Innate Immune Effectors in Mycobacterial Infection

Tuberculosis, which is caused by infection with Mycobacterium tuberculosis (Mtb), remains one of the major bacterial infections worldwide. Host defense against Mtb is mediated by a combination of innate and adaptive immune responses. In the last 15 years, the mechanisms for activation of innate immunity have been elucidated. Toll-like receptors (TLRs) have been revealed to be critical for the recognition of pathogenic microorganisms including mycobacteria. Subsequent studies further revealed that NOD-like receptors and C-type lectin receptors are responsible for the TLR-independent recognition of mycobacteria. Several molecules, such as active vitamin D3, secretary leukocyte protease inhibitor, and lipocalin 2, all of which are induced by TLR stimulation, have been shown to direct innate immune responses to mycobacteria. In addition, Irgm1-dependent autophagy has recently been demonstrated to eliminate intracellular mycobacteria. Thus, our understanding of the mechanisms for the innate immune response to mycobacteria is developing.


Introduction
In humans, tuberculosis is one of deadly infectious diseases. Indeed, approximately 2 million tuberculosis patients die every year. The risk of disease is also increased by emergence of acquired immune deficiency syndrome and development of multidrug-resistant mycobacteria [1]. Therefore, it is important to understand the host defense mechanisms against mycobacteria. Inhalation of aerosols containing Mycobacterium tuberculosis (Mtb) causes tuberculosis. After inhalation, Mtb invades alveolar macrophages to enter into the host and establish the infection. The host, in turn, ignites defense responses through sequential activation of immunity, a combination of innate and adaptive immune systems. In the adaptive phase of immune responses, the importance of Th1/IFN-γ-mediated responses in mycobacterial infection has been well established [2]. In contrast, although macrophages are the major target of invasion by Mtb, how the innate arm of immunity mediates host defense against mycobacteria had long remained unknown. However, the mechanisms behind innate immune responses have been revealed in the past 15 years following the identification and characterization of pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs) [3]. Furthermore, it has been elucidated that TLR-dependent activation of innate immunity controls the development of adaptive immune responses [4]. The involvement of PRRs other than TLRs in the recognition of mycobacteria has also been revealed. In addition to the induction of adaptive immune responses, the PRR recognition of mycobacteria induces expression of several effector molecules participating in the innate host responses. The role of these innate effector molecules in mycobacterial infection is being elucidated. PRR-independent mechanisms for mycobacterial killing, such as autophagy, have also been revealed. In this paper, we will describe recent advances in our understanding of effectors that mediate innate immune responses against mycobacteria.
2 Clinical and Developmental Immunology TLRs ( Figure 1). Genomic DNA from a Mycobacterium bovis strain, bacillus Calmette-Guérin (BCG), have an ability to augment NK cell activity and induce type I IFNs from murine spleen cells and human peripheral blood lymphocytes. The immunostimulatory activity of mycobacterial DNA was ascribed to the presence of palindromic sequences including the 5 -CG-3 motif, now called CpG motif [5], and now known to activate TLR9 [6]. The mycobacterial cell wall consists of several glycolipids. Among these, lipoarabinomannan (LAM) lacking mannose end capping, lipomannan (LM), and phosphatidyl-myo-inositol mannoside (PIM) are recognized by TLR2 [7,8]. The 19-kDa lipoprotein of Mtb also activates macrophages via TLR2 [9,10]. TLR4 is also presumed to recognize mycobacterial components.
The in vivo importance of the TLR-mediated signal in host defense to Mtb was highlighted in studies using mice lacking MyD88, a critical component of TLR signaling. MyD88-deficient mice are highly susceptible to airborne infection with Mtb [11][12][13]. In contrast to mice lacking MyD88, mice lacking individual TLRs are not dramatically susceptible to Mtb infection. Susceptibility of TLR2-deficient mice to Mtb infection varies between different studies [14,15], while TLR4-deficient mice do not show high susceptibility to Mtb infection [16,17]. A report demonstrates that TLR9-deficient mice are susceptible to Mtb infection and mice lacking both TLR2 and TLR9 are more susceptible [18]. These findings indicate that multiple TLRs might be involved in mycobacterial recognition. However, a recent report using mice lacking TLR2/TLR4/TLR9 indicated that these triple KO mice show a milder phenotype than MyD88deficient mice [12]. Therefore, more intensive examination is required to reveal whether TLRs or molecules other than TLRs activating MyD88 mediate innate immune responses to mycobacterial infection. This study also demonstrated that Th1-like adaptive immune responses are induced even in Mtb-infected MyD88-deficient mice [12]. Therefore, the TLR/MyD88-independent component of innate immunity is involved in the induction of adaptive immune responses during mycobacterial infection. The TLR/MyD88-independent response might be induced by other PRRs described below.

Non-TLRs in Mycobacterial Infection
Several recent findings have indicated that PRRs other than TLRs evoke innate immune responses [19]. These include RIG-I-like receptors, NOD-like receptors (NLRs), and C-type lectin receptors. Among these PRRs, NOD-like receptors and C-type lectin receptors have been implicated in the innate recognition of mycobacteria ( Figure 2).
NOD2 is a member of NLRs that recognize muramyl dipeptide (MDP), a core component of bacterial peptidoglycan, in the cytoplasmic compartment. Macrophages from NOD2-deficient mice show a defective cytokine production after Mtb infection [20]. Similarly, mononuclear cells of individuals homozygous for the 3020insC NOD2 mutation show a defective cytokine response after stimulation with Mtb [7]. Activation of the NOD2-mediated pathway is induced by stimulation with live Mtb, but not by heat-killed Mtb [8]. Live Mtb, which is localized in the phagosomal compartment within macrophages, stimulates the cytosolic NOD2 pathway by inducing phagosomal membrane damage [21]. The NOD2 ligand MDP is N-acetylated in most bacteria. However, MDP is N-glycolylated by N-acetyl muramic acid hydroxylase (NamH) in mycobacteria. Analyses using M. smegmatis namH mutant and NOD2-deficient mice showed that N-glycolyl MDP is recognized by NOD2. In addition, N-glycolyl MDP is the more potent NOD2 activator than N-acetyl MDP [22]. Thus, NOD2 contributed to the recognition of mycobacteria.
Several members of the NLR family, such as NLRP1, NLRP3, and IPAF, induce assembly of the inflammasome, which leads to caspase-1-dependent secretion of IL-1β and IL-18 [23]. The involvement of IL-1β and IL-18 in mycobacterial infection was demonstrated in studies using knockout mice [24][25][26][27]. A recent study demonstrated that mycobacteria inhibit the inflammasome-dependent casapase-1 activation leading to defective IL-1β production [28]. The inhibition of caspase-1 activation has further been shown to be mediated by an Mtb gene, zmp1, which encodes a putative Zn 2+ metalloprotease. Thus, Mtb has a strategy that evades the inflammasome-mediated innate immune responses.
C-type lectin receptors, such as mannose receptor, were originally reported to mediate phagocytosis of mycobacteria [29]. Another C-type lectin receptor, DC-SIGN, has been shown to recognize mycobacteria, and thereby modulate the function of dendritic cells [30][31][32]. Recognition of mycobacteria by dectin-1 has been shown to induce gene expression such as TNF-α, IL-6, and IL-12 [33,34]. In addition, macrophage inducible C-type lectin (Mincle) has recently been shown to recognize trehalose-6,6 -dimycolate (TDM: also called cord factor), a mycobacterial cell wall glycolipid that is the most studied immunostimulatory component of Mtb [35,36], thereafter modulating macrophage activation. Thus, several C-type lectin receptors are involved in the recognition of mycobacteria.
CARD9 is involved in the signaling pathways of several PRRs including TLRs, NOD-like receptors, and FcRγassociated C-type lectin receptors through association with Bcl-10 and MALT. Therefore, it is not surprising that CARD9-deficient mice are highly susceptible to Mtb infection. However, interestingly the high susceptibility of CARD9-deficient mice to the infection has been shown to be excessive inflammatory responses due to defective production of the immunosuppressive cytokine IL-10 [37]. Mincle is a member of C-type lectin receptors associated with FcRγ [38]. Accordingly, TDM-induced immune responses are mediated by the signaling pathway activating CARD9 [36,39].
TLRs and C-type lectin receptors are expressed on the plasma membrane or the endosomal/phagosomal membrane, whereas NOD-like receptors are expressed within the cytoplasm. Indeed, distinct patterns of TLR-and NODlike receptor-mediated gene expression profiles have been demonstrated in infection with intracellular bacteria [40]. Thus, several PRRs recognize mycobacteria in distinct sites within the host cells (macrophages) to synergistically induce effective host defense responses.

Effectors for Mycobacterial Killing
The recognition of mycobacteria by several PRRs induces the expression of several genes that mediate host defense ( Figure 3). Among these gene products, vitamin D receptor (VDR) and Cyp27b1, a 25-hydroxyvitamin D 3 1-α-hydroxylase that catalyzes inactive provitamin D into the bioactive form of vitamin D (1, 25 (OH) 2 D 3 ), have been shown to be induced by TLR2 ligands in human macrophages [41].
Stimulation of macrophages with 1, 25 (OH) 2 D 3 induces the expression of the antimicrobial peptide cathelicidin, and thereby enhances the antimycobacterial killing activity [42]. In addition to cathelicidin, the small cationic antimicrobial peptide defensin mediates innate immune responses to Mtb [43,44]. Experimental infection of the lung epithelial cell line A549 with Mtb strongly induces production of human β-defensin HBD-2, which leads to Mtb killing [43]. HBD-2 expression has also been shown to be induced by TLR2 [45]. Gene expression analyses of the lung of mycobacteriainfected mice have identified several TLR-dependent genes that are involved in innate immune responses during mycobacterial infection. These genes include Slpi, encoding secretory leukocyte protease inhibitor (SLPI), and Lcn2, encoding lipocalin 2 (Lcn2). SLPI is a secreted protein composed of two cysteine-rich whey acidic protein (WAP) domains [46][47][48]. SLPI was named after its presence in secretions and its function as a serine protease inhibitor. SLPI was originally shown to mediate wound healing [49,50]. SLPI is produced by bronchial and alveolar epithelial cells as well as alveolar macrophages and is secreted into the alveolar space at the early phase of mycobacterial respiratory infections. Recombinant mouse SLPI effectively inhibits the in vitro growth of BCG and Mtb through disruption of the mycobacterial cell wall structure. Cationic residues within the WAP domains of SLPI are essential for the disruption of mycobacterial cell walls. Moreover, SLPI-deficient mice are highly susceptible to mycobacterial infection [51]. The mechanism by which SLPI attaches to the membrane of mycobacteria has been elucidated. SLPI recognizes mannancapped lipoarabinomannans and phosphatidylinositol mannoside, which are conserved in mycobacteria. Thus, SLPI might act as a PRR in order to bind to the mycobacterial membrane [52].
Lcn2 (also known as neutrophil gelatinase-associated lipocalin, 24p3, or siderocalin) was originally identified in the granules of human neutrophils. Lcn2 is a member of the lipocalin protein family and able to bind to small hydrophobic molecules, siderophore. It is a bacterial molecule made in iron-limited environment and facilitates iron uptake by bacteria [53][54][55][56][57][58]. The expression of Lcn2 is increased in macrophages of LPS-treated mice [59]. In addition, it is secreted into the alveolar space by alveolar macrophages and epithelial cells during the early phase of respiratory mycobacterial infection. Lcn2 inhibits in vitro growth of Mtb by binding the mycobacterial siderophore carboxymycobactin, thereby sequestering iron uptake. Moreover, Lcn2-deficient mice are highly susceptible to intratracheal infection with Mtb. Lcn2 is internalized into alveolar epithelial cells by endocytosis and colocalized with mycobacteria within the cells. Therefore, Lcn2 presumably sequesters iron uptake of mycobacteria within epithelial cells and thereby inhibits their intracellular growth. Within macrophages, the endocytosed Lcn2 and mycobacteria show distinct patterns of subcellular localization, which might allow growth of mycobacteria within macrophages [60]. Thus, Lcn2, which is secreted into the alveolar space during the early phase of mycobacterial infection, is endocytosed into alveolar epithelial cells, thereby inhibiting mycobacterial growth [61].

Autophagy in Mycobacterial Infection
Phagocytosis of myobacteria and PRR-dependent recognition of mycobacteria activate several effector functions in macrophages (Figure 4). Maturation of phagosomes is a crucial step in the elimination of intracellular bacteria. The natural-resistance-associated macrophage protein (Nramp1), which is encoded by Slc11a1, is thought to mediate transportation of divalent cations in the phagosomal membrane and thereby sequesters iron (Fe 2+ ) from mycobacteria to enhance bacterial killing by macrophages [62]. Polymorphisms of the SLC11A1 gene have been associated with susceptibility to several infectious diseases, Clinical and Developmental Immunology  including tuberculosis [63,64]. However, in vivo studies have shown that Nramp1-deficient mice are not more susceptible than wild-type mice to infection with virulent Mtb [65]. Thus, the role of Nramp1 in mycobacterial infection is still controversial. This might be due to the presence of other killing mechanisms for mycobacteria in macrophages. Indeed, autophagy has recently been shown to be involved in host defense against several intracellular pathogens that reside within phagosomes [66]. Autophagy was originally identified as a homeostatic mechanism for the catabolic reaction of cellular constitutes [67,68]. It has been demonstrated that autophagy mediates innate immune responses against mycobacteria by promoting phagolysosomal maturation within macrophages [69,70]. Autophagy is induced by IFN-γ-dependent induction of a member of the immunity-related p47 guanosine triphosphatases (IRG) family, LRG47 (also known as Irgm1) in murine macrophages [69]. The importance of LRG47 in resistance to Mtb infection was demonstrated in LRG47-deficient mice, which show high susceptibility to infection [71]. A subsequent study demonstrated that stimulation of macrophages with the TLR4 ligand LPS leads to the MyD88-independent induction of autophagy, which enhances mycobacterial colocalization with the autophagosomes. Since LPS stimulation induces expression of LRG47, the TLR signaling establishes a close relationship between innate immunity and autophagy in mycobacterial infection [72]. In humans, the most equivalent gene to murine Irgm1 is IRGM. IRGM has also been implicated in the induction of autophagy in mycobacteria-infected human macrophages [73]. Irgm1 has been shown to associate with the mycobacterial phagosome by interacting with phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P(2)) and PtdIns(3,4,5)P(3) [74]. The connection of the IRG family of proteins with autophagy has been further demonstrated in an alternative intracellular infection model. In this study, Irgm3 (also known as IGTP) has been implicated in autophagy induction in macrophages infected with Toxoplasma gondii [75].
p62 (also called A170 or SQSTM1) directly binds to cytosolic polyubiquitinated proteins and thereby induces their autophagic clearance [76,77]. It has also been shown that p62 targets intracellular Salmonella typhimurium decorated by ubiquitinated proteins to induce autophagy [78]. In the case of mycobacteria residing in the phagosome, p62 delivers cytosolic ubiquitinated proteins to autophagolysosomes where they are proteolytically processed to products that are able to kill mycobacteria [79]. In accordance with this finding, it has been shown that mycobacterial killing by ubiquitin-derived peptides is enhanced by autophagy [80].

Human Genetics in Tuberculosis
In addition to the intensive studies using murine models, considerable advances have been made in our understanding of the susceptibility to Mtb infection in humans through the identification of mutations and polymorphisms of innate immunity-related genes in tuberculosis patients. As described above, polymorphisms of the SLC11A1 gene are associated with tuberculosis. Subsequent studies identified a significant distinction between tuberculosis patients and healthy controls in TLR2 Arg753Gln polymorphism genotype, indicating that the TLR2 polymorphism influences the susceptibility of Mtb infection [82]. VDR polymorphisms have also been implicated in the susceptibility of Mtb infection [83]. These studies suggest that several genes, which have been revealed to be critical in innate responses in mouse models of Mtb infection, regulate Mtb infection in humans.

Conclusion
Since the discovery of TLRs at the end of the 20th century, rapid advances have been made in our understanding of the mechanisms for activation of innate immunity. Accordingly, innate immunity has been revealed to have a pivotal role in host defense against mycobacteria. The TLRindependent mechanisms for the innate immune response to mycobacteria have also been elucidated. The emergence of multidrug-resistant Mtb is now a major public health problem all over the world. In this context, it is highly critical to develop a new strategy for the treatment of Mtb-infected patients that supplements the conventional antimycobacterial chemotherapeutic drugs. More precise understanding of the innate immune response to Mtb will pave the way for the development of an effective drug that targets the host innate immunity for the treatment of tuberculosis.