate immune system Priscila Carneiro Campos, Marco Tulio Ribeiro Gomes, Gabriela Guimar~aes, Miriam Maria Silva Costa Franco, Fernanda Martins Marim, Sergio Costa Oliveira*
Q4
Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Av. Antonio Carlos 6627, Pampulha, 31270-901 Belo Horizonte, MG, Brazil Received 30 June 2014; accepted 20 August 2014
Abstract Immunity against Brucella abortus depends on the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Signaling pathways triggered by Brucella DNA involves TLR9, AIM2 and possibly STING and MAVS. Herein, we review the advances in B. abortus DNA sensing by host innate immune receptors and the progress in this field. © 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur.
Keywords: Brucella abortus; Innate immunity; TLRs; Cytosolic sensors; Bacterial DNA
1. Introduction The innate immune system is the first line of defense against invading pathogens and other danger signals, acting through the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) [1]. These PRRs sense foreign molecules shared by a large group of microbes. For example, many Toll-like receptors (TLRs), NOD-like receptors (NLRs) or RIG-I receptors (RLRs) recognize lipopolysaccharide, peptidoglycan or RNA motifs that are expressed by different bacteria or viruses but not by the host [2]. Other receptors, however, are activated by common molecules expressed by microbes and/or the host if these molecules are located at unphysiological cellular compartments. The DNA, for instance, is a molecule present in the pathogen and it is highly abundant in the nucleus of host cells.
* Corresponding author. Tel./fax: þ55 31 34092666. E-mail addresses: [email protected], [email protected] (S.C. Oliveira).
Its presence in other cell compartments including endosomes and the cytosol activates DNA recognition systems to detect DNA derived from invading pathogens (DNA PAMPs) and disturbed self (DNA DAMPs) [3]. Unmethylated CpG DNA (abundant in many pathogen genomes) and classical B form double-stranded DNA (dsDNA) are examples of motifs which have the ability to stimulate immune responses through DNA sensors such as TLR9 on endosomal membranes [4]; and cGAS (Cyclic GMP-AMP synthase), AIM2 (absent in melanoma 2) and RNA polymerase III [5] in the cytosol, respectively. These sensors signal through specific adapter molecules including MyD88, STING (stimulator of interferon genes), MAVS (mitochondrial antiviral-signaling protein) and ASC (apoptosis-associated speck-like protein containing a CARD) to stimulate production of proinflammatory cytokines or type I IFN, or to activate inflammasomes [6e9]. Several groups have described DNA-sensing receptors involved in innate immune response to bacterial species, such as Mycobacterium tuberculosis [10], Listeria monocytogenes [11e13], Francisella tularensis [11,14] and Brucella abortus [15e16]. Our research group is currently identifying host
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receptors that recognize B. abortus-derived DNA in a murine model. B. abortus is a Gram-negative facultative intracellular bacterium, that causes brucellosis, with pathological manifestations of arthritis, endocarditis, and meningitis in humans, and in cattle it leads to abortion and infertility resulting in serious economic losses to the livestock industry [17]. This pathogen resist killing by neutrophils, it replicates inside macrophages and dendritic cells (DCs), maintaining a long lasting interaction with host cells. Immunity against Brucella infection involves mainly Th1 cells and CD8þ T lymphocytes, gamma-interferon (IFN-g) and tumor necrosis factor alpha (TNF-a) production and activated macrophages and DCs [18e19]. We have identified Brucella DNA as a major bacterial component to induce type I IFN and our study revealed that this molecule operates through a mechanism dependent on RNA polymerase III to be sensed probably by an unknown receptor via the adapter molecule STING [20]. It is also possible that STING acts as a DNA sensor itself and the mitochondrial protein MAVS could be involved in the RNA pol III and/or STING-mediated responses. Besides, we determined that AIM2, which senses Brucella DNA in the cytosol, is partially required for caspase-1 activation and IL-1b secretion [16]. This review summarizes the current knowledge about host cell response to microbial DNA, with emphasis on recent advances on B. abortus-derived DNA sensing pathways and we discuss the new findings in this field. 1.1. The role of the endosomal receptor TLR9 in sensing B. abortus DNA TLRs were the first PRRs to be identified and since then the most well characterized. TLRs are type I integral membrane proteins with ectodomains containing leucine-rich repeats for PAMP recognition, a transmembrane domain, and a cytosolic Toll/IL-1 receptor (TIR) domain responsible for transducing signals to downstream adapters including TRIF and MyD88 [21]. There are 5 identified TLRs involved in nucleic acid sensing: TLR3, TLR7, TLR8, TLR9, and TLR13, although TLR8 is not functional in mice and TLR13 has not been found in humans yet [22e24]. These receptors are responsible for sensing a diversity of pathogen-derived molecules. The sensing of nucleic acids performed by these TLRs which reside within the endoplasmic reticulum induces its traffic to endosomal compartment in the presence of stimuli [25]. TLR3 senses dsRNA, TLR7/TLR8 sense ssRNA and TLR9 senses unmethylated cytosine-guanosine (CpG) DNA motifs that are common for bacterial but rare in mammalian genomes [26,27]. The adaptor protein MyD88 plays a pivotal role in the cell signaling initiated upon TLRs (except for TLR3) or IL-1R family activation. PAMPs generally induce TLR dimerization leading the recruitment of MyD88 followed by the interleukin-1 receptorassociated kinase (IRAK) family activation [28]. This process generates multiple cellular responses including induction of inflammatory cytokines secretion through activation of nuclear factor (NF)-kB and AP-1 transcription factors [29]. However, activation of TLR7 and TLR9 alternatively might elicit the production of type-I IFNs by activating the IFN regulatory
factor-7 (IRF-7) [27]. Since the adaptor MyD88 and IRAK proteins show this central role in cellular response to PAMPs, it is conceived that these molecules participate in the innate immune response against pathogens. Regarding B. abortus infection, it was shown that MyD88/ and IRAK4/ mice are inefficient to control infection and macrophages or DC derived from these mice produce low levels of proinflammatory cytokines in response to this pathogen [30e32]. The essential role attributed to MyD88 and IRAK-4 proteins argues to the participation of TLRs during the control of B. abortus infection. Initially, it was shown that TLR2 plays no role controlling infection in mice [30]. Subsequently, it was shown by our group that TLR6 is important for host resistance since knockout mice for this receptor are more susceptible to B. abortus infection [33]. Regarding TLR9, it was demonstrated that this receptor is implicated, at least at early phases of infection, in host resistance to B. abortus in mice [31]. Furthermore, it was shown that bacterial DNA extracted from heat killed B. abortus induces IL-12 production by dendritic cells dependent on TLR9. Moreover, DNA methylation abrogates the production of inflammatory cytokines by splenocytes, suggesting that stimulatory property of DNA resides in CpG motifs that are not methylated [15]. As mentioned before, the activation of TLR9 signaling cascades also induces type I IFN secretion which might trigger expression of the interferon-inducible resistance proteins at late time points. For instance, murine macrophages infected with B. abortus produce interferon-inducible resistance proteins such as the members of the IRG (immunity-related GTPases) family. Remarkably, albeit this induction requires the adaptor molecule MyD88 it does not involve TLR2/TLR4/ TLR5/TLR9 [34]. In addition, our group demonstrated that type I IFN secretion induced by B. abortus or its DNA is independent of TLR2/TLR4/TLR9 recognition but requires the adaptor molecule MyD88. This pathway leads to type-I IFN secretion and regulation of IRG family proteins expression independent on TLR signaling but engaging MyD88, which suggests the participation of an unknown receptor sensing B. abortus and its DNA [20]. Although it seems that TLR9 is not required for type-I IFN induction, the role of TLR9 to control B. abortus infection and inflammatory cytokines production was well documented as described before [31]. In addition, we suggest that inflammatory cytokine signaling pathway, MAPKs (ERK1/2, p38 and JNK) and p65 NF-kB phosphorylation are impaired in TLR9/ macrophages activated by B. abortus or its DNA (unpublished results). Taken together, these results suggest that B. abortus DNA contains motifs that act as an immune cell activator, and the sensing of this molecule by TLR9 orchestrates a robust immune response that helps to control B. abortus infection. 1.2. Cytosolic molecules involved in sensing B. abortus DNA In addition to the endosomal TLR-dependent nucleic acid sensing pathway, cytosolic nucleic acid sensors are also
Please cite this article in press as: Campos PC, et al., Brucella abortus DNA is a major bacterial agonist to activate the host innate immune system, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.010
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involved in the recognition of pathogen-derived DNA. One of these pathways involves AIM2 that leads to inflammasome activation which via caspase-1 controls the catalytic cleavage of the pro-forms of the cytokines IL-1b and IL-18 [11,35]. A second pathway involves other sensors like MAVS and STING and activation of the TANK-binding kinase (TBK1)-Interferon Regulatory Factor (IRF)-3 signaling pathway to trigger transcriptional induction of type I IFN genes and NF-kB activation to produce proinflammatory cytokines (Fig. 1) [6,9,12,36]. AIM2 is a cytosolic dsDNA sensor which belongs to the hematopoietic interferon-inducible nuclear HIN200 protein family (also called PYHIN protein family) characterized by Nterminal pyrin (PYD) domain and a C-terminal hematopoietic interferon-inducible nuclear antigen with 200 amino acid repeats (HIN200) domain [36,37]. Although all PYHIN family members can bind to DNA, only AIM2 interacts with ASC through homotypic PYDePYD domains and is primarily localized in the cytoplasm [11]. In this context, AIM2 binds to dsDNA via its HIN200 domain and oligomerizes with ASC to initiate the formation of a caspase-1-activating inflammasome, leading to the secretion of proinflammatory cytokines, including IL-1b and IL-18 [9,27,36,38].
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Some studies have demonstrated that AIM2 is required for the host defense against DNA viruses such as vaccinia virus and murine cytomegalovirus (MCMV) [9,11,36,39] and against intracellular bacteria such as F. tularensis [11,40e42], L. monocytogenes [11,43,44], M. tuberculosis [45], Mycobacterium bovis [46] and B. abortus [16]. AIM2 is critical in stimulating proinflammatory responses during F. tularensis infection since AIM2/ mice presented rapid mortality, decreased production of IL-18 and increased microbial load [11,41,47]. L. monocytogenes infection is sensed by multiple inflammasomes that cooperatively coordinate a strong caspase-1 activation and proinflammatory response. Consequently, AIM2 acts in a redundant manner with other inflammasomes (NLRP3 and NLRC4) in sensing L. monocytogenes, with a prevalent involvement of the AIM2inflammasome [11,43e44]. Moreover, another study showed that AIM2/ mice were highly susceptible to intratracheal infection with M. tuberculosis and infected AIM2/ macrophages showed severely impaired secretion of IL-1b and IL-18 as well as activation of the inflammasome, determined by caspase-1 cleavage [45]. A more recent study that virulent M. bovis induces AIM2-inflammasome activation in macrophages
Fig. 1. Overview of B. abortus DNA recognition pathways. B. abortus DNA is sensed by TLR9 through a MyD88-dependent endosomal pathway leading to induction of proinflammatory cytokines. Additionally, bacterial DNA induces type I interferon through an unknown receptor in a MyD88-dependent but TLRindependent pathway. Two different DNA motifs, AT-rich dsDNA and dsDNA, can be recognized by cytosolic receptors. RNA polymerase III (RNA pol III) detects AT-rich dsDNA and converts it into triphosphorylated RNA (pppRNA). The pppRNA is recognized by RIG-I which in turn interacts with the mitochondrial protein MAVS. This route culminates in the production of proinflammatory cytokines and type I interferon. The AT-rich dsDNA can also be recognized by cGAS/ cGAMP, interacting with STING and inducing type I interferon. Recent reports suggest a direct interaction between MAVS and STING, indicating an alternative pathway of cytosolic DNA sensing. Alternatively, bacterial dsDNA is sensed by AIM2-inflammasome in the cytosol leading to IL-1b secretion. Solid arrows are related to pathways involved in B. abortus DNA-mediated response already described (Huang et al., 2005; Macedo et al., 2008; de Almeida et al., 2011; Gomes et al., 2013). Dashed arrows represent B. abortus DNA-related pathways currently under investigation. Colored molecules correspond to proteins cited in this review. Please cite this article in press as: Campos PC, et al., Brucella abortus DNA is a major bacterial agonist to activate the host innate immune system, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.010
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further emphasizes the potential for AIM2-inflammasome in host defense against mycobacteria [46]. Recently, our research group showed that ASC inflammasomes, mainly AIM2 sensing DNA and NLRP3, are essential for the secretion of caspase-1-dependent IL-1b and for the resistance of mice to infection with B. abortus [16]. In vitro data showed that bacterial DNA induced caspase-1 activation and IL-1b secretion in WT cells but not in ASC and caspase-1 KO macrophages. Moreover, it was observed a significant reduction in IL-1b secretion and caspase-1 activation in AIM2/ macrophages transfected with B. abortus DNA, indicating an important role of AIM2 in recognizing B. abortus genomic DNA and further caspase-1 activation and IL-1b secretion. Additionally, consistent with the in vitro findings, AIM2, ASC, and caspase-1 deficient mice showed a reduced resistance to B. abortus at four weeks postinfection, determined by bacterial burden in the spleens. Therefore, these studies establish AIM2 as an important antimicrobial sensor and a key determinant of protective immunity to pathogens. AIM2 definitely acts as a sensor of B. abortus DNA and plays a critical role in caspase1-mediated cytokine processing during infection [16]. Another DNA sensing pathway involves the Stimulator of Interferon Genes (STING, also referred to as TMEM173, MPYS and MITA) that is a transmembrane-spanning protein located in the endoplasmic reticulum (ER) and upon exposure to cytosolic DNA it translocates to certain perinuclear sites. It is expressed in many cell types such as macrophages, dendritic cells, T cells, endothelial, epithelial cells and select fibroblasts [48,49]. STING functions as a dimer and it contains 4 transmembrane helices and a globular carboxy-terminal domain (CTD) that protrudes into the cytosol. The very C-terminal tail (CTT) of this domain maintains STING in an autoinhibited state [50,51]. STING is essential for IFN-b induction by exogenous DNA ligands and also by various DNA pathogens after infection [48]. Burdette et al. first demonstrated that STING acts as a direct molecule sensor for cyclic diguanylate monophosphate (c-di-GMP) and other cyclic dinucleotides (which function as conserved signaling molecules in bacteria) [52]. Sun et al. demonstrated that cGAMP synthase (cGAS) is a cytosolic DNA sensor and also reveal that cGAS generates the second messenger cyclic-GMP-AMP (cGAMP), which binds to active STING [53]. It was unclear whether STING is directly involved in the sensing of DNA species, or whether upstream molecules facilitate this purpose, however, recent evidence indicates that STING also associates with dsDNA directly [54]. These findings suggest that STING act not only as an adapter, but can also function as a receptor that participates in DNA sensing directly. STING activates and recruits TANK-binding kinase 1 (TBK1) to phosphorylate interferon regulatory factor 3 (IRF3) and it enables IKK to phosphorylate IkBa, leading to IkBa degradation. Subsequently, IRF3 dimer and NF-kB translocate into the nucleus to induce the production of type I IFNs and other proinflammatory cytokines [55,56]. Some studies have demonstrated that STING is required for effective innate immune signaling processes. Macrophages from mutant mouse strain showed that STING is essential for
the type I IFN response to both c-di-GMP and c-di-AMP and for response to L. monocytogenes in vivo [57]. It has additionally been shown that M. tuberculosis triggers the STING/ IRF3 pathway, since macrophages derived from STING/ mice were unable to activates IRF3 translocation when infected with M. tuberculosis [10]. Barker et al. showed that primary mouse lung fibroblast (MLFs) from STING/ mice were unable to mount a type I IFN response to Chlamydia trachomatis and produced more Chlamydia infectious units than wild-type MLFs. In addition, they also showed that Chlamydia synthesizes c-di-AMP during infection [58]. Previous studies reported by our research group showed that B. abortus-induced IFN-b is dependent on STING. Realtime RT-PCR analysis demonstrates that RAW 264.7 macrophages transfected with siRNA directed against STING exhibited a significant decrease in IFN-b mRNA levels during B. abortus infection (55%) or upon stimulation with bacterial DNA (93%) compared to siRNA non-transfected cells. This result demonstrates that IFN-b expression induced by B. abortus-derived DNA requires STING as an adapter [20]. Moreover, additional results performed by our research team corroborates that STING plays an important role in the response to infection with B. abortus in vitro. Macrophages derived from STING/ mice showed reduction in IL-12 cytokine production, when infected with the bacteria or stimulated with bacterial DNA (unpublished data). The role of STING in innate signaling pathways leading to type I IFN and proinflammatory cytokines production, as well as its contribution to control B. abortus infection is under investigation in our laboratory. The molecule STING has also been reported to be a MAVS-interacting protein, in a mitochondrial complex [48]. Although targeted to the endoplasmic reticulum, STING interacts with the adapter MAVS which, in turn, it may activates TRAF3/TBK1/IKKi and IRF3/IRF7, resulting in expression of type I IFN and also leading to activation of NF-kB pathway [59,60]. MAVS (mitochondrial antiviral-signaling receptor, also known as IPS -1, VISA or Cardif) is involved in DNA sensing by the retinoic acid-inducible gene-I (RIG-I) pathway through the RNA polymerase III (RNA pol III) activity. This enzyme converts AT-rich dsDNA into uncapped 50 triphosphate-bearing RNA, which serves as an agonist for RIG-I. The protein MAVS functions downstream of RIG-I and upstream of IkB and IRF3 phosphorylation [61]. MAVS was initially described as an intracellular molecule involved in viral double-stranded RNA sensing. It contains an N-terminal CARD-like domain that interacts with the CARD of RIG-I and melanoma differentiation-associated gene-5 (MDA-5) [62e65]. Since that, several investigators had investigated the role of this molecule in different viral infections [66,67]. Nevertheless, only few studies have been reported about its role in the recognition of bacterial nucleic acid and the results are controversial. Previous reports suggested that IFN induction by L. monocytogenes occurs in a MAVS-independent, and thereby cytosolic RIG-I independent way [68]. However in 2010, Buss et al. described a role of MAVS in mediated IFN-b response and control of
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Chlamydophila pneumonia replication [69]. Additionally, Hagmann et al. have shown that siRNA-mediated knock-down of MAVS extensively inhibited type I IFN production in A549 cells (a lung epithelial cell line) infected with L. monocytogenes but did not in THP-1 cells [70], indicating that the role of MAVS in inducing type I IFN production could be cell type-dependent. Since MAVS is a common adapter molecule to different cytosolic receptors from RLR families (RIG-I and MDA-5), it is an important target molecule to be studied during B. abortus infection. We are now carrying on experiments to define the role of MAVS on B. abortus nucleic acid sensing. Preliminary results from our group implicated the adapter molecule MAVS in Brucella nucleic acid recognition. However, further studies are required to define MAVS function during B. abortus infection. 2. Concluding remarks
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There is a considerable amount of evidence that indicates the capacity of Brucella sp to avoid or interfere with components of the host innate immune responses that plays a critical role in their virulence. It has been suggested that Brucella has developed a stealth strategy through PAMPs reduction, modification and hiding, to ensure low stimulatory activity and toxicity for cells. This strategy allows Brucella to reach its replication niche before activating antimicrobial mechanisms by host immune responses. However, inside the host cells, Brucella releases vital molecules for the bacteria such as its DNA. Then, Brucella DNA is sensed by endosomal TLR9 and by cytosolic AIM2-inflammasome to induce proinflammatory cytokine production that contributes to host control of infection. Additionally, in this review we discuss the potential role of the adapter molecules STING and MAVS in cytosolic Brucella DNA sensing. However, further studies are required to elucidate this complex circuit by which the host innate immune system recognizes Brucella-derived nucleic acids. Acknowledgments
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This work was supported by grants from CNPq, CNPq/ CONICET, FAPEMIG, FAPEMIG/CNPq (PRONEX), CAPES/PNPD, CNPq/MAPA, CNPq/REPENSA and INCTVacinas. References [1] Vilaysane A, Muruve DA. The innate immune response to DNA. Semin Immunol 2009;21:208e14. [2] Medzhitov R, Janeway Jr CA. Innate immunity: the virtues of a nonclonal system of recognition. Cell 1997;91:295e8. [3] Ablasser A, Hertrich C, Wassermann R, Hornung V. Nucleic acid driven sterile inflammation. Clin Immunol 2013;147:207e15. [4] Wagner H. Toll meets bacterial CpG-DNA. Immunity 2001;14:499e502. [5] Chiu YH, Macmillan JB, Chen ZJ. RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway. Cell 2009;138:576e91. [6] Ishii KJ, Coban C, Kato H, Takahashi K, Torii Y, Takeshita F, et al. A toll-like receptor-independent antiviral response induced by doublestranded B-form DNA. Nat Immunol 2006;7:40e8.
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Please cite this article in press as: Campos PC, et al., Brucella abortus DNA is a major bacterial agonist to activate the host innate immune system, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.010
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Please cite this article in press as: Campos PC, et al., Brucella abortus DNA is a major bacterial agonist to activate the host innate immune system, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.08.010
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