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Brazilian Journal of Medical and Biological Research

Print version ISSN 0100-879XOn-line version ISSN 1414-431X

Braz J Med Biol Res vol.42 no.4 Ribeirão Preto Apr. 2009

http://dx.doi.org/10.1590/S0100-879X2009000400002 

Braz J Med Biol Res, April 2009, Volume 42(4) 312-322 (Review)

Genetic polymorphisms in vitamin D receptor, vitamin D-binding protein, Toll-like receptor 2, nitric oxide synthase 2, and interferon-γ genes and its association with susceptibility to tuberculosis

A.C.C.S. Leandro1, M.A. Rocha1, C.S.A. Cardoso2 and M.G. Bonecini-Almeida1

1Laboratório de Imunologia e Imunogenética, 2Serviço de Nutrição e Dietética, Instituto de Pesquisa Clínica Evandro Chagas, FIOCRUZ, Rio de Janeiro, RJ, Brasil

Abstract
Introduction
References
Correspondence and Footnotes


Abstract

Mycobacterium tuberculosis kills more people than any other single pathogen, with an estimated one-third of the world's population being infected. Among those infected, only 10% will develop the disease. There are several demonstrations that susceptibility to tuberculosis is linked to host genetic factors in twins, family and associated-based case control studies. In the past years, there has been dramatic improvement in our understanding of the role of innate and adaptive immunity in the human host defense to tuberculosis. To date, attention has been paid to the role of genetic host and parasitic factors in tuberculosis pathogenesis mainly regarding innate and adaptive immune responses and their complex interactions. Many studies have focused on the candidate genes for tuberculosis susceptibility ranging from those expressed in several cells from the innate or adaptive immune system such as Toll-like receptors, cytokines (TNF-α, TGF-β, IFN-γ, IL-1b, IL-1RA, IL-12, IL-10), nitric oxide synthase and vitamin D, both nuclear receptors and their carrier, the vitamin D-binding protein (VDBP). The identification of possible genes that can promote resistance or susceptibility to tuberculosis could be the first step to understanding disease pathogenesis and can help to identify new tools for treatment and vaccine development. Thus, in this mini-review, we summarize the current state of investigation on some of the genetic determinants, such as the candidate polymorphisms of vitamin D, VDBP, Toll-like receptor, nitric oxide synthase 2 and interferon-γ genes, to generate resistance or susceptibility to M. tuberculosis infection.

Key words: Tuberculosis; Vitamin D; Vitamin D-binding protein; Toll-like receptor; Nitric oxide synthase 2; IFN-γ


Introduction

Tuberculosis, primarily caused by Mycobacterium tuberculosis, continues to be an important public health problem despite the existence of national and international tuberculosis control programs. Recent data from the World Health Organization show that about 8-10 million new cases arise annually and eventually 2-3 million die of the disease every year. Tuberculosis is one of the major infections that cause disease and death worldwide. It is estimated that one-third of the World's population is infected with M. tuberculosis, but that only one in ten (10%) of those infected ever develop clinical disease.

The central question is if tuberculosis patients are inherently susceptible to the disease or if disease development is caused by specific environmental factors. Clearly, environmental factors such as poor economic conditions, malnutrition, stress, and overcrowding play a role in determining the susceptibility to tuberculosis in human populations. It is known that genetic and non-genetic factors of both the bacterium and the host have impact on the immune response to M. tuberculosis. The interaction between these different factors is unknown as well as the impact on disease development. Thus, the increasing interest in understanding the role of human genetic factors in the immune system controlling susceptibility/resistance to infectious diseases is of great importance for tuberculosis research because this will allow a genetic dissection of anti-mycobacterial immunity and should open new possibilities for preventive and therapeutic measures.

Vitamin D

When skin is exposed to sufficient UVB light (290 to 320 nm), the hormonal cascade for the endogenous production of vitamin D is activated. The prohormone (7-dehydrocholesterol) is converted into pre-vitamin D3 (25-hydroxy-VD3 or 25(OH)2D3) and then to 1,25 dihydroxy-D3 (1,25(OH)2D3 or vitamin D3) within keratinocytes. Sunlight also induces the expression of vitamin D receptors (VDR). Circulating 25(OH)2D3 is the major form of vitamin D, with concentrations 1000 times higher than 1,25(OH)2D3, the active form of vitamin D. The conversion of 25(OH)2D3 to 1,25(OH)2D3 is controlled mainly by parathyroid hormone, calcium and phosphate levels. The extent of this conversion regulates the effect of vitamin D on bone, gut and kidney. The importance of sunlight has been observed throughout the history of tuberculosis treatment as an alternative treatment of pulmonary and cutaneous tuberculosis since as early as 1849. During the 13th Workshop consensus for vitamin D nutritional guidelines, held in Victoria, British Columbia in 2007, it was pointed out that half of the elderly in North America and two-thirds of the elderly in the rest of the world do not have enough circulating vitamin D to maintain healthy bone density through calcium metabolism, thus increasing their risk of fractures, major cancers, problems of tooth attachment, and of reduced muscle strength. Research reported at that meeting showed that adults must maintain a much higher blood vitamin D level than children, i.e., approximately 75 nM/L. Low levels of circulating vitamin D were reported for an elderly Brazilian population, which has a deficiency (25 nM/L; 15.4%) or insufficiency (25-50 nM/L; 41.9%) of vitamin D (1). To date, there are no reports regarding the serum level of vitamin D in the Brazilian population comparing age, gender, sun exposure, food intake, and related disease. However, the general consensus is that a desirable serum concentration of vitamin D is 50 nM (or 20 ng/mL). A recent nutritional analysis of food intake demonstrated vitamin D deficiency in all major geographic regions of Brazil that could result in up to a 6-fold decrease in presumable normal serum levels of vitamin D, independent of socio-economic level (2). The correlation between serum level of vitamin D and risk for tuberculosis latent infection or disease development has been described in African immigrants living in Australia (3). Lower mean vitamin D levels were observed in latent infection compared with individuals with no M. tuberculosis infection and lower in tuberculosis patients. Higher vitamin D levels were associated with lower probability of any M. tuberculosis infection (4). These results and other studies in sunny countries have shown that the existence of vitamin D deficiencies might be related to some risk factors, such as inadequate food source, sunshine exposure, sun block use, religion, pollution and, finally, skin color (ethnicity). These factors explained how it was possible in sunny countries to detect a vitamin D deficiency (4). This deficiency associated with the existent environmental conditions and parasitic infection seemed to explain the increased risk for tuberculosis development (4).

Vitamin D metabolites are transported in the blood by vitamin D-binding protein (VDBP) and are cleared from the blood by the liver, where they are converted into 25-hydroxyvitamin D3 (calcidiol) and released back into the blood as the major circulating form of vitamin D. 25(OH)2D3 is taken up by the kidneys, where it undergoes further conversion into the most active hormonal form of vitamin D (1,25(OH)2D3), and is released back into the blood.

Recently, the role of vitamin D has been emphasized not only in bone mineralization and in anti-tumor activity but also, a large body of work has accumulated supporting an important role for vitamin D in multiple organ systems as well as physiologic and molecular processes. VDR are distributed ubiquitously, and by binding with its receptor, vitamin D initiates a series of events that can affect cellular proliferation and differentiation, inflammation, the immune and endocrine systems, insulin resistance, and lipid metabolism (5). Vitamin D metabolites are important immunomodulatory hormones, which activate monocytes and suppress lymphocyte proliferation, immunoglobulin production, and cytokine synthesis. Moreover, this hormone has been shown to act as both an up-regulating agent during natural immunity via the enhancement of phagocytosis by monocyte/macrophage populations and a down-regulator during acquired immune response via an inhibitory effect on MHC class II expression by professional antigen-presenting cells (6). Apparently 25(OH)2D3 has no direct antimycobacterial action, but its active metabolite 1,25(OH)2D3 is able to block the maturation of myeloid dendritic cells (DCs) by reducing the expression of co-stimulatory molecules. This inhibits secretion of IL-12 and increases IL-10 production as well as inhibits the secretion interferon-γ (IFN-γ) and IL-2 by T cells and stimulates Th2 cell development with increased production of IL-4, IL-5, and IL-10 (7). Thus, these data indicate that the 1,25(OH)2D3 does not mediate protection via those cytokines. It is known that the modulation of 1,25(OH)2D3 by binding to nuclear VDR up-regulates protective innate host responses, including the induction of nitric oxide synthase (NOS2). However, the mechanisms that regulate the expression of NOS2 and the role of 1,25(OH)2D3 in tuberculosis infection are not known. Some investigators have reported that the human macrophage-like cell line acquires the ability to produce substantial amounts of nitric oxide (NO) on stimulation with lipopolysaccharide or 1,25(OH)2D3 in the absence of activating factors such as IFN-γ. This suggests that 1,25(OH)2D3 acts to suppress the growth of M. tuberculosis in these cells and that vitamin D-induced NO production may play a role in the host defense against human tuberculosis (8). In addition to the NO production induced by 1,25(OH)2D3, this metabolite also up-regulates the expression of 1α-hydroxylase, the enzyme that metabolizes 25(OH)2D3 to 1,25(OH)2D3 due to ligation of Toll-like receptor (TLR)2/1 in macrophages stimulated by M. tuberculosis antigens and up-regulates the cathelicidin gene. 1,25(OH)2D3 regulates the expression and activation of antimicrobial peptides in keratinocytes and monocytes in the epidermis and provides a stimulus for rapid production of a chemical antimicrobial shield with the expression of LL-37, a human antimicrobial peptide belonging to the cathelicidin family (9). Cathelicidins are relevant to defense against microbes including Gram-positive and -negative bacteria as well as fungi and certain viruses and it is increased with increasing concentrations of 1,25(OH)2D3 (9). The addition of a VDR antagonist VAZ (ZK159222) inhibited the induction of cathelicidin mRNA by greater than 80%, and consequently the antimicrobial activity was reduced by about 70% (10). The increase in TLR2/1 enabled the cells to respond to microbial stimulation and also amplify the synthesis of 1,25(OH)2D3 (Figure 1). The active metabolite of vitamin D, 1,25(OH)2D3, suppresses growth of M. tuberculosis in vitro and this effect may be facilitated by TLR in vivo (10). Thus, there is an elegant system of control of innate immunity by 1,25(OH)2D3. The most reasonable explanation suggests that vitamin D stimulates the immune cells to resist M. tuberculosis infection by mediating protection against tuberculosis by `nonclassical' mechanisms.

Vitamin D receptor

Vitamin D exerts its action through VDR, a nuclear hormone receptor when activated by its ligand 1,25(OH)2D3, that is present on monocytes and activated T and B lymphocytes (Figure 1) (5). Extensive data indicate that there are two mechanisms of action. One involves the activation of nuclear VDR and transcriptional regulation of many vitamin D-responsive genes. The other involves activation of non-genomic signal transduction pathways in target cells. This second mechanism is likely to involve a membrane VDR. A candidate form of VDR that is a 64.5-kDa protein from chick epithelium has been described recently, which specifically binds 1,25(OH)2D3 and is responsible for some of the rapid cellular actions of 1,25(OH)2D3 (11). Is it possible that this membrane receptor for 1,25(OH)2D3 acts with ligand-binding properties that are different from those of the nuclear/cytosol receptor? Studies suggest that the ligand-receptor complex mediates the signal transduction of the hormone via opening of voltage-gated Ca21 channels to initiate the biological response(s) (12).

The VDR gene is located on the long arm of chromosome 12 (12q12-14) and is composed of 10 exons, the first of which is not transcribed (13). The VDR gene is a candidate locus for susceptibility to different diseases, such as prostate cancer, inflammatory bowel disease, osteoporosis and tuberculosis, due to allelic variation that affects the activity of the receptor and subsequent downstream vitamin D-mediated effects such as calcium absorption, excretion, and modulation of cellular proliferation and differentiation. The variation in the ability to synthesize vitamin D, including polymorphisms in the VDR gene, may be a contributing factor to increase tuberculosis susceptibility. Four of these VDR polymorphisms can be distinguished by digestion with restriction enzymes. Three of them do not change the translated protein (TaqI, ApaI and BsmI). The polymorphisms recognized by BsmI (BB, Bb and bb) and ApaI (AA, Aa and aa) are located in intron 8 of the VDR gene and the one recognized by TaqI (TT, Tt and tt) is located in exon 9, that leads to a silent codon change, with ATT and ATC, both coding for isoleucine and has been associated with increased VDR mRNA stability. The genotype tt, detected with the TaqI restriction enzyme, is associated with decreased risk of tuberculosis. The fourth VDR polymorphism, recognized by FokI restriction enzyme (FF, Ff and ff), results from a CT transition that creates an alternative initiation codon (ATG), three codons from the downstream start site. The VDR encoded by the f allele from the FokI restriction enzyme recognition is increased in length by three amino acids, and the transcription of this allele is 1-7 times less efficient than the F allele and can alter the amount of VDR produced (13).

The association or not of VDR polymorphism with tuberculosis in several geographic areas was discussed by Lewis et al. (14). They evaluated VDR gene polymorphism in different ethnic population studies published prior to August 2004. The tt genotype was underrepresented in tuberculosis patients from Gambia (15) and India (16), but in patients from Peru (17) the Tt and FF genotypes were associated with faster sputum culture conversion after initiation of therapy. The same was seen in China (18), where the FF genotype was associated with resistance to tuberculosis. After this meta-analysis description other results appeared describing VDR polymorphism in Chinese Tibetans (19) in which FokI polymorphism of the VDR gene might be associated with tuberculosis, but there was no evidence that the TaqI polymorphism was associated with the disease in this population. A larger study conducted by Babb et al. (20) with a South African population reported that the VDR genotypes might affect the severity of disease or the ability of the treated cases to recover (positive sputum conversion or smear stain to negative), but no association was demonstrable between tuberculosis and VDR polymorphisms. When they used diplotypes and haplotypes to analyze the results, only a weak association was detect between the VDR haplotypes and tuberculosis susceptibility. They found that the FokI-ApaI-TaqI haplotype (`FAT') tended to be associated with tuberculosis and may be a risk factor, whereas the `FaT' haplotype is probably protective. Regarding the role of VDR polymorphisms in disease caused by Mycobacterium leprae, Goulart et al. (21) reported that there was a higher risk of leprosy when individuals carry the combination of tt genotype from TaqI polymorphisms in the VDR gene and negative Matsuda test, demonstrating a possible synergistic role of these two variables in leprosy susceptibility via effects on cellular immunity in a Brazilian population.

While there is evidence that vitamin D promotes macrophage killing of M. tuberculosis, the effector mechanisms are not clear, and the association of VDR polymorphisms with susceptibility to tuberculosis remains unproved. As described in Lewis et al. (14), larger studies are required to determine whether VDR polymorphisms play a role in genetic susceptibility to tuberculosis worldwide. A sample size of more than 2000 cases and controls is required to demonstrate if VDR polymorphism is associated with tuberculosis. This large sample size would be necessary to have enough power to detect specific polymorphisms related to tuberculosis, because any association will be diluted due to the extended linkage disequilibrium with the specific functional allele. Most studies conducted until now were in ethnically and geographically different populations, thus, they were underpowered and impossible to reach any conclusions by examining the alleles separately. The VDR polymorphisms contain many more single nucleotide polymorphisms (SNPs) than the four normally measured and it is necessary to conduct family-based studies that will allow research to construct reliable haplotypes and eliminate the possibility of population stratification and other risk factors such as gender, HIV status, occupation, and environment issues.

Vitamin D-binding protein

VDBP is a multifunctional, highly expressed, polymorphic serum protein that was initially named the group-specific component of serum (Gc-globulin). VDBP, a 458-amino acid polymorphic serum protein, is the major plasma carrier of vitamin D3 and its metabolites and ensures that vitamin D is transported to the liver, 25(OH)2D3 to the kidney, and 1,25(OH)2D3 to target cells and organs. VDBP is encoded by the Gc gene, a member of a multigene cluster that includes albumin and α-fetoprotein genes, located at chromosome 4q11-q13. Sequence variations at codons 416 and 420 in exon 11 of the Gc gene give rise to major electrophoretic variants of VDBP, termed Gc1 fast (Gc1F), Gc1 slow (Gc1S) and Gc2 (22). These variants differ by amino-acid sequence, as well as by attached polysaccharides. Combinations of the three VDBP or Gc variants result in six common circulating phenotypes: Gc1F/Gc1F, Gc1F/Gc1S, Gc1S/Gc1S, Gc1F/Gc2, Gc1S/Gc2, and Gc2/Gc2 (23). DBP polymorphism (Gc phenotype) is related to the VDBP concentration and vitamin D status, as described by Lauridsen et al. (23). These authors showed a strong correlation of higher, intermediate and lower circulating levels of 25(OH)2D3 and 1,25(OH)2D3 with Gc1-1, Gc1-2 and Gc2-2 phenotypes, respectively, in Danish Caucasian postmenopausal women population. In addition to storing and transporting vitamin D3 hormones, VDBP also affects their pharmacokinetics (24) and plays a role in the sustained effective conversion of cutaneously derived vitamin D3 into 25(OH)2D3. In addition to transporting vitamin D3, VDBP has multifunctional properties, including an important role in macrophage activation that is distinct from its vitamin D-binding ability. Variations in this property could affect the functioning of the immune system, as shown for DBP knockout mice that exhibited an impaired immune response to bacterial infections (24). VDBP has been shown to enhance the leukocyte chemotactic activity of activated complement peptides, which are the precursor of the macrophage-activator factor (MAF). Indeed Gc-MAF influences macrophage chemotaxis (24).

A role of DBP polymorphism in autoimmune diabetes mellitus and infectious disease in Polynesia and Japan (25) has been suggested. Until now, only one study evaluated the DBP phenotype in tuberculosis patients and no differences were seen among patients and the control group. In that study, a 33% frequency of Gc2 in tuberculosis patients was slightly but not significantly higher than in the control group (26%), and this elevation was at the expense of both Gc1F and Gc1S alleles (26). Further studies are necessary to understand the physiological role of VDBP and its phenotypes on susceptibility to tuberculosis and other diseases.

The Toll-like receptors

The TLRs represent a group of single-pass transmembrane receptors observed from Drosophila to mammals. They are expressed on innate immune cells and are the key sensors for a broad spectrum of pathogen-derived molecules, and are critical in shaping host-pathogen interactions (Figure 1). All bacteria contain ligands for many TLRs and the ligation of TLRs by these pathogen-specific ligands initiates a signal transduction pathway in the host cell that culminates in the activation of nuclear factor-kappa B (NF-κB) and the induction of cytokines and chemokines (27), which are crucial for eliciting the adaptive immune response against pathogens. The production of IL-12, TNF-α and NO is induced mostly by macrophages soon after innate recognition of mycobacteria through TLRs (28). TLRs have been recognized to be the principal mediators of the pro-inflammatory signal, TNF-α, induced by whole M. tuberculosis (28). Indeed, TLR2 has been shown to be involved in response to mycobacteria, specifically to the 19-kDa lipoprotein and lipoarabinomannan from M. tuberculosis. Interestingly, the triacylated mycobacterial lipopeptide is recognized by dimmers of TLR2 and TLR1, whereas similar diacylated compounds are recognized by dimmers of TLR2 and TLR6 (29). However, recent data point to the deactivation of macrophage cytokine induction by M. tuberculosis proteins. ESAT-6, a member of a unique family of proteins present in M. tuberculosis, probably attenuates the innate immune response by dampening the production of the IL-12 p40, TNF-α and NO (30). Recently, it has been shown that ESAT-6 and some peptides were able to dampen TLR2 signaling by preventing assembly of the cytosolic MyD88-dependent signaling scaffold (31). However, other members of the TLR family could be activated by M. tuberculosis, with or without TLR2 interaction. DNA from mycobacteria contains stimulatory CpG motifs that activate TLR9 (31) and up-regulate Th1 response.

The role of TLR in resistance to M. tuberculosis was suggested initially by the fact that MyD88-deficient mice are more susceptible to M. tuberculosis infection (31) and by the observation that TRL2/TLR1 reduced the viability of intracellular M. tuberculosis in human monocytes and macrophages, but not in monocyte-derived DCs (10). Liu et al. (10) also reported that TLR induces up-regulation of the VDR, 1α-vitamin D hydroxylase (enzyme that converts inactive to active vitamin D) and CYP27B1 gene expression in monocytes and macrophages. These actions permit the macrophages to internalize serum VDBP-bound 25(OH)2D3 from the extracellular fluid by facilitated endocytosis and use that 25(OH)2D3 as substrate for the upregulated CYP27B1 (expression gene of 1α-vitamin D hydroxylase). Because VDR is functional when exogenous 1,25(OH)2D3 is added, it was hypothesized that TLR2/1 induction of CYP27B1 and the conversion of 25(OH)2D3 to 1,25(OH)2D3 by 1α-vitamin D hydroxylase could represent a key step of the TLR pathway. Because TLR affects the interaction of innate and adaptive immune response against M. tuberculosis, a TLR2 agonist has been used as "natural adjuvant" to enhance Th1-induced response, inducing pulmonary IFN-γ secreting-CD4+ T cell (32), or in prime-boost vaccine, inducing antigen specific IFN-γ and IL-2 responses in mice (33). The structural integrity of TLR signaling components is essential for immunological protection from infection. Changes in structure of TLR signaling molecules that result from SNPs are often associated with susceptibility to various infectious diseases. The fact that TLR2-deficient mice are highly susceptible to M. tuberculosis suggests that polymorphisms that affect TLR2 expression or structure may impair the susceptibility to tuberculosis in the human host. Approximately 89 SNPs have been reported in the TLR2 gene; 26 of them are in the 5'-untranslated region, 17 are in the 3'-untranslated region, 29 are located in intronic parts of the gene, and 17 modify bases of the third exon of TLR2. Six non-synonymous SNPs of the TLR2 gene change amino acids in the cytosolic part of this receptor. However, only two have been linked to reducing NF-κB activation and increasing risk of infection. The first consists in a change of C to T replacing arginine (Arg; R) with tryptophan (Trp, W) at position 677, abolishing the binding with MyD88 with TLR2. This specific polymorphism located within the bb loop of TLR2 (Arg677Trp) abolishes activation of NF-κB in response to M. tuberculosis (34), resulting in decreased IL-12 serum level production by 677W carriers. The second functional TLR2 variant consists of a G to A substitution, which substitutes an arginine for glutamine at position 753. The TLR2 753Q seems to be associated with an increased risk of developing tuberculosis for carriers the AA and AG genotypes (35). Recently, Thuong et al. (36) described a strong association of SNP T597C TLR2 with susceptibility to military tuberculosis patients from Vietnam. Further association was described among Koreans regarding the microsatellite polymorphisms in intron II or TLR2 (37). In addition, TLR1 polymorphism in a non-synonymous region (I602S) could be associated with TLR1/2 heterodimer binding sites to mycobacterial lipopeptide, since individuals with 602II genotype produced substantially more IL-6 than those with the 602SS variant (38). However, these preliminary studies need to be repeated on a large scale in distinct populations. Currently, the polymorphism in TLR2 might be an important risk factor for disease progression, and that the functional consequences of different alleles may explain some of the variation in cytokines and NO expression and clinical forms of the disease. Understanding gained from knowledge of the effects of different alleles can contribute to the design of new therapeutic strategies including vaccines.

Nitric oxide synthase 2

NO is a free radical and second messenger that has been shown to be important in the development of several diseases, including tuberculosis. NO plays a major role in the pulmonary host-defense mechanism in response to infections and is implicated in bacteriostatic as well as bactericidal processes. NO is formed when the guanine group of the essential amino acid L-arginine is split forming NO and L-citrulline. The reaction is catalyzed by a class of enzymes called NO synthases (NOS). NO is produced by three different NOS: NOS1, NOS2 and NOS3. NOS1 is constitutively expressed due to fluctuations of Ca2+ and produces relatively small amounts of NO. The alveolar macrophages induced by mycobacteria are capable of producing TNF-α, IL-1β. These cytokines along with IFN-γ produced by T-cells can induce NO via action of NOS2 (Figure 1). Thus, the NO and radical nitrogen intermediate can kill and/or inhibit intracellular pathogens like mycobacteria. In contrast to murine models of tuberculosis, there is greater controversy about the role of NO killing or limiting the growth of M. tuberculosis in humans. It has been proposed that NO produced by tuberculosis-infected human macrophages and by epithelial cells is also antimycobacterial against M. tuberculosis (10). Nevertheless, a report from our group indicates that the alveolar macrophages from the lungs of patients with tuberculosis express NOS2 in potentially mycobactericidal amounts and that this NOS2 can kill mycobacteria in vitro (39). NOS2 expression is not usually detected in human macrophages from cell lines or macrophages derived in vitro from blood monocytes of health humans. However, alveolar and tissue macrophages from the lungs of tuberculosis patients show high levels of functional NOS2. Thus, NOS2 may represent a pivotal mechanism that protects against tuberculosis. Investigation is hampered by difficulty in estimating in vivo production of NO, but genetic studies provide a potential means of examining the relationship between NOS2 expression and disease outcome. NOS2 is encoded by a polymorphic gene known as NOS2A at chromosome 17q11.2-q12. Several SNPs and microsatellite polymorphisms have been described. NOS2 gene is located in a region that has been linked to susceptibility to tuberculosis. It is known how important the biological and genetic action of NOS2 is in the immune system, because of that; the SNPs in this gene have been reported in many populations around the world. The SNPs in the promoter region of the encoding gene (-954GC, -1173CT, -1659 AT) have been shown to increase NO synthesis (40). This region in the human gene is situated from -0.7 to -2.6 kb upstream of the transcription start and contains important DNA motifs for binding of NF-κB, activator protein 1, signal transducer and activator of transcription protein 1, and NF-κB repressing factor (41). Another important region lies between -2.5 and -950 kb upstream, and is characterized by a number of polymorphic elements like SNPs and repeat regions. It is this region in which various mutations are associated with NOS2 expression in vivo (40). The -954GC variant was originally reported in a highly endemic African malaria area (42), suggesting that this mutation originated as a consequence of selective pressure of Plasmodium. The G allele has been shown to be absent from Caucasian populations (42) as well as from the Peruvian population (43). In Mexicans, in whom the frequency of G allele was ≈5%, this SNP was not associated with tuberculosis (44). There are no further reports regarding NOS2 polymorphism frequency in Brazilian populations. Further work is in progress to determine if NOS2A gene polymorphism is associated with tuberculosis in this population.

Interferon-γ

IFN-γ is essential to a relationship between innate and adaptive immunity in combating M. tuberculosis infection (Figure 1). Active tuberculosis is characterized by lower peripheral blood mononuclear cell production of IFN-γ than latent infection, and local and systemic IFN-γ levels correlate with the severity of disease. Perhaps the most relevant mechanism is the activation of mononuclear phagocytes by the cytokines, particularly by IFN-γ. This cytokine is produced as a consequence of antigenic activation by natural killer (NK) and T cells, especially T helper 1 (Th1), and its production is increased by antigen-presenting cell-derived cytokines such as IL-12 and -18. IFN-γ has regulatory effects in both innate and adaptive immune responses, which include activation of mononuclear phagocytes and NK cells, increased expression of major histocompatibility complex I and II, induction of CD8+ lymphocyte maturation into cytotoxic cells, and promotion of the immunoglobulin isotype switch in B cells towards immunoglobulin G1 and G3 in humans (45).

Production of IFN-γ is genetically controlled, and there are two well-known polymorphisms in the IFN-γ gene. It has been reported that the 12 CA repeat microsatellite allele in the noncoding region of the first intron is associated with a high level of in vitro cytokine production (46). The same group has also reported complete linkage disequilibrium between the 12 CA repeat allele and the presence of the T allele at position +874 T/A IFN-γ from the translation start site. This polymorphism lies within a binding site for the transcription factor NF-κB, and electrophoretic mobility shift assays showed specific binding of NF-κB to the allele sequence containing the +874 IFN-γ T allele (47). As this transcription factor induces IFN-γ expression, +874T IFN-γ T and A alleles probably correlate with high and low IFN-γ expression, respectively (48). In addition, Ottenhoff et al. (49) discovered several families with Mendelian susceptibility to mycobacterial disease that has mutations in one of two subunits of the IFN-γ receptor gene (IFN-γ R1 and IFN-γR2). More recent data suggested that a more common polymorphism at position +874 is associated with risk of tuberculosis in different populations (48,50). IFN-γ acts as a regulator of gene expression through activation by a receptor complex comprising two subunits, each encoded by a different gene: IFN-γR1 on chromosome 6, region q23-24 and IFN-γR2 on chromosome 21 region q22.1-22.2. Homodimers of IFN-γ interact with both receptor proteins, leading to receptor dimerization. None of the three molecules (two receptor proteins and the cytokine itself) play a redundant role in ligand-activated receptor signaling.

The AA genotype in +874T/A region is associated with increased tuberculosis susceptibility in Spanish and South African populations (48,50), but not in Croatian and North American populations (51,52). Few studies have been conducted in the Brazilian admixed population regarding IFN-γ polymorphisms at position +874 and its correlation, if any with infectious diseases. Amim et al. (53) showed an important association in +874 AA genotype with the occurrence of active tuberculosis disease, independent of tuberculosis skin test status. However, the selection of these tuberculosis cases (from the Southwest region of Brazil) and healthy controls (from the Central-west region of Brazil) may cause a misunderstanding, because the Brazilian population is considered admixed. The genetic background between these two geographic areas should be different and may interfere in the genetic background analysis and results.

Perhaps lower IFN-γ secretion (inferred from the lack of T at +874 position) might influence replication of M. tuberculosis, thus influencing the outcome of the tuberculosis diagnosis (51) and disease. The association of IFN-γ genotype +874 AA with disease in several populations confirms a significant role of genetic variation at the IFN-γ locus and provides more detailed understanding of the genetic mechanisms underlying the association with disease. The absence of association with IFN-γ R2 polymorphism in the African population was interesting, but firm conclusions cannot be drawn without additional studies. Furthermore, the association of disease with IFN-γ R1 polymorphisms in the same population was novel, and together, these findings support the hypothesis that a genetically determined variation in both IFN-γ production and responsiveness influences the disease. Thus, despite the remarkable importance of IFN-g production in M. tuberculosis growth control, published studies can only suggest that polymorphism in this gene might influence susceptibility to tuberculosis in some populations.

On the other hand, an important IFN-γ inducer may interfere in the multigenic tuberculosis resistance or susceptibility. IL-12, a heterodimeric pro-inflammatory cytokine produced by monocytes, macrophages, DCs and B lymphocytes, modulates Th1 differentiation, having a clear interface in IFN-γ production. The importance of IL-12 as an IFN-γ inducer lies not only in its high efficacy at low concentration but also in its synergism with many others activating stimuli. IL-12-induced IFN-γ production requires the presence of low concentrations of TNF-α and IL-1, which are usually produced in an autocrine manner by T and NK cells. The gene for the IL-12 p40 subunit is located on chromosome 5q 31.1-33.1. The SNP in the gene responsible to express this subunit was first described by Hall et al. (54). They showed a frequency of 60, 36.9, and 3.1% for genotypes AA, AB and BB, respectively, in a Caucasian population in the United Kingdom. Recently, several reports have described the IL-12B polymorphism in black and white North American populations with no tuberculosis susceptibility correlation. However, four SNPs described at positions 641 A-G, 684 C-T, 1094 T-C, and 1132 G-C can cause three missense variants (Q214R, M365T and G378R) and one synonymous substitution in the extracellular domain of the IL-12Rβ1 gene (55). These investigators reported that the association of R214-T365-R378 allele (allele 2) is over-expressed in Japanese tuberculosis patients with the homozygosis for R214 - T365 - R378 (the 2/2 allele) being significantly associated with tuberculosis. In this manner, IFN-γ and IL-12 gene polymorphisms and further protein expression may play an important role during the innate and adaptive immune response to M. tuberculosis infection and disease, driving a protective long life latent infection or disease installation. Further investigations are necessary to identify the haplotype distribution in IFN-γ and IL-12 gene polymorphism.

Cross talk between TLR2, NOS2, IFN-γ, VDR and DBP gene expression and immunity to M. tuberculosis

It is known that genetic defects can lead to increased risk of mycobacterial infections (56). Thus, the identification of the genes where mutations lead to extreme susceptibility will help us to identify essential components of the innate and/or adaptive human immune defense to M. tuberculosis. Several SNPs or defined haplotypes have been studied to identify possible candidate(s) allele(s) that may cause subtle changes in genetic function that could account for individual variation in susceptibility to tuberculosis.

For this reason, many studies described in this review have looked for an association of tuberculosis susceptibility with polymorphisms in genes encoding specific elements of the immune system thought to be important in controlling mycobacterial infection.

One of the first lines of defense of the immune response is the recognition and uptake of microorganisms by phagocytes and several different pattern recognition receptors in these cells including the TLR. Pathogen binding to specific TLRs or combinations of TLRs may recruit different adaptor proteins allowing a specific signaling cascade and gene activation programs. A central function of TLRs is to directly activate acute anti-microbial defense systems (57). These receptors, after recognition, can stimulate signalizing pathways that activate the innate immune response, cytokine production and the process of adaptive immunity. Activation of TLRs from specific mycobacterial lipoproteins can stimulate macrophages to express inflammatory cytokines (TNF-α, IL-6, IL-12 and IFN-γ) and induction of anti-microbial genes, such as the expression of NOS2 and final production of NO (57). The activation of TLRs can also up-regulate the expression of vitamin D receptor and vitamin D-1-hydroxylase genes (enzyme that metabolizes 25(OH)2D3 to 1,25(OH)2D3), leading to induction of the antimicrobial peptide cathelicidin and killing of intracellular M. tuberculosis (8). In addition, 1,25(OH)2D3, by itself, can modulate the immune response by its nuclear receptor (VDR), where it up-regulates protective immune host defense by induction of NOS (58) and cathelicidin (9) as well as down-regulation of IFN-γ gene expression by down-regulating its promoter (59). It is known that 1,25(OH)2D3 is present 1000-fold less in peripheral blood than 25(OH)2D3, and the release of these forms in the granuloma space by VDBP may modulate T cell responses. The induction of 1,25(OH)2D3 can inhibit T cell proliferation and suppression of B cell immunoglobulin production initially by inhibition of IL-2, GM-CSF and IFN-γ (7). However, 1,25(OH)2D3 suppresses Th1 cytokine profile favoring Th2 cells and can activate regulatory T cells (7), modulating the host response to M. tuberculosis infection. On the other hand, the TLR signaling in macrophages and DC leads to secretion of IL-12 that skews the resultant T-cell response towards Th1 phenotype. Th1 cells secrete IFN-γ, which has multiple functions in activating macrophages and enhances their phagocytic and microbicidal abilities (60). IFN-γ is, also, critical for further expression and production of NOS2 in adaptive response (39) (Figure 1). In addition, IFN-γ secreted by T cells inside the granuloma potentiates the up-regulation of 1α-hydroxylase and inhibits the key enzyme in 1,25(OH)2D3 inactivation (24-hydroxylase) (58).

Therefore, it is possible to suggest a link between TLR, vitamin D (receptor and transporter) and specific cytokines expressed in the immune response, such as IL-12 and IFN-γ. Consequentially, possible defects in these molecules caused by specific polymorphisms might be associated with susceptibility to tuberculosis (34,35,37).

Conclusions

Several gene polymorphisms are being described in association with human tuberculosis susceptibility; however, few of them are clearly described in a genetically distinct population. Tuberculosis incidence, socio-economic status, nutritional condition, exposition grade, cultural aspects, bacilli virulence, M. tuberculosis strain distribution, access to healthcare and medication should also be considered. There is always a need for better understanding of the immunopathology of tuberculosis through the knowledge of the precise genetic mechanisms controlling human infectious diseases. Furthermore, SNP description alone will not be sufficient to describe susceptibility to tuberculosis in a broad diverse population, and thus, functional gene studies need to be done. A real challenge is to associate candidate genes with a biologically plausible mechanism that explains the epidemiological data for tuberculosis in which only 10% of the infected individuals will develop tuberculosis. We believe that from the genes involved in M. tuberculosis control, TLR2, NOS2, IFN-γ and vitamin D, their carrier and their receptor may generate a potent resistant haplotype and could be highlighted as powerful tools for understanding the interplay between innate and adaptive host resistance. The relationship between M. tuberculosis virulence strains and host genetic variability will further help us to design new vaccines and immunotherapy.


Figure 1. Scheme of the innate and adaptive immune response phases against mycobacteria. The figure identifies all polymorphic proteins involved during and after entrance of mycobacteria into the cell. The first step achieved by the mycobacteria is entering the macrophages (MΦ) (1). After this step, the MΦ are activated by cytokines (IFN-γ) to kill the mycobacteria (2). This phase is marked by important polymorphisms like nitric oxide production inside the phagocyte (3). If the MΦ survives, the adaptive immune system is activated. This system establishes the granuloma, an important structure formed to contain the dissemination of the mycobacteria. The diagram illustrates the MΦ, giant cells (GC-epithelial cells fusion), CD4, CD8 T and natural killer cells (NK) harboring intracellular mycobacteria from the centre of granuloma (4 and 5). The IFN-γ activates monocytes from circulation to MΦ, which starts to kill the intracellular mycobacteria via reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) (6). The MΦ associated with the other antigen presenting cells (GC) present antigens to T cells and activate them to produce a variety of cytokines and chemokines. The chemokines recruit additional cells from circulating to kill the mycobacteria. Granulomas are a result of CD-mediated delayed type hypersensitivity reaction within parenchymal tissues (7). Accordingly, the same system that is responsible for bacterial growth decrease (host defense) is also intrinsically associated with tissue damage through granuloma formation and necrosis. If you have any disturbance in this system, you could be more susceptible to tuberculosis development. 1,25(OH)2D3 and the vitamin D receptors (VDR) then together induce the expression of the gene encoding the human antimicrobial peptide LL-37. Circulating monocytes are activated by TLR2/1 agonists present on specific microbes. The genes encoding VDR and CYP27B1 are induced. CYP27B1 converts 25(OH)2D3 from the circulation to 1,25(OH)2D3, joins with VDR and activates the gene encoding LL-37, leading to an increase in cellular LL-37 and enhanced microbicidal activity of the phagocyte (8). IL = interleukin; IFN-γ = interferon-γ; TNF-α = tumor necrosis factor α; TLR = Toll-like receptor; MBL = mannose binding lectin; HLA = human leukocyte antigen; Th1 = T helper 1; TGF-β = tumor growth factor β; VDBP = vitamin D-binding protein; NRAMP-1 = natural resistance-associate macrophage protein 1. M-CSF = macrophage colony-stimulating factor.

[View larger version of this image (134 K JPG file)]


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Correspondence and Footnotes

Address for correspondence: A.C.C.S. Leandro, FIOCRUZ, Av. Brasil, 4365, 21045-900 Rio de Janeiro, RJ, Brasil. Fax: +55-21-2598-9988. E-mail: crisasantiago@gmail.com

Research supported by the Programa de Desenvolvimento Tecnológico em Insumos em Saúde (PDTIS), Instituto de Pesquisa Clínica Evandro Chagas (IPEC), Oswaldo Cruz Foundation, Rio de Janeiro, RJ, Brazil. A.C.C.S. Leandro is the recipient of a PhD scholarship from CAPES. Received March 24, 2008. Accepted February 16, 2009.

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