Globally, HIV is a formidable public health challenge. This life threatening disease is characterized by dilapidating cellular immunity and impaired humoral immunity to some extent (Fauci and Lane 2005). The depletion of CD4+ T-cells and rapid virus replication during HIV-infection leads to a chronic persistent immune activation and HIV progression (Sulkowski. 1998). The impaired cellular immunity limits the ability of the host to control development of chronic inflammations and results in development of OIs, which is the hallmark of HIV-infection (Cohen and Walker 2001). This overwhelming of immune system eventually results in AIDS, associated with high morbidity and mortality (Sodora and Silvestri 2008). HIV-infection is known as the most potent of all known risk factors for LTBI reactivation (Sharma. 2005). Therefore, HIV-infected individuals in TB endemic nations (with high background prevalence of LTBI) not only have increased susceptibility towards M.tb infection but once infected have elevated risk of progression to an active TB disease (Selwyn.1989). HIV and TB infections when present together, due to their individual devastating effect results in higher morbidity and mortality as compared to each of them when present separately (Dye and Williams 2010). TB elevates the cellular markers of immune activation on T-lymphocytes and stimulates viral replication. This facilitates faster disease progression, development of AIDS and reduced human life expectancy. However, it has been observed that HIV-infected individuals exhibit high degree of variability in progressing to profound immunodeficiency and its associated complications. While most infected individuals develop AIDS within 10 years of infection, others may progress to immunodeficiency within 1-5 years of infection, though few remain immunocompetent even after 20 years of infection (O’Brian and Nelson 2004). It has been also been reported that AIDS patients demonstrate variability in responsiveness to anti-retroviral drugs. All these findings reveal that there is inter-individual variability in susceptibility, transmission and progression to HIV-infection. (Mallal. 2002, Rauch. 2008, Mahungu. 2009)    It has been indicated that besides the knowledge of HIV morphology, anatomy, modes of transmission and virus immunopathogenesis; it is also important to investigate the prognostic determinants responsible for generating genetic variability within a HIV-positive individual since it might aid in development of potential therapeutics or vaccines against HIV.  Since, genetic factors are important in determining individual susceptibility towards various infectious diseases and generating variations in rate of disease progression, investigations were carried out at molecular level. On detailed analysis of human DNA, it was discovered that changes in the sequence of nucleotide are common at a specific region of human genome and were determinant for genetic polymorphism. It was discovered that genetic polymorphisms are most common at regions of genome which encodes protein involved in defence mechanism of the host (Bochud. 2007). These findings led to the implication that genetic mutation occurring within the genes of immune system may be responsible for influencing individual susceptibility towards variety of infectious agents; hence, these should be investigated.  It is well established that as soon as human body is invaded by any pathogen, innate immune system acting as first line of defence system provides natural resistance to the intruders. The first few hours or the days of infection are very crucial in determining the fate of pathogen and establishment of infection. Once pathogen is established within the host, progression to clinical disease occurs. During this period innate immunity coordinates non-specific defence mechanism within the host, however, soon after adaptive immune system comes in picture and facilitates more specific immune response against the pathogen. It was therefore, contemplated that mutation within the elements of immune system may provide insight into the underlying mechanism responsible for causing variability in humans towards infectious pathogen.

TLRs, as key component of innate immune system have the capability to sense the invading pathogen through differential recognition of PAMPs. They are responsible for eliciting innate immune effectors through production of inflammatory cytokines (Medzhitov and Janeway 2000). The triggering of TLRs stimulates TLR induced transcription factor NF-kB and results in activation of adaptive immunity of the host (Kawai and Akira. 2005, Bafica . 2004). Recently, occurrence of genetic polymorphism or mutations has been reported in TLRs. Studies have demonstrated that mutations in TLRs are capable of influencing signal transduction molecules which may result in increased or decreased susceptibility to various bacterial and viral infections (Schroder and Schumann 2005). Several studies have also highlighted the association between TLR polymorphisms and increased susceptibility to or protection against several infectious diseases (Ogus. 2004, Tal. 2004, Hawn.2003). Since, variability in disease progression rate, susceptibility to OIs (associated complications) and progression to profound immunodeficiency or AIDS is quite evident in HIV-positive individuals, Therefore, it was hypothesized that TLR polymorphism might influence disease progression in HIV-infected individuals. Present study was planned with the aim to investigate common SNPs in TLRs (TLR2, TLR4, and TLR9) in HIV-infected individuals with and without TB co-infection and on healthy subjects so as to examine the influence of genetic variability in TLRs on HIV disease progression. The study also explored the mRNA expression of above mentioned TLRs among subjects.The demographic characteristics of participants demonstrated that BMI and CD4 cell counts were found to be increased among HIV+ patients and significantly decreases in HIV+ LTBI patients and were lowest among HIV+TB patients. However, the viral load demonstrated opposite pattern and was increased among HIV+TB patients and lowest in HIV+ patients. These findings implicated that HIV and TB when present together weakens the immunity of host (evident through low CD4 cell count and low BMI).

5.1 TLR polymorphisms5.1.1 TLR2 polymorphismIt has been reported that the most common polymorphism occurring within TLR2 gene among Asian and African population is Arg677Trp. The occurrence of this polymorphism modulates the function of intracellular domain of TLR2 protein, this altered protein fails to bind with MyD88 and results in low production of cytokines especially interleukin-2 (IL-2). This polymorphism causes amino acid change from arginine to tryptophan (Kang and Chae 2001). Studies have demonstrated the role of TLR2 polymorphism in association with various infectious disease caused by bacteria containing TLR agonists. A study from Korea revealed association of TLR2 polymorphism with lepromatous leprosy (Kang and Chae. 2001). The occurrence of TLR2 polymorphism in Tunisia was found to be associated with increased susceptibility to TB (Ben-Ali. 2004). In Turkey, a study demonstrated association of TLR2 polymorphism with recurrent bacterial infections in children (Kutukculer. 2007). TLR2 polymorphism in Taiwan was associated with increased susceptibility to PTB (Chen. 2010).  However, contrary to all these findings, majority of studies have failed to show that TLR2 polymorphism influences individual susceptibility to various infectious diseases and their progression rate. A study from Korea revealed that TLR2 polymorphism was not associated with increased susceptibility to lung disease caused by nontuberculous mycobacteria (Ryu YJ. 2006). Supporting, these findings another study from Korea also demonstrated lack of TLR2 polymorphism in patients with bacteraemia (Yoon et al. 2006). It was reported from

Turkey, that children suffering from rheumatic fever lacked TLR2 Arg677Trp polymorphism (Berdeli. 2005). It was also shown that TLR2 Arg677Trp polymorphism was absent from Turkish population with chronic periodontitis (Berdeli. 2007). On examining genomic DNA samples from healthy Chinese individuals it was revealed that TLR2 SNP was absent from healthy individuals (Cheng. 2007). An Indian study also failed to exhibit association of TLR2 polymorphism with increased prevalence of TB (Biswas. 2009).On investigating TLR2 polymorphism in the present study, significant difference was observed in ‘CC’ and ‘CT’ genotypic frequencies of TLR2 gene between a newly diagnosed sputum smear positive PTB patients and HIV+ patients. However, the allelic frequencies between these two groups of patients were not significantly different. Moreover, no significant difference was observed on comparing allelic and genotypic frequencies of TLR2 gene between healthy subjects and patient groups, therefore, it was indicated that TLR2 polymorphism does not influence individual susceptibility towards HIV and TB in Indian ethnic population.  The results of our study are in strong agreement with previous study from Indian subcontinent, which demonstrated lack of TLR2 polymorphism with increased prevalence of TB (Biswas. 2009).  These findings suggested that TLR2 polymorphism is absent from Indian ethnic population. After considering variable results with regard to TLR2 polymorphism it can also be proposed that genetic polymorphism may vary according to race, ethnicity or various other conditions. Another possibility for observing such a trend in our study is that there may be presence of other polymorphism within TLR2 gene, which might have counteracted or abolished the ability of TLR2 polymorphism to influence disease susceptibility. The findings of our study also indicated that TLR2 polymorphism among Indian population may not be responsible for causing any significant alteration in structure and function of TLR2 protein and as a result does not increase or decrease the host susceptibility towards various infectious diseases.

5.1.2 TLR4 polymorphismTLRs are expressed on variety of cells such as macrophages, dendritic cells, endothelial cells and smooth muscle cells. Among all known TLRs, the first and best-studied is TLR4. TLR4 after differential recognition of PAMPs and binding with its ligand leads to stimulation of cytoplasmic adaptor protein MyD88. This results in activation of key transcription factor, NF-kB, which is involved in expression of various inflammatory cytokines (Chow. 1999). Thus, TLR4-dependent pathway within the host is responsible for inducing Th1 immune response against the invading pathogen. It has been discovered that presence of Asp299Gly polymorphism within TLR4 gene leads to substitution of aspartic acid with glycine at position 299 which eventually results in altered TLR4 extracellular domain (Arbour . 2000). In the present study, statistically significant difference was observed in TLR4 allelic frequencies between healthy subjects and HIV+TB patients. It was observed that TLR4 allelic frequencies also differ significantly between healthy subjects and PTB Cat-I patients. Statistical significant difference was also observed in TLR4 allelic frequency between HIV+ patients and HIV+TB patients. A significant decrease in frequency of ‘AA’ genotype was observed in HIV+TB patients as compared to healthy subjects. On the contrary, AG and GG genotypes were significantly higher among HIV+TB patients as compared to healthy subjects. The GG genotype was exclusively found in HIV+TB group.These findings led to the implication that presence of TLR4 Asp299Gly polymorphism is associated with increased susceptibility to TB both among HIV-negative and HIV-positive individuals. There are several possible explanations for association of TLR polymorphism

with increased susceptibility to TB among HIV positive subjects in our study. TLR4 is known to trigger the interferon (α, β, γ) which tends to inhibit viral replication. However, occurrence of TLR4 polymorphism results in conformational changes in extracellular domain of TLR4, which further causes, altered interaction of TLR4 with the ligand of invading pathogen. Thus, impairing the ability of TLR4 to generate Th1 immune response (or activate interferon and therefore, resulting in increased viral replication). The defective release of pro-inflammatory mediators (cytokines) and more pronounced CD4 cell counts loss (declined cellular immunity) within the host predisposes HIV+ patient towards TB. The impaired immune response not only increases the susceptibility of host towards TB infection but also aggravates the infection already present in the host and results in severe forms of TB.  TLR4 polymorphism primarily leads to development of TB in those HIV+ patients who have more evident CD4 cell count loss. Thus, development of active TB in HIV+ patients seems to involve the effect of TLR4 polymorphism on the CD4 T-cell counts. In other words a CD4+ T-cell count is the most important prognostic factor in HIV for the development of OIs, such as TB, as demonstrated in present study. There may be other factors also to explain the observed low number of CD4+T cells in HIV+ patients bearing the TLR4 Asp299Gly polymorphism.  Studies carried out to investigate the association of TLR4 polymorphism with various infectious diseases such as Gram-negative septic shock (Newport. 2007), Gram-negative infections (Casanova. 2002), C. albicans infection (Morre. 2002), legionnaire's disease (Thomas et al. 2005), meningococcal disease (Read . 2001), periodontal disease (Folwaczny. 2004), and malaria (Mockenhaupt . 2005) produced conflicting results. It was also discovered that TLR4 polymorphism was associated with inflammatory disorders such as asthma (Qureshi. 1999) and sarcoidosis (Pabst . 2006). Several studies have recently proposed microbial translocation from the gut flora to circulation and have explained it as the major cause of systemic activation. It has been proposed that this effect is probably mediated through TLRs-dependent signalling pathways (Bafica . 2004, Brenchley . 2006). On one hand, it has been projected that TLR4 polymorphisms may be capable of modulating the systemic inflammatory response to translocated lipopolysaccharide (LPS) as several studies suggest a lower inflammatory response in individuals bearing Asp299Gly polymorphism (Ferwerda. 2008). On the other hand, it has been proposed that HIV RNA might also influence the response to microbial translocation through TLR4 signaling and may promote the cycle of immune activation (Lester. 2009). It has been demonstrated that TLR polymorphism is associated with decreased signaling response to LPS and decreased airway response to inhaled bacterial LPS (Arbour et al. 2000). It has also been revealed that TLR4 plays a crucial role in generating the immune reaction against infectious microorganisms and is also involved in the inflammatory processes of several other inflammatory diseases, such as atherosclerosis or diabetes mellitus (Shcroder. 2005). A recent study conducted in north Indian population also demonstrated the association of Asp299Gly TLR4 polymorphism with susceptibility to TB (Najmi. 2010). Although, this study supported the results of present study that presence of TLR4 polymorphism confers conformational change in TLR4 and affects its binding with the ligand (LPS) of invading pathogen. This leads to impaired immune response which increases the host susceptibility to M.tb.  However, it is to be noted that all the participants of this study were HIV-seronegative. Ours is the first study from India which has identified association of TLR4 polymorphism with TB among HIV-positive patients.     There are few other studies which have evaluated the role of TLR4 polymorphisms in HIV-1 infected population. (Papadopoulos. 2010) have demonstrated that HIV+ patients with normal or moderately decreased CD4+ T-cell counts demonstrated no major differences in

terms of infections or presence of TLR4 polymorphism. However, individuals with CD4+ T-cell counts <100 cells/mm3 and bearing TLR4 polymorphism had significant risk of developing severe infection. This effect is mainly attributed to the attenuation of an effective adaptive cellular response due to low CD4 T-cell counts and relative increase in the importance of innate host defense. These results are in agreement with the result of our study which also showed increased frequency of TLR4 polymorphism in patients who were severely affected with HIV-TB co-infection. It has been suggested that TLR4 Asp299Gly polymorphism may facilitate more intense viral replication with concomitant CD4 cell loss and increased susceptibility to infection from M. tuberculosis (Ferwerda. 2007). These findings also support the results of the present study. (Pine. 2009) demonstrated that TLR4A299Gly polymorphism is associated with elevated viral load levels in HIV-1 infected white population; however, the result of present study did not find any association of TLR4 Asp299Gly polymorphism with increased viral load among HIV-positive patients.Previous studies have also demonstrated defective cytokine release by cells of individuals with TLR4 polymorphisms (Ferwerda . 2008) which goes well with the findings of present study because it has been explained that it could have served as an effective mechanism responsible for increasing host susceptibility towards various infectious diseases, particularly TB.

5.1.3 TLR9 polymorphismTLR9 are expressed exclusively in the intracellular compartment, as membranes of endosomes (Kawai and Akira 2006). TLR9 are involved in recognition of cytidine-phosphate-guanosine (CpG) DNA motifs (microbial nucleic acids) of bacteria and viruses. (Hemmi. 2000). TLR9 have a prominent role in generating host defense mechanism. They interact with several adaptor proteins through their intracellular domain, this result in activation of transcription factor which further leads to inflammatory cytokine production and activates adaptive immunity of the host.  TLR9 are known to play an important role in HIV pathogenesis. HIV results in formation of double stranded DNA containing CpG DNA motifs during the process of reverse transcription. These double stranded DNA are transferred from cytoplasm to the nucleic acid of the host cell and integrates with the host cell DNA. The debris of phagocytised HIV infected cells containing proviral DNA resulted in activation of macrophages and TLR9 mainly located in the endosomal compartment. This process results in production of inflammatory cytokines and generating host defence mechanism (Bafica e. 2004, Kawai and Akira 2006). In TLR9 gene, increased frequency of ‘A’ allele and ‘AA’ genotype in 47 HIV+ patients who progressed to active TB/AIDS as compared to 306 HIV+ patients (no TB/AIDS) suggest that there might be increased risk of development of active TB within two year of serodiagnosis in HIV+ patients having ‘AA’ genotype in TLR9. This is the first study which investigates the role of TLRs polymorphism in the prediction of development of active TB in HIV+ patients with and without LTBI, after the initial two year of serodiagnosis. Till now, no data is available in the literature to compare these findings with others.The results of present study indicate that TLR9 polymorphism has role in HIV pathogenesis as described above. However, presence of TLR9 polymorphism must have altered their intracellular domain or resulted in low expression of TLR9 which may lead to decreased antiviral response against the pathogen and can increase susceptibility to HIV or may enhance viral replication and leads to development of OIs such as TB.

Results of the present study were consistent with the results of previous study carried out in Spain. This study demonstrated presence of TLR9 1635A/G polymorphism among HIV-positive patients (Soriano-Sarabia . 2008). However, our study was carried out in five study groups (HIV, HIV+LTBI, HIV+TB, PTB Cat-I and healthy subjects) with large sample size while previous study by Soriano-Sarabia. , Only recruited HIV+ patients and healthy subjects, in small numbers. Several other studies have also demonstrated association of TLR9 polymorphism with various infectious and inflammatory disorders. (Bochud. 2006 and Pine. 2009) demonstrated association of TLR9 polymorphism with HIV-infection. (Tao. 2007) demonstrated that TLR9 polymorphism among Japanese was associated with systemic lupus erythematosus. It has also been demonstrated that TLR9 polymorphism were significantly associated with transmission of HIV-I infection from infected mothers to their children (Ricci. 2010).

5.2 Disease progressionGaining knowledge about the prognostic determinants or factors influencing disease progression can aid the search of new therapeutic avenues. The CD4+ T cell count is known as the most significant predictor of HIV disease progression. The PVL or HIV-RNA level is another important predictor of HIV disease progression due to its inverse relationship with CD4+ T cell count pattern (increase in PVL results in rapid decline of CD4+ T cell count). Therefore, in order to study disease progression in the present study both of these parameters (immunological and virological) were recorded during two years follow-up time period. As there were increasing evidences for role of TLRs in HIV disease pathogenesis, it was planned to investigate the influence of TLRs genotypic variability on HIV disease progression. Findings of the present study indicates no influence of genetic variability in TLRs (TLR2, TLR4, TLR9) on CD4 cell count and PVL pattern of different HIV+ patient groups before and after starting their treatment (ATT and ART/only ART) during two year of follow-up time period. These results are at discordance with the previously reported study by Pine et al. 2009 in longitudinal sero-incident cohort of white population which showed significantly higher viral load and decreased CD4+T-cell count over a period of 4.2 years in HIV+ patients with TLR4 Asp299Gly polymorphism. In another retrospective study in Netherland (Papadopoulos. 2010) association of TLR4Asp299Gly polymorphism with decreased CD4+T- cell count and significantly increased risk of occurrence of severe infections was found. There is evidence which suggest that TLR4 Asp299Gly polymorphism is associated with the course of HIV-1 infection. It was demonstrated (Ferwerda . 2007)in Tanzanian patients that TLR4 Asp299Gly polymorphism might be involved in the intense viral replication with concomitant CD4 cell loss and increased susceptibility to infection from M. tuberculosis. (Pine.. 2009)  investigated that 1635G minor allele of TLR9 gene was associated with lower viral load set point and slower progression compared to different allele. In 2008 Soriano-Sarabia also provided the consistent results as they demonstrated the association of 1635G allele with slow progression in seroprevalence, dynamic therapeutic cohort. They discovered that TLR9 (1635 AA) genotype is significantly associated with lower CD4+T-cell count and higher HIV viral load at the time of patient’s enrolment in the cohort and also with the HIV clinical disease progression. They analyzed the association between TLR9 (1635A/G) SNP and the development of AIDS-related events and death in treated patients. However, these observations lacked concordance with the findings of Swiss HIV cohort study, in which the 1635G allele of TLR9 gene was more frequent in rapid progression as compared to other HIV+ patients enrolled in the study (Bochud. 2007). The median follow-up time before the initiation of HAART was 6.0 years and they assessed the CD4+T-cell count decay among patients naive for HAART.

It is important to highlight that study design of present study and that of previous were different. Soriano-Sarabia. 2008 have analyzed the association between TLR9 (1635A/G) SNP and the development of AIDS-related events and death in treated patients while we have investigated the influence of TLRs genotypic variability on CD4+T cell decline pattern in asymptomatic HIV+ and HIV+LTBI patients with in the two year time period. The possible explanation could be that patients with the TLR9 (1635 AA) genotype showed both lower CD4+T cell count and higher HIV viral load at their entry in the cohort whereas in our study all the patients whose results were analyzed were ART naïve patients with high CD4+T cell count and low viral load at the time of their entry in the cohort. In contrast to our results (Pine. 2009) have demonstrated that TLR4 Asp299Gly polymorphism is significantly associated with elevated viral load in HIV+ patients, in the total follow-up period of 4.2 years after starting on HAART. They have also showed that TLR9 1635G allele is associated with lower viral load and slower progression of the disease in HIV+ patients. These results are consistent with another study conducted by Soriano-Sarabia. 2008, which reflected that patients with AG and GG genotypes of TLR9 have decreased viral load in their plasma at the time of entry in the study and had slower disease progression rate as compared to those who had AA genotype.

5.3 TLR mRNA expressionThe results in this study indicates that TLRs were differentially expressed in chronic HIV-1 infected patients with CD4+T-cell count >200 cells/mm3 and in subjects with more advanced disease with CD4+T cell count <200 cells/mm3. In our study, fold increased in the mRNA expression of TLR2 and TLR4 in patients with advanced HIV disease with CD4+T-cell count <200 cells/mm3 and co-infected with TB as compared to healthy subjects. In chronic HIV-infected patients with CD4+T-cell count >200 cells/ml, the fold increase in the mRNA expression of TLR9 was observed as compared to healthy subjects. However, the difference in the expression of TLRs was not statistically significant.It has been observed previously that patients having different viral infections show changes in their TLR expression (Riordan. 2006, Dolganiuc . 2006). Previously reported study (Lester. 2008) also showed that TLR2 and TLR4 mRNA expression is increased in HIV+ patients with CD4 cell count <200 cells/ml than to those having CD4 count >200 cells/ml. These results are in agreement with our results. Another study (Scagnolary. 2009) in Italy showed reduced expression of TLR4 and TLR9 in HIV+ patients failing to respond to ART as compared to healthy subjects.

5.4 Strengths and limitations of the studyTo our knowledge this is the first study among Asian population which focused on TLR polymorphism among HIV-seropositive subjects and investigates its association with HIV disease progression. This study is unique in terms of study design. The different study groups included in the study are ART and ATT naïve HIV+ patients with no evidence of LTBI or active TB, HIV+ patients with LTBI, HIV+ patients with active TB. The study also recruited PTB Cat-I patients as disease control and healthy subjects as normal control. The sample size of different study groups recruited in the study was relatively large as compared to previous studies. In the present study HIV+ patients with and without LTBI were actively followed-up to look for the development of active TB, to our best knowledge this is the first study in which TLR polymorphism was investigated among breakdown TB cases and among patients who did not develop active TB. The influence of genetic variability in TLR was investigated on CD4+T-cell count and PVL pattern and compared between breakdown TB cases and patients who did not develop active TB. In line with previous studies, present study demonstrated strong association between TLR4 polymorphism and susceptibility to TB among HIV+ patients (Ferwerda. 2007). Our study

also demonstrated significant association between TLR9 with susceptibility to active TB breakdown among HIV+ patients with and without LTBI.  However, our study was limited by several factors. First, the CD4+T-cell counts at each time point (six monthly) were not available for some of the HIV+ patients with and without LTBI during the two year follow-up period. The reason being majority of these patients were asymptomatic, ART and ATT naïve with good CD4+T cell counts. These patients do not appreciate the significance of visiting the hospital for follow-up visit. Moreover, social stigma attached to the disease also prevented their visit to the ART centre for CD4 cell count estimation.  Second, the plasma viral load (PVL) of every six month was not available for HIV+ patients of the study. Although, quantification of HIV-RNA is important due to its inverse relationship with CD4+T-cells and strong role in predicting rate of HIV disease progression, however, in resource constrained settings like ours, majority of patients could not afford this test because of its prohibitive cost. Furthermore, national guidelines also does not recommend this test and patients are primarily monitored on the basis of CD4+ T cell count. Third limitation of this study is that the seroconversion time point of HIV+ patients were also not available with us and two year follow-up is too short a period for studying disease progression. In order to carry out well planned disease progression study it is important to have information on time of seroconversion and the follow-up period should be longer.