Toll-like Receptor 2 Gene Polymorphism, Serum Cytokines and Susceptibility to Disease Severity or Treatment Response of Pulmonary Tuberculosis
Toll-like Receptor 2 Gene Polymorphism, Serum Cytokines and Susceptibility to Disease Severity or Treatment Response of Pulmonary Tuberculosis
Infection with Mycobacterium tuberculosis remains at epidemic levels globally. Innate and
 adaptive immune responses evolve as protective mechanisms against mycobacterial infection in
 humans. Toll-like receptors (TLRs) are transmembrane proteins characterized by an
 extracellular leucine-rich domain that participates in ligand recognition and an
 intracellular tail. TLRs are the first defense system to detect potential pathogens, initiate
 immune responses and form the crucial link between innate and adaptive immune systems.
 Stimulation of TLR initiates a signaling cascade that involves a number of proteins, such as
 MyD88 and IL-1 receptor-associated kinase. This signal cascade leads to NF-κB activation,
 which induce the secretion of pro-inflammatory cytokines.

 TLR2 is a family of TLR family and has been reported to be the principle mediator of
 macrophage activation in response to mycobacterium. Growing amounts of data suggest that the
 ability of certain individuals to respond properly to TLR ligands may be impaired by single
 nucleotide polymorphisms (SNPs) within TLR genes, resulting in an altered susceptibility to,
 or course of, infectious disease. The genetic polymorphism of TLR2 (arginine to glutamine
 substitution at residue 753 (Arg753Gln)) has been associated with a negative influence on
 TLR2 function, which may, in turn, determine the innate host response to mycobacteria. In
 addition, another polymorphism (Arg677Trp) of the TLR2 was reported to be associated with
 susceptibility to tuberculosis in Tunisian patients. Moreover, in Mycobacterium leprosy
 patients with TLR2 mutation (Arg677Trp), production of IL-2, IL-12, IFN-gamma, and TNF-alpha
 by M. leprae-stimulated peripheral blood mononuclear cell were decreased compared with that
 in groups with wild-type TLR2.

 To date, there have been no studies of the association of SNPs of TLR2 with cytokine profiles
 and clinical outcomes on M. tuberculosis. We hypothesize that polymorphisms in the TLR2 are
 associated with :

 1. increased prevalence of active pulmonary TB infection,

 2. altered levels of pro-inflammatory and anti-inflammatory cytokines in serum,

 3. clinical outcomes and presentations. We thus design a prospective case-control study to
 test this hypothesis. The frequency of TLR2 polymorphisms in both pulmonary TB patients
 and healthy controls will be determined by polymerase chain reaction-restriction
 fragment length polymorphism. Serial serum levels of IL-12, IFN-γ, and IL-10 in
 pulmonary TB patients with or without TLR2 polymorphisms will be measured by enzyme
 linked immunosorbent assay. Relationships between TLR2 polymorphisms and serum cytokines
 dynamics or clinical outcomes will be analyzed.
Infection with Mycobacterium tuberculosis (TB) remains at epidemic levels globally. One third
 of the word's population is infected with this organism, making tuberculosis the most
 prevalent infectious disease. Annually, 8 million people contract the disease, and there are
 2 million deaths worldwide each year, with increasing prevalence predicted over the next
 several decades.(1) In Taiwan, there are 24,161 reporting cases registered in the national
 tuberculosis database, and 17,660 were diagnosed as confirmed TB cases in 2004. The annual
 incidence rate of newly TB cases is 74.11 per 100,000 people.

 Immunity Against Mycobacteria M. tuberculosis is a unique pathogen in that once infection
 occurs, even in the face of in intact host immune system it is not eradicated but establishes
 a chronically persistent, or latent, state. Viable organisms remain sequestered by the host's
 immune system, though they fail to progressively replicate. Subsequently, in approximately 15
 % of those latently infected, the infection may reactivate with the development of overt,
 progressive, pulmonary disease. Impairments in the immune system may linked to reactivation,
 but much remains to be discovered about control of infection by M. tuberculosis.

 Innate and adaptive immune responses evolve as protective mechanisms against infectious
 organisms in humans. Phagocytosis of relatively small numbers of organisms initiated the host
 response in the alveolar cells.CD14 and toll-like receptors (TLRs) are examples of pattern
 recognition receptors that detect antigenic molecules on the surface of bacteria and
 mycobacteria.(2) The family of TLRs is capable of recognizing conserved microbial patterns
 including components of bacterial cell wall, microbial nucleic acids, and bacterial motility.
 TLRs are the first defense system to detect potential pathogens, initiate immune responses
 and form the crucial link between innate and adaptive immune systems. TLRs also play an
 import role in the pathophysiology of infectious diseases, inflammatory diseases such as
 Crohn's disease and atherosclerosis, possibly play a role in autoimmune disease.( 3) TLRs are
 transmembrane proteins characterized by an extracellular leucine-rich domain that
 participates in ligand recognition and an intracellular tail that contains a conserved region
 called the Toll interleukin 1 receptor (IL-1R) homology domain.(4) Stimulation of TLR
 initiates a signaling cascade that involves a number of proteins, such as MyD88 and IL-1
 receptor-associated kinase.(5) This signal cascade leads to NF-κB activation, which induce
 the secretion of pro-inflammatory cytokines. TLR2 has been reported to be the principle
 mediator of macrophage activation in response to mycobacteria. TLR2 expression is found
 primarily on alveolar macrophages and epithelial cells type Ⅱwithin tuberculous granulomas.
 Due to its ability to recognize Mycobacterium tuberculosis and its components, the expression
 of TLR2 at the site of disease is critical. (6, 7)

 Single nucleotide polymorphisms of Toll-like receptors and susceptibility to pulmonary
 tuberculosis Growing amounts of data suggest that the ability of certain individuals to
 respond properly to TLR ligands may be impaired by single nucleotide polymorphisms (SNPs)
 within TLR genes, resulting in an altered susceptibility to, or course of, infectious
 disease.TLR2 is a member of the TLR family.(8) Animal studies have shown that TLR2-knockout
 mice are more susceptible to septicemia due to Staphylococcus and Listeria monocytogenes,
 meningitis due to Streptococcus pneumoniae, and infection with Mycobacterium tuberculosis,
 suggesting the functional TLR2 polymorphisms may impair host response to a certain spectrum
 of microbial pathogens.(9) Mycobacterium tuberculosis infects 2 billion people globally, yet
 only 10 % develop clinical disease. The identification of factors that predispose to disease
 could aid the development of new therapies and vaccines.

 The genetic polymorphism of TLR2 (arginine to glutamine substitution at residue 753
 (Arg753Gln)) has been associated with a negative influence on TLR2 function, which may, in
 turn, determine the innate host response to mycobacteria. In a recent cohort study, this
 polymorphism was demonstrated to influence the risk of developing tuberculosis in Turkey
 patients.(10) In addition, another polymorphism (Arg677Trp) of the TLR2 was reported to be
 associated with susceptibility to tuberculosis in Tunisian patients (11), as well as
 lepromatous leprosy(12). More recently, polymorphisms in CD 14 and TLR2 are demonstrated to
 be associated with increased prevalence of infection in critical ill adults (13). TLR2 gene
 Arg753Lin polymorphism is also strongly associated with rheumatic fever in children.
 Moreover, this polymorphism is a risk factor for coronary restenosis.(14)

 Dynamics of cytokine generation by inflammatory cells and clinical outcomes TLRs mediate the
 activation of cells of the innate immune system leading to dynamic functions including direct
 anti-microbial activity, induction of cytokine secretion, triggering dendritic cell
 maturation, and triggering apoptosis. Macrophage phagocytosis of M. tuberculosis is
 accompanied by activation of the transcription factor NF-κB and secretion of inflammatory
 mediators that play an important role in granuloma formation and immune protection. Once
 antigen-presenting cells (alveolar macrophage or dendritic cell) have processed the engulfed
 mycobacterial protein, they present the antigens in the context of major histocompatibility
 complex (MHC) class Ⅱ surface molecules to naïve CD4+ lymphocytes. The antigen-presenting
 cell produce IL-12 to bias the immune reaction to T helper 1 (Th1) and IL-1, which stimulate
 the CD4+ lymphocytes to produce IL-2. The net result is the rapid clonal expansion of
 specific CD4+ Th1 lymphocyte, which produce interferon gamma (IFNγ), a cytokine that activate
 the macrophage that have engulfed mycobacteria to become mycobactericidal. It has been
 demonstrated that a 19-kDa lipoprotein of M.tb triggered cells to activate NF-κB and secret
 IL-2 in a TLR-2 dependent pathway(15).On the other hand, The Th2 cytokines may play roles in
 mycobacterial inflammation as well. IL-10, produced by monocytes, macrophage, and
 lymphocytes, is upregulated after mycobacterail infection, and downregulates IFN-γ
 production. Secretion of IL-10 will favor the activation of a Th2 response which is incapable
 of destroying intracellular pathogens. Activation of human monocyte derived dendritic cells
 with M.tb 19 kDa lipoprotein results in the preferential secretion of IL-12 over IL-10 (16,
 17).

 The balance between IFN-γ and IL-10 production may determine wheather effective immunity is
 established or anergy supervenes in any infected patient, and may influence clinical outcome.
 Levels of IFN-γ are higher in moderate disease than advanced diaseses, whereas advanced cases
 showed higher IL-12, and TNF-alpha compared with cases of moderate TB. In most patients,
 decreased interferon-γ production by PBMC seems to be a transient response because it is
 significantly increased in most active TB patients during and following successful therapy.
 In the TB patients with a systemic reaction, both IL-12 and IFN-γproduction by monocytes
 after challenge with a virulent M.tb strain were significantly reduced compared to PPD
 reactor group. Bronchoalveolar lavarge fluid levels of IFN-γ was also correlated with disease
 grading and decreased after anti-TB chemotherapy(18).However, some patients remain anergic in
 vivo and in vitro after chemotherapy, and the underlying biochemical mechanisms for T cell
 anergy in modulating protection or pathology in TB needs further clarification.(19)

 Toll-like receptor 2 mutation and the profiles of cytokines The production of IL-6 and IL-10
 from dendritic cells in response to M.tuberculosis is principally dependent on TLR2 (20). On
 the other hand, M. tuberculosis can induce IL-12 production in the absence of either TLR2 or
 TLR4. In leprosy patients with TLR2 mutation (Arg677Trp), production of IL-2, IL-12,
 IFN-gamma, and TNF-alpha by M. leprae-stimulated peripheral blood mononuclear cell were
 decreased compared with that in groups with wild-type TLR2. However the cells from patients
 with the TLR2 mutation showed significantly increased production of IL-10. These results
 suggest that TLR2 signal pathway plays a critical role in the alteration of cytokine profiles
 in PBMC from patients with mycobacterial infection. ( 21)

 In summary, TLR2 polymorphisms have been shown to be associated with susceptibility to
 tuberculosis in Turkey and Tunisian people. These polymorphisms have been demonstrated to
 affect cytokine production by monocytes in vitro. To date, there have been no studies of the
 association of SNPs of TLR2 with serum cytokine profiles and clinical outcomes on M.
 tuberculosis infection. We hypothesize that polymorphisms in the TLR2 are associated with :

 1. increased prevalence of active pulmonary TB infection,

 2. altered levels of pro-inflammatory and anti-inflammatory cytokines in serum,

 3. clinical outcomes and presentations. We thus design a prospective case control study to
 test this hypothesis. The frequency of TLR2 polymorphisms in both pulmonary TB patients
 and healthy controls will be determined and compared by polymerase chain
 reaction-restriction fragment length polymorphism. Serial serum levels of IL-12, IFN-γ,
 and IL-10 in pulmonary TB patients with or without TLR2 polymorphisms will be measured
 by enzyme linked immunosorbent assay at initial presentation, 2months and 6 months after
 anti-TB drugs treatment. Relationships between TLR2 polymorphisms and serum cytokines
 dynamics or clinical outcomes will be analyzed.
Inclusion Criteria:

 -  a) findings on CXR that are compatible with presentations of Mycobacterium
 tuberculosis b) clinical symptoms, such as fever, body weight loss, night sweating,
 chest pain and chronic cough, that indicate active infection of pulmonary tuberculosis
 (TB) c) microbiological diagnosis by sputum smear and culture, bronchoalveolar lavage
 fluid culture, or DNA probe examination.

 d) Resolution on CXR with anti-TB regimens e) Written informed consent form prior to
 participation into this study

 Exclusion Criteria:

 -  a) concurrent active disease of other chronic illnesses, such as lung cancer, chronic
 bronchitis and bronchial asthma b) poor physical conditions that make any examination
 infeasible c) participation in another trial with use of an investigated drug within
 on month d) use of corticosteroid or immunosuppressant drugs