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ISSN : 2287-7991(Print)
ISSN : 2287-8009(Online)
Journal of the Preventive Veterinary Medicine Vol.37 No.4 pp.185-192
DOI : https://doi.org/10.13041/jpvm.2013.37.4.185

The key roles of toll-like receptor (TLR) for intracellular survival of Brucella

Suk Kim1,3,†, Dae Geun Kim1, Hannah Leah Tadeja Simborio1, Alisha Wehdnesday Bernardo Reyes1, Wongi Min1, Hu Jang Lee1, Jin Ju Lee2
1College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Republic of Korea
2Animal and Plant Quarantine Agency, Anyang 430-757, Republic of Korea
3Institute of Agriculture and Life Science, Gyeongsang National University, Jinju 660-701, Republic of Korea
Received 15 September 2013, Revised 30 October 2013, Accepted 15 November 2013

Abstract

Brucella spp. are facultative intracellular pathogens that have the ability to survive and multiply in professional andnonprofessional phagocytes and cause abortion in domestic animals and undulant fever in humans. Brucella species cansurvive in a variety of cells, including macrophages and their virulence and chronic infections are thought to be due to theirability to avoid the killing mechanisms within macrophages. Inhibition of phagosome-lysosome fusion has been proposed asa mechanism for intracellular survival of Brucella in professional and nonprofessional phagocytes. Toll-like receptors (TLRs) arepart of a skillful system for detection of invasion by microbial pathogens. Recognition of microbial components by TLRs triggerssignaling pathways that promote expression of genes and regulate innate immune responses. Recent studies for the interactionbetween TLRs-Brucella have indicated the importance of control of Brucella infection. Here, we review selected aspectsof TLRs-Brucella interaction, which may be helpful to understanding the mechanism of Brucella pathogenesis.

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INTRODUCTION

 Brucellosis known as undulant fever in humans is a major zoonotic disease that causes a serious debilitating disorder in humans and abortion and sterility in domestic animals.

 Brucella spp. are small Gram-negative and facultative intracellular bacteria, which can multiply within professional and nonprofessional phagocytes [21, 22]. The genus Brucella consists of six well-recognized species classified according to host preference - B. melitensis (sheep and goats), B. abortus (cattle), B. suis (hogs), B. ovis (sheep), B. canis (dogs) and B. neotomae (wood rats) [37]. In recent years, Brucella has been recovered from several marine mammals, including cetaceans and pinnipeds. These marine isolates belong to two potential new species, B. pinnipedialis and B. ceti [27]. A new species of Brucella, B. microti, was recently isolated from wild common voles suffering from a systemic disease [32, 59]. B. melitensis, B. abortus and B. suis strains cause abortion and infertility in their natural hosts - goats and sheep, cattle and swine, respectively. Humans can also acquire a severe, debilitating febrile illness known as Brucellosis, resulting from contact with infected animals or their products [48]. B. ovis, a natural pathogen of sheep, primarily causes epididymitis and infertility in rams [11].

 B. canis infection causes abortion and infertility in dogs [23, 67]. Although B. ovis and B. canis are important in animals, human infection with B. canis is rare [18], and human infection with B. ovis has not been reported. B. neotomae, a Brucella strain that infects only desert wood rats is not known to be associated with clinical disease in any host species.

 Contrary to other intracellular pathogens, Brucella species do not produce exotoxins, antiphagocytic capsules or thick cell walls, resistance forms or fimbriae and do not show antigenic variation [25]. The ability of Brucella to avoid the killing mechanisms within macrophages is thought to be the key aspect of their virulence [58, 64].

 In brucellosis, respiratory, digestive, and genital tracts are the most common point of entry for both animals and humans. Brucella enter phagocytic cells at an unknown cellular site and spread throughout the body by the regional lymph nodes. Brucella show high tropism in macrophages; in particular, the monocytes and liver, spleen, mammary glands, and reproductive tracts are the main tissues of preference. Bacterial resistance to host immune response and the debilitated health status of the host is the main cause of chronic brucellosis [42].

 A tenth of the total Brucella will survive to avoid phagocytosis and penetrate cell membrane for intracellular growth; macrophages are the most important for achievement of a successful infection. During the infection, interference with macrophage function is observed, namely the inhibition of IFN-g [6] and TNF-a expression [14], and the reduction of antigen presentation and subsequent T cell activation [26]. Brucella inside dendritic cells (DC) contributes to chronic infection and induced low levels of proinflammatory cytokines and increased MHC II expression [57]. Placental trophoblasts produce erythritol during the last trimester and increase the carbon source for Brucella. Brucella caused abortion or stillbirth of infected foetus by inducing placental damage [62] and targeting giant trophoblasts [35]. Brucella have also been reported in other cell types and were studied with cell models and lines, including human pulmonary epithelial cells [24], caprine uterine epithelial cells [43], human osteoblastic cells [20], murine neurons [28], bovine and human polymorphonuclear [33] and many other cells lines. In addition, surprisingly, extracellular brucellae were observed on the 21st day post infection [56].

When unopsonized B. melitensis, B. abortus and B. suis strains internalize into macrophages and epithelial cells, the Brucella-containing vacuoles (BCVs) enter into an intracellular trafficking pathway, leading to development of specialized membrane-bound compartments [1, 15, 36, 45, 50, 54] known as replicative phagosomes [36] or brucellosomes [38]. Interactions between the O-chain of the smooth LPS of these Brucella strains and lipid rafts on the surface of macrophages have been shown to be important for mediating entry into host cells in a manner leading to development of the replicative phagosome [52]. During the initial stages of intracellular trafficking of BCVs, these compartments suffer temporary interactions with lysosomes [60], resulting in their acidification [1, 51]. These vacuoles begin to interact extensively with the endoplasmic reticulum [15]. Eventually their intracellular pH rises to a level that allows intracellular replication of Brucella. During development of the replicative phagosome in epithelial cells, BCVs acquire properties resembling those of autophagosomes [50], however, this does not appear to be the case during development of BCVs in macrophages [15]. Studies employing the human monocytic cell line THP-1 and B. abortus strains opsonized with hyperimmune IgG have also shown that when Brucella internalize host macrophages in this manner, the resulting BCVs also undergo temporary association with the lysosomal compartment and become acidified, however, these BCVs do not interact extensively with the ER [7]. An obvious potential benefit of this altered intracellular trafficking is that limiting the fusion of BCVs with lysosomes minimizes exposure of these bacteria to the bactericidal proteins that reside in these intracellular compartments [7].

 Toll-like receptors (TLRs) are the best characterized pattern recognition receptors (PRRs). Receptor-ligand interaction via TLRs leads to production of antimicrobial peptides and proinflammatory cytokines through NF-κB, mitogen-activated protein kinase (MAPK) and other signaling pathways [34]. As a result, TLR signaling is critical to development of the host innate immune response, including recruit- ment of DCs and T effector cells, and upregulation of MHC I and II on antigen presenting cells (APCs) and by extension adaptive immunity against infection. In Brucellosis, many studies have reported that TLRs play important role in control of Brucella infection. In this review, we will discuss the key roles of TLRs including immune response, signal transduction cascade, and phagocytic pathway for Brucella infection within host cells.

GENERAL ASPECTS OF TLRs FOR MICROBES

 TLRs are single-pass type I transmembrane-spanning proteins characterized by a single intracellular Toll/interleukin-1 (IL-1) receptor (TIR) domain and multiple extracellular leucine-rich repeats (LRRs), which is responsibile for binding to ligands [44]. TLRs recognize and are activated by a small collection of microbe-derived molecules (Fig. 1). Through studies of targeted mutants, among 13 paralogous TLRs, 10 in humans and 12 in mice, the diverse mode of ligand recognition of individual TLRs was determined, except for TLR8, TLR10 (only present in humans) and TLR11–13 (only present in mice). Lipopeptides and other components of Gram-positive bacterial cells activate TLR2 in conjunction with either TLR1 or TLR6; accessory protein MD-2 is necessary for recognition LPS of TLR4; flagellin is detected by TLR5; poly I:C, a double-stranded RNA (dsRNA) analog, is detected by TLR3; unmethylated DNA and CpG-oligodeoxynucleotides (CpG-DNA) are detected by TLR9, in which Granulin and high mobility group (HMG) B proteins have been proposed to deliver CpG-DNA to TLR9 through an ability to bind simultaneously to both CpG-DNA and TLR9; and single-stranded RNA and its synthetic analogs resiquimod, imiquimod, and loxoribine activate TLR7. Despite the variation in modes of ligand recognition, all known TLR dimer structures show the same arrangement, with the two carboxy-terminal tails closely juxtaposed and the amino termini at opposite ends of the dimer [2, 9, 10, 44]. This conformation may be required in order to bring the intracellular TIR domains into close proximity for initiation of signaling. Toll-like receptor (TLR) activation can induce cell-intrinsic antimicrobial activity. For example, activation of TLR2 and TLR4 can lead to assembly of NADPH oxidase as well as relocalization of mitochondria to the bacteria-containing phago- some, leading to a burst of reactive oxygen and nitrogen species within this compartment [39, 65, 69]. Evidence suggests that TLR signaling can cause rapid acidification of the phagosome in which TLR signaling has occurred, likely through recruitment of vacuolar-ATPase subunits to the phagosomal membrane [3, 9, 10, 63]. These activities increase the antimicrobial capacity of the phagosome, although some bacteria have actually coopted these signals for use in regulation of their virulence programs. Detection of microbial ligands by TLRs can also induce expression and secretion of antimicrobial peptide (AMPs), such as β-defensins and cathelicidin, further supporting the role of TLR-mediated detection in cell-intrinsic antimicrobial activity [53, 55, 61]. However, bacteria have attempted to avoid TLR signaling by altering their surface structures, interfering with TLR signaling pathways or escaping, inhibiting, or subverting phagocytosis [2]. Brucella spp. are recognized by TLR2, TLR4 and TLR9, which identify lipopolysaccharide (LPS), lipoproteins and bacterial DNA, respectively [46].

Fig. 1. TLR signaling elicits inflammation. Pathogens, such as bacteria, may enter the host through a breached epithelial barrier, leading to activation of macrophage TLRs by pathogen-derived molecules, including LPS. TLR signaling leads to the production of inflammatory cytokines, which act near the site of infection to recruit neutrophils and induce neutrophil production of antimicrobial molecules, including peptides, reactive oxygen species (e.g. H2O2 and superoxide anion), and leukotrienes. Cytokines also travel throughout the body and induce systemic effects. Both macrophages and neutrophils act to limit infection by phagocytosis of pathogens [44].

THE ROLES OF TLRs IN BRUCELLA INFECTION

 Involvement of TLR2 and TLR4 in recognition of Brucella was reported in several studies. The former was proposed to induce secretion of TNF-α, IL-6, IL-12 and IL-10 in peritoneal macrophages stimulated by B. abortus lipoproteins, such as Omp16 and Omp19 [29], the major Brucella TLR2 ligands responsible for pro-inflammatory response, however, no role was observed in control of the pathogen in vivo (Fig. 2) [46]. Involvement of TLR4 was demonstrated in B. melitensis resistance in cooperation with TLR9, which has been shown to play a more prominent role during Brucella infection [41]. Interaction of TLR4 with non-canonical Brucella LPS induces activation of NF-κB, expression and its interaction with lumazine synthase from Brucella spp. stimulates maturation of dendritic cells [8] and increases expression of co-stimulatory molecules and major histocompatibility class II as well as production of IL-6, TNF-α, and IL-12p70 [46].

Fig. 2. TLR signaling in response to Brucella in macrophages or dendritic cells. Recognition of Brucella's pathogen-associated molecular patterns (PAMPs) by TLR2 (Outer membrane proteins, Omp16 and Omp19), TLR4 (Brucella LPS, Brucella lumazine synthase- BLS) and TLR9 (Brucella-DNA) activates intracellular signaling via MyD88 resulting in the activation of NF-κB, MAPKs and production of proinflamatory cytokines, mostly important IL- 12. A possible role for subversion of TLR signaling by Brucella producing TIR domain-containing proteins is detailed (Btp1 and TcpB). Btp1 acts in TLR2 and TcpB interacts with MyD88 interfering in TLR2 and TLR4 signaling. Solid arrows represent the important TLR pathways to control Brucella infection. Discontinuous arrows represent TLRs and type I IFN receptor signaling which are not necessary for Brucella clearance. Barred circle represents the impairment of TLR signaling by Btp1 and TcpB proteins [46].

Absence of MyD88 protein during Brucella infection impairs maturation of dendritic cells and production of IL-12 and TNF-α in macrophages and dentritic cells [68], and reduces levels of inflammatory chemokines RANTES (CCL5), MCP-1 (CCL2), and MIP-1α (CCL) [41]. This molecule is required for development of IFN-γ producing T cells and control of brucellosis [41], suggesting that Th1 response induced during the infection is regulated by a MyD88-dependent pathway [46]. In addition, the molecule is used by other inflammatory signaling pathways, including IL-1 and IL-18 [3]. However, no role of IL-18 in control of murine brucellosis was observed [46].

 Brucella appears to interfere in TLR signaling through production of inhibitory homologues of Toll/interleukin-1 receptor (TIR) domain, such as B. abortus Btp1, which acts in TLR2 signaling down-modulating maturation of dendritic cells and secretion of pro-inflammatory cytokines [57] and B. melitensis TcpB, which has been shown to interact with MyD88 in vitro, impeding TLR2 and TLR4 signaling and secretion of pro-inflammatory cytokines [16]. These proteins then suppress innate immunity and cause increased virulence [46].

 The key roles of TLRs in Brucella infection are as follows:

TLR2

 The role of TLR2 in Brucella infection remains controversial. Some studies have suggested that TLR2 is not required for control of Brucella infection in mice [13, 17, 68]. However, other studies have indicated that TLR2 is important for clearance of Brucella from the lung following aerosol exposure [49], cytokine production such as TNFα and IL-12 [29, 30, 41, 68, 70], MHC-II expression [5] and down regulation of the type I receptor for the Fc portion of IgG (FcγRI, CD64) [6].

TLR4

 The role of TLR4 in Brucella infection also remains in dispute. Some studies have suggested that TLR4 is required for control of Brucella replication in mice [13, 17, 41], while others have reported that TLR4 is not involved [4, 68]. Lee et al. [40] reported that TLR4 linked Janus kinase 2 (JAK2) plays an essential role in phagocytosis of B. abortus by macrophages. TLR4-associated activation of JAK2 in early cellular signaling events plays a pivotal role in B. abortus-induced phagocytic process in macrophages (Fig. 3), implying the pathogenic significance of JAK2-mediated entry [41]. However, they play diverse roles in the Brucella antigen specific antibody production and antibody class switching [49].

Fig. 3. Diagram illustrating the phagocytic signaling pathway initiated by TLR4-linked JAK2 activation during the internalization of B. abortus into macrophage. The interaction of B. abortus with TLR4 induces the activation of Cdc42 GTPase and JAK2, and the subsequent activation of PI3K and MAPKs promotes actin polymerization. This event contributes to the phagocytosis of B. abortus by macrophage. Lines with arrows denote an activating reaction and dotted line denotes uncertainty of the reaction [40].

TLR6

 TLR6 is an important component for triggering an innate immune response against B. abortus. TLR6 is recruited to the macrophage phagosome, where it recognizes bacterial peptidoglycan and lipoproteins [47]. TLR6 plays a role in recognition of bacterial diacylated lipopeptides such as MALP2, however, it is not essential for cytokine production in response to triacylated lipopeptides. TLR6 cooperates with TLR2 in sensing Brucella and further activates NF-κB signaling in vitro. However, TLR6, not TLR2, is required for efficient control of B. abortus infection in vivo [19].

TLR9

 TLR9 plays a role in control of B. abortus infection in mice [41, 68]. In addition, heat-killed B. abortus induces expression of IL-12 by dendritic cells, which is partially mediated by TLR9 [31]. TLR9 plays a significant role in preventing replication of B. ovis in vivo, however, only MyD88 is required for wild type levels of inflammation [66].

CONCLUSION

 Throughout this study, we described the interaction between Brucella and TLRs, including interaction of specific molecules (ligands), immune response and signal cascade, and strategies for control. This review may be helpful to understanding the pathogenic and defense mechanisms of Brucellosis. In addition, understanding of TLRs mediated control of intracellular parasitic bacterial infection would be helpful to eradication of these diseases.

ACKNOWLEDGEMENTS

 The work was supported by iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries) (112012-3), Ministry for Food, Agriculture, Forestry and Fisheries, Korea.

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