Cellular recognition of mycobacteria Accepted Article Receptor-mediated recognition of mycobacterial pathogens 1 Kate E. Killick1*, Clíona Ní Cheallaigh2, Cliona O’Farrelly2, Karsten Hokamp3, David E. MacHugh1,4 and James Harris5 1 Animal Genomics Laboratory, UCD School of Agriculture and Food Science, University College Dublin, Belfield, Dublin 4, Ireland. 2 School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland. 3 4 Smurfit Institute of Genetics, Trinity College, Dublin 2, Ireland UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland 5 Faculty of Medicine, Nursing and Health Sciences, Southern Clinical School, Monash Medical Centre, Monash University, Clayton, Victoria, Australia. * Corresponding author: killickk@tcd.ie Phone number: +353-1-7166256 Fax: +353-1-7161103 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/cmi.12161 1 This article is protected by copyright. All rights reserved. Accepted Article Cellular recognition of mycobacteria Summary Mycobacteria are a genus of bacteria that range from the non-pathogenic Mycobacterium smegmatis to Mycobacterium tuberculosis, the causative agent of tuberculosis in humans. Mycobacteria primarily infect host tissues through inhalation or ingestion. They are phagocytosed by host macrophages and dendritic cells. Here, conserved pathogen-associated molecular patterns (PAMPs) on the surface of mycobacteria are recognised by phagocytic pattern recognition receptors (PRRs). Several families of PRRs have been shown to non-opsonically recognise mycobacterial PAMPs, including membranebound C-type lectin receptors, membrane-bound and cytosolic Toll-like receptors and cytosolic NOD-like receptors. Recently, a possible role for intracellular cytosolic PRRs in the recognition of mycobacterial pathogens has been proposed. Here, we discuss current ideas on receptor-mediated recognition of mycobacterial pathogens by macrophages and dendritic cells. Introduction Mycobacterium is a genus of Actinobacteria that includes more than 50 different species, ranging in virulence from the non-pathogenic M. smegmatis to the causative agent of tuberculosis (TB) in humans, M. tuberculosis, (Table 1). Infection with mycobacteria most commonly occurs through inhalation or ingestion of bacilli. The bacilli are phagocytosed by host macrophages and dendritic cells (DCs) at the site of infection, for example by alveolar macrophages in M. tuberculosis-infected lungs or by intestinal macrophages in animals infected with M. avium subsp. paratuberculosis (MAP). Mycobacteria have evolved a range of mechanisms to circumvent phagosome maturation, preventing lysosomal degradation, and 2 This article is protected by copyright. All rights reserved. Cellular recognition of mycobacteria Accepted Article are therefore able to both survive and replicate inside the host phagosome (Ni Cheallaigh et al., 2012, Welin et al., 2012). In order for mycobacteria to be phagocytosed by macrophages and DCs, the pathogen is first recognised by PRRs on the host cell (Gordon, 2002, Medzhitov et al., 2000). This is achieved through the recognition of highly conserved molecular structures found on the surface of pathogens, often critical for microbial survival, termed pathogen-associated molecular patterns [PAMPs] (Akira et al., 2006). Several families of PRRs exist, all of which are capable of recognising a different repertoire of PAMPs; including membrane-bound and cytosolic Toll-like receptors (TLRs) and cytosolic NOD-like receptors (NLRs) and RIG-I like receptors [RLRs] (Jo, 2008). This review examines some of the receptors that are involved in the non-opsonic recognition of mycobacteria by macrophages and DCs. C-type lectin receptors C-type lectin receptors are a family of membrane-bound calcium-dependent receptors that recognise carbohydrate-rich molecules. Four C-type lectin receptors have been closely associated with responses to mycobacteria: the mannose receptor, DC-SIGN, Mincle and Dectin-1 (Figure 1). Mannose receptors Mannose receptors (MRs), encoded by the mannose receptor C type 1 and 2 (MRC1 and MRC2) genes, recognise lipoarabinomannan (LAM) and mannosylated LAM (ManLAM) glycolipids on the cell wall of mycobacteria and other microbes (Martinez-Pomares, 2012). Early work demonstrated a role for MRs in the recognition of virulent strains of M. tuberculosis (Schlesinger et al., 1996). More recent research has found that stimulation of human monocyte-derived macrophages (MDM) with either virulent M. tuberculosis or 3 This article is protected by copyright. All rights reserved. Cellular recognition of mycobacteria Accepted Article ManLAM resulted in upregulation of peroxisome proliferator-activated receptor γ (PPARγ) with a simultaneous increase in production of IL-8 and prostoglandin E2, as well as an increase in COX-2 expression. This was independent of NF-κB activation and TLR2 expression, which indicates the employment of an MR-specific signalling pathway. Interestingly, infection with BCG induced less PPARγ and was mediated via NF-κB activation. This indicates that MR-mediated PPARγ expression and the downstream signalling it initiates are specific to virulent M. tuberculosis (Rajaram et al., 2010). Macrophage MRs may be targeted by mycobacteria to enhance survival within host macrophages. Binding of ManLAM by MRs has been shown to limit phagosome-lysosome fusion, favouring mycobacterial persistence within macrophages while blocking MRs during infection with virulent M. tuberculosis increased phagosome-lysosome fusion (Kang et al., 2005). While recognition of ManLAM by MRs may aid mycobacterial survival within the host macrophage, it is not clear whether differential binding via MRs is responsible for differences in virulence between strains. It has been shown that mannose capping of LAM was not crucial for mycobacterial survival in macrophages (Afonso-Barroso et al., 2012); murine bone marrow-derived macrophages (BMDM) were infected with mutant forms of M. bovis BCG and H37Rv M. tuberculosis lacking the mannose cap of LAM. No difference in the ability of these mutant strains to survive and proliferate in macrophages compared to the parental strains of M. bovis BCG and M. tuberculosis was observed (Afonso-Barroso et al., 2012). It also remains to be determined whether the differential signalling induced by virulent and non-virulent strains of mycobacteria (Rajaram et al., 2010) are responsible for differences in phagosome maturation. MRs have also been shown to be involved in the uptake of MAP (Souza et al., 2007). However, polymorphisms in MRC1 have been associated with susceptibility to TB and leprosy, suggesting that MR may have an important 4 This article is protected by copyright. All rights reserved. Cellular recognition of mycobacteria Accepted Article functional role in the immune response to mycobacteria (Azad et al., 2012, Wang et al., 2012, Zhang et al., 2013). DC-SIGN Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC- SIGN), encoded by the CD209 gene, is a type II transmembrane C-type lectin receptor found on the surface of immature DCs (Tailleux et al., 2003). DC-SIGN-specific antibodies have been shown to block DC interactions with M. bovis BCG. The receptor also interacts with ManLAM, found on the surface of M. tuberculosis, but not with non-mannosylated arabinofuranosyl-terminated LAM (AraLAM), found on the surface of M. smegmatis (Geijtenbeek et al., 2003). It has yet to be determined whether mannose capping of LAM affects the interaction between mycobacteria and DCs. DC-SIGN appears to play a role in mycobacterial-induced immune suppression, as it mediates ManLAM-induced production of the anti-inflammatory cytokine IL-10 in DCs and inhibits LPS-induced DC maturation/activation (Wu et al., 2011). Interestingly, cyanovirin-N (CV-N), a mannose binding lectin, has been found to compete with both MRs and DC-SIGN to bind LAM and inhibits the recognition of M. tuberculosis by DCs but not macrophages, indicating that DCs are of lesser importance in the recognition of M. tuberculosis than macrophages. Despite this, CV-N did not inhibit M. tuberculosis infection in a mouse model, suggesting that mannose-dependent C-type lectins, such as DC-SIGN and MRs, may not be essential during the early stages of murine M. tuberculosis infection. These findings demonstrate the functional redundancy of C-type lectin PRRs in the recognition of mycobacterial pathogens (Driessen et al., 2012). Interestingly, despite these findings, it has been hypothesised that a variant of the CD209 gene contributes to susceptibility to TB infection (Vannberg et al., 2008, Zheng et al., 5 This article is protected by copyright. All rights reserved. Cellular recognition of mycobacteria Accepted Article 2011). However, a meta-analysis of the CD209 336A/G polymorphism and TB susceptibility that integrated ten independent association studies found no evidence for a genetic association at this locus (Miao et al., 2012). The apparent discrepancies between these studies could be due to differences in genotype distributions of CD209 variants in Asian or African populations compared to other human populations. Consequently, population-specific associations of susceptibility variants with TB infection could exist. It may be possible to clarify this by studying larger samples of geographically distinct human populations. Mincle The C-type lectin receptor Mincle, encoded by the C-type lectin domain family 4, member E gene (CLEC4E), is expressed on the surface of macrophages (Matsumoto et al., 1999). In 2009, Ishikawa and colleagues demonstrated Mincle to be an essential receptor for trehalose-6, 6-dimycolate (TDM), the mycobacterial cord factor (Ishikawa et al., 2009). Recently, Mincle has been shown to play a role in CARD9-dependent signalling and inflammasome activation of IL-1β and subsequent induction of a TH17 response (Shenderov et al., 2013). Upon exposure to M. bovis BCG, alveolar macrophages from Mincle-deficient mice demonstrated significantly reduced proinflammatory cytokine profile, decreased leukocyte infiltration and increased pulmonary and extrapulmonary bacterial load compared to wild type mice (Behler et al., 2012). However, in another study, both low and high dose infection with H37Rv a similar granulomatous, TH1 and TH17 response in Mincle deficient mice to that in wild-type controls. As well as suggesting that Mincle may play a different role in the response to different strains of mycobacteria, these results also indicate that the receptor is dispensible in an in vivo model of M. tuberculosis infection. It is probable that other receptors can compensate for Mincle deficiency and that other PRRs recognise TDM (Heitmann et al., 2013). Indeed, macrophage C-type lectin (MCL), thought to have arisen 6 This article is protected by copyright. All rights reserved. Cellular recognition of mycobacteria Accepted Article from a gene duplication of Mincle, has been reported to recognise TDM (Miyake et al., 2013), as has the scavenger receptor macrophage receptor with a collagenous structure (MARCO) (Bowdish et al., 2009), discussed below. Dectin-1 Dectin-1, encoded by the C-type lectin domain family 7, member A gene (CLEC7A), has been shown to play a role in the recognition of the non-tuberculous M. abscessus (Shin et al., 2008). A more recent study using human MDM has demonstrated that Dectin-1 is required for activation of the NLRP3 inflammasome in response to infection with M. abscessus (Lee et al., 2012). Dectin-1 has also been shown to play a role in infection with M. tuberculosis. A study by Lee and colleagues demonstrated that M. tuberculosis H37Rv induced the expression of Dectin-1 in A549 alveolar epithelial cells in a TLR2-dependent manner (Lee et al., 2009). Moreover, Dectin-1 is required for TLR2-mediated production of TNF-α by murine BMDM in response to infection with the attenuated mycobacterial strains M. bovis BCG and M. tuberculosis H37Ra, as well as the avirulent M. smegmatis and M. phlei, although this was not the case for the virulent M. avium and M. tuberculosis H37Rv strains (Yadav et al., 2006). However, a role for Dectin-1 in M. tuberculosis-induced production of IL-12p40 in splenic DCs, a cytokine subunit previously shown to be important in granuloma formation , has also been proposed (Rothfuchs et al., 2007). Interestingly, Dectin-1 activation by M. tuberculosis has been shown to promote a mixed TH1/TH17 response; driving the secretion of IL-1, IL-23, TNF-α and IL-6 by monocyte-derived DC, which in turn drives IFN-γ and IL-17 secretion by CD+ T cells in vitro(Zenaro et al., 2009). Co-stimulation of MR or DC-SIGN, on the other hand, inhibits the secretion of these cytokines and drives a predominantly TH1 phenotype in CD4+ Cells (Zenaro et al., 2009). TH1-type responses, typified by the secretion of IFN-γ, TNF-α and IL7 This article is protected by copyright. All rights reserved. Cellular recognition of mycobacteria Accepted Article 12 are essential for protective immunity to M. tuberculosis (Harris et al., 2010). The role of IL-17 in M. tuberculosis infection is not clear, although studies suggest that high numbers of IFN-γ/IL-17-producing T lymphocytes correspond with more severe disease and low responders (Jurado et al., 2012). However, IL-17 may have an important role to play in protective responses following vaccination (Chatterjee et al., 2011, Gopal et al., 2012). Thus, targeting Dectin-1 during vaccination may represent a therapeutically advantagous approach. However, a study by Marakalala and colleagues detected no differences in pulmonary cytokine expression between wild type and Clec7a-/- mice infected with M. tuberculosis H37Rv (Marakalala et al., 2011), suggesting that Dectin-1 may be functionally redundant for proinflammatory cytokine production during M. tuberculosis infection in vivo. Scavenger receptors Scavenger receptors (SRs) are expressed on the cell surface of mammalian monocytes and macrophages and recognise a wide range of ligands. SR sub-group A consists of SR-AI, SR-AII and MARCO, while SR sub-group B consists of SR-B1 and CD36 [Figure 1] (Peiser et al., 2002). It has been demonstrated that SR-A-deficient mice infected with M. tuberculosis H37Rv survive significantly longer than their wild type cohorts (Sever-Chroneos et al., 2011), suggesting that recognition by these receptors may favour subversion of the immune response by the bacilli. Evidence exists for a role of the SR-B1 as a macrophage receptor involved in BCG recognition. However, this was shown to be only a minor role in in vivo, possibly due to the ability of other PRRs to compensate for the SR-B1 function (Schafer et al., 2009). Similarly, mice deficient in CD36 were found to be less susceptible to infection with M. bovis BCG, while Cd36-/- macrophages demonstrated increased intracellular killing of M. tuberculosis H37Rv and M. marinum in vitro (Hawkes et al., 2010). The mechanism through which CD36 deficiency confers greater protection against infection is not clear, but it does not involve differences in phagocytic uptake, macrophage apoptosis, TNF-α or IL-10 8 This article is protected by copyright. All rights reserved. Cellular recognition of mycobacteria Accepted Article production, or the generation of reactive oxygen or nitrogen intermediates (Hawkes et al., 2010). MARCO has been found to be involved, at least in part, in the recognition of TDM (Bowdish et al., 2009). However, mice deficient in MARCO show no differences in response to acute or chronic infection with M. tuberculosis compared to WT controls (Court et al., 2010). Interestingly, in this same study, no difference in response to infection were observed in CD36-/-, SRA-/-, or MR-/- mice, in contrast to the studies discussed above. The reasons for these differences are not clear, but there may be some functional redundancy within the different classes of SR. However, a polymorphism in MARCO has been shown to be associated with susceptibility to TB infection in a Gambian population (Bowdish et al., 2013). Nod-like receptors There are two human NOD genes, NOD1 and NOD2, both of which detect peptidoglycan, a structural component essential to the bacterial cell wall (Kanneganti et al., 2007). NOD-2 also senses muramyl dipeptide (MDP), which is a common cell wall component of Gram-negative and Gram-positive bacteria (Girardin et al., 2003). A role for NOD-2 in the recognition of M. tuberculosis was first demonstrated using BMDM and DCs from Nod2-/- mice. NOD-2 was found to be required for optimal production of proinflammatory cytokines in response to M. tuberculosis in vitro (Gandotra et al., 2007), suggesting that NOD-2 is at least one of the receptors required for sensing of the bacillus. Moreover, Nod2-/- mice have been shown to display decreased immunopathology and T cell recruitment to the lungs upon infection with M. bovis BCG, coupled with higher bacterial burdens and increased mortality. In this model, M. bovis BCG-infected alveolar macrophages from Nod2-/- mice released significantly less TNF-α than wild type cells (Divangahi et al., 2008). Similarly, Brooks and colleagues studied the NOD-2 receptor in both human MDM 9 This article is protected by copyright. All rights reserved. Cellular recognition of mycobacteria Accepted Article and alveolar macrophages in response to infection with M. tuberculosis. Survival of the bacteria within Nod2-/- macrophages was higher than in controls, suggesting that NOD-2 plays an important role in the recognition and control of mycobacterial infection (Brooks et al., 2011). In addition, activation of NOD-2 with MDP in human alveolar macrophages infected with M. tuberculosis has been shown to increase intracellular control of bacterial growth and lead to the recruitment of proteins linked with autophagy to the bacteria-containing phagosome/autophagosome (Juarez et al., 2012). It is well established that autophagy represents an important mechanism for the intracellular killing of mycobacteria by macrophages (Castillo et al., 2012, Gutierrez et al., 2004). These data highlight the possibility of a PRR-dependent mechanism for the activation of autophagy, unlike previous studies that have shown that autophagic killing of mycobacteria is regulated by cytokines— particularly IFN-γ (Gutierrez et al., 2004, Harris et al., 2007). However, the role of macrophage-derived cytokines, such as TNF-α and IL-1, which can also induce autophagy, was not assessed in this study (Harris, 2011, Harris, 2013). In a case-control study of an African-American population in the United States, three common nonsynonymous polymorphisms in the NOD2 gene were significantly associated with a genetic susceptibly to TB infection (Austin et al., 2008). An association has also been found between TB susceptibility and the NOD2 synonymous Arg5878Arg SNP in the Chinese Han population (Zhao et al., 2012). This association was further validated in a larger Chinese population (Pan et al., 2012). Genetic associations between allelic variants of NOD2 and susceptibility to infection have also been reported for other mycobacterial species. For example, a highly significant association (P < 0.0001) with MAP infection for genotypes at a nonsynonymous SNP in the bovine NOD2 gene has been reported in a mixed population of domestic cattle (Pinedo et al., 2009) and in Holstein-Friesian cattle (Ruiz-Larranaga et al., 10 This article is protected by copyright. All rights reserved. Cellular recognition of mycobacteria Accepted Article 2010). In addition, it has been proposed that several NOD2 polymorphisms are associated with susceptibility to leprosy, an infectious disease of the peripheral nerves and skin, caused by the mycobacterial pathogen M. leprae (Berrington et al., 2010) [Figure 1]. RIG-I-like receptors and cytosolic DNA sensors Retinoic acid-induced gene (RIG)-I-like receptors (RLRs) are a family of PRRs located in the cytoplasm. The involvement of RLRs in the detection of intracellular bacteria has been previously demonstrated (Chiliveru et al., 2010, Monroe et al., 2009). Moreover a RIG-I signalling pathway was over-represented in a genome-wide transcriptomics study of M. bovis-infected bovine MDM (Magee et al., 2012). A murine cytosolic DNA sensor, IFI204, has also been shown to recognise M. tuberculosis and activates the STING/Tbk1/Irf3 axis, resulting in IFN-β secretion (Manzanillo et al., 2012). This pathway is activated by multiple receptors, including NLRs, TLRs and RLRs (Barber, 2011). The STING/Tbk1 axis has been shown to be required for targeting of M. tuberculosis to the autophagosome (Watson et al., 2012). Interestingly, Irf3-/- mice are resistant to infection with M. tuberculosis, suggesting that cytosolic DNA receptors may be specifically targeted by the bacteria to promote their intracellular survival (Manzanillo et al., 2012). A role for intracellular PRRs in the recognition of mycobacterial pathogens may also tally with recent evidence that M. tuberculosis has the potential to escape from the phagosome (Harriff et al., 2012, Watson et al., 2012). Further research in this area may reveal the full extent to which intracellular PRRs are involved in the sensing of mycobacterial pathogens. Toll-like receptors Toll-like receptors (TLRs) are a conserved family of ten human (TLRs 1-10) and twelve murine (TLRs 1-9 and TLRs 11, 12, 13) PRRs that have a fundamental role in nonopsonic phagocytosis and recognition of intracellular ligands (Kawai et al., 2010). TLRs 1, 2, 11 This article is protected by copyright. All rights reserved. Cellular recognition of mycobacteria Accepted Article 4, 5 and 6 are located on the cell surface, while TLRs 3, 7, 8 and 9 are located intracellularly, mainly on the endoplasmic reticulum (ER) membrane. TLR-10 is an orphan member of the human TLRs, expressed on B cells and plasmacytoid DCs and non-functional in mice (Hasan et al., 2005). TLRs have been shown to play a role in the cellular response to mycobacterial pathogens (Basu et al., 2012). Interestingly, whereas live M. tuberculosis activates NF-κB via TLR-2 and TLR-4, heat-killed M. tuberculosis only activates through TLR-2. Expression of a dominant-negative TLR-2 construct in RAW 264.7 macrophages consistently blocked M. tuberculosis-induced NF-κB activation by 70–80%, whereas expression of a dominantnegative TLR-4 construct blocked NF-κB activation by 30–40%. Co-expression of both TLR2 and TLR-4 dominant-negative constructs resulted in almost complete absence of NF-κB activation (Means et al., 2001). Moreover, a TLR-4 inhibitor inhibited mycobacteria-induced TNF-α production and apoptosis in both RAW 264.7 cells and primary human alveolar macrophages (Means et al., 2001). A role for TLR-2 and TLR-4 in the induction of apoptosis has subsequently been confirmed by other groups (Lopez et al., 2003, Sanchez et al., 2010). TLR-2 is also involved in the production of IL-1β following infection by M. tuberculosis (Kleinnijenhuis et al., 2009). In human cells, stimulation of TLR-2 triggers a pathway that involves up-regulation of the vitamin D receptor and the vitamin D-activating enzyme CYP2R1, transcription of cathelicidin and beta 4 defensin, and culminates in the co-localisation of cathelicidin and mycobacteria and intracellular killing of bacteria (Liu et al., 2006). Similarly, autophagy in response to the mycobacterial lipoprotein LpqH is TLR1/2- and vitamin D receptordependent (Shin et al., 2010). TLR-2 may also be involved in mycobacterial inhibition of host immune responses; Noss and colleagues reported that inhibition of MHC-II antigen processing by the 19 kDa mycobacterial lipoprotein was TLR2-dependent (Noss et al., 2001). 12 This article is protected by copyright. All rights reserved. Cellular recognition of mycobacteria Accepted Article Further work demonstrated that this was mediated via TLR2-dependent inhibition of IFN-γregulated HLA-DR protein and mRNA expression (Gehring et al., 2003). It has subsequently been shown that exposure of the murine J774 macrophage cell line to the TLR2 ligands 19 kDa mycobacterial lipoprotein or zymosan, but not the TLR4 ligand LPS, inhibits IFN-γinduced killing of M. bovis BCG in a TLR2-dependent manner (Arko-Mensah et al., 2007). In addition, mycobacterial CpG motifs have been found to be recognised by TLR-9 (Carvalho et al., 2011), and classical TLR-9 signalling has been shown to be blocked by MAP in bovine monocytes (Arsenault et al., 2013). Genetic associations between TLR gene polymorphisms and TB susceptibility has been reviewed previously (Azad et al., 2012). Briefly, TLR2 gene polymorphisms and TB, particularly extrapulmonary TB, have been reported for a range of different human populations in Africa and Asia (Chen et al., 2010, Motsinger-Reif et al., 2010). No studies have yet shown an association between TLR4 polymorphisms and TB in HIV-negative patients; however, a link between a TLR4 SNP and TB susceptibility in HIV-infected individuals has been reported (Pulido et al., 2010). Two studies have also reported an association between a TLR9 polymorphism and TB (Velez et al., 2010, Kobayashi et al., 2012). A study on a Columbian population found no association between TB susceptibility and polymorphisms in either TLR2, TLR4 or TLR9. However, this study may have been underpowered to detect minor associations (Sanchez et al., 2012). In addition, SNPs within the TIR domain containing adaptor protein gene (TIRAP, or Mal), which encodes a signalling adaptor protein downstream of TLR-2 and TLR-4, have also been associated with TB susceptibility in some studies (Selvaraj et al., 2010, Zheng et al., 2011). The importance of TLRs in infection with M. tuberculosis in the murine model, particularly the key role of TLR-2 has previously been demonstrated (Bafica et al., 2005, Drennan et al., 2004, Mayer-Barber et al., 2010). However, these results do vary by model: 13 This article is protected by copyright. All rights reserved. Cellular recognition of mycobacteria Accepted Article Hölscher and colleagues report that Tlr2/4/9 triple knockout mice infected with 100 cfu H37Rv have normal production of TNF-α, IL-12p40, normal CD4+ and CD8+ reponses to stimulation with M. tuberculosis peptides, and no difference in bacterial burden at 150 days compared to wild type mice (Holscher et al., 2008). These conflicting results may be due to different H37Rv exposure times. MyD88 is a signalling adaptor protein downstream of all TLRs, with the exception of TLR-3; it is also involved in other signalling pathways including IFN-γ and IL-18 (Schneider et al., 2010, Sun et al., 2006). In addition, Myd88-/- mice show a markedly increased susceptibility to infection with M. tuberculosis, which is thought to primarily reflect its role in IL-1β signalling (Holscher et al., 2008, Mayer-Barber et al., 2010). With regards to other mycobacterial infections, genetic variants of the TLR1 gene have been identified as a major determinant of leprosy susceptibilty (Wong et al., 2010). DNA sequence polymorphisms in TLR2 and TLR4 have also been associated with susceptibility to M. leprae infection (Hart et al., 2012). In addition, susceptibility to MAP infection in cattle has been associated with polymorphisms in the bovine TLR1, TLR2 and TLR4 genes [Figure 1] (Mucha et al., 2009). Conclusion Host macrophage and DC recognition of mycobacterial pathogens is a complex process: different populations of myeloid immune cells use different receptors or combinations of receptors and act in conjunction with other molecules to successfully identify and phagocytose mycobacteria, while the bacteria themselves may preferentially target specific receptors to manipulate the host response and promote their own survival. Understanding these processes will help us to better understand the co-evolution of host immune genes and mycobacterial genomes. The development of effective vaccines against 14 This article is protected by copyright. All rights reserved. Cellular recognition of mycobacteria Accepted Article mycobacterial diseases will be paramount to the future of human and animal health. Deciphering the mycobacterial-specific mechanisms for evading macrophage and DC recognition will be be key to this, including a greater knowledge of the relationship between different PRRs and mycobacterial disease prevalence across global populations. Future studies targeting different combinations of receptors may lead to a better appreciation of the complex interactions between phagocyte and mycobacterium. Ultimately, increasing our understanding of innate immune recognition of mycobacterial pathogens will aid in our abilty to treat and prevent mycobacterial diseases. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements Work that led to this review was supported by Investigator Grants from Science Foundation Ireland (Nos: SFI/01/F.1/B028 and SFI/08/IN.1/B2038), a Research Stimulus Grant from the Department of Agriculture, Food and the Marine (No: RSF 06 405) and a European Union Framework 7 Project Grant (No: KBBE-211602-MACROSYS). KEK is supported by the Irish Research Council (IRC) funded Bioinformatics and Systems Biology thematic PhD programme. COF is supported by a Research Stimulus Grant from the Department of Agriculture, Fisheries and Food (2006 06/340). JH was supported by Science Foundation Ireland (SFI) as part of the Immunology Research Centre, SFI Strategic Research Cluster (07/SRC/B1144). CNC is supported by the Health Research Board, Ireland and the Health 15 This article is protected by copyright. All rights reserved. Cellular recognition of mycobacteria Accepted Article Service Executive, Ireland, under the National SpR Academic Fellowship Programme. We thank Prof. Stephen Gordon (UCD) and Dr. David Magee (UCD) for valuable comments. 16 This article is protected by copyright. All rights reserved. Accepted Article Cellular recognition of mycobacteria References Afonso-Barroso, A., Clark, S.O., Williams, A., Rosa, G.T., Nobrega, C., Silva-Gomes, S., et al. (2012). Lipoarabinomannan mannose caps do not affect mycobacterial virulence or the induction of protective immunity in experimental animal models of infection and have minimal impact on in vitro inflammatory responses. Cell. Microbiol. Akira, S., Uematsu, S. and Takeuchi, O. (2006). Pathogen recognition and innate immunity. Cell 124, 783-801. Arko-Mensah, J., Julian, E., Singh, M. and Fernandez, C. (2007). 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Zhao, M., Jiang, F., Zhang, W., Li, F., Wei, L., Liu, J., et al. (2012). A novel single nucleotide polymorphism within the NOD2 gene is associated with pulmonary tuberculosis in the Chinese Han, Uygur and Kazak populations. BMC Infect. Dis. 12, 91. 29 This article is protected by copyright. All rights reserved. Cellular recognition of mycobacteria Accepted Article Zheng, R., Zhou, Y., Qin, L., Jin, R., Wang, J., Lu, J., et al. (2011). Relationship between polymorphism of DC-SIGN (CD209) gene and the susceptibility to pulmonary tuberculosis in an eastern Chinese population. Hum. Immunol. 72, 183-186. 30 This article is protected by copyright. All rights reserved. Accepted Article Cellular recognition of mycobacteria Tables Table 1: Overview of important mycobacterial species Table 2: Pattern recognition receptors (PRRs) and mycobacterial infections Figure legends Figure 1: Pattern recognition receptors (PRRs) involved in the non-opsonic recognition of mycobacterial pathogens. PRRs and their cellular locations are shown. Gene symbols are indicated in grey text and PRR genes with sequence variants associated with resistance to various mycobacterial infections are also indicated. These infections are tuberculosis caused by M. tuberculosis, leprosy caused by M. leprae and Johnne’s disease in cattle caused by M. avium subsp. paratuberculosis. 31 This article is protected by copyright. All rights reserved. Accepted Article Cellular recognition of mycobacteria Table 1: Overview of important mycobacterial species Mycobacterial group Mycobacterial species Disease Host species Primary tissue/cell infected M. tuberculosis M. tuberculosis H37Rv Virulent strains Tuberculosis M. tuberculosis Erdman Avirulent strains M. tuberculosis H37Ra Human Non-pathogenic Human (high incidence in HIVpositive patients) M. africanum Mycobacterium tuberculosis complex Human (other hosts are unknown) M. canettii M. caprae This article is protected by copyright. All rights reserved. Tuberculosis 32 Humans, cattle, goats Alveolar macrophage Accepted Article Cellular recognition of mycobacteria Mycobacterium avium complex (MAC) M. leprae Nontuberculous mycobacteria (NTM) M. pinnipedii Seals (Pinnipeds) M. microti Voles M. bovis Bovine tuberculosis Alveolar macrophage Cattle, humans and other mammals Avirulent strains M. bovis BCG Vaccine against tuberculosis M. avium Opportunistic infections M. intracellulare Circulating monocytes Opportunistic infections Found in soil and water but can infect humans, particularly AIDS patients Infection mainly occurs via the gastrointestinal tract but occasionally occurs via the lungs M. avium subspecies paratuberculosis Johne’s disease Cattle Intestinal macrophages M. leprae Leprosy Human Macrophages and Schwann cells M. marinum Skin infections in people persistently exposed to saltwater or fresh water, disseminated infection in immunecompromised patients Found in soil and water but can infect humans Macrophages This article is protected by copyright. All rights reserved. 33 Accepted Article Cellular recognition of mycobacteria M. abscessus Chronic lung disease in immune-compromised patients M. smegmatsis Can cause primary lesions in immunecompromised patients This article is protected by copyright. All rights reserved. 34 Macrophages Soil, water and plants Macrophages Accepted Article Cellular recognition of mycobacteria Table 2: Pattern recognition receptors (PRRs) and mycobacterial infections Gene symbol/s Ligand/s MRC1 MRC2 Lipoarabinomannon (LAM) Mannosylated LAM (ManLAM) DC-SIGN CD209 ManLAM, DNaK, Glyceraldehyde-3 phosphate dehydrogenase (GAPDH), IprG Mincle CLEC4E Protein/s Mannose receptors Trehalose-6, 6-dimycolate (TDM) This article is protected by copyright. All rights reserved. PRR/PAMP interactions reported for other microbes Cellular location Gene variation and susceptibility/resistance Cell surface Association between MRC1 polymorphism and susceptibily to TB and leprosy Yes Cell surface Association between CD209 polymorphism and susceptibility to TB None reported Cell surface No associations reported None reported 35 Accepted Article Cellular recognition of mycobacteria Dectin-1 CLEC7A Scavenger receptors:SRAI MSR1 SR-AII MARCO MARCO SCARB1 SR-B1 CD36 Oligosaccharide ligands Cell surface No associations reported Yes Wide variety of ligands Cell surface Polymorphism in MARCO has been shown to be associated with susceptibility to TB Yes Cytoplasm Conflicting evidence for an association between CARD15 polymorphisms and susceptibility to TB Association between CARD15 polymorphisms and susceptibility to leprosy in humans and to MAP infection in cattle Association between CARD15 polymorphisms and leprosy Yes CD36 NOD2 TLR1 CARD15 Muramyl dipeptide TLR1 Peptidoglycan and lipoproteins (forms a heterodimer with TLR2) This article is protected by copyright. All rights reserved. Association between TLR1 polymorphisms and susceptility to leprosy Yes Cell surface TLR1 polymorphisms have also been associated with susceptibility to MAP infection in cattle 36 Accepted Article Cellular recognition of mycobacteria TLR2 TLR4 TLR2 TLR4 Lipoproteins and mycoplasma Lipopolysaccharide (LPS) Association between TLR2 polymorphisms and susceptibility to TB (particulary extrapulmonary TB), leprosy and MAP infection in cattle Cell surface Association between TLR4 polymorphisms and TB susceptibility in HIV-positive patients (no association shown for HIV-negative patients) Cell surface Yes Yes TLR4 polymorphisms associated with susceptibility to leprosy and MAP infections in cattle TLR9 TLR9 CpG motifs on bacterial and viral DNA Cytoplasm Association between TLR9 polymorphisms and TB susceptibility RIG-I DDX58 Viral double-stranded RNA Cytoplasm No associations reported This article is protected by copyright. All rights reserved. 37 Yes Yes Accepted Article Extracellular TLR-1 TLR-2 TLR-4 TLR-9 TLR1 TLR2 TLR4 TLR9 Mannose receptor, C type 1 Dectin-1 Mincle DC-SIGN MRC1 Toll-like receptors CLEC7A CLEC4E RIG-I NOD2 NOD2 Intracellular M. leprae SR-A1 SR-A2 MARCO MSR1 MSR1 MARCO SCARB1 C-type lectin receptors DDX58 M. tuberculosis CD209 M. avium subsp. paratuberculosis SR-B1 CD36 CD36 Scavenger receptors Leucin-rich repeat region Carbohydrate recognition domain Cystein-rich domain Collagenous domain Cytoplasmic TIR domain CARD domain Type II fibronectin domain Helicase domain Immunoreceptor ITAM domain NACHT domain 23-amino acid tandem repeat region Alpha helical coiled domain