Intracellular signalling cascades regulating innate immune responses to Mycobacteria: branching out from Toll-like receptors

Authors


*E-mail hayoungj@cnu.ac.kr; Tel. (+82) 42 580 8243; Fax (+82) 42 585 3686.

Summary

Toll-like receptors (TLRs) recognize Mycobacterium tuberculosis (Mtb) or Mtb components and initiate mononuclear phagocyte responses that influence both innate and adaptive immunity. Recent studies have revealed the intracellular signalling cascades involved in the TLR-initiated immune response to mycobacterial infection. Although both TLR2 and TLR4 have been implicated in host interactions with Mtb, the relationship between specific mycobacterial molecules and various signal transduction pathways is not well understood. This review will discuss recent studies indicating critical roles for mycobacteria and mycobacterial components in regulation of mitogen-activated protein kinases and related signal transduction pathways that govern the outcome of infection and antibacterial defence. To better understand the roles of infection-induced signalling cascades in molecular pathogenesis, future studies are needed to clarify mechanisms that integrate the multiple signalling pathways that are activated by engagement of TLRs by both individual mycobacterial molecules and whole mycobacteria. These efforts will allow for the development of novel diagnostic and therapeutic modalities for tuberculosis that targets the intracellular signalling pathways permitting the replication of this nefarious pathogen.

Introduction

One-third of the global population is infected with Mycobacterium tuberculosis (Mtb), the causative agent of pulmonary tuberculosis (TB). Improvement in TB vaccines and therapy for infected patients hinge on knowledge of the immune responses and how they are modulated (Abel et al., 2002). Viable mycobacteria and mycobacterial components are potent activators of monocytes, macrophages and dendritic cells (DCs). In the primary phase of infection, mycobacteria encounter and activate antigen-presenting cells such as macrophages and DCs. These cells provide both microbicidal mechanisms and antigen presenting function to elicit T cell responses to Mtb. If infection is established, Mtb interferes with immune effector mechanisms and becomes resistant to elimination by the host's immune system, causing a latent infection (Zahrt, 2003). Pattern recognition receptors (PRRs) expressed on macrophages and other leucocytes activate signalling cascades that play a fundamental role in phagocytosis and other host defence mechanisms (Flynn et al., 1995). Well-studied PRRs include the mannose receptor and Toll-like receptors (TLRs). Following opsonization, complement receptors also play important roles in the interaction of Mtb with host cells.

Toll-like receptors are key sensors of mycobacterial infections and are known to play an important role in the innate immune response. TLRs detect a broad spectrum of pathogen-derived molecules, and are critical in shaping host–pathogen interactions. Numerous studies in vitro and in vivo have shown that whole mycobacteria or mycobacterial components act as agonists for TLRs (Pai et al., 2004; Quesniaux et al., 2004a; Bafica et al., 2005). Mycobacterium avium is a classically established TLR2 agonist (Yoshimura et al., 1999). The outcome of Mycobacterium bovis bacillus Calmette–Guerin infection (BCG) following intraperitoneal inoculation of mice was shown to be dependent on TLR2 and minimally on TLR4 (Heldwein et al., 2003). Various mycobacterial proteins and lipids have been individually demonstrated to be involved in TLR-dependent signalling cascades and innate immune responses (Table 1).

Table 1.  TLR recognition of mycobacterial components.
Mycobacterial componentsTLR usageSpeciesReference
19 kDa lipoprotein (LpqH)TLR2M. tuberculosis, M. bovisBrightbill et al. (1999); Noss et al. (2001)
27 kDa lipoproteinTLR2M. tuberculosisHovav et al. (2004)
33 kDa lipoproteinTLR2M. lepraeKrutzik et al. (2003)
38 kDa glycolipoproteinTLR2, TLR4M. tuberculosisJung et al. (2006)
Ara-LAMTLR2M. smegmatisWieland et al. (2004)
GPLTLR2M. aviumSweet and Schorey (2006)
HSP65TLR4M. tuberculosisBulut et al. (2005)
HSP70TLR2, TLR4M. tuberculosisBulut et al. (2005)
LipomannanTLR2M. tuberculosis, M. bovisQuesniaux et al. (2004b)
LprA lipoproteinTLR2M. tuberculosis, M. bovisPecora et al. (2006)
LprG lipoproteinTLR2M. tuberculosis, M. bovisGehring et al. (2004)
Man-LAMM. tuberculosis, M. bovis, M. kansasiiQuesniaux et al. (2004b)
PE_PGRS33TLR2M. tuberculosisBasu et al. (2007)
PIM 2, PIM 6TLR2M. tuberculosis, M. bovis, M. smegmatisGilleron et al. (2003)
PILAMTLR2M. smegmatisHeldwein and Fenton (2002)
Soluble tuberculosis factorTLR2M. tuberculosisMeans et al. (1999)

Understanding the molecular mechanisms following TLR engagement by mycobacteria is of great importance. TLRs modulate the induction of hundreds of host genes through a complex network of signalling that allows for the appropriate response to a microbial pathogen (Lasker and Nair, 2006). The mitogen-activated protein kinase (MAPK) pathways are crucially involved in macrophage signalling induced by mycobacteria via TLRs (Pathak et al., 2004; Yadav et al., 2004; Yang et al., 2007). Recent studies have focused on the critical role of MAPK-dependent innate immune and inflammatory responses, which ultimately affect antimycobacterial defence (Tse et al., 2002; Pathak et al., 2004; Yadav et al., 2004; Yang et al., 2007).

Current studies on the pathways preceding or following MAPK signalling underscore the importance of TLR-mediated activation of innate immunity following mycobacterial infection. Virulence of the mycobacterial strain affects the degree of signalling. Indeed, regulation of MAPK signalling is altered in TB patients, and mounting evidence indicates that this signalling has key roles in immunologically relevant pathophysiological processes during human TB infection. A better understanding of pathogen tactics and of the immune system's defensive armoury is needed to improve prevention and therapy strategies. In the present review, we summarize the current knowledge regarding the regulation of host immune responses to mycobacterial infection by intracellular signalling pathways induced by various mycobacterial components, including proteins and lipids.

The role of different TLRs during mycobacterial infection

Toll-like receptors play a pivotal role in the induction of an innate immune response to various infectious agents, including Mycobacteria spp. TLR2 was classically recognized as a principal inducer of the proinflammatory signal, tumour necrosis factor (TNF)-α, induced by whole Mtb (Underhill et al., 1999). Several lines of evidence have suggested the protective role of TLR2 in mycobacterial infection. TLR2-deficient mice are impaired in host resistance and neutrophil responses to the infection with virulent M. avium strain (Feng et al., 2003). Recent studies using a human-like infection model show that certain TLR knockout mice are more susceptible than wild-type mice at an early stage of respiratory tract infection; in particular, TLR2–/– mice are more susceptible than TLR4–/– or wild-type mice (Tjarnlund et al., 2006). In addition, defective capability of intracellular killing, preferentially in TLR2–/– macrophages, was correlated with impaired production of TNF-α (Tjarnlund et al., 2006), which is vital for containment of mycobacterial infections (Roach et al., 2002). Recent studies by Liu et al. (2006) revealed a mechanism whereby TLR2/1 functions in regulating responses to infection with Mtb. They showed that TLR2/1 signalling mediated by Mtb augments expression of the vitamin D receptor and the vitamin D hydroxylase thereby leading to increased expression of antimicrobial peptides (Liu et al., 2006).

TLR4 may contribute to resistance to Mtb infection, but consensus has not been reached on this issue. Studies using an intranasal infection model with live Mtb demonstrate that TLR4 plays a protective role in host defence against murine pulmonary TB in vivo, as reflected by an increased mortality and mycobacterial load in the lungs of mice with a non-functional TLR4 (Branger et al., 2004). In addition, an increased susceptibility to Mtb was reported in C3H/HeJ mice which have a non-functional TLR4, as reflected by an enhanced mycobacterial outgrowth and an increased mortality (Abel et al., 2002). In contrast, TLR4-deficient animals are indistinguishable from wild-type controls in a model of M. avium infection (Feng et al., 2003). Importantly, Reiling et al. (2002) highlighted the effect of inoculum sizes in studies assessing the relative importance of TLR4 and TLR2 in resistance to airborne Mtb infection. High-dose aerosol Mtb challenge revealed TLR2-, but not TLR4-deficient mice to be more susceptible than control mice, whereas TLR2- and TLR4-deficient mice were as resistant as control mice at a low-dose challenge with Mtb (Reiling et al., 2002). Whereas molecules that generate TLR2-dependent signals are entirely dependent upon myeloid differentiation primary response protein 88 (MyD88), TLR4 generates both MyD88-dependent and MyD88-independent signalling. It is possible, then, that host defence could be affected by TLR4 in a MyD88-independent manner. In summary, the role of TLR4 in Mtb infection in vivo remains unclear, with results depending on the infection model, strains of Mtb and inoculum size.

TLR9 also plays a role in response to Mtb. Mice lacking both TLR9 and TLR2 were significantly more susceptible to Mtb infection than mice with single deficiency of either TLR2 or TLR9 (Bafica et al., 2005), indicating a significant role for TLR9 and cooperation between TLR9 and TLR2 in host defence to mycobacterial infection. In addition, induction of interleukin (IL)-12 and CD86 levels expression by DCs infected with M. bovis BCG is not exclusively dependent on TLR2, and is abrogated only in TLR2/4/9-deficient DCs, supporting a pivotal role of TLR9 in the recognition of M. bovis BCG by murine DCs (von Meyenn et al., 2006). Taken together, these data suggest a role for signalling by multiple TLRs in protection against mycobacterial infection, particularly in the acute phase of respiratory tract infection (Tjarnlund et al., 2006), consistent with observations with MyD88-deficient mice (below).

The role of intracellular signalling components downstream of the TLR in mycobacterial infection

Full activation of signal transduction via TLRs requires the cooperation of a number of signalling adaptors, including MyD88 and the adaptors of the MyD88-independent pathway (Akira and Hoshino, 2003). MyD88-deficient mice do not produce inflammatory cytokines, such as TNF-α, IL-6 and IL-12, in response to most TLR ligands (Akira et al., 2001). MyD88 is essential to the signalling pathways of all TLR family members that initiate the production of inflammatory cytokines with the exceptions of TLR3 and the MyD88-independent component of TLR4 signalling, which require Toll/interleukin-1 receptor (TIR) domain-containing adaptor-inducing interferon-β (TRIF) (Akira and Hoshino, 2003). Therefore, TLR4 can utilize either the MyD88/TIR domain-containing adaptor protein (TIRAP) or TRIF/TRIF-related adaptor molecule (TRAM) adaptor pairs to generate distinct responses (Akira and Takeda, 2004). The latter pathway is critical to the induction of DC maturation and activation of IFN-regulated factor-3, which also induces late activation of nuclear factor-κB (NF-κB) (Fig. 1).

Figure 1.

A schematic representation of the molecules implicated in MAPK activation following mycobacterial agonist stimulation. Stimulation with mycobacterial ligands recruits TIR domain-containing adaptors, including MyD88 and TIRAP, to the receptor, leading to formation of IRAK and TRAF6 complexes. Although TLR2 utilizes MyD88, whereas TLR4 utilizes both MyD88 and a TRIF-dependent signalling cascade, the so-called MyD88-independent pathway. The resultant responses include the induction of proinflammatory genes and type-I IFNs (see text). TRAF6 activates the TAK1 complex, resulting in the phosphorylation of NEMO and activation of the NF-κB pathways. Freed NF-κB translocates into the nucleus and initiates the expression of proinflammatory cytokine genes. Simultaneously, TAK1 activates the MAPK cascades, leading to the activation of AP-1, which is critical for the induction of cytokine genes. Subsequent steps include activation of the MAPK kinases such as the MKK6 and MAPK. The MAPK can directly phosphorylate various transcription factors to initiate the expression of proinflammatory cytokine genes. Negative regulators, IRAK-M and CATERPILLER, modulate TLR signalling through interference with IRAK-1 function, resulting in the repression of TLR signalling induced by mycobacterial stimulation. MD, myeloid differentiation protein-2; TRAF6, TNF receptor-associated factor 6; TOLLIP, Toll interacting protein; IRF, interferon regulatory factor; TAK, TGF-β-activated kinase; CATERPILLER, CARD, transcription enhancer, R[purine]-binding, pyrin, lots of leucine regions.

The increased susceptibility of MyD88-deficient mice to Mtb infection has been shown in a low-dose aerosol infection (Scanga et al., 2004). Of interest, MyD88-deficient mice fail to control M. avium growth and succumb to infection after 9–14 weeks (Feng et al., 2003). Profound immune response impairments were seen in the induction of proinflammatory cytokines (TNF-α, and IL-6) and Th1-associated cytokine (IFN-γ) by MyD88–/– mice infected with M. avium (Feng et al., 2003). Fremond et al. observed that MyD88 deficiency leads to profound innate immune defects and increased fatality to Mtb infection despite adaptive immune responses (Fremond et al., 2004), supporting an essential role for MyD88 in Mtb infection. On the other hand, Sugawara et al. found that MyD88-null mice were not highly susceptible to Mtb infection, but they developed granulomatous pulmonary lesions with neutrophil infiltration (Sugawara et al., 2003). Furthermore, some studies suggest that gene expression in macrophages infected with Mtb is largely MyD88-independent (Shi et al., 2003), although other studies have implicated MyD88-dependent signalling in control of macrophage and DC functions. Responses to Mtb may be greatly influenced by innate immune receptors other than MyD88-coupled TLRs, such as nucleotide binding oligomerization domain (NOD) receptors. Additional research is needed to define the precise function of MyD88-independent pathways and the contributions of other innate immune receptors in antimycobacterial defence.

The TLR family recognizes a diverse spectrum of microbial ligands. Ligand recognition results in the recruitment of the TIR-containing adaptor molecule MyD88, which in turn leads to an association with the IL-1 receptor-associated kinase-1 (IRAK-1) and IRAK-4 (Suzuki et al., 2002; Janssens and Beyaert, 2003). A model for TLR signalling in response to mycobacterial infection is shown in Fig. 1. IRAK harbours a death domain and a serine/threonine kinase domain, and four members have been identified in the IRAK family: IRAK-1, IRAK-2, IRAK-M and IRAK-4 (Li et al., 2002). Previous studies have suggested that IRAK-4 is central to the TLR signalling cascade and acts upstream of IRAK-1 (Takeda and Akira, 2004). Studies of patients with Mendelian susceptibility to mycobacterial disease have revealed that mutations in the IRAK-4 gene are associated with an increased susceptibility to bacterial infection, and patients possessing these mutations are unresponsive to TLR ligands (Picard et al., 2003). In addition, NF-κB essential modulator (NEMO) and IRAK-4 were found to be important in IL-12 production and subsequent IFN-γ production, which is crucial in establishing protective immunity to mycobacteria infection in humans and mice (Feinberg et al., 2004).

Upon stimulation of TLRs, IRAK-4 associates with IRAK-1. Introduction of a dominant negative form of IRAK-4 results in impaired IRAK-1 activation (Li et al., 2002). Unlike IRAK-1 and IRAK-4, IRAK-M is catalytically inactive and preferentially expressed in monocytes and macrophages. IRAK-M-deficient mice produce increased amounts of inflammatory cytokines in response to TLR ligands (Lomaga et al., 1999). Pathak et al. showed that Mtb Mannosyl-capped lipoarabinomannan (Man-LAM) suppresses lipopolysaccharide-induced IL-12 p40 expression and promoter activity in murine RAW264.7 macrophages through an IRAK-M-mediated pathway (Pathak et al., 2005).

A negative regulator of both TLR and tumour necrosis factor receptor pathways is the CATERPILLER (CLR, also Monarch-1) protein. This protein interferes with IRAK-1 function, resulting in the repression of TLR signalling (Williams et al., 2005). Downregulation of Monarch-1 parallels the increase in IL-6 cytokine expression seen in response to virulent Mtb infection (Williams et al., 2005). Taken together, these data indicate that IRAK-M and Monarch-1 are responsible for the negative regulation of mycobacteria-induced macrophage activation and TLR signalling pathways (Fig. 1).

Mycobacterial agonists of TLRs

The interaction between Mtb, various TLRs is not fully understood, but it appears that whole mycobacteria or distinct mycobacterial components may interact with different members of the TLR family (Table 1). Initially, a soluble, heat-stable, protease-resistant mycobacterial fraction was reported to signal through TLR2, whereas heat-labile components associated with the cell wall were reported to signal through TLR4 (Means et al., 1999). The mycobacterial 19 kDa lipoprotein (LpqH) has been well characterized and shown to signal through TLR2 to induce cell activation, apoptosis and mycobacterial killing [Aliprantis et al., 1999; Brightbill et al., 1999; Noss et al., 2001; Thoma-Uszynski et al., 2001; Gehring et al., 2004 (LprG); Pecora et al., 2006 (LprA)]. Recent reports indicate that the 38 kDa glycolipoprotein acts through both TLR2 and TLR4 to induce the activation of proinflammatory cytokine responses during mycobacterial infection (Jung et al., 2006). Of note, recent work has shown that PE_PGRS33, one member of the PE_PGRS (polymorphic GC-rich sequence) family of Mtb H37Rv, signals through TLR2. Moreover, clinical isolates show variations in sequence that are associated with varying levels of TNF-α release by the respective PE_PGRS33 proteins (Basu et al., 2007). The mycobacterial heat shock protein (HSP) 65 signals exclusively through TLR4, whereas HSP70 is known to signal through TLR2 and TLR4 (Bulut et al., 2005). Both mycobacterial HSPs induce NF-κB activation in a MyD88-, TIRAP-, TRIF- and TRAM-dependent manner (Bulut et al., 2005). TLR9 has been shown to play a role in host defence to infection with Mtb, bacterial DNA, the presumptive mycobacterial agonist for TLR9, is also implicated as a TLR ligand of significance in mycobacterial infection.

The mycobacterial envelope contains a wide array of chemically diverse lipids and glycolipids that likely mediate specific host interactions and have been shown to possess potent biological activity against eukaryotic cells in vitro (Glickman and Jacobs, 2001). Macrophages and DCs have been shown to be activated via TLR2 by LAM from rapidly growing mycobacteria (Ara-LAM) (Jones et al., 2001), lipomannan from slow-growing mycobacteria Mtb, M. bovis BCG (Quesniaux et al., 2004b) and the phosphatidylinositol mannosides (PIM)2 and PIM6 membrane anchors (Jones et al., 2001; Gilleron et al., 2003). Very recent studies have demonstrated that the highly expressed surface glycopeptidolipids (GPL) of M. avium can activate the NF-κB pathway as well as MAPK p38 and Jun N-terminal kinase (JNK) pathways in a TLR2-dependent, but not TLR4-dependent, manner (Sweet and Schorey, 2006). Elucidating the mycobacterial protein recognition and signalling pathway activation will reveal the key immunological processes induced by this important human pathogen and help in the rational design of more effective vaccines and adjuvants.

The role of MAPKs: essential components of TLR signal transduction networks activated in response to mycobacteria

Primary stimuli, such as mycobacterial products or whole mycobacteria, act through the TLRs to trigger the MAPK pathways, leading to activation of the transcription factors, including NF-κB and activator protein-1 (AP-1) (Wang et al., 2001; Aleman et al., 2004; Pathak et al., 2004; Pennini et al., 2006; Yang et al., 2006). Three major groups of distinctly regulated MAPK cascades are known in humans to cause altered gene expression: extracellular signal-regulated kinases (ERK), the JNK and the p38 MAPK. The MAPK signalling pathways are activated upon mycobacterial infection, and have been implicated in mycobacterial pathogenesis (Yadav et al., 2004). Macrophage activation in response to infection or inflammatory stimuli initiates intracellular signalling pathways that carry the signal needed to activate the production of inflammatory mediators.

Previous studies in mice and humans have linked the p38 MARK pathway to the production of mycobacteria-induced IL-10 as well as other cytokines (Hasan et al., 2003; Song et al., 2003). In addition, ERK1/2 signalling was shown to be involved in increasing the expression of TNF-α in human macrophages (Surewicz et al., 2004; Yadav et al., 2004). It is of interest that MAPK activation is differentially regulated upon macrophage infection with pathogenic and non-pathogenic mycobacteria (Roach and Schorey, 2002). Murine bone marrow-derived macrophages infected with the non-pathogenic Mycobacterium smegmatis induced significantly elevated MAPK activity and TNF-α production relative to SmT M. avium 724-infected cells (Roach and Schorey, 2002). Similarly, virulent SmO M. avium 2151-infected macrophages have elevated MAPK activity and produce significantly higher TNF-α compared with virulent SmT-infected cells (Tse et al., 2002). Furthermore, M. avium ssp. paratuberculosis causes a chronic intestinal infection and chronic wasting disease termed paratuberculosis or Johne's disease and shows greater expression of IL-10 and less TNF-α expression in macrophages than M. avium ssp. avium (Weiss et al., 2002). Collectively, the modulation of MAPK pathways leading to the suppression of antimycobacterial cytokines and the increase of macrophage inhibitory cytokines may contribute to the virulence of mycobacteria pathogenesis.

Macrophages have several mechanisms to control mycobacterial infections. The MAPK signalling pathways are involved in the regulation of well-known antimycobacterial activities, such as phagosome acidification, apoptosis and the presentation of antigens through MHC class II expression. Previous studies have suggested that signalling through the p38 MAPK pathway may provide a means by which the mycobacteria attenuate the antimycobacterial function of macrophages. The p38 MAPK has been implicated in the arrest of Mtb phagosome maturation (Fratti et al., 2003). The p38 MAPK has been associated with a reduction in the recruitment of endosome and phagosomal membrane-tethering molecules. Pharmacological blocking of p38 MAPK activity caused an increase in phagosome acidification (Fratti et al., 2003). Recently, Souza et al. (2006) demonstrated that blockade of the p38 MAPK pathway results in an increase in phagosome acidification and a significant increase in the ability of monocytes to kill ingested M. avium ssp. paratuberculosis organisms. In addition, Pennini et al. showed that signalling through ERK1/2 and p38 both contribute to inhibition of CIITA by 19 kDa lipoprotein, a well-characterized mycobacterial TLR2 agonist (Pennini et al., 2006). Thus, the p38 MAPK and ERK1/2 pathways play an essential regulatory role over the antimicrobial function of macrophages and presentation of antigens by infected macrophages, potentially contributing to evasion of host immune responses. Further studies are needed to elucidate the role of individual kinase pathways involved in the molecular events controlling host defence to mycobacterial infection.

The players upstream of MAPK signalling

Several pathways have been reported to act upstream of the MAPKs implicated in the regulation of macrophage immune response to mycobacteria (Fig. 2). Classically, ERK is activated by MAP kinase kinase (MKK) and MKK2, JNK by MKK4 and MKK7 and p38 MAPK by MKK3, MKK4 and MKK6 (Raingeaud et al., 1996; Pearson et al., 2001). In both M. smegmatis- and M. avium-infected macrophages, ERK1/2 activation was dependent on the calcium/calmodulin/calmodulin kinase II pathway (Yadav et al., 2004). In addition, stimulation of the cyclin adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway in M. smegmatis-infected cells was required for prolonged ERK1/2 activation and the increased TNF-α production observed in macrophages infected with non-pathogenic M. smegmatis (Yadav et al., 2004). Pathak et al. (2004) reported that Ras associates with TLR2 and activates the ERK pathways in M. avium-challenged RAW264.7 macrophages. However, our recent studies showed that for the most part, Ras is not involved in Mtb-induced ERK1/2 activation or TNF-α expression in various monocyte and macrophage types (Yang et al., 2007), suggesting that the involvement of Ras in signalling processes may be cell-type dependent.

Figure 2.

Conceptual model of the signal transduction cascade initiated in macrophages by mycobacteria. Engagement of macrophage receptors by mycobacteria or mycobacterial components initiates a series of intracellular responses by the macrophage, including the activation of the ERK1/2 pathways. In macrophages infected with mycobacteria, various upstream signalling molecules are activated to stimulate the ERK1/2 pathways as well as transcription factors downstream of the ERK1/2. Studies have shown that activation of the MAPK is essential for the production of various immune effector molecules and that the degree of ERK1/2 activation is correlated with the virulence of the infecting mycobacteria. MEKK, MAP/Erk kinase kinase; MEK, mitogen-activated protein kinase.

Our recent studies have shown that atypical protein kinase C (PKC) ζ plays an essential role in the upstream regulation of ERK1/2 activation and TNF-α expression in response to Mtb in various monocytes and macrophages, including primary human monocytes, monocyte cell lines (THP-1 and U937) and primary murine macrophages (Yang et al., 2007). In addition, PKCζ associates with TLR2 after Mtb stimulation of primary human monocytes and monocytic cells (Yang et al., 2007). Yadav et al. showed that sphingosine kinase (SPK) activity is required for ERK1/2, but not p38, MAPK activation following M. avium or M. smegmatis infection of murine macrophages (Yadav et al., 2006). The SPK functions along with phosphatidylinositol-specific phospholipase C (PI-PLC) and conventional PKC to mediate ERK1/2 activation (Yadav et al., 2006). Coupled with the studies from our laboratory, this suggests that PKC families function as upstream kinases to transmit TLR signalling directly into the MAPK signalling pathway during mycobacterial infection.

Mycobacteria also trigger other intracellular signalling cascades, such as phosphatidylinositol 3-kinase (PI3K) (Maiti et al., 2001). Recently, we demonstrated that PI3K activity is required for Mtb-induced phosphorylation of the 70 kDa S6 protein kinase 1 and ERK1/2 activation (Yang et al., 2006). Moreover, PI3K activity was dependent on the SPK/PI-PLC/PKC pathway in murine macrophages infected with non-pathogenic M. smegmatis or M. avium (Yadav et al., 2006). In this study, PI3K activity was not dependent on the MAPK activation, and vice versa, indicating that the PI3K and MAPK pathways are parallel (Yadav et al., 2006). The discrepancy in PI3K–MAPK cross-talk between two studies may be attributable to the different cell types and mycobacterial strains used, indicating that signalling cross-talk is cell type- and mycobacterial strain-dependent. Previous reports have shown that PI3K negatively regulates the activity of p38 MAPK (Fukao et al., 2002). During exposure to Mtb, PI3K inhibition enhances p38 MAPK activation and the activity of apoptosis signal-regulating kinase 1, an upstream regulator of p38 (Yang et al., 2006). Consistent with these findings, inhibition of PI3K or a lack of PI3K upregulates p38 activity in DCs (Fukao et al., 2002). These data highlight the complexity of mycobacterial signalling mechanisms, and the diversity that can exist in the regulation of signalling pathways by a given mycobacterial strain or component.

The players downstream of MAPK signalling

Classically, the MAPK pathways were known to drive the transcription of inflammatory genes. The promoter regions of inflammatory genes have binding sites for a limited set of transcription factors (Saklatvala, 2004). JNKs phosphorylate and activate c-Jun and activating transcription factor 2, which are components of the AP-1 binding complexes. ERK activates C/EBPβ and AP-1, and all three MAPKs can phosphorylate Ets transcription factors such as ETS-like kinase protein-1 (Elk-1) and serum response factor accessory protein-1 (Kracht and Saklatyala, 2002) The p38 pathway is involved in transcriptional control, but the precise mechanisms of control remain unclear. Several transcription factors can be phosphorylated by p38 (Ono and Han, 2000; Kracht and Saklatyala, 2002), and downstream kinases may also be involved. Mitogen- and stress-activated kinases (MSK) 1 and 2 activate the cAMP response element binding protein (CREB) (Wiggin et al., 2002) and the p65 subunit of NF-κB (Vermeulen et al., 2003).

The involvement of CREB in TNF-α promoter activity in macrophages following a mycobacterial infection was demonstrated by Roach et al. (2005). They showed that M. smegmatis infection leads to significantly higher induction of CREB phosphorylation in macrophages than in induction observed by M. avium 724-infected cells, and that this phosphorylation is dependent on p38 MAPK and PKA activation (Roach et al., 2005). Another study by the same group showed that the diminished TNF-α and nitric oxide synthase 2 promoter activity in macrophages infected with virulent M. avium as compared with those infected with M. smegmatis may be due to pronounced differences in the Ets/Elk and NF-κB promoter activities (Lee and Schorey, 2005). Moreover, the Ets/Elk, but not the NF-κB, transcriptional response was dependent on ERK1/2 and correlated with the requirement for ERK1/2 in TNF-α promoter activity (Lee and Schorey, 2005).

Furthermore, Pathak et al. showed that the NF-κB and CRE elements of the cyclooxygenase-2 (COX-2) promoter play critical roles in M. avium-induced COX-2 gene expression (Pathak et al., 2004). They also demonstrated that TLR2 contributes to M. avium-induced COX-2 gene induction and both ERK and p38 MAPK activation converge to regulate the activation of MSK1. Of note, MSK1 activation is essential for M. avium-triggered phosphorylation of the transcription factor CREB and NF-κB-dependent gene expression (Pathak et al., 2004). Collectively, these studies demonstrate that the MAPKs and downstream nuclear kinase MSK1 are necessary for regulation of the mycobacteria-driven expression of inflammatory genes by transcription factors. Controlling the activity of MSK1 may therefore have potential benefits in restricting the survival of pathogenic mycobacteria in macrophages (Pathak et al., 2004).

Genetic and immunologic analysis of TLR signalling in human TB

Structural integrity of TLR signalling components is essential for immunological protection to infection. Changes in structure of TLR signalling molecules that result from single nucleotide polymorphism (SNPs) and are often associated with susceptibility to various infectious diseases. A SNP located within the bb loop of TLR2, Arg677Trp, is strongly correlated with lepromatous leprosy (Kang and Chae, 2001). This human TLR2 polymorphism abolished activation of NF-κB in response to Mycobacterium leprae and Mtb (Bochud et al., 2003). The Arg753Gln (TLR2 SNP) impairs TLR2 signalling and is associated with susceptibility to TB (Ogus et al., 2004). These results suggest that TLR2 polymorphisms are associated with susceptibility to TB, demonstrating essential roles for TLR2 in human mycobacterial infection. The importance of TLR4 SNPs remains unresolved. Future studies including a careful analysis of polymorphisms of TLRs will uncover the genetic basis of disease susceptibility and outcome of infection.

More research on host defence and immunopathogenesis mechanisms of Mtb is urgently needed in order to develop a new vaccine and adjunctive immunotherapy for TB. After infection with Mtb, proinflammatory cytokines TNF-α, IL-1 and IL-6 are secreted primarily by monocyte-derived macrophages (Giacomini et al., 2001). Upregulated production of these cytokines is seen in monocytes and macrophages isolated from TB patients, especially in advanced cases (Lee et al., 2003; Jung et al., 2006; Lee et al., 2006). Recent studies suggest the possibility of a primary abnormality in monocyte and macrophage regulation of signalling activation during the response to mycobacterial components. Our studies on the immune response to various mycobacterial protein antigens, such as the 38 kDa glycolipoprotein, 30 kDa antigen and secreted MTB12 protein, indicate the activation of ERK1/2 and p38 MAPK, and subsequent cytokine secretion was greater in monocytes from active pulmonary TB patients than in monocytes isolated from healthy control subjects (Jung et al., 2006; Lee et al., 2006).

The importance of the MAPK cascade during TB pathogenesis is further illustrated by the finding that apoptosis of peripheral neutrophils from patients with active TB induced by whole Mtb via TLR2 involves the p38 MAPK pathway (Aleman et al., 2004). Moreover, these studies demonstrate a correlation between increased apoptosis in peripheral neutrophils from TB patients and a high expression of phosphorylated p38 in circulating neutrophils (Aleman et al., 2004). This mechanism has yet to be confirmed at the site of infection, but it suggests that when the bacterial burden is low, peripheral mononuclear cells and/or neutrophils from TB patients could detect non-opsonized Mtb via TLR2, activate the MAPK pathways and induce apoptosis or proinflammatory responses by activated cells (Aleman et al., 2004; Jung et al., 2006; Lee et al., 2006). In addition, other studies have demonstrated that TLR signalling by Mtb or its 19 kDa lipoprotein induces apoptosis of macrophages (Lopez et al., 2003; Krutzik and Modlin, 2004). Although further work is needed to clarify whether these findings can be generalized to patients with different clinical stages and sites of infection, altered signal transduction mechanisms that would result in aberrant regulated cytokine dynamics or antimycobacterial activities in human TB should be actively investigated.

Other PRRs act in concert with TLRs in mycobacterial infection

Receptors other than TLRs also provide important contributions to the host response to Mtb, and the C-type lectins are included in this group. Man-LAM from virulent Mtb, which mainly has anti-inflammatory effects, functions through its interaction with the mannose receptor and DC-specific intercellular adhesion molecule 3-grabbing non-integrin, also termed DC-SIGN (Nigou et al., 2001; Geijtenbeek et al., 2003). In addition, dectin-1 belongs to the C-type lectin receptor family and is the major β-glucan receptor that affects production of TLR2-mediated TNF by macrophages infected with M. smegmatis, M. bovis BCG, and Mtb H37Ra, but not virulent M. avium 724 and Mtb H37Rv (Yadav and Schorey, 2006). Furthermore, dectin-1 facilitated IL-6, regulated upon activation, normal T-cell expressed and secreted, and granulocyte colony-stimulating factor production by mycobacteria-infected macrophages (Yadav and Schorey, 2006).

NOD2, a susceptibility gene for Crohn's disease (Hugot et al., 2001; Ogura et al., 2001), is an intracellular protein containing leucine-rich repeats similar to those found in TLRs. NOD2 is the intracellular sensing molecules responsible for recognition of bacterial peptidoglycans from both Gram-positive and Gram-negative bacteria, through its interaction with muramyl dipeptide (Girardin et al., 2003a), whereas NOD1 recognizes peptidoglycans of Gram-negative bacteria only (Girardin et al., 2003b). Recent studies demonstrate that NOD2 and TLRs are essential for effective and synergistic activation of the proinflammatory cytokine production by Mtb infection through non-redundant recognition mechanisms of Mtb (Fewerda et al., 2005). These studies demonstrate that host cells sense the presence of Mtb using multiple recognition systems in which TLRs cooperate with other receptors. Further research is needed to elucidate the role of PRRs other than TLRs and their roles in infection models.

Concluding remarks

Toll-like receptor family members contribute significantly to the inflammatory and innate immune responses involved in host defence against mycobacterial infection. Recent studies have reported that whole mycobacteria and distinct mycobacterial components may interact with different TLR family members, and it has become clear that MAPK signalling plays an essential role in modulating a variety of host cellular responses to mycobacterial agonists. Studies on upstream regulators and downstream effectors of the MAPK pathways underscore the importance of individual components in TLR-mediated activation of signalling cascades. It will be important to understand how the regulators of MAPK pathways influence the outcome of mycobacterial infection and the difference between infection induced by virulent or avirulent mycobacteria. The identification of the key components in these responses may reveal novel targets for TB therapy. In humans, two polymorphisms in the exon part of TLR2, which attenuate receptor signalling, are associated with susceptibility to TB. Finally, an appreciation of the inflammatory mechanisms of MAPK activation by TLR signalling will identify the role of TLR-dependent MAPK pathways in the immunopathogenesis and protective immunity to TB. Knowledge of the molecular details underlying this signalling process is critical to understanding immunological pathogenesis and protection from TB. In the future, it will be important to clarify additional mechanisms of MAPK pathway regulation, through either NOD or other yet uncharacterized signalling pathways.

Acknowledgements

This work was supported by Grant R01-2005-000-10561-0 (2005) from the Basic Research Program of the Korea Science and Engineering Foundation and NIH Grants AI35726, AI069085 and AI34343.

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