Combination antiretroviral therapy (ART) has led to a dramatic reduction in morbidity and mortality of individuals infected with HIV. Successful clinical outcomes are dependent on adequate recovery of CD4+ T cells following ART . Several studies  have shown that ∼30% of patients fail to restore CD4+ T-cell counts more than 500 cells/μl despite years of suppressive therapy. There is now increasing evidence to show that patients failing to achieve CD4+ T-cell counts more than 500 cells/μl are at increased risk of developing serious non-AIDS events , including cardiovascular disease (CVD), hypertension, liver disease, non-AIDS malignancies and neurocognitive impairment . Recent studies  suggest that normalization of CD4+ T-cell counts should be defined as more than 900 cells/μl.
HIV disease progression is associated with increased adaptive and innate immune activation , which is multifactorial including increased expansion of CMV-specific T cells  and persistent HIV RNA at low levels . However, a major contributing factor to immune activation is the depletion of CD4+ T cells in gut-associated lymphoid tissue during acute HIV infection [9–11] leading to increased microbial translocation. Upon initiation of ART, microbial translocation is reduced as measured by reduction in the circulating levels of lipopolysaccharide (LPS), but this rarely reaches the levels of HIV-uninfected controls [9,10,12,13]. A case–control study  that compared CD4+ T-cell nonresponders (CD4+ T-cell counts <200 cells/μl after 24 months of suppressive ART) versus full responders (>400 cells/μl after 24 months of suppressive ART) showed that LPS was significantly increased in nonresponders.
Toll-like receptor 4 (TLR4) and CD14 expressed on macrophages, monocytes, dendritic cells (DCs) and neutrophils  are the main cellular sensors of LPS . Binding of LPS to these receptors induces a signalling cascade leading to activation of NF-κB, and eventual secretion of proinflammatory cytokines . CD14 can also be found in plasma in a soluble form , and soluble CD14 (sCD14) is elevated in HIV-infected individuals despite long-term suppressive ART . Elevated sCD14 has also been associated with CVD, neurological abnormalities, lymphoma and mortality in ART-treated HIV-infected individuals . Collectively, these data demonstrate that host responses to elevated LPS levels could influence the rate of CD4+ T-cell recovery post-ART and consequently to development of important clinical end points in HIV disease.
Polymorphisms in the genes encoding for CD14 and TLR4 have been shown to determine responsiveness to LPS . A common single-nucleotide polymorphism (SNP) found at position −260 of the CD14 promoter (in which a C → T change occurs) enhances the transcriptional activity of CD14, which is associated with an increase in high-density membrane-bound CD14 on monocytes as well as elevated levels of sCD14 in tissue culture supernatants along with other proinflammatory cytokines such as TNF-α and IL-6 [20,21]. Others have shown evidence for an association between the CD14 159TT genotype and inflammatory conditions, including chronic periodontitis , myocardial infarction , Crohn's disease  and susceptibility to brucellosis .
The main TLR4 SNPs that have been studied include A+896G (rs4986790) and C+1196T (rs4986791) . Compared with noncarriers, carriers of A+896G/C+1196T showed blunted inflammatory responses to inhaled LPS  as well as reduced plasma IL-6, IL-1β and C-reactive protein levels in response to LPS administration ex-vivo . Others have shown that both these TLR4 SNPs were associated with reduced prevalence of diabetic neuropathy in type 2 diabetes  and that carriers of these SNPs were more susceptible to severe bacterial infections [29,30].
In this study, we hypothesized that SNPs in the TLR4 and CD14 genes will have an impact on CD4+ T-cell recovery in HIV-infected individuals following initiation of ART secondary to a change in response to LPS. Using multivariate analysis, we examined the clinical and biological factors associated with restoration of CD4+ T cells and found that both TLR4 and CD14 SNPs were independently associated with enhanced long-term CD4+ T-cell recovery.
Patients were recruited from the Alfred Hospital and the Melbourne Sexual Health Centre (Melbourne, Australia) based on previously published inclusion criteria . All plasma and peripheral blood mononuclear cells (PBMCs) were collected at pre-ART and one time point at least 12 months after the initiation of ART. The clinical specimens were processed by standard protocols within 24 h of collection. Demographic and clinical parameters such as date of birth, sex, HIV-exposure category, diagnosis of AIDS-defining illnesses (ADIs) before initiation of ART, coinfection with hepatitis B and C viruses and initial ART-treatment regimen were obtained from the Victorian HIV Service Database of the Alfred Hospital. All CD4+ T-cell counts and plasma HIV RNA results were also collected longitudinally from ART initiation to the most recent data available. All participants provided written informed consent, and the protocols were approved by the Research and Ethics Committee of Alfred Health.
CD14 and Toll-like receptor 4 genotyping
DNA was obtained from cryopreserved PBMCs. Genotyping of a common polymorphism in CD14, C-260T (rs2569190) and two common polymorphisms in the TLR4 gene, A+896G (rs4986790) and C+1196T (rs4986791), was performed using the Sequenom MassARRAY iPLEX (Agena Bioscience, San Diego, California, USA) platform at the Australian Genome Research Facility.
Plasma lipopolysaccharide and soluble CD14
Microbial translocation was estimated by measuring LPS levels using a commercial chromogenic limulus amebocyte lysate assay (Lonza Walkersville Inc., Walkersville, Maryland, USA) and sCD14 levels using a Quantikine sCD14 ELISA (R&D Systems, Minneapolis, Minnesota, USA) as previously described . All samples were assayed in duplicate, and samples from the same patient (i.e. pre-ART and post-ART samples) were always assayed on the same plate to minimize interassay variation. In addition, two samples were used as internal quality control for each assay. The results of each plate were only included if the coefficient of variation of the two quality control samples was less than 15%.
Genotype frequencies were tested for Hardy–Weinberg equilibrium (HWE) by the χ2 test. The association between TLR4 and CD14 SNPs, LPS and sCD14 was analysed using the Kruskal–Wallis test with a post-hoc two-tailed Mann–Whitney U test. Linear mixed model (LMM) regression analysis was used to adjust for differences in the LPS levels across the genotypes tested. Plasma levels of LPS and sCD14 of a paired sample (pre-ART and post-ART) were compared using the Wilcoxon signed-rank test. Piecewise regression by LMM was used to determine the association between TLR4 and CD14 SNPs with the rate of CD4+ T-cell recovery at two different time periods separately (i.e. baseline to 3 months and 3rd month onwards) after ART initiation. The association between TLR4 and CD14 SNPs and other clinical parameters to CD4+ T-cell counts post-ART were first assessed using univariate LMM. Variables with P less than 0.2 were considered as candidates and included in the multivariate analysis. Variables with P less than 0.05 were considered as independent predictors. As CD4+ T-cell count was an outcome variable that was significantly skewed and was resistant to sensible transformation (log, square-root transformation), the use of maximum likelihood estimation was precluded. In this case, generalized estimating equation (GEE) was used in the LMM analysis.
Patient demography and clinical characteristics
We recruited 156 HIV-infected individuals, and DNA was available from 125 patients. Cohort demographics and clinical details of the patients are described in Table 1. After initiation of ART, all patients had viral suppression (plasma HIV RNA < 400 copies/ml) throughout the follow-up period except three patients who had a single episode of virological failure (plasma HIV RNA > 400 copies/ml). The success rate of amplification to determine the genotype was 100%. Of the 125 patients examined, 54 were heterozygous and 34 homozygous for the CD14 −260T allele; and 12 heterozygous for the TLR-4 SNPs. There were no patients homozygous for the TLR4 SNPs. This resulted in an allele frequency of 0.49 for CD14 SNP (χ2 test P = 0.13) and 0.048 for TLR4 SNPs (χ2 test P = 0.573) that was in HWE. Both TLR4 SNPs A+896G and C+1196T were cosegregated.
Suppressive antiretroviral therapy for 12 months resulted in significant decrease of plasma lipopolysaccharide and soluble CD14 levels
The effects of long-term suppressive ART on HIV viral load, CD4+ T-cell count microbial translocation and innate immune activation were assessed in plasma samples of patients before and at one time point after at least 12 months of suppressive ART. We showed that both LPS and sCD14 levels were significantly reduced following ART (both P < 0.0001) (Wilcoxon signed rank test, data not shown), consistent with multiple prior studies [9,13,31]. We found a weak correlation between sCD14 and LPS levels (r = 0.2044; P = 0.0222) pre-ART (Fig. 1a) and also between pre-ART CD4+ T-cell count with LPS (r = −0.195; P = 0.029) and sCD14 (r = −0.404; P < 0.0001) (Fig. 1b and c). Pre-ART HIV viral load was not correlated with plasma levels of LPS, whereas it correlated with the levels of sCD14 (r = 0.329; P = 0.0002) and inversely with CD4+ T-cell count (r = −0.268; P = 0.003) (Fig. 1d–f).
Single-nucleotide polymorphisms in CD14, but not Toll-like receptor 4, were associated with increased soluble CD14 levels postantiretroviral therapy
We next compared the levels of CD4+ T-cell count, LPS, sCD14 and HIV viral load in plasma among different CD14 and TLR4 genotypes. We found that CD4+ T-cell count and sCD14 in the post-ART samples were significantly different amongst the different CD14 genotypes (Kruskal–Wallis test; P = 0.024 and 0.006, respectively) but not in the pre-ART samples. A post-hoc analysis showed that individuals with the genotype CC compared to CT and TT had higher CD4+ T-cell counts (P = 0.013 and 0.058, respectively; Fig. 2a and 5).
Genotype CC and CT were associated with lower levels of plasma sCD14 as compared with TT (P = 0.0026 and 0.083, respectively), whereas the difference between CC and CT was NS (P = 0.6) (Fig. 2a). Using a regression model adjusted for LPS levels post-ART and years on ART, the CC genotype compared with the CC and CT genotypes was independently associated with lower sCD14 levels [hazard ratio −0.11, 95% confidence interval (CI) −0.13 to −0.077; P < 0.001].
The CD14 genotypes were not associated with CD4+ T-cell count, plasma sCD14 and HIV viral load pre-ART and LPS pre-ART and post-ART (Fig. 2a and b). The TLR4 SNPs, A+896G and C+1196T were not associated with these four variables pre-ART or post-ART (Fig. 2a and b). Together, we concluded that only the presence of CD14 CC genotype was associated with lower sCD14 levels post-ART.
The correlation of soluble CD14, lipopolysaccharide levels and CD4+ T-cell counts depends on the CD4+ and plasma HIV viral load
As the plasma levels of LPS, sCD14 and CD4+ T-cell count were poorly correlated, we speculated if this could be influenced by the degree of immune recovery. We therefore performed a post-hoc analysis stratified by baseline CD4+ T-cell levels and plasma HIV viral load and showed that the levels of LPS and sCD14 were more strongly correlated in individuals with baseline CD4+ T-cell count less than 100 cells/μl (r = 0.622; P = 0.0009) and baseline HIV viral load more than 5 log/ml (r = 0.385; P = 0.002). In this cohort, a similar correlation was not observed among individuals with baseline CD4+ cell count more than 100 cells/μl and HIV viral load less than 5 log/ml. We also found that the levels of LPS and sCD14 were more strongly correlated in individuals with CD14 −260TT (r = 0.484; P = 0.0037) and TLR4 +896AA (r = 0.246; P = 0.008), the two genotypes that were associated with an increased responsiveness towards LPS stimulation (Fig. 2c, c1).
Single-nucleotide polymorphisms in CD14 and Toll-like receptor 4 were independently associated with enhanced CD4+ T-cell recovery
To assess the independent influence of CD14 and TLR4 SNPs on CD4+ T-cell recovery following suppressive ART, we performed multivariate piecewise regression by LMM, controlling for clinical and biological factors that have previously been shown to be associated with CD4+ T-cell reconstitution. In general, recovery of CD4+ T cells during viral suppression occurs in two phases . During the first 3 months of ART, the number of CD4+ T cells rapidly increases because of T-cell homeostasis [33,34], improved thymic output  and redistribution of T cells into peripheral circulation , followed by a slower phase of T-cell repopulation that takes up to several years post-ART and is largely driven by T-cell proliferation and enhanced thymic production . Considering the biphasic pattern of CD4+ T-cell recovery, LMM was performed at two different time periods following initiation of ART, that is from baseline to 3 months and 4 months onwards, as we had previously reported .
In unadjusted analyses from baseline to 3 months, multiple factors were significantly associated with early CD4+ T-cell recovery (Table 2). In adjusted analyses, baseline CD4+ T-cell count (β-coefficient = 0.87; 95% CI = 0.779–0.966; P < 0.001) was associated with enhanced early CD4+ T-cell recovery, whereas receiving a protease inhibitor regimen (β-coefficient = −38.81; 95% CI = −67.39 to −10.23; P = 0.008) was associated with decreased early CD4+ T-cell recovery (Table 2).
Next, we investigated the determinants associated with late CD4+ T-cell recovery that is 4 months and beyond. Unadjusted analyses are shown in Table 3. In an adjusted model, multiple clinical parameters remained significant including baseline CD4+ T-cell count, age at ART initiation, receiving trimethoprim and sulfamethoxazole therapy at the time of initiation of ART, post-ART LPS level, pre-ART and post-ART sCD14 levels, previous ADIs, sex, positive for HBV antigen, mode of HIV transmission as well as both CD14 and TLR4 genotype. Our preliminary analysis showed that individuals with CD14 −260CC compared with CT and TT had a higher CD4+ T-cell count [β-coefficient 117 (P = 0.002) and 87 (P = 0.0026), respectively]. Therefore, TT and CT were combined and compared with CC in subsequent analyses. The pooled β-coefficient of CD14 −206CC genotype compared with CT/TT was independently associated with enhanced late CD4+ T-cell recovery (β-coefficient = 65.71; 95% CI = 42.51–88.92; P < 0.001). In the same model, the TLR4 SNPs (AG/CT), as compared with the genotype AA/CC, were also independently associated with enhanced late CD4+ T-cell recovery (β-coefficient = 111.87; 95% CI = 78.33–159.41; P < 0.001) (Table 3).
In the present study, we sought to investigate the relationship of TLR4 and CD14 SNPs with LPS and sCD14, and CD4+ T-cell recovery in HIV-infected individuals who were on suppressive ART. We found significant associations between TLR4 and CD14 SNPs with the trajectory of late recovery of CD4+ T-cell counts following ART using LMM. Using a multivariate analysis we found that the TLR4 genotype +896AG/+1196CT and CD14 genotype −260CC were independently associated with enhanced late CD4+ T-cell recovery post-ART. Finally, we showed that plasma LPS and sCD14 were significantly correlated in HIV-infected participants with a CD4+ T-cell count less than 100 cells/μl, HIV viral load more than 5 log/ml, and in individuals carrying the CD14 −260TT and TLR4 +896AA SNPs, the genotypes associated with an increased responsiveness or sensitivity towards LPS stimulation.
In this study, we assessed the changes in CD4+ T-cell counts over time after initiation of ART as a continuous variable using GEE analysis, controlling for time-varying covariates. Although several others have used Cox regression to study CD4+ T-cell recovery [39,40], we believe that the LMM approach is more robust because it takes into account the heterogeneity in both the rate and magnitude of CD4+ T-cell count increases following ART [2,41].
The TLR4 SNPS in exon 3 – A+896G and C+1196T – are common nonsynonymous SNPs with replacement of an aspartic acid (Asp) residue with glycine (Gly) at amino acid position 299 (Asp299Gly) and threonine with an isoleucine at position 399 (Thr399Ile), respectively . Both SNPs are known to affect the extracellular domain of TLR4. Nonetheless, the precise mechanism whereby these SNPs affect LPS signalling is not clearly understood. A recent study  suggested that the SNPs could interfere with TLR4 dimerization and therefore prevent the assembly of intracellular docking platforms for adapter recruitment leading to the hyporesponsiveness to LPS stimulation. In line with this, we demonstrated in a multivariate model that carriage of TLR4 +896AG/+1196CT was associated with greater late CD4+ T-cell reconstitution, independent of LPS levels. However, as TLR4 +896AG was less prevalent (9.6%), we also felt that the SNP effect might have been masked by the effect of the more prevalent SNP, that is CD14 −260CC (29.6%) in the univariate analysis. Therefore, the TLR4 +896AG SNP only showed its effect after the adjustment in a multivariate model.
The SNP in the CD14 gene, C-260T (also known as C-159T), is a common genetic polymorphism that occurs in the promoter region of the CD14 and is reported to occur in ∼48% of whites . This SNP (C → T) reportedly reduces the binding affinity of suppressor proteins to its binding domain and hence enhances the promoter activity of CD14. Consistent with this finding, we found that the CC genotype was associated with lower plasma levels of sCD14 (and TT genotype with higher sCD14), independent of LPS. Interestingly, we only observed this in plasma collected post-ART. This potentially could be due to the high level of immune activation pre-ART that may have masked the effects of CD14 C-260T SNPs.
We have recently examined the relationship of these same polymorphisms and CVD in a cohort of individuals on ART recruited in Malaysia . We are currently examining the relationship of these polymorphisms to CD4+ cell count recovery but given that this cohort is entirely Asian and they initiated ART late (median CD4+ cell count = 63 cells/μl compared with median CD4+ cell count = 202 cells/μl in this manuscript), we anticipate that the relationship between the polymorphisms and CD4+ cell count recovery that we describe here may be quite different. Ideally, the relationship between the polymorphisms described here and CD4+ cell count recovery should be examined in a replicate cohort of similar ethnicity which we are currently planning.
There were a number of limitations in our study. First, although this was a longitudinal study, patients were enrolled post-ART, and therefore samples were obtained at varying time points following ART initiation. We adjusted for time between initiating ART and sample collection in the multivariate model, but our results could have been confounded by loss to follow-up or death, although death is now significantly reduced in HIV-infected individuals on ART . Second, only a small fraction of our patients were carriers of the rare allele for TLR4 SNPs, and therefore this analysis had less statistical power. Although there is a plausible biological basis to support our findings of the genetic association between TLR4 and CD14 SNPs and recovery of CD4+ T cells following ART, given the small sample size, these findings require confirmation in an independent cohort of similar ethnicity. Lastly, the CD4+/CD8+ T-cell ratio has previously been shown to be associated with T-cell activation, senescence, exhaustion  as well as a strong predictor of HIV disease progression, CD4+ T-cell recovery  and ageing-related diseases [50,51]. In this cohort, baseline CD8+ cell count data were available only from a limited number of patients and therefore was not included in the analyses.
In summary, we demonstrated enhanced late CD4+ T-cell recovery in HIV-infected individuals receiving ART with SNPS in TLR4 +896AG/+1196CT and CD14 −260CC. Taken together, these data suggest that apart from microbial translocation, genetic polymorphisms that alter the host response to LPS stimulation could also be an important determinant of CD4+ T cells reconstitution following ART. The current study provides the first evidence to show that CD4+ T-cell recovery in HIV-infected individuals on ART is associated with genotypes related to LPS metabolism. These findings, once repeated in an independent cohort, could potentially inform novel interventions to enhance CD4+ cell count recovery in those at the greatest risk.
The authors are grateful to all the participants, clinical and laboratory staff of the Alfred Hospital and Melbourne Sexual Health Centre for assistance with patient recruitment, specimen collection and cooperation. We also thank Victorian HIV Blood and Tissue Bank and the Victorian Infectious Diseases Reference Laboratory for access to stored samples. We thank Pushparaj Velayudham for his assistance and Reena Rajasuriar for helpful discussions and review of the manuscript.
Grant support: The work was supported by a grant from the High Impact Research-Ministry of Higher Education, Malaysia (UM.C/625/1/HIR/MOHE/MED/01), University of Malaya Research Grant (UMRG) (RG448–12HTM) by the Health and Translational Medicine Research Cluster to E.M.S. S.R.L. is supported by the National Health and Medical Research Council of Australia and the Delaney AIDS Research Enterprise (DARE), National Institutes of Health (U19 AI096109).
Author contributions: Y.K.Y., E.M.S. and S.R.L. conceived and designed the experiments. Y.K.Y. performed the experiments. Y.K.Y. and T.S. analysed the data. A.S., C.K.F., J.E., J.H., P.C. and A.K. contributed reagents/materials/analysis tools. Y.K.Y., E.M.S., T.S. and S.R.L. wrote the manuscript.
Conflicts of interest
There are no conflicts of interest.
Presented in part: 7th IAS Conference on HIV Pathogenesis, Treatment and Prevention, 30 June–3 July 2013, Kuala Lumpur, Malaysia. Abstract number: WEPE453.
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