Correspondence: Dr Marius Trøseid, Department of Infectious Diseases, Oslo University Hospital, Ullevål, Postboks 4950 Nydalen, 0424 Oslo, Norway. Tel: 92440240; fax: 22119181; e-mail: email@example.com
The aim of the study was to test the hypothesis that microbial translocation, quantified by levels of lipopolysaccharide (LPS) and subsequent monocyte activation [soluble (sCD14)], is associated with hypertension in HIV-infected individuals.
In this exploratory substudy, 42 patients were recruited from a larger, longitudinal HIV-infected cohort study on blood pressure. LPS and sCD14 levels were measured retrospectively at the time of nadir CD4 cell count, selecting untreated HIV-infected patients with both advanced immunodeficiency and preserved immunocompetence at the time of nadir. Patients with later sustained hypertension (n = 16) or normotension (n = 26) throughout the study were identified. LPS was analysed using the Limulus Amebocyte Lysate colorimetric assay (Lonza, Walkersville, MD) and sCD14 using an enzyme-linked immunosorbent assay (ELISA). Nonparametric statistical tests were applied.
In the HIV-infected patients [median (interquartile range) age 42 (32–46) years; 79% male and 81% Caucasian], LPS and sCD14 levels were both negatively correlated with nadir CD4 cell count. Plasma levels of LPS (P < 0.001) and sCD14 (P = 0.024) were elevated in patients with later hypertension compared with patients with normotension. There was a stepwise increase in the number of patients with hypertension across tertiles of LPS (P = 0.001) and sCD14 (P = 0.007). Both LPS and sCD14 were independent predictors of elevated blood pressure after adjustment for age and gender. For each 10-unit increase in LPS (range 66–272 pg/ml), the increment in mean blood pressure in the first period of blood pressure recording was 0.86 (95% confidence interval 0.31–1.41) mmHg (P = 0.003).
As LPS and sCD14 were both independently associated with elevated blood pressure, microbial translocation may be linked to the development of hypertension.
Non-AIDS-related morbidities such as hypertension, cardiovascular disease (CVD), malignancy, and renal, liver and bone diseases have emerged as increasing clinical problems in HIV-infected patients . In fact, non-AIDS-related mortality today exceeds AIDS-related mortality in populations with access to antiretroviral therapy (ART) . A premature ageing process has been suggested to occur in HIV-infected individuals for which several contributing factors have been proposed, including viral replication, drug toxicity, lifestyle factors, and persistent immune activation with increased cell proliferation and apoptosis as well as elevated levels of pro-inflammatory markers .
Primary HIV infection is characterized by massive T-cell depletion in the gastrointestinal mucosa with subsequent enhanced translocation of bacterial products such as lipopolysaccharide (LPS) and flagellin from the intestinal lumen into the systemic circulation [3, 4]. LPS is a potent inducer of immune response and inflammation through the innate immune system. Soluble CD14 (sCD14) is a marker of monocyte activation and is shed from monocytes upon LPS stimulation . Microbial translocation has been suggested to be a major driver of HIV-associated immune activation through stimulation of Toll-like receptors (TLRs) . Measures of T-cell-related immune activation independently predict disease progression and mortality in patients receiving and not receiving ART [6, 7]. Moreover, gut epithelial barrier dysfunction  as well as high levels of sCD14  predicts mortality in HIV infection. Immune activation and microbial translocation are reduced, but often not normalized despite prolonged effective ART with achievement of viral suppression [4, 10-12]. Importantly, increased cardiovascular risk has been linked to lack of CD4 cell count restoration despite effective ART [13, 14], which in turn is associated with low nadir CD4 cell counts , persistent microbial translocation  and immune activation .
Hypertension occurs frequently in HIV-infected populations, and is a major cause of myocardial infarction , non-AIDS-related mortality , and cardiovascular and all-cause mortality . In the large D:A:D (Data Collection on Adverse Effects of Anti-HIV Drugs) study, predictors of new-onset hypertension such as older age, male gender and higher body mass index (BMI) were similar to those in the general population, but markers related to immune activation were not included in this study . Other studies have shown an association between low nadir CD4 cell counts and the development of hypertension after initiation of ART [19, 20], although the mechanisms have not been addressed. Whether immune activation and microbial translocation play a role in the relationship between immunodeficiency and hypertension is not known. Our group has previously identified low nadir CD4 cell count (i.e. <50 cells/μL) as a predictor of sustained hypertension in the present HIV-infected cohort , and in this substudy we wanted to explore microbial translocation as a possible pathogenetic link. Thus, we chose to include patients with previous severe immunodeficiency as well as patients with preserved immunocompetence throughout their ART-naïve course of HIV infection. The aim of the present study was to test the hypothesis that markers of microbial translocation and subsequent monocyte activation before initiation of ART could predict hypertension in HIV-infected individuals.
Materials and methods
Study design and population
In this exploratory substudy, 42 patients were recruited from a larger longitudinal HIV-infected cohort study on blood pressure (BP) (n = 434) . Plasma levels of LPS and sCD14 were measured retrospectively at the time of nadir CD4 cell count (T0) (Fig. 1), selecting both patients with earlier advanced immunodeficiency and patients with preserved immunocompetence, as reflected by low [<50 cells/μL (lowN)] or high [>200 cells/μL (highN)] nadir CD4 cell count. Exclusion criteria for the substudy were diabetes mellitus, ART exposure at T0 or known hypertension [systolic blood pressure (SBP) ≥ 140 mmHg and/or diastolic blood pressure (DBP) ≥ 90 mmHg or use of antihypertensive drugs] based on hospital records at T0. Patients with available plasma samples at T0 with sustained hypertension (HT) or sustained normotension (NT) throughout the longitudinal study were recruited to both the highN and lowN groups. Availability of plasma samples turned out to be a major limitation for recruitment, and all eligible patients with available samples in the lowN group (HT and NT) as well as the highN HT group were included. Because matching across the two nadir strata was not possible, we chose to increase the number in the highN NT group to increase statistical strength. Recruitment to the highN NT group was performed in a random fashion. A total of 16 HT patients (lowN, n = 10) and 26 NT patients (lowN, n = 5) were included in the substudy. Written informed consent was obtained before inclusion in the longitudinal study. The study was performed in accordance with the Declaration of Helsinki. A control group of 15 HIV-negative Caucasian subjects were recruited by advertisement in local newspapers. It should be noted that the controls were not specifically matched for risk factors for hypertension, although the age and gender distribution was similar (Table 1). One-third of the control group had hypertensive BP readings at recruitment, but none used antihypertensive drugs.
Table 1. Characteristics of the controls and the HIV-infected patients at the time of nadir CD4 cell count (T0) and of the HIV-infected patients according to sustained hypertension status during time periods 1 and 2
Characteristics at T0
Controls (n = 15)
HIV-infected patients (n = 42)
Sustained NT (n = 26)
Sustained HT (n = 16)
Data are given as number (%) or median (interquartile range) for skewed distributions.
BMI, body mass index; eGFR, estimated glomerular filtration rate; SBP, systolic blood pressure; DBP, diastolic blood pressure; LPS, lipopolysaccharide; sCD14, soluble CD14; NT, normotension; HT, hypertension; NA, not applicable; T0, the time of nadir CD4 cell count.
aRecorded during time period 1.
bAvailable data for n = 30.
cWithin 1 year from the time of nadir.
dAvailable data for n = 37; missing in the normotension (n = 2) and hypertension (n = 3) groups.
Standardized BP was recorded during two prospectively scheduled time periods; time period 1 (T1) and time period 2 (T2). The time interval between the median dates of T1 and T2 was 2.7 [interquartile range (IQR) 2.5–3.1] years. BP was measured at three separate clinical visits days to months apart during both time periods (Fig. 1). The time interval between the first and the last visits during T1 and T2 was 0.5 (IQR 0.3–1.4) and 0.5 (0.3–0.8) years, respectively. BP measurement was performed in duplicate 2 min apart using a semiautomatic oscillometric device (Omron M4; Matsusaka Co. Ltd, Matsusaka, Japan) with appropriate cuff size according to the upper arm circumference. BP was measured in a relaxed sitting position in a quiet room after five minutes of rest by well-trained nurses. The average of six SBP and DBP measurements during T1 and T2, respectively, was used for statistical analyses. Patients with sustained hypertension, defined as hypertension at both T1 and T2 (treated hypertension at T1, n = 2/16; at T2, n = 6/16), and patients with sustained normotension were thereby identified. Mean arterial pressure (MAP) was calculated as DBP + 1/3 (SPB − DBP).
Plasma samples were collected with standard venipuncture as per clinical practice, and fasting state had been recommended but was not recorded. The samples were stored at −20°C until analysis.
LPS was analysed using the Limulus Amebocyte Lysate colorimetric assay (Lonza, Walkersville, MD) according to the manufacturer's instructions, with the following modifications: samples were diluted 10-fold to avoid interference with background colour, and preheated to 68°C for 12 min prior to analyses to dissolve immune complexes, as previously described . sCD14 was analysed using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (R&D, Minneapolis, MN).
Demographic, clinical and laboratory data at T0, T1 and T2 were obtained from the hospital records, the local HIV database and the case report forms (CRFs) at inclusion at T1 and T2. Nadir CD4 cell count was recorded as the lowest CD4 cell count in each individual's history. The estimated glomerular filtration rate (eGFR) was calculated according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula .
Skewed data (LPS, sCD14 and CD4 cell count) were log-transformed, and performance of parametric bivariate correlation analyses with calculation of Pearson's coefficient (r) was justified in analyses including all the HIV-infected patients. Otherwise nonparametric statistics were used, and Spearman's coefficient (rho) was calculated in bivariate correlation analyses. Between-group differences were evaluated with the Mann–Whitney U-test. A χ2 linear-by-linear association test was used for exploring linear trend in categorical data. Predictors of BP at T1 and T2, respectively, were explored using multivariate linear regression analyses. Statistical assumptions for the use of the linear regression model were satisfied. Because of the small sample size, the models included only age, gender, and LPS or sCD14, respectively, as independent variables. However, models with four independent variables were also created, adding a fourth variable to the original model, one at a time. The independent variables were chosen based on clinical relevance or association with outcome in univariate analyses (P < 0.1). A significance level of 0.05 was used. The statistical analyses were performed with spss software, version 20.0 (SPSS Inc., Chicago, IL).
Characteristics of the controls and the HIV-infected patients at T0, including stratification of the 16 HT and 26 NT patients at T0, are given in Table 1. The HIV-infected study population included 19% non-Caucasians, and they had higher triglyceride levels, lower cholesterol and lower BMI compared with the Caucasian controls. All patients were ART-naïve at T0, whereas 50% and 64% were receiving ART at the time of inclusion at T1 and T2, respectively.
In the control group, LPS and sCD14 plasma levels did not correlate with age, BMI, BP or triglycerides (data not shown). Plasma levels of LPS correlated with sCD14 in the HIV-infected population (Pearson's r=0.48; P = 0.001), but not in the control group. In the HIV-infected study population, LPS and sCD14 were both negatively correlated with nadir CD4 cell count (r = −0.57; P < 0.001 and r = −0.66; P < 0.001, respectively). A diagnosis of AIDS within 1 year of nadir occurred in two-thirds of the lowN patients.
Elevated plasma levels of LPS and sCD14 in HIV-infected subjects with hypertension
Plasma levels of LPS and sCD14 were strongly correlated in patients with HT (Spearman's rho=0.62; P = 0.011), but not in patients with NT or controls (Fig. 2a). Plasma levels of LPS (P < 0.001) and sCD14 (P = 0.024) at T0 were elevated in patients with HT compared with those with NT, and in both HT and NT patients compared with controls (P < 0.001 for all) (Fig. 2b and c). Furthermore, there was a stepwise increase in the number of HT subjects through tertiles of LPS (P = 0.001) and sCD14 (P = 0.007) (Fig. 3).
LPS and sCD14 as independent predictors of elevated blood pressure
In univariate analyses, plasma LPS correlated with MAP at T1 and T2 (r = 0.57; P < 0.001 and r = 0.48; P = 0.001, respectively) (Fig. 4), with SBP at T1 and T2 (r = 0.52; P < 0.001 and r = 0.43; P = 0.004, respectively), and with DBP at T1 and T2 (r = 0.57; P < 0.001 and r = 0.46; P = 0.002, respectively). Furthermore, sCD14 correlated with MAP at T1 and T2 (r = 0.39; P = 0.012 and r = 0.38; P = 0.013, respectively) (Fig. 4) as well as with DBP at T1 and T2 (r = 0.45; P = 0.003 and r = 0.41; P = 0.007, respectively), but not significantly with SBP either at T1 (r = 0.27; P = 0.083) or T2 (r = 0.29; P = 0.060). Univariate linear regression analyses for MAP at T1 are presented in Table 2.
Table 2. Univariate and multivariate linear regression analyses with mean blood pressure at time period 1 as outcome; also presented are adjusted β coefficients for lipopolysaccharide (LPS) and soluble CD14 (sCD14), respectively, when a fourth variable was added to the multivariate regression model
Characteristics at T0
Unadjusted unstandardized coefficient β [95% CI]
Model with LPS
Model with sCD14
Adjusted unstandardized coefficient β [95% CI]
Adjusted unstandardized coefficient β [95% CI]
ART, antiretroviral therapy; BMI, body mass index; CI, confidence interval; eGFR, estimated glomerular filtration rate.
aRecorded during time period 1.
LPS (+10 pg/mL)
sCD14 (+100 ng/ml)
Age (+1 year)
Basic model with additional adjustment for each of the following variables
Adjusted unstandardized coefficient β [95% CI] for LPS (+10 pg/mL)
Adjusted unstandardized coefficient β [95% CI] for sCD14 (+100 ng/ml)
Both LPS and sCD14 remained independent predictors of MAP at T1 after adjustment for age and gender (Table 2). Moreover, for each 10 pg/ml increase in LPS (range 66–272 pg/ml), the increment in BP at T1 was 1.03 [95% confidence interval (CI) 0.23–1.83] mmHg for SBP (P = 0.013) and 0.78 (95% CI 0.29–1.26) mmHg for DBP (P = 0.002) after adjustment for age and gender. For each 100 ng/ml increase in sCD14 (range 135–4218 ng/ml), the increment was 0.40 (95% CI 0.10–0.70) mmHg for DBP (P = 0.004) after adjustment, but with no independent association with SBP (P = 0.101). Similarly, for each 10 pg/ml increase in LPS, the increment in BP at T2 was 0.75 (95% CI 0.20–1.29) mmHg for MAP (P = 0.009), 0.97 (95% CI 0.15–1.80) mmHg for SBP (P = 0.022) and 0.63 (95% CI 0.15–1.11) mmHg for DBP (P = 0.011) after adjustment for age and gender. For each 100 ng/ml increase in sCD14, the increment in BP at T2 was 0.40 (95% CI 0.10–0.70) mmHg for both MAP and DBP (P = 0.008 and P = 0.007, respectively) and 0.50 (95% CI 0.00–1.00) mmHg for SBP (P = 0.036) after adjustment for age and gender. The multivariate regression models including LPS and sCD14 yielded R2 of 0.17–0.29 and 0.19–0.28, respectively.
When a fourth variable was added to the multivariate regression model, i.e. ethnicity, viral load, nadir CD4 cell count, triglyceride concentration, BMI, eGFR, glucose concentration, smoking or current ART, plasma levels of LPS and sCD14 remained independent predictors of MAP at both T1 (Table 2) and T2. In addition, LPS independently predicted SBP and DBP, whereas sCD14 independently predicted DBP but not SBP at T1 and T2 (data not shown).
In the present study, LPS and sCD14 levels were measured at the time of nadir CD4 cell count, selecting both patients with advanced immunodeficiency and preserved immunocompetence at the time of nadir. LPS and sCD14 levels were both independent predictors of later BP levels and were associated with subsequent sustained hypertension. These findings could not be explained by cardiometabolic risk factors, viral load, nadir CD4 cell count or later use of ART. Our data suggest that microbial translocation and immune activation might contribute to increased BP and the development of hypertension.
The significant association between LPS and hypertension found in this study can be explained by several mechanisms. First, LPS is well known to induce endothelial dysfunction , among other mechanisms by activating the endothelium through a receptor complex consisting of TLR4, CD14 and myeloid differentiation (MD)-2 , and by inducing apoptosis of endothelial cells . Endothelial dysfunction and impaired arterial elasticity are present throughout the atherosclerotic process , and an association of LPS with carotid atherosclerosis has been described in the general population  as well as in HIV-infected individuals . In non-HIV-infected cohorts, we and others have shown that pro-inflammatory cytokines  and sCD14 are associated with arterial stiffness  which is mechanistically linked to hypertension.
In HIV-infected individuals, viral replication has been associated with endothelial dysfunction . Furthermore, increased LPS levels are associated with activated T cells , consistent with a recent study reporting that a predominance of such cells was associated with arterial stiffness in HIV-infected women . Taken together, these findings suggest that microbial translocation and subsequent activation of macrophages and T cells may contribute to HIV-associated endothelial dysfunction and arterial stiffness, which over time may cause hypertension.
Other mechanisms may also be involved. LPS has been shown to activate the sympathetic nervous system in experimental settings , and high autonomous nervous activity and elevated levels of noradrenaline have been linked to impaired ART response and enhanced replication of HIV . Furthermore, the renin-angiotensin system is involved in LPS-induced endothelial dysfunction, and can be reversed by the angiotensin-converting enzyme-inhibitor enalapril .
Nadir CD4 cell count was associated with LPS and sCD14 in our study. In fact, microbial translocation could explain the reported independent association between low nadir CD4 cell count and the development of hypertension [19-21]. However, other mechanisms could also be at play. Hypertension has previously been related to inflammation [37, 38]. Immune activation is linked to HIV progression, and could be driven by factors other than microbial translocation, for example viral coinfections, other opportunistic infections and amplified HIV replication during immunodeficiency . Dyslipidaemia during advanced HIV infection  could have an adverse effect on endothelial cells, as could viral toxicity . Furthermore, the combination of severe immunodeficiency and subsequent ART-mediated immune reconstitution could possibly have an adverse influence on BP [19, 20].
There are limitations in this study that need to be acknowledged. First, the small sample size increases the risk of statistical type II errors. However, the risk of type I errors is lower, and the significant association between microbial translocation and elevated BP levels is likely to be reliable. Secondly, the study was not originally designed for the purpose of exploring a link between microbial translocation and hypertension. Consequently, the study lacks systematic BP measurements at T0. Because of limited availability of plasma samples in all groups except the NT highN group, the HT and NT patients could not be matched across the two nadir strata, either in number or in other characteristics. However, adjusting for relevant covariates in linear regression analyses did not alter the main result. Thirdly, measurement of LPS has many pitfalls, including technical difficulties and possible interference from plasma turbidity . A fasting state was advised but not formally recorded, and thus post-prandial hypertriglyceridaemia could have influenced the LPS levels, in particular in samples with elevated triglycerides. Furthermore, storage of plasma samples over a long period of time could possibly have an adverse influence. Fourthly, the controls were recruited with the purpose of adding context to the data. Because of the lack of matching between the HIV-infected cohort and controls, the comparisons should be interpreted with caution. Finally, although all samples were run in duplicate, inter- and intra-assay variability cannot be excluded.
Our study also has several strengths. The close association between LPS and BP was supported by the finding of a similar predictive power for sCD14, which is pathogenetically linked to LPS . The longitudinal design in the assessment of hypertension status, the thorough validation of BP measurements, and the use of standardized BP readings and sustained hypertension as predefined endpoints are additional strengths.
The majority of patients with a low nadir CD4 cell count reached nadir at the time-point at which ART was introduced (1996). Thus, the study population does not reflect the situation today, and this study should therefore be considered a proof of concept study. Prospective studies should be performed in populations on stable ART in which non-AIDS-related complications are an increasing clinical problem .
Hypertension is a non-AIDS-related complication which deserves particular clinical attention. As our data show that hypertension was closely associated with markers of microbial translocation in HIV-infected individuals, a key aspect of HIV pathogenesis may be linked to the development of hypertension. We propose that the gastrointestinal mucosal barrier may present as a potential therapeutic target for the prevention of future hypertension and long-term cardiovascular complications in HIV infection, and this needs to be explored in future studies.
We thank the patients for their participation. We acknowledge the skilled assistance of the staff at the Outpatient Clinic of Infectious Diseases at Oslo University Hospital, Ullevål. Financial support was received from Signe and Albert Bergsmarken's fund for investigation of kidney diseases, the HIV fund of the Department of Infectious Diseases at Oslo University Hospital, Ullevål and the South-Eastern Norway Regional Health Authority and Novo Nordisk Foundation.