ORIGINAL ARTICLE Year : 2023  |  Volume : 12  |  Issue : 1  |  Page : 10-16

Influence of genetic variability in toll-like receptors (TLR 2, TLR 4, and TLR 9) on human immunodeficiency virus-1 disease progression

Gaurav Kaushik1, Richa Vashishtha2
1 School of Allied Health Sciences, Sharda University, Greater Noida, Uttar Pradesh; Department of Medicine, All India Institute of Medical Sciences, New Delhi, India
2 Department of Medicine, All India Institute of Medical Sciences, New Delhi, India

Date of Submission04-Nov-2022Date of Decision25-Dec-2022Date of Acceptance18-Jan-2023Date of Web Publication14-Mar-2023

Correspondence Address:
Gaurav Kaushik
School of Allied Health Sciences, Sharda University, Greater Noida, Uttar Pradesh
India

Source of Support: None, Conflict of Interest: None

Check

DOI: 10.4103/ijmy.ijmy_190_22

Background: It has been demonstrated that toll-like receptors (TLR2), TLR4, and TLR9 which were initially known for recognizing bacterial products are involved in the detection of viral components. It was planned to undertake a prospective longitudinal study among ethnically homogeneous antiretroviral treatment and antitubercular treatment naïve human immunodeficiency virus (HIV)-positive patients representing the north Indian population. The aim of the study was to investigate the influence of TLR2, TLR4, and TLR9 polymorphism in HIV disease progression. Methods: The present study was designed to investigate genetic polymorphism in TLRs (TLR2, TLR4, and TLR9) among HIV-infected patients with and without TB coinfection. The study population consisted of two groups: (i) HIV-positive patients without TB infection and disease (n = 223, HIV-positive patients); (ii) HIV-positive patients with latent tuberculosis infection (LTBI) (n = 150, HIV-positive LTBI patients). These participants were of either gender between 18 and 60 years of age and treatment naïve for both TB and HIV. HIV-positive and HIV-positive LTBI patients were longitudinally followed up for t2 years to study HIV disease progression. Results: On comparing TLR2 and TLR4 allelic and genotypic frequencies between 306 HIV-positive patients (no TB/AIDS) and 47 HIV-positive patients progressed to active TB/AIDS, no significant difference was observed between the two groups. The frequency of “A” allele in TLR9 was found to be significantly increased in 47 HIV-positive patients who progressed to active TB/AIDS (61.7%) as compared to 42.16% in 306 HIV-positive patients (no TB/AIDS), (P < 0.001). Furthermore, a significantly increased frequency of “AA” genotype in TLR9 was observed in 47 HIV-positive patients progressed to active TB/AIDS (55.32%) as compared to 20.26% in HIV-positive patients (no TB/AIDS). Conclusion: Findings of the present study revealed that genetic variability in TLR9 may influence HIV disease progression. The AA genotype in TLR9 may be associated with progression to TB/AIDS for 2 years in HIV-positive patients.

Keywords: HIV, polymorphism, toll-like receptors, tuberculosis

How to cite this article:
Kaushik G, Vashishtha R. Influence of genetic variability in toll-like receptors (TLR 2, TLR 4, and TLR 9) on human immunodeficiency virus-1 disease progression. Int J Mycobacteriol 2023;12:10-6
How to cite this URL:
Kaushik G, Vashishtha R. Influence of genetic variability in toll-like receptors (TLR 2, TLR 4, and TLR 9) on human immunodeficiency virus-1 disease progression. Int J Mycobacteriol [serial online] 2023 [cited 2023 Mar 15];12:10-6. Available from: https://www.ijmyco.org/text.asp?2023/12/1/10/371658   Introduction 

Worldwide, HIV resulted in 37.7 million prevalent cases, 1.5 million incident cases, and 680,000 deaths.[1] In India, 0.22% (23.48 lakhs) of people were reported human immunodeficiency virus (HIV) seropositive in 2019.[2],[3]

Globally, tuberculosis is a leading cause of death with an estimated 1.5 million casualties yearly worldwide. It has been estimated that up to one-third of the world's population has been infected with TB.[4] In India, 2.64 million incident cases of TB were reported in 2019.[5] However, 26.9 lakh people were died as reported in the Indian study.[6]

The presence of TB bacilli within the host in dormant or nonreplicating state is known as latent tuberculosis infection (LTBI). It has been observed that, out of the large number of people exposed to Mycobacterium tuberculosis, only 30% acquire TB infection and the remaining 70% remain uninfected. Of these 30% infected individuals, 10% develop active TB disease while in the remaining 90%, the infection is contained by the host defense system.[7] In HIV-negative individuals with LTBI, the lifetime risk of developing clinical TB ranges from 10% to 20% and half of this risk is during the first 2 years of infection, i.e., 50% of these will develop the disease within the first 2 years of getting infected.[8]

It has been demonstrated that innate immune response plays an important role in HIV disease pathogenesis, disease progression, and susceptibility to TB.[9] The key component of the innate immune system, TLRs are responsible for activating the signaling pathway which regulates HIV replication and disease progression. In other words, the latent and chronic stages of HIV infection are dependent on TLR-mediated pathways.[10] Immune regulatory molecules frequently exhibit genetic variability or mutations, which affect how susceptible an individual is to infectious diseases.[6]

It was noted that the majority of the studies were carried out in cohorts of Western Europe ancestry and very limited studies have investigated the role of mutations in TLRs in susceptibility to the HIV population. Thus, it was planned to undertake a prospective longitudinal study among ethnically homogeneous antiretroviral treatment (ART) and ATT naïve HIV-positive patients representing the north Indian population. The hypothesis of the study was that SNPs within these TLRs (TLR2, TLR4, and TLR9) may account for variability in susceptibility to HIV infection and influence HIV disease progression rate. Therefore, the main aim of the study was to determine the association between common SNPs in TLRs and HIV disease progression. The objective of the study was to investigate the influence of genetic variability in TLRs (TLR2, TLR4, and TLR9) on HIV-1 disease progression in patients with and without TB coinfection. For this purpose of the study, HIV positive and HIV positive with LTBI patients were followed up for 2 years.

  Materials and Methods 

Study setting and design

The study protocol was approved by the Institutional Ethics Committee (Ref. No.:A-35/05.05.2008) of the All India Institute of Medical Sciences (AIIMS), New Delhi.

Patients consent form

Written informed consent was obtained from all the study participants before enrollment in the study. The present study was prospective longitudinal components.

Study population

The present study was designed to investigate genetic polymorphism in TLRs (TLR2, TLR4, and TLR9) among HIV-infected patients with and without TB coinfection. The study population consisted of two groups: (i) HIV-positive patients without TB infection and disease (n = 223, HIV-positive patients) and (ii) HIV-positive patients with LTBI (n = 150, HIV-positive LTBI patients).

These participants were of either gender between 18 and 60 years of age and treatment naïve for both TB and HIV.

Study procedure

The demographic and clinical information of all the recruited participants was captured in the standardized questionnaire. Baseline body weight and height were measured in all the participants. Details of smoking, alcoholism, substance abuse, family history of TB, etc., were also collected.

Biological samples

Approximately, 6–8 ml of peripheral venous blood was obtained aseptically in the ethylenediaminetetraacetic acid vials from all the study participants for molecular study/genomic DNA extraction and RNA isolation.

Follow-up and treatment of recruited patients

In the present study, HIV-positive and HIV-positive LTBI patients were longitudinally followed up for 2 years to study HIV disease progression. During the 2-year follow-up period, these participants were asked to visit the hospital every 6 months (6 months, 12 months, 18 months, and 24 months time points). Patients who did not turn up for scheduled follow-up visits were contacted telephonically. At every follow-up visit, patients were meticulously examined for the presence of active TB. Immunological (CD4 cell count estimation every 6th month) and virological responses (plasma viral load [PVL] at baseline and the end of 2 years) were also estimated in these patients during the follow-up period.

Molecular techniques and protocols

DNA extraction

Genomic DNA was extracted from peripheral blood using a DNA blood maxi kit (QIAamp, Qiagen, Germany). All centrifugation steps were carried out at 15°C–25°C. The protocol was used as per the manufacturer's instructions.

Genotyping for the toll-like receptor polymorphisms

The TLR genotypes for TLR2, TLR4, and TLR9 genes were assessed using polymerase chain reaction (PCR) – restriction fragment length polymorphism analysis by Eppendorf thermocycler for PCR amplification (Eppendorf AG 22331, Hamburg, Germany). Specific primers purified by polyacrylamide gel electrophoresis were obtained from Bio Basic Inc, East Markham, Canada.

Statistical analysis

Data are presented as mean ± standard deviation/median (interquartile range). Qualitative characteristics such as genotypic and allelic frequencies were compared using Chi-square (χ[2]) test. All analysis was performed using Stata version 12.0 (Stata Corporation, College Station, TX, USA). Two-sided P < 0.05 was considered to be statistically significant.

  Results 

This study was undertaken to investigate the genetic variability in TLRs (TLR2, TLR4, and TLR9) among HIV-1 infected patients with and without TB coinfection. Two different study groups (HIV-positive patients and HIV-positive LTBI patients, were genotyped for three SNPs (rs121917864 in TLR2, rs4986790 in TLR4, and rs352140 in TLR9) followed by comparative analyses.

The objectives of the study were to explore the influence of genetic variability in these TLRs on HIV disease progression. Therefore, 223 HIV-positive patients and 150 HIV-positive LTBI patients were longitudinally followed-up prospectively for 2 years and the influence of genetic variability in TLRs was examined among two groups.

During the follow-up of 223 HIV-positive patients and 150 HIV-positive LTBI patients, 25 HIV-positive and 22 HIV-positive LTBI patients progressed to active TB/AIDS and 11 HIV-positive and 9 HIV-positive LTBI patients were initiated on ART. The demographic and baseline characteristics of these two study groups are provided in [Table 1]. For analysis, HIV-positive and HIV-positive LTBI patients who did not progress to active TB/AIDS (n = 306) were compared with the HIV-positive patients progressed to active TB/AIDS (n = 47) and HIV-positive patients initiated on ART (no TB), (n = 20).

Table 1: Baseline characteristics of human immunodeficiency virus-positive patients (no tuberculosis/acquired immunodeficiency syndrome), human immunodeficiency virus-positive patients progressed to active tuberculosis/acquired immunodeficiency syndrome and human immunodeficiency virus-positive patients initiated on antiretroviral treatment (no tuberculosis)

Click here to view

The CD4 cell count and PVL have been expressed as the median and interquartile range (IQR), data are shown in [Table 2]. The HIV-positive TB patients had the lowest CD4 cell counts (median 283 cells/mm[3]) and highest PVL (median 5.01 log10 copies/ml) as compared to other patient groups (P < 0.01).

Table 2: Comparison of toll-like receptor 2, 4, and 9 allelic and genotypic frequencies between 306 human immunodeficiency virus-positive patients (no tuberculosis/acquired immunodeficiency syndrome) and 47 human immunodeficiency virus-positive patients progressed to active tuberculosis/acquired immunodeficiency syndrome

Click here to view

All HIV-positive LTBI patients were TST positive, performed by 5 TU and PPD (SPAN Diagnostics Ltd, Surat, India). Induration >5 mm was considered a positive reaction.

The mean BMI of 306 HIV-positive patients (20.41 ± 3.51 kg/m[2]) at baseline was slightly higher than that of 47 HIV-positive patients who progressed to active TB/AIDS (19.38 ± 2.22 kg/m[2]), however, similar to HIV-positive patients initiated on ART (no TB), 20.5 ± 3.31. The baseline CD4 cell count of 306 HIV-positive patients (no TB/AIDS), (438.5 [356–568] cells/mm[3]) was higher than that of 47 HIV-positive patients who progressed to active TB/AIDS, (361 [285–471] cells/mm[3]), and 20 HIV-positive patients initiated on ART (no TB), (339.5 [272–426]). The PVL of 20 HIV-positive patients initiated on ART (no TB), (4.85 [3.92–5.83] log10 copies/ml) was higher than the baseline PVL of 47 HIV-positive patients who progressed to active TB/AIDS (4.67 [3.73–5.21] log10 copies/ml), and 306 HIV-positive patients (no TB/AIDS) (3.97 [3.18–4.86] log10 copies/ml).

The median time (range) to the diagnosis of TB in 47 HIV-positive patients who progressed to active TB/AIDS was 11 (range 8–16) months; however, in 20 HIV-positive patients who progressed from pre-ART to ART, the median time (range) of progression was 12 (range 6–24) months. Of 47 HIV-positive patients who progressed to active TB/AIDS, 14 (29.79%) developed PTB, while the remaining 33 (70.21%) developed EPTB.

Analysis of toll-like receptors allelic and genotypic frequencies

TLR2, TLR4, and TLR9 allelic and genotypic frequencies were compared between 306 HIV-positive patients (this group includes 187 HIV-positive and 119 HIV-positive LTBI patients who did not progress to TB/AIDS) and 47 HIV-positive patients progressed to active TB/AIDS and 20 HIV-positive patients initiated on ART.

On comparing TLR2 and TLR4 allelic and genotypic frequencies between 306 HIV-positive patients (no TB/AIDS) and 47 HIV-positive patients progressed to active TB/AIDS, no significant difference was observed between the two groups.

The frequency of “A” allele in TLR9 was found to be significantly increased in 47 HIV-positive patients who progressed to active TB/AIDS (61.7%) as compared to 42.16% in 306 HIV-positive patients (no TB/AIDS), (P < 0.001). A significant increased frequency of the “AA” genotype in TLR9 was observed in 47 HIV-positive patients progressed to active TB/AIDS (55.32%) as compared to 20.26% in HIV-positive patients (no TB/AIDS), (P < 0.001). However, the frequency of AG genotype was significantly decreased in HIV-positive patients progressed to active TB/AIDS (12.77%) as compared to 43.8% in HIV-positive patients (no TB/AIDS), P < 0.001. No significant difference in GG genotype was observed between the two groups [Table 2].

TLRs allelic and genotypic frequencies were also compared between 306 HIV-positive patients (no TB/AIDS) and 20 HIV-positive patients initiated on ART (no TB). No significant difference was observed in allelic and genotypic frequencies of TLR2, TLR4, and TLR9 between the two groups [Table 3].

Table 3: Comparison of toll-like receptor 2, 4, and 9 allelic and genotypic frequencies in 306 human immunodeficiency virus-positive patients (no tuberculosis/acquired immunodeficiency syndrome) and 20 human immunodeficiency virus-positive patients initiated on antiretroviral treatment (no tuberculosis)

Click here to view

On comparing TLRs allelic and genotypic frequencies between 47 HIV-positive patients who progressed to active TB/AIDS and 20 HIV-positive patients initiated on ART (no TB), no significant difference in TLR2, TLR4, and TLR9 allelic and genotypic frequencies was observed between two groups [Table 4].

Table 4: Comparison of toll-like receptor 2, 4, and 9 allelic and genotypic frequencies in 47 human immunodeficiency virus-positive patients progressed to active tuberculosis/acquired immunodeficiency syndrome and 20 human immunodeficiency virus-positive patients initiated on antiretroviral treatment (no tuberculosis)

Click here to view

  Discussion 

TLRs, as a key component of the innate immune system, have the capability to sense the invading pathogen through differential recognition of PAMPs. Recently, the occurrence of genetic polymorphism or mutations has been reported in TLRs. Studies have demonstrated that mutations in TLRs influence signal transduction molecules, resulting in increased or decreased susceptibility to various bacterial and viral infections.[11] Several studies have also highlighted the association between TLR polymorphisms and increased susceptibility or protection against several infectious diseases.[12]

HIV-seropositivity is the most potent of all known risk factors for LTBI reactivation and TB is the foremost and the most common opportunistic infection occurring among HIV-seropositive individuals. The impact of HIV-TB coinfection is bidirectional and they are often referred to as “cursed duet.” The consequence of two diseases occurring together in synergy is greater as compared to either of them present alone.[13] 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.[14]

HIV-infected individuals exhibiting a high degree of variability in susceptibility to associated complications and progression to profound immunodeficiency have encouraged researchers to focus attention toward defining immunological correlates of protection and/or disease progression. In particular, it is important to have an understanding of the host genetic factors and their interaction with the immune and virological factors, as this could yield important information on the immunopathogenesis of HIV infection.

Large cohort and candidate gene-based studies have highlighted the importance of host determinants comprising a number of immune response genes that contribute toward differential vulnerability of individuals to HIV/AIDS and TB. There are compelling evidence that genetic variants significantly influence HIV viral load set point, rate of CD4 T cell decline, susceptibility to specific AIDS-defining illnesses, and response to antiviral therapy.[15],[16]

It has been observed that variability in disease progression rate, susceptibility to OIs, development of 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. The present study was planned to examine the influence of genetic variability in TLRs (TLR2, TLR4, and TLR9) on HIV disease progression.

In the present study, particular attention was paid toward evaluating the influence of genetic factors on disease transmission and progression. Accordingly depending on the availability and follow-ups, ethnically homogeneous HIV-positive population (HIV positive and HIV-positive LTBI were included.

Limitations of the study

Our study was limited by several factors. First, the CD4-positive T-cell count and PVL at each time point (6 monthly) were not available for some of the HIV-positive patients with and without LTBI during the 2 years follow-up. The reason being the majority of these patients were asymptomatic, ART and ATT naïve with good CD4+ T cell counts, and therefore, they do not appreciate the significance of visiting the hospital for follow-up visits. Another limitation of this study was that the seroconversion time point of HIV-positive patients was also not available with us and 2 years follow-up is too short a period for studying disease progression. To carry out a well-planned disease progression study, it is important to have information on the time of seroconversion and the follow-up period should be longer.

  Conclusion 

The present study determines the importance of TLR polymorphisms as genetic markers for disease susceptibility and progression in the Indian population. In spite of the appreciably high number of HIV-infected people in the Indian subcontinent, this objective had not been addressed comprehensively in previous studies. The findings of the present study revealed that genetic variability in TLR9 may influence HIV disease progression. The AA genotype in TLR9 may be associated with progression to TB/AIDS for 2 years in HIV-positive patients.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 

  References 
1.Global Report: UNAIDS Report on the Global AIDS Epidemic; 2020.  
    2.HIV Facts & Figures. NACO Report; 2019.  
    3.Kaushik G, Vashishtha R. Messenger RNA expression of toll-like receptors (TLR2, TLR4, and TLR9) in HIV-1 infected patients with and without tuberculosis co-infection. Int J Mycobacteriol 2022;11:293-8.  
[PUBMED]  [Full text]  4.Nguyen H, Gazy N, Venketaraman V. A role of intracellular toll-like receptors (3, 7, and 9) in response to Mycobacterium tuberculosis and Co-Infection with HIV. Int J Mol Sci 2020;21:6148.  
    5.WHO. Global Tuberculosis Report; 2019.  
    6.Kaushik G, Vashishtha R, Tripathi H, Yadav RN. Genetic polymorphism of toll-like receptors in HIV-I infected patients with and without tuberculosis co-infection. Int J Mycobacteriol 2022;11:95-102.  
[PUBMED]  [Full text]  7.Carranza C, Pedraza-Sanchez S, de Oyarzabal-Mendez E, Torres M. Diagnosis for latent tuberculosis infection: New alternatives. Front Immunol 2020;11:2006.  
    8.Menzies NA, Swartwood N, Testa C, Malyuta Y, Hill AN, Marks SM, et al. Time since infection and risks of future disease for individuals with Mycobacterium tuberculosis infection in the United States. Epidemiology 2021;32:70-8.  
    9.de Martino M, Lodi L, Galli L, Chiappini E. Immune response to Mycobacterium tuberculosis: A narrative review. Front Pediatr 2019;7:350.  
    10.Kawasaki T, Kawai T. Toll-like receptor signaling pathways. Front Immunol 2014;5:461.  
    11.Maglione PJ, Simchoni N, Cunningham-Rundles C. Toll-like receptor signaling in primary immune deficiencies. Ann N Y Acad Sci 2015;1356:1-21.  
    12.Zhang Y, Liu J, Wang C, Liu J, Lu W. Toll-like receptors gene polymorphisms in autoimmune disease. Front Immunol 2021;12:672346.  
    13.Dye C, Williams BG. The population dynamics and control of tuberculosis. Science 2010;328:856-61.  
    14.Sodora DL, Silvestri G. Immune activation and AIDS pathogenesis. AIDS 2008;22:439-46.  
    15.Claireaux M, Robinot R, Kervevan J, Patgaonkar M, Staropoli I, Brelot A, et al. Low CCR5 expression protects HIV-specific CD4+ T cells of elite controllers from viral entry. Nat Commun 2022;13:521.  
    16.Esbjörnsson J, Jansson M, Jespersen S, Månsson F, Hønge BL, Lindman J, et al. HIV-2 as a model to identify a functional HIV cure. AIDS Res Ther 2019;16:24.  
    

 
 

  [Table 1], [Table 2], [Table 3], [Table 4]