Microbes and Infection 16 (2014) 991e997 www.elsevier.com/locate/micinf

Innate immune sensing of nucleic acids from mycobacteria Lívia Harumi Yamashiro a,b, S
ergio Costa Oliveira c, Andr
e B
afica a,b,* a

Laboratory of Immunobiology, Department of Microbiology, Immunology and Parasitology, Brazil b Pharmacology Graduate Program, Federal University of Santa Catarina, Brazil c Laboratory of Immunology and Infectious Diseases, Federal University of Minas Gerais, Brazil Received 9 July 2014; accepted 4 September 2014 Available online 2 October 2014

Abstract Endosomal and cytosolic receptors engage recognition of mycobacterial-derived nucleic acids (MyNAs). In contrast, virulent mycobacteria may utilize nucleic acid recognition pathways to escape the host immune system. This short review will summarize the mechanisms by which MyNAs are sensed and how they influence host protective responses. © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Acid nucleics; Sensors; Mycobacteria; Tuberculosis

1. Introduction The innate immune system has evolved effective strategies to control different classes of infectious agents through a limited and invariant repertoire of receptors. The pattern recognition receptors (PRRs) [33] expressed on cells such as macrophages and dendritic cells recognize molecular structures known as pathogen-associated molecular patterns (PAMPs). PRRs, whether expressed on the cell surface, in endosomal compartments or present in the cytoplasm, sense a wide array of microbial and self ligands such as lipopolysaccharide (LPS), mannose-rich oligosaccharides, flagellin, nucleic acids and heat shock proteins (HSPs) (reviewed in Ref. [27]; in Ref. [40]. Recognition of PAMPs by PRRs activates multiple signaling cascades, which leads to the induction of costimulatory molecules and secretion of interferons (IFNs), proinflammatory cytokines and chemokines. Early studies have led to the discovery and characterization of several PRR families present in distinct cellular compartments (reviewed in * Corresponding author. Department of Microbiology, Immunology and Parasitology, UFSC, PO Box 476, Florian
opolis 88040-900, Brazil. E-mail address: [email protected] (A. B
afica).

Ref. [27]; in Ref. [40]. However, sensing of pathogen-derived ligands in endosomal and cytosolic compartments is now being elucidated. Moreover, a number of reports have demonstrated how intracellular PRRs recognize pathogenderived nucleic acids [6,70]. While the field of cytosolic DNA sensing has seen a rapid development in the past few years, new insights into how established PRRs (e.g. Toll-like receptors-TLRs and RIG-like receptors-RLRs) engage with and discriminate microbial RNA have recently emerged (reviewed in Ref. [76]. The innate immune system's ability to detect RNA and DNA via endosomal and cytosolic PRRs is an essential mechanism to generate protective immune responses to pathogens. The immunostimulatory activity of foreign DNA or RNA has been known for ~50 years [28,42]. It was further established that unmethylated CpG-DNA motifs stimulate receptors present in endosomal compartments [20], whereas classical B form double-stranded (ds)DNA signals in the cytosol [70]. Receptors involved in recognition of foreign RNA have also been identified [35,51,86]. However, most studies have focused on how nucleic acid receptors induce immune responses against virus. Novel data has emerged from the literature suggesting that these receptors may be involved in host responses to a variety of infectious agents.

http://dx.doi.org/10.1016/j.micinf.2014.09.006 1286-4579/© 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

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Mycobacterium tuberculosis (Mtb), the causative agent of deadly tuberculosis (TB) [83], is sensed by several PRRs, initiating signaling events that ultimately results in activation of innate and adaptive immune responses. Receptors from the TLR [4,10,47], Nod-like receptor (NLR) [17,44] and C-type lectin-like receptor (CLR) [49,57] families are known to be involved in recognition of Mtb, throughout the interaction with mycobacterial cell wall components as well as secreted molecules. It has also been described several families of innate receptors that engage mycobacterial-derived nucleic acids (MyNAs) such as TLR and inflammasome platforms. Such receptors are expressed in many cells. However, most studies have utilized cell populations such as antigen presenting cells (APCs) to define possible interactions of mycobacteria and PRRs. Macrophages and dendritic cells, two different types of APCs, are targets of mycobacteria [63,82] and play a role in sensing MyNAs. It has been shown that these cell populations present differential expression of PRRs [26,75] which could influence their responses when exposed to MyNAs upon pathogen infection. Of interest, following mycobacteria uptake by APCs, MyNAs could be released due to pathogen degradation or during mycobacteria replication, which is associated with virulence factors present in the studied mycobacteria strains. In virulent strains such as Mtb, region of difference 1 (RD1), has been described as a key genomic sequence involved in preventing phagosomal maturation [25]. In contrast, attenuated mycobacterial strains such as BCG lack RD1 and, therefore, cannot avoid the phagolysosomal compartment. A detailed description of the differences between virulent and avirulent mycobacterial strains can be found elsewhere [54,58,69]. This short review will summarize the mechanisms by which MyNAs are sensed and how they influence host protective responses. 2. Mycobacterial DNA sensing Although DNA isolated from mycobacteria was demonstrated to contain immunostimulatory effects in mammalian cells 30 years ago [65,72], the role of TLR activation by purified mycobacterial DNA was only demonstrated 20 years later [4,45,79]. Toll-like receptors are the best characterized membranebound PRR family which have their recognition domain facing towards the extracellular space or the endosomal lumen. These receptors can detect microbial surface molecules or pathogen-derived nucleic acids in the endocytic compartment. The endosomal TLRs, namely TLR3, TLR7, TLR8 and TLR9, recognize nucleic acids such as those derived from phagocytosed microbes or infected apoptotic cells [2,19]. Synthetic CpG-DNA similar from those sequences found in bacterial DNA activates macrophages and DCs [32,68] via a specific receptor, TLR9 [20]. Following ligand binding, a signaling platform involving TLR dimerization is initiated, which is succeeded by a downstream signaling cascade that in turn upregulates the expression of cytokines and chemokines [1,34].

Earlier experiments indicated that human monocytic cells, known to play a major role in the response to mycobacteria, present functional TLR9 receptors [43]. have demonstrated that human monocytes display increased responsiveness to CpG stimulation. Similar observations were reported by two other groups which shown that a monocytic cell line, THP-1, can be stimulated by CpG immunostimulatory sequences [41,61]. Interestingly, TLR9 has been detected in lung granulomas from TB patients [14] and it was demonstrated that human monocyte-derived macrophages [37] as well as pulmonary macrophages [23,37] express TLR9. The direct involvement of TLR9 in mycobacteria infections has been investigated. Several studies [4,7,29,37] have shown that TLR9 senses purified mycobacterial DNA and may play a role in the immune responses to mycobacteria in vivo. Bonemarrow-derived macrophages or dendritic cells exposed to Mtb DNA displayed increased production of pro-inflammatory cytokines such as IL-12 and TNF [4]. Similar findings were observed in human alveolar macrophages exposed to BCG DNA [37] or bone-marrow-derived macrophages treated with Mycobacterium avium DNA [7]. Furthermore [29], have demonstrated that TLR9 is involved in granuloma formation, a key immune-mediated event to control Mtb proliferation [62]. Taken together, these observations indicate that TLR9 is a sensor of mycobacterial DNA and participates in antimycobacteria immune responses in vivo. However, the role for this receptor in murine models of mycobacterial infection has been evidenced by some [4,7] but not all studies [22]. In the case of Mtb infection [4], have demonstrated that TLR9deficient mice are susceptible to a high dose inoculum of this bacterial pathogen. Moreover, a second PRR previously shown to be involved in the control of chronic Mtb infection, TLR2 [12,46], was found to cooperate with TLR9 in host resistance to Mtb infection using a murine model [4]. These studies suggest the importance of in vivo recognition of pathogen-derived DNA and lipids in the generation of optimal immune responses against mycobacteria. In contrast, H€ olscher and colleagues reported that TLR2/4 double- and TLR2/4/9 triple-deficient mice are able to efficiently control low dose aerosol infection with Mtb [22]. Causes for such variable outcomes to Mtb infection are unclear, but they could be related to different experimental conditions, Mtb strains, dose of infection, or perhaps to differences in commensal microbiota. The importance of TLR9 regarding mycobacteria infection extends to pulmonary granulomatous response [29]. In that study, TLR9-deficient mice displayed altered granuloma formation and impaired migration of CD4þ T cells into PPDinduced granulomas [29]. Follow up studies have determined that lack of TLR9 results in altered Th17 cytokine profile, reduction of granuloma-associated myeloid DCs, and a profound impairment of delta-like 4 (dll4) Notch ligand expression [30]. Altogether, these results implicate TLR9 as a M. tuberculosis DNA sensor, which is involved in optimal immune responses to mycobacteria in mice. The recognition of bacterial DNA has also been documented in experiments using a number of mycobacteria species. For instance, DNA from M.

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avium [7], M. avium subsp. paratuberculosis [3], Mycobacterium marinum [48] or Mycobacterium bovis attenuated BCG [23,37,79] have been found to trigger TLR9-dependent responses. As a result of the involvement of TLR9 in the recognition of MyNAs in humans, one could speculate that genetic variations present in its encoding genes correlate with susceptibility to TB. Indeed, TLR9 polymorphisms have been found to be associated with predisposition to develop TB in Mexican [73], Indonesian and Vietnamese [38] and African-American [78] patient cohorts, suggesting this receptor influences TB pathogenesis in human populations. However, it remains unknown whether this effect is due to failure of direct recognition of mycobacterial DNA. Although all the aforementioned studies report recognition of MyNAs by TLR9, it may be possible that Mycobacterium spp. inhibits TLR9 signaling as a survival strategy. In support of this idea, a recent study by Ref. [3] has demonstrated that infection with M. avium subsp. paratuberculosis blocks TLR9 responsiveness elicited by CpG oligodeoxynucleotides (ODNs), highlighting the complex pathogen-host interaction as well as the plasticity of TLR9 signaling [3]. Interestingly, several studies have suggested that TLR9 is not the exclusive sensor for mycobacterial DNA [59,64,84], adding a piece of complexity to this recognition system. Recently, a new type of inflammasome, known as absent in melanoma (AIM)2 inflammasome, has been identified. Inflammasomes are multiprotein oligomer platforms, responsible for activating inflammatory processes through caspase-1 processing and secretion of IL-1b and IL-18. AIM-2 inflammasome is composed of caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and AIM2, a cytosolic receptor for double-strand (ds) DNA [15,24]. Recognition of dsDNA by AIM2 activates the AIM2 inflammasome and induces the maturation of pro-IL-1b to IL-1b. AIM2 inflammasome has been reported to be involved in the secretion of mature IL-1b during infection with several cytosolic bacterial pathogens including Francisella tularensis [15] and Listeria monocytogenes [74]. Recent results have demonstrated that AIM2 activation is also involved in the recognition of several mycobacteria strains. Infection of dendritic cells and macrophages by Mycobacterium smegmatis, Mycobacterium fortuitum, Mycobacterium kansasii [64] or M. bovis [84] leads to activation of AIM2 inflammasome which increases IL-1b and IFN-b expression as well as caspase-1 activation. However, conflicting outcomes regarding the role of AIM2 in Mtb infection have been observed. Although a recent report suggests that Mtb limits IFN-b production in infected host cells, leading to inhibition of the AIM2 inflammasome [64], a different study points to the involvement of this sensor on the activation of inflammasome and host immune responses [59]. [59] have worked with AIM2 deficient mice and found an increased mortality as well as impairment of IL-1b and IL-18 production upon Mtb infection. These authors also demonstrate colocalization of Mtb DNA and AIM2, suggesting Mtb DNA indeed escapes the phagosome. Thus, the role of AIM2

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inflammasome in mycobacterial infection may be important following translocation of mycobacteria to the cytosol of host cells [84]. Although a number of studies support the notion that mycobacterial DNA is recognized by different nucleic acids sensors and, therefore, play important roles in bacterial infectivity, how DNA is released and exposed during Mtb infection remains unknown. The cytosolic DNA, which activates nucleic acid sensors, may have at least 3 sources: (1) DNA produced by the breakdown and digestion of killed mycobacteria by lysosomal enzymes shortly after entering into the macrophages; (2) DNA from live mycobacteria escaping from the phagosome into the cytosol and (3) extracellular DNA (eDNA) naturally occurring on the surface of the bacterium during growth, and thus preexisting prior to infection. Indeed, eDNA can play important roles in bacterial physiology, most notably biofilm formation [81]. Furthermore, eDNA has been identified within outer membrane vesicles secreted by various gram-negative and gram-positive bacterial species [13] and has recently been found to be encapsulated within mycobacterial membrane vesicles [53]. Studies have also shown that M. tuberculosis, Mycobacterium leprae [77], M. marinum [67] and M. bovis [84] may translocate from the phagosome into the cytosol. Nonetheless, the exact mechanism by which mycobacteria deliver their DNA into the cytosol is unclear. A recent study, however, showed that Mtb eDNA is exposed to the host cytosol during macrophage infection, activating TBK1 and eliciting type I IFN production [80]. The authors conclude that Mtb triggers a cytosolic surveillance pathway via exposure of eDNA to the cytosol, by showing that host molecules required for cytoplasmic sensing of DNA (IFI204, STING, and TREX1) are also involved in the response to Mtb. This response was shown to be independent of TLR/NOD signaling and distinct from cytoplasmic RNA sensing molecules. 3. Mycobacterial RNA sensing Recognition of RNA by the innate immune system needs to specifically differentiate non-self-RNA from self-RNA. This necessity is due to the fact that during infection, several host RNA species (e.g. messenger RNA, transfer RNA, ribosomal RNA, microRNA, and other small regulatory RNAs) are localized with microbial RNAs on the cytosolic cellular compartment. Accordingly, cytosolic sensors must distinguish specific microbial features to avoid activation by host molecules that would otherwise elicit autoimmune responses. Despite this outward challenge, efficient detection of foreign RNA in the cytosol is essential for innate immunity (reviewed in Ref. [76]. Although the role for RNA sensing during viral infection has been extensively studied, the importance of this pathway upon bacterial exposure has recently been demonstrated [56,60]. A possible role for the existence of sensing of mycobacterial RNA during infection has been suggested by earlier studies [8]. The authors have shown that BCG activates double-stranded (ds) RNA-activated protein kinase (PKR),

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which induces cytokine expression following infection. In addition, BCG stimulates phosphorylation and activation of PKR, suggesting that mycobacterial RNA is recognized by a PKR-dependent manner. PKR is mostly known as an intracellular virus detector due to its involvement in the recognition of dsRNA [9]. Upon binding of dsRNA, PKR dimerizes and autophosphorylates to become an active protein kinase [52], that would downstream lead to inhibition of translation [11], apoptosis and antimicrobial responses [85]. However, the exact mechanisms by which PKR elicits antimycobacterial responses remain to be elucidated. Among TLRs, TLR3, TLR7 and TLR8 are the ones shown to be involved in sensing of RNA and are located in intracellular endosomal/lysosomal compartments [36]. This localization is critical to limit their function in detecting phagocytosed ribonucleic acids, thereby preventing detection of endogenous cellular RNAs. TLR3 is an intracellular sensor [2] that recognizes viral and host RNA. Anti-viral innate immune responses mainly associated with the production of type I IFN and inflammatory cytokines are elicited upon TLR3 triggering. Interestingly, it has been recently demonstrated an essential role for TLR3-PI3K/AKT axis during BCG infection [5]. The authors shown that TLR3 senses mycobacterial RNA and triggers IL-10 production by macrophages, which in turn inhibits IL-12p40 synthesis, leading to disease exacerbation [5]. Other receptors expressed on endosomal compartments such as TLR7 and TLR8 can be activated by bacterial RNA [19,39]. Because TLR8 is highly expressed on monocytes/

macrophages [18], this receptor would also be a candidate utilized by the human innate system to recognize bacteria. Indeed, a genetic analysis of 149 polymorphisms across all known TLRs and associated adaptor proteins in active TB patients showed that TLR8 polymorphisms were associated with disease [10]. In addition, TLR8 mRNA was upregulated in patients with active TB [10], suggesting that this receptor may play a role in immunity to TB. Taken together, these observations suggest that RNA sensors may play a role in the regulation of immune responses to mycobacterial infection and could serve as important molecules in the innate antimycobacterial defense. 4. Sensing of nucleic acid-associated pathways as a mycobacteria evasion strategy Once inside macrophages, intracellular pathogens such as Mtb use a large array of strategies to evade or counteract host immune responses. Central to Mtb effective immune avoidance is its ability to modulate the early innate inflammatory response and prevent the establishment of adaptive T cellmediated immunity [16]. For example, pathogens can abrogate antigen presentation capacity, thus reducing T-cellmediated immune responses such as priming of T cells [71]. Blockade of innate inflammatory cytokines is a different example used by mycobacteria to control antigen presentation [55], suggesting that evasion of the innate immune system is an important survival strategy found in pathogens such as Mtb.

Fig. 1. Pathways for recognition of Mycobacterium spp. derived nucleic acids. Mycobacteria-derived nucleic acids can be found either extracellularly or in the cytosol, being actively secreted or as a result of impaired ability to clear exogenous DNA. After recognition, intracellular signaling cascades are activated, which eventually will lead to the activation of transcription factors. After transcription, several cytokines as well as interferons are synthesized (see text for details). Mtb: M. tuberculosis; Msg: M. smegmatis; Mft: M. fortuitum; Mka: M. kansasii; Mbv: M. bovis; Mav: M. avium; Mav subsp.: M. avium subsp. paratuberculosis; Mmn: M. marinum; dsDNA: double-strand DNA; dsRNA: double-strand RNA; eDNA: extracellular DNA.

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Molecular sensing of MyNAs is part of the recognition mechanisms used by the host to contain infection and to trigger the release of important mediators. Nevertheless, throughout pathogen-host co-evolution, it is possible that Mtb may utilize these pathways to undermine host defenses. Experiments showed that Mtb inhibits induction of IFN-a/b and antigen cross processing by murine DCs in response to TLR2 and 9 agonists expressed by Mtb, affecting the response to pathogen [66]. Furthermore, inhibition of IFN-b production and signaling through Mtb AIM2 activation was also demonstrated to be another immune evasion mechanism [64]. An alternative evasion mechanism by mycobacteria could be related with a possible differential capacity of DNA from a variety of strains to trigger TLR9 signals. Using human macrophages derived from monocytes or alveolar samples [37], have demonstrated that DNA from virulent mycobacteria strains induces lower macrophage activation when compared to DNA from avirulent bacteria strains. They have also suggested that attenuated TLR9 activation contributes to the insufficient host response against this pathogen [37]. This evasion mechanism may be utilized by another mycobacteria from the same genus, M. avium subsp. paratuberculosis, which can cause tuberculosis in cattle. M. avium subsp. paratuberculosis inhibits classical TLR9-mediated responses despite increased TLR9 expression and maintenance uptake of CpG [3], suggesting that the infection changes the responsiveness of foreign DNA. It remains to be elucidated whether pathogenic mycobacteria specifically modulates sensing of nucleic acids associated pathways as an immune evasion mechanism. 5. Concluding remarks Following infection, it is likely that both mycobacterial DNA and RNA gain intracellular access, influencing important molecular hubs involved in immune responses to this pathogen. The described pathways associated with recognition of MyNAs are summarized in Fig. 1. These pathways may take place in bacteria-containing phagosomes, in the cytosol and the host cell membrane. Several questions regarding the biology of recognition of MyNAs merit further investigation. 1. How and where MyNAs become available and are sensed by PRRs? 2. Do pathogenic mycobacteria produce inhibitory nucleic acids which decrease macrophage activation? In addition, since Mtb is also found in the extracellular milieu in the granuloma [21], it would be interesting to investigate the interplay of eDNA and the complex cellular network involved in the control of this important human pathogen. Mycobacterial-derived nucleic acids may also be utilized in clinical settings. For that matter, it has long being known the proinflammatory properties of BCG in therapy against bladder cancer [31] and it might be possible that sequences of mycobacterial DNA could induce such effect in vivo, as seen for CpG therapy [50].

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Acknowledgments The authors wish to thank Prof. Ant^onio Rothfuchs/MTC/ KI for his critical comments and valuable contributions to this brief review. AB received financial support from NIH-GRIP (TW008276), HHMI-ECS (55007412), CNPq (478668) and CAPES (23038.021356). LHY received scholarship from CNPq and AB and SCO are CNPq-PQ fellows. References [1] Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell 2006;124:783e801. [2] Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. Recognition of double-stranded RNA and activation of NF-kappaB by toll-like receptor 3. Nature 2001;413:732e8. [3] Arsenault RJ, Li Y, Maattanen P, Scruten E, Doig K, Potter A, et al. Altered toll-like receptor 9 signaling in Mycobacterium avium subsp. paratuberculosis-infected bovine monocytes reveals potential therapeutic targets. Infect Immun 2013;81(1):226e37. [4] Bafica A, Scanga CA, Feng CG, Leifer C, Cheever A, Sher A. TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis. J Exp Med 2005;202(12):1715e24. [5] Bai W, Liu H, Ji Q, Zhou Y, Liang L, Zheng R, et al. TLR3 regulates mycobacterial RNA-induced IL-10 production through the PI3K/AKT signaling pathway. Cell Signal 2014;26(5):942e50. [6] Bhat N, Fitzgerald KA. Recognition of cytosolic DNA by cGAS and other STING-dependent sensors. Eur J Immunol 2014;44(3):634e40. [7] Carvalho NB, Oliveira FS, Dur~aes FV, de Almeida L a, Fl
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