The development of the typical comorbidities of aging which currently affects people living with HIV/AIDS (PLWHA) can be partially ascribed to the persistent immune activation and chronic inflammation characterizing these individuals. The aim of this study was to analyze the effect exerted by combined antiretroviral therapy (cART) administration on plasma levels of HMGB1 (high mobility group box protein-1), AGEs (advanced glycation end products), their soluble receptor sRAGE, cytokines, C-reactive protein (CRP), and some metabolic markers in asymptomatic PLWHA. Analyses were performed longitudinally in 30 PLWHA, before and about 6–12 months after cART initiation. We observed that lower levels of AGEs in post-cART group were accompanied by an increase of CRP and triglyceride levels already in the early months of therapy. Because of the current ever-earlier recommendations to start cART and its prolonged use, these and other markers should be investigated in order to monitor and postpone the appearance of non-AIDS comorbidities in PLWHA.
Although the HIV epidemics affect more than 36 million individuals worldwide, with a global prevalence of almost 1% [
However, the great challenge today is to delay the onset and severity of non-AIDS-defining comorbidities, which appear much earlier in HIV-infected individuals [
Advanced glycation end products (AGEs) are a consequence of oxidative stress which occur during normal physiological conditions such as aging. However, if they are not excreted in the urine or their formation is exacerbated as in the case of diabetes hyperglycemia and other diseases, they accumulate in the vascular tissue, causing harmful effects [
It has also been observed that an alternative mRNA splicing which removes RAGE transmembrane domain [
High mobility group box protein-1 (HMGB1) is another key factor in promoting inflammation. It can be actively secreted following different stimuli, such as cytokines, endotoxins, and pathogen-associated molecular patterns (PAMPs) [
Few studies have investigated the role of these markers in HIV infection and how their expression is altered following cART initiation. Considering the need for better understanding of inflammatory mechanisms and cellular activation in this patients, as well as their influence on the “premature aging” phenomenon, the aim of this study was to compare the levels of HMGB1, AGEs, sRAGE, and some inflammatory biomarkers in the plasma of asymptomatic PLWHA before cART and approximately six months to one year after its introduction.
This is a longitudinal study involving 30 HIV-infected subjects, attended at the Specialist Outpatient Service for Infectious Diseases “Domingos Alves Meira” (SAEI-DM), at the Botucatu Medical School Complex (FMB)-UNESP, in São Paulo state, Brazil. This service covers about 600 people from Botucatu city and surrounding area and, for this study, 150 people (those cART-naïve) were interviewed, with only 30 of them being included after applying the exclusion criteria. The collection of data and biological samples was carried out in two stages, before therapy initiation (M0) and approximately six to twelve months after this intervention (M1), according to the patient regular return in the service. This research project was approved by the local Research Ethics Committee and was conducted between the years 2012 and 2015.
The inclusion criteria were age between 20 and 50 years, no previous cART administration, and signing an informed consent form.
Patients excluded were those with any of the following conditions: use of vitamin supplements, cancer history (current or previous), anorexia, morbid obesity, diabetes
Sociodemographic and clinical data were collected by interviews and from the patients’ medical records. Most of the participants were males (60.0%) aged approximately 34 years (±8.2), heterosexual (66.6%), and single (36.6%). The mean duration of HIV infection, considering its diagnosis, was 2.2 years (±3.2), and 70% of subjects showed a CD4+ T cells count lower than 500 cells/mm3 in the first collection, with
Furthermore, 30.0% were active smokers, 23.3% practiced intense physical activity, and 6.6% were taking anxiolytics and/or antidepressants.
Results from the following tests were collected from the medical records: plasma HIV viral load (VL), CD4+ T cells count, glucose, triglycerides, total cholesterol, and C-reactive protein (CRP). Adherence to cART was considered, at first, based on the reports of patients and subsequently verified with the records of medicine withdrawal, at the pharmacy service.
Twelve milliliters of blood were collected into an EDTA containing tube from each patient included in the study. The sample was maintained in a cooled and dark environment for 2-3 hours and then centrifuged at 1,500 rpm (
A sandwich-type immunoenzymatic assay (ELISA) was performed, using 100
A commercial ELISA kit (
Using 100
Cytokine measurement was performed by flow cytometry (
Continuous variables were expressed as mean (ME) and standard deviation (SD) and categorical variables by frequency and percentage (%). We used Poisson test or gamma distribution for nonparametric variables and negative binomial or one-way ANOVA with subsequent Tukey-Kramer post hoc test for those parametric data. We also performed Pearson correlations among the continuous clinical variables: AGE, sRAGE, HMGB1, glucose, triglycerides, total cholesterol, CRP, cytokines, and CD4+ T cells.
All calculations were adjusted for age, sex, and tobacco use, practice of intense physical activity, and use of anxiolytics and/or antidepressants.
Significant differences were considered when
In this study, we observed an increase in the patients’ CD4+ T counts (
Clinical data of 30 PLWHA before and after cART initiation.
Variable | M0 (MEAN ± SD) | M1 (MEAN ± SD) | |
---|---|---|---|
CD4+ T count (cells/mm3) | | | <0.0001 |
HIV VL (RNA copies/mm3) | | | 0.019 |
Undetectable HIV VL | — | 80.0% | — |
cART regimen – NRTI + NNRTI | — | 83.3% | — |
cART regimen – NRTI + PI/r | — | 16.7% | — |
PLWHA: people living with HIV/AIDS; cART: combined antiretroviral therapy; VL: viral load; NRTI: nucleoside reverse transcriptase inhibitor; NNRTI: nonnucleoside reverse transcriptase inhibitor; PI/r: protease inhibitors reinforced with ritonavir; M0: before cART initiation; M1: after cART initiation.
Among the PLWHA studied, 83.3% were under a therapeutic regimen consisting of the nonnucleoside reverse transcriptase inhibitors (NNRTI), and 80.0% had undetectable VL at M1. At the moment of this second collection, the participants were asked about adherence to therapy, which was reported by 86.6% of them. The average of VL measurements in pre-cART was 126,550.8 (±69,756.2) RNA copies/mm3. At M1, 80.0% of the patients had undetectable VL; in the remaining 20% patients, VL was 30,813.3 (±27,463.9) RNA copies/mm3. Viral load differences between M0 and M1 reached statistical significance (
Differences in triglyceride serum levels were observed as well (
Mean of blood glucose measurements, total cholesterol, and triglycerides of 30 PLWHA, before and after cART initiation. PLWHA: people living with HIV/AIDS; cART: combined antiretroviral therapy; M0: before cART initiation; M1: after cART initiation.
After cART introduction, AGEs plasma levels (
Plasma levels of RAGEs, AGEs, and HMGB1 of 30 PLWHA, before and after cART initiation. PLWHA: people living with HIV/AIDS; cART: combined antiretroviral therapy; AGEs: advanced glycation end products; sRAGE: soluble receptor of AGEs; HMGB1: high mobility group box protein-1. M0: before cART initiation; M1: after cART initiation.
CRP levels also increased after six months of cART (
Changes in plasma levels of cytokines and CRP of 30 PLWHA, before and after cART initiation. PLWHA: people living with HIV/AIDS; cART: combined antiretroviral therapy; IL: interleukin; CRP: C-reactive protein; M0: before cART initiation; M1: after cART initiation.
After the Pearson correlation among the studied variables, we only observed that IL-10 level was positively correlated with IL-8 and CRP and CD4+ T cell count was negatively correlated with IL-6 and IL-8.
The risk of progression to AIDS and death are related to high viremia and low CD4+ T lymphocyte level [
In order to delay the decrease in CD4+ T cells to very low levels, current recommendations are to begin treatment as soon as the diagnosis of HIV infection is confirmed, regardless of CD4+ T counts. Despite this knowledge, 26% of Brazilians have a very late diagnosis, when their CD4+ T count is below 200 cells/mm3 and, as a result, they also begin treatment later [
Despite the benefits of cART in the stabilization of HIV infection, there is evidence that the therapy is accompanied by some adverse effects, especially considering its long-term administration [
Early aging has been associated with and explained by the persistent immune activation and inflammation shown by HIV-infected individuals due to residual HIV replication in lymphoid tissues, the intense microbial translocation that may occur, the metabolic imbalance, and the instability of the cytokine profile and of the redox status, among others [
No significant statistical differences were observed in IL-6, IL-8, and IL-10 levels production here, although IL-6 has presented a slightly lower value and the IL-10 levels were more prevalent after cART initiation. Other authors reported a decrease in IL-6 synthesis after therapy introduction, considering a longer intervention, of one to two years [
In the present study, the CRP levels of all patients were normal or slightly high, considering the reference value at the two moments studied; however, a significant increase in its synthesis was observed after treatment. Thus, these results do not seem to suggest a contribution from cART to inflammation decrease during the study period. Actually, our hypothesis is that CRP elevation may be associated with the increase of parameters related to metabolic syndrome, such as triglycerides, which in our study also showed higher values after therapy. Similar results were found by Masiá et al. [
Nevertheless, another study [
The present research investigated other three poorly studied parameters: AGEs and HMGB1, which are involved in the induction of cellular activation and trigger inflammatory responses, and sRAGE, which plays the opposite effect.
After cART initiation, AGEs plasma levels decreased significantly. Little is known about AGEs concentrations and their effect on the pathogenesis of HIV infection, especially in vivo. To our knowledge, this is the first study to evaluate the effect of cART on the levels of AGEs in asymptomatic PLWHA. However, different reports suggest that their high levels are related to the development of various comorbidities in non-HIV-infected individuals, mainly aging-related complications, such as cataract formation, atherosclerosis [
Differences in sRAGE levels before and after cART introduction were not observed in the present study, possibly because of the short interval analyzed. After all, firstly AGEs activate the inflammatory cascade with consequent production of IL-6, and it is probably only after such induction that sRAGE increase in the circulation occurs [
In the general population, alterations in levels of blood have been associated with diseases typical of aging, especially those related to metabolic syndrome [
High systemic levels of HMGB1 also are found in many chronic proinflammatory conditions, including diabetes
Although the present study has analyzed a small sample and does not present data on the dietary inquiries of patients, which may also influence the levels of AGEs [
Finally, we concluded that, in the first months of cART, there was a decrease in glucose pathway oxidation, reducing AGEs soluble especially in individuals with initial CD4+ T <500 cells/mm3, which would indicate lower immune activation and an improvement in the inflammatory status of these patients. However, the analysis of other inflammatory markers did not show such benefit since the levels of inflammatory cytokines were not modified and those of CRP increased over the same period. Another negative aspect was the increase in triglycerides in this short period. Because HMGB1 and sRAGE levels were not modulated by initial therapy, other pattern-recognition receptors and other products responsible for cell activation and inflammation could be more informative in the analyses of chronic inflammation in HIV-infected individuals.
Therefore, analysis of other markers and further studies with larger cohorts and in the long term are important to clarify the influence of drug treatment on the mechanisms of activation, inflammation, and metabolic changes that affect the PLWHA. Thence, despite the incontestable benefits of cART in controlling HIV replication, some strategies could be useful in preventing/delaying the onset of non-AIDS-defining illnesses, such as physical exercises practice, a balanced diet, and other healthy lifestyle habits.
The authors declare that they have no conflict of interests.
This work was supported by grants from the Brazilian Agency “CAPES” (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), to Karen Ingrid Tasca, Caio Cavassan de Camargo; from “Reitoria/UNESP, to Juliana Trindade Caleffi; and from FAPESP” (Fundação de Amparo à Pesquisa do Estado de São Paulo) to Mariana Gatto (number 2010/13922-5). The authors want to acknowledge the efforts of the personnel from EAP/UNESP, especially Professor Dr. José Eduardo Corrente for thr help with the statistical analysis. The authors thank all the people from Tropical Disease Department, Botucatu Blood Center, Laboratory of Tropical Diseases and Experimental Research Unit (FMB/UNESP), especially Professor Dra. Alexandrina Sartori, Professor Dra. Cilmery S. Kurokawa, Maria Regina Moretto, and Silvia R. T. Estevan for all help. The authors also thank the patients and employees of Specialized Outpatient Service of Infectology “Domingos Alves Meira.”