HIV/AIDS MAJOR ARTICLE Association of Toll-Like Receptor 4 Asp299Gly and Thr399Ile Polymorphisms with Increased Infection Risk in Patients with Advanced HIV-1 Infection Antonios I. Papadopoulos,1 Bart Ferwerda,2 Anastasia Antoniadou,1 Vissaria Sakka,1 Lambrini Galani,1 Dimitra Kavatha,1 Periklis Panagopoulos,1 Garyphalia Poulakou,1 Kyriaki Kanellakopoulou,1 Jos W. M. van der Meer,2 Evangelos J. Giamarellos-Bourboulis,1 and Mihai G. Netea2 1 4th Department of Internal Medicine, University of Athens Medical School, Athens, Greece; and 2Department of Internal Medicine and Center for Infectious Diseases, University St Radboud, Nijmegen Medical Center, Nijmegen, the Netherlands Background. The Toll-like receptor 4 (TLR4) is an essential component of the innate immune response to various microorganisms. We investigated the association between TLR4 polymorphism and the risk of acquiring severe infections, in patients with human immunodeficiency virus (HIV)–1 infection. Methods. The presence of TLR4 Asp299Gly and Thr399Ile single nucleotide polymorphisms (SNPs) was determined in a cohort of 199 HIV-1 infected patients and evaluated in relation to the occurrence of various infections. Results. One hundred seventy-two patients were homozygous for the wild-type genotype; 22 patients (11%) were heterozygous for both SNPs; 4 were heterozygous for 1 polymorphism; 1 patient was heterozygous for the Asp299Gly SNP and homozygous for the Thr399Ile SNP. Of individuals with a nadir CD4 cell count of !100 cells/ mm3, those who carried both SNPs, compared with those who carried the wild-type genotype, demonstrated a 13-fold increase in the odds ratio (OR) of any serious infection (OR, 6.33 vs OR, 1.83, P p .043). Conclusions. This study suggests an association between the presence of TLR4 Asp299Gly and Thr399Ile polymorphisms and the occurrence of serious infections in HIV-1 infected patients with a history of nadir CD4 cell count of !100 cells/mm3. Human immunodeficiency virus type 1 (HIV-1) infection is characterized by a gradual dysfunction, mainly in cell-mediated immunity but also in humoral immunity, attributable to CD4 lymphocyte depletion, leading to high levels of HIV RNA and development of opportunistic disease [1]. Chronic persistent immune activation has been implicated in the pathogenesis of HIV infection and the progression to AIDS [2]. An intact innate immune system may play a significant role in controlling chronic inflammation and may influence the occurrence of opportunistic diseases [3, 4]. Received 10 December 2009; accepted 4 April 2010; electronically published 3 June 2010. Reprints or correspondence: Dr Antonios I. Papadopoulos, 4th Dept of Internal Medicine, Attikon University Hospital, 1 Rimini St, Athens 12462, Greece ([email protected]). Clinical Infectious Diseases 2010; 51(2):242–247 2010 by the Infectious Diseases Society of America. All rights reserved. 1058-4838/2010/5102-0018$15.00 DOI: 10.1086/653607 242 • CID 2010:51 (15 July) • HIV/AIDS Toll-like receptors (TLRs) are transmembrane receptors that activate cells of the innate immune systems upon recognition of pathogen-associated molecular patterns [4–6]. When activated, TLRs interact with various adaptor proteins that orchestrate a series of cascade reactions leading to induction of suppression of genes involved in the inflammatory process [5–8]. At the present time, 10 TLRs (TLR1-TLR13) have been identified in humans [9, 10]. One of the best-studied TLRs is TLR4, which recognizes lipopolysaccharide of gramnegative bacteria, mannans of fungal pathogens, ligands of Mycobacterium tuberculosis and Plasmodium species, as well as endogenous ligands, such as fibronectin, oxidized low density lipoproteins, and heat shock proteins [11, 12]. The interaction of lipopolysaccharide with TLR4, through the soluble protein MD2, initiates a downstream intracellular signaling transduction through adaptor molecules that induces production of inflammatory cytokines such as tumor necrosis factor–a, in- terleukin 1b, interleukin 6, and interleukin 8 [9, 13]. TLR4 plays a role not only in the immune reaction against infectious microorganisms but also perhaps in the inflammatory processes of other inflammatory diseases, such as atherosclerosis or diabetes mellitus [14]. Studies in mice and humans have demonstrated an increased susceptibility to infection when mutations in genes encoding TLRs or downstream signaling proteins exist [14, 15]. Several single nucleotide polymorphisms (SNPs) in various TLR genes, such as TLR2, TLR4, TLR5, TLR6, TLR9, and TLR10, have been associated with disease susceptibility [14]. TLR4 SNPs have been studied more extensively. Among them, 2 nonsynonymous SNPs are more prevalent, an A/G transition at SNP rs4986790 that causes the substitution of aspartic acid by glycine at amino acid position 299 (Asp299Gly) and a C/T transition at SNP rs4986791 that causes the substitution of threonine by isoleucine at amino acid position 399 (Thr399Ile). Some studies have suggested an association of those SNPs with susceptibility to various conditions and diseases, including infections with gram-negative bacteria, septic shock, endotoxemia, disseminated candidiasis, invasive aspergillosis, respiratory syncytial virus infection, meningeal disease, bacterial vaginosis, premature birth, brucellosis, coronary heart disease, asthma, hematogenous osteomyelitis, and Plasmodium falciparum infections [11–14, 16–19]. Other studies have shown that carriage of both SNPs is associated with decreased incidence of atopy and hay fever as well as protection against legionnaires disease and atherosclerosis [14, 20, 21]. Only a few recent studies have investigated the role of TLR SNPs in the susceptibility to infections of patients infected with HIV. The TLR9 1635 A/C polymorphism has been associated with low CD4 cell count, increased viral load, and more rapid clinical progression [22]. Another study confirmed these findings and extended to the 1174G/A SNP in TLR9 that was also associated with the rapid progressor phenotype [23]. A higher susceptibility to active tuberculosis has been shown in HIVinfected patients in Tanzania who carry the TLR4 Asp299Gly SNP [24]. We hypothesized that polymorphisms in TLR4 genes may be associated with an increased susceptibility to severe infections in HIV-infected patients. To test this hypothesis, we analyzed the frequencies of the 2 common TLR4 SNPs, Asp299Gly and Thr399Ile SNP of the TLR4 gene, in a Greek cohort of patients with HIV infection. METHODS Patients. The study population consisted of 199 white, HIV1 infected adults ⭓18 years old, who were recruited from a cohort of patients attending the AIDS Unit of the 4th Department of Internal Medicine of the University of Athens (Greece), at University General Hospital Attikon, a tertiary care center. Blood specimens were obtained during patients’ regular visits after enrollment. Patients lost to follow-up or with missing data concerning demographics, clinical evolution, antiretroviral therapy administration, or laboratory parameters were excluded. Data were extracted from our electronic database, which is comprised of patients for whom clinical data were available since 1986. The following data were recorded: age, sex, stage of HIV infection, nadir CD4 cell count, maximum HIV-RNA levels, status with regard to highly active antiretroviral therapy (naive or experienced), and history of documented infectious diseases. The stage of the disease was recorded in accordance with the expanded European AIDS case definition [25]. Important infections that occurred at the time of or after the diagnosis of HIV-1 infection were also recorded. Included were serious opportunistic infections that fulfilled the case definition of the Centers for Disease Control and Prevention, such as Pneumocystis jirovecii pneumonia, tuberculosis due to M. tuberculosis or Mycobacterium avium complex, cerebral toxoplasmosis, cytomegalovirus retinitis, progressive multifocal leukoencephalopathy due to JC virus, salmonella bacteremia, and bacterial pneumonia due to Streptococcus pneumoniae or Pseudomonas aeruginosa [26]. Other important infectious diseases, such as syphilis or community-acquired pneumonia, were also recorded. Community-acquired pneumonia was diagnosed in accordance with previously defined criteria [27]. Written informed consent was obtained from all patients. The study was approved by the ethics committee of Attikon University Hospital. All clinical research was conducted in accordance with the principles of the Helsinki Declaration. Laboratory procedures. Routine CD4 cell count was determined using conventional flow cytometry with fresh blood samples from all patients. Plasma HIV-1 RNA was also routinely measured by the quantitative branch DNA method (Roche Diagnostic Systems), in accordance with the manufacturer’s instruction. TLR4 genotyping DNA was extracted from whole blood in accordance with the manufacturer’s instructions from the isolation kit Puregene (Gentra Systems). Polymerase chain reaction (PCR) assays were performed using 50 ng of genomic DNA at a final volume of 27 ml with 50 mM of MgCl2 (Invitrogen), 20 mM of deoxynucleotide triphosphates (Invitrogen), and 1 mM of Taq polymerase (Invitrogen). For the Asp299Gly polymorphism, the sense primer 5’-ATA CTT AGA CTA CTA CCT CCA TG and the antisense primer AGC CTT TTG AGA GAT TTG AGT-3’ were used. PCR consisted of 1 initial denaturation phase of 95C for 5 min followed by 40 cycles; each cycle consisted of 1 annealing step of 95C for 1 min, 1 polymerization step of 52C for 1 min, and 1 elongation step of 72C for 1 min. Then, another cycle of 72C for 7 min was performed before termination. Ten ml of the PCR product were digested HIV/AIDS • CID 2010:51 (15 July) • 243 Table 1. Characteristics of a Cohort of 199 Patients Infected with Human Immunodeficiency Virus–1 and Studied in Relation to Single Nucleotide Polymorphisms of Toll-Like Receptor 4 Wild type (n p 172) Characteristic Male-to-female ratio Age,a mean years SD Stage C HAART-naive–to–HAART-experienced ratio a Nadir CD4 cell count, mean cells/mm3 SD a Maximum viral load, median copies/mL Asp299Gly HT + Thr399Ile HT (n p 22) Asp299Gly HT (n p 2) Thr399Ile HT (n p 2) Asp299Gly HT + Thr399Ile homozygous (n p 1) 142:30 18:4 2:0 1:1 0:1 41.4 11.3 32 30:142 38.9 9.6 4 3:19 30.0 1.4 0 0:2 50.0 1.4 0 1:1 30 0 0:1 237.4 141.3 252.2 130.8 196.0 19.7 364.5 78.5 222 66,391 74,400 50,856 184,697 46,900 Note. HAART, highly active antiretroviral therapy; HT, heterozygous; SD, standard deviation. a Difference between groups not significant by analysis of variance. after incubation for 60 min at 37C with 0.5 units of the restriction enzyme NCO1 (BioLabs). The digestion product was evaluated after 2% agarose gel electrophoresis and ethidium bromide staining. For the Thr399Ile polymorphism, the sense primer 5’-GCT GTT CTC AAA GTG ATT TTG GGA and the antisense primer CAC TCA TTT GTT TCA AAT TGG AAT G-3’ were used. PCR consisted of 1 initial denaturation phase of 95C for 4 min followed by 35 cycles; each cycle consisted of 1 annealing step of 95C for 30 sec, 1 polymerization step of 56C for 30 sec, and 1 elongation step of 72C for 30 sec. Then, another cycle of 72C for 7 min was performed before termination. Ten ml of the PCR product was digested after incubation for 60 min at 37C with 0.5 units of the restriction enzyme Hinf1 (BioLabs). The digestion product was evaluated after 2% agarose gel electrophoresis and ethidium bromide staining. Statistical analysis. Quantitative characteristics were expressed by their mean value standard deviation (SD) for normally distributed values and by their median value for nonnormally distributed values. A nadir CD4 cell count !100 cells/ mm3 was defined as a common risk for the advent of any infection and was evaluated by the Mantel-Haenzel statistic. Odds ratios (ORs) and 95% confidence intervals (CIs) for the advent of an infection were estimated. Comparisons of ORs were calculated by the Breslow-Day and Tarone tests. RESULTS The study included 199 HIV-1 infected patients, who were assessed for the presence of TLR4 Asp299Gly and Thr399Ile polymorphisms. The characteristics of the patients at study entry are presented in Table 1. The study population comprised 163 males (82%) and 36 females (18%), with a mean age (SD) of 4111 years. Thirty-six patients (18%) were in stage C. The distribution of TLR4 polymorphisms is also shown in Table 1; 172 (86%) patients were bearing the wild-type (WT) 244 • CID 2010:51 (15 July) • HIV/AIDS genotype; 22 patients (11%) were heterozygous for both Asp299Gly and Thr399Ile SNP; 2 patients were heterozygous for the Asp299Gly SNP alone; 2 patients were heterozygous for the Thr399Ile SNP alone; and 1 patient was heterozygous for the Asp299Gly SNP and homozygous for the Thr399Ile SNP. Further statistical analysis was focused on patients bearing the WT genotype and on patients heterozygous for both SNPs. Overall, 92 infections occurred during the disease history of 55 patients. These infections occurred in 6 (27%) of the 22 patients heterozygous for both SNPs and in 49 (28%) of the 172 patients bearing the WT genotype (Table 2) (not significant). No difference was found between carriers of the WT genotype and patients heterozygous for both SNPs with regard to intake of prophylaxis medications, namely trimethoprimsulfamethoxazole (P p .378), macrolides (P p .459), and azoles (P p .615), or with regard to history of antipneumococcal vaccination (P p .552). Among patients bearing the WT genotype, the OR for the advent of an infection for patients with a history of nadir CD4 cell count of !100 cells/mm3, compared with patients with a history of nadir CD4 cell count ⭓100 cells/mm3, was 1.83 (95% CI, 1.14–2.93). This OR was changed to 6.33 (95% CI, 2.24– 17.89) when analysis comprised patients carrying both SNPs of TLR4. A statistically significant difference was found between the ORs of the 2 populations (P p .043 by the Breslow-Day test; P p .043 by the Tarone test) (Table 3). No other cut-off of CD4 cell counts was determined to be associated with an impact of carriage of TLR4 SNPs. Among the 27 cases of community-acquired pneumonia, S. pneumoniae was defined with certainty as a cause in 3 patients, although we cannot completely exclude it as an etiological agent in more of the cases. All 3 patients were bearing only WT alleles for the studied SNPs. No legionnaires disease was recorded. The time of seroconversion was known for only 18 patients of the entire cohort; 15 were heterozygous for both SNPs and 3 bore both WT alleles. Of the 15 patients heterozygous for Table 2. History of Infections and Measures of Prophylaxis in 199 Human Immunodeficiency Virus–Infected Patients in Relation to Single Nucleotide Polymorphisms of the Toll-Like Receptor 4 Gene No. (%) of patients Variable Wild type (n p 172) Asp299Gly HT + Thr399Ile HT (n p 22) History of infections Pneumocystis jirovecii pneumonia 11 (6.4) 2 (9.1) 0 (0) 0 (0) 0 (0) Community-acquired pneumonia Salmonellosis 25 (14.5) 1 (0.6) 2 (9.1) 1 (4.5) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Tuberculosis Mycobacterium avium complex 9 (5.2) 1 (0.6) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) Toxoplasmosis Cytomegalovirus 1 (0.6) 2 (1.2) 1 (4.5) 2 (9.1) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) PML Candidiasisa Syphilis Any Prophylaxis measure TMP-SMX Macrolides Azoles Pneumococcal vaccination Asp299Gly HT (n p 2) Thr399Ile HT (n p 2) Asp299Gly HT + Thr399Ile homozygous (n p 1) 2 (1.2) 1 (4.5) 0 (0) 0 (0) 0 (0) 18 (10.5) 11 (6.4) 49 (28) 1 (4.5) 1 (4.5) 6 (27) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 35 (20.3) 2 (9.1) 0 (0) 0 (0) 0 (0) 15 (8.7) 4 (2.3) 76 (44.2) 1 (4.5) 0 (0) 10 (45.4) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (50) 0 (0) 0 (0) 0 (0) Note. HT, heterozygous; PML, progressive multifocal leukoencephalopathy; TMP-SMX, trimethoprim-sulfamethoxazole. a Oropharyngeal or esophageal. both SNPs, 10 (66.7%) had started highly active antiretroviral therapy at the time of seroconversion (P p .350 ) and 2 (13.3%) developed an infection (P p .686). Of the 3 patients who bore both WT alleles, 3 (100%) had started highly active antiretroviral therapy at the time of seroconversion (P p .350) and none developed an infection (P p .686). Nine malignant neoplasms occurred (6 cases of B-cell nonHodgkin lymphoma and 3 cases of anal carcinoma), all among patients bearing both WT alleles (5%). The difference between patients bearing only the WT genotype and those heterozygous for both SNPs was not significant (P p .330). DISCUSSION To our knowledge, this study represents the first effort to demonstrate how a haplotype containing 2 commonly segregated polymorphisms of the human TLR4 gene, the Asp299Gly and the Thr399Ile SNPs, influences the occurrence of serious infections in patients with advanced HIV-1 infection. Results revealed that the carriage of both SNPs was associated with an increased risk for the advent of infections. Studies to evaluate the role of TLR4 polymorphisms in the course of HIV-1 infection have been limited [24]. In line with Table 3. Toll-Like Receptor 4 (TLR4) Asp299Gly and Thr399Ile Polymorphisms in 194 Human Immunodeficiency Virus–1 Infected Patients Stratified according to CD4 Cell Count and the History of Any Infection Presence of TLR4 single nucleotide polymorphisms, by nadir CD4 cell count No. of patients History of infection, no. (%) of patients Yes No Wild type !100 cells/mm3 ⭓100 cells/mm3 Asp299Gly HT + Thr399Ile HT !100 cells/mm3 ⭓100 cells/mm3 36 16 (44%) 20 (56%) 136 33 (24%) 103 (76%) 3 3 (100%) 0 (0%) 19 3 (16%) 16 (84%) HIV/AIDS • CID 2010:51 (15 July) • 245 this, we have observed that in HIV-infected patients with normal or moderately decreased CD4 cell counts, no major differences in terms of infections have been observed in relation to the presence of TLR4 polymorphisms. However, in individuals with CD4 cell counts !100 cells/mm3, those patients bearing TLR4 polymorphisms had a significant increase in the risk of occurrence of a severe infection. This effect is likely attributable to the attenuation of an effective adaptive cellular response due to low CD4 cell counts and to an increase in the relative importance of innate host defense. Furthermore, the in vitro activation and signaling through TLR2, TLR4, and TLR9 might facilitate HIV replication. Increased expression of TLR2, TLR3, and TLR4 on the cell membrane of monocytes has been demonstrated in advanced stages of HIV-1 infection and especially in patients with a CD4 cell count !200 cells/ mm3 [22, 28]. Moreover, microbial translocation from the gut flora to circulation has recently been considered to be a major cause of systemic activation, and this effect is probably mediated through TLRs-dependent signaling [29, 30]. Subsequently, on the one hand, TLR4 polymorphisms may modulate the systemic inflammatory response to translocated lipopolysaccharide, with many (although by no means all) studies suggesting a lower inflammatory response in individuals bearing the polymorphisms described in the present study [13]. On the other hand, HIV RNA may also influence the response to microbial translocation through TLR4 signaling and may promote the cycle of immune activation [31]. It may be tempting to speculate whether the presence of TLR4 SNPs would prove to be protective against this circle of infection-inflammation, but this remains to be demonstrated. This generalized activation may ultimately lead to exhaustion of the immune system and to the subsequent development of opportunistic and other infections. If the latter hypothesis is true, it may in part explain the present findings. Scant evidence exists about a role of TLR4 SNPs in the course of HIV-1 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 [24]. However, a more direct effect of TLR4 polymorphisms on the release of proinflammatory mediators, induced by ligands from the invading pathogen, may be an alternative explanation for the effects found, because defective cytokine release by cells of individuals with TLR4 polymorphisms has been reported by some, although not all, of the studies [13]. Three main limitations of this study may be recognized: (1) the retrospective nature of data collection, (2) the limited number of patients with CD4 cell count !100 mm3 bearing both TLR4 SNPs, and (3) the fact that all major infections were collectively analyzed. The relatively low number of each type of infection precluded us from analyzing separately the role of 246 • CID 2010:51 (15 July) • HIV/AIDS TLR4 polymorphisms on each specific infection. However, our cohort consisted of a homogenous white population with similar demographic characteristics, with a long and consistent follow-up. The patients were also consecutively tested at random, with no selection bias. The presented findings suggest a role of TLR4 SNPs for the advent of serious infections during the progression of HIV-1 infection. A point that one should always bear in mind is that factors other than SNPs, namely CD4 cell count and viral load, impinge considerably on the physical course of HIV-1 infected individuals. Additional casecontrol studies in the future, with HIV-negative patients bearing serious infections, may provide a concise conclusion about the role of TLR4 SNPs for the advent of serious infections among HIV-infected individuals with low CD4 cell counts. In conclusion, this study suggests an association between TLR4 haplotypes segregated together and an increased risk of serious infections in patients with HIV-1 infection. The present results may influence future strategies concerning disease surveillance and antibiotic prophylaxis of opportunistic infections. 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