Chronic immune activation is a central feature of progressive HIV-1 infection, including increased T-cell activation [1,2] and increased levels of proinflammatory cytokines and chemokines . Furthermore, the degree of immune activation might be a better predictor of disease progression than plasma viral load . However, the pathogenic mechanisms remain to be determined.
The gastrointestinal immune system seems to play a central role in this process. A damaged gastrointestinal tract results in translocation of microbial products that are likely to stimulate the innate immune system through Toll-like receptors (TLRs) . Such microbial translocation has been demonstrated, with increased plasma lipopolysaccharide (LPS) levels in progressive HIV-1 disease, which were decreased but not normalized by antiretroviral therapy (ART) .
High mobility group box-1 protein (HMGB1) is a DNA-binding protein that is released from damaged or necrotic cells to the extracellular milieu, in which it may act as a potent proinflammatory marker by stimulating cytokine expression in monocytes . Of note, HMGB1 per se does not seem to have proinflammatory activity, but has a high affinity to form complexes with bacterial substances such as LPS and CpG-DNA in vitro. Such complexes might bind various receptors, including TLR4 and TLR9, and promote a large variety of inflammatory and immunological responses [8–10]. We have recently reported elevated plasma HMGB1 levels in HIV-1 patients, in particular among patients with advanced disease .
To date, little is known about a potential interaction of LPS and HMGB1 in HIV-1 infection. The aims of our study were, therefore, to investigate the combined effect of these substances on viral replication and disease progression, and whether effective ART could reduce the levels of LPS and HMGB1.
Study design and patients
This was a retrospective study of patients (n = 42) treated with ART since 2004 and 19 healthy controls. Patients were recruited based on available plasma samples as well as virologic response after 2-year ART. Five patients were excluded from the study due to poor quality of the plasma samples. Thirty-two individuals had undetectable viral load and 10 had detectable viremia (nonresponders). Although the distribution of age and sex was similar (median age 37 years, 50% women), the control group consisted of only white patients, whereas the HIV-1 cohort included black (n = 17), Oriental (n = 6) and white (n = 19) patients, all living in Sweden. The study was approved by the Regional Ethics Committee.
Plasma HIV-1 RNA quantification and CD4+ T-cell counts
Measurements of plasma HIV-1 RNA (COBAS Amplicor test; Roche Molecular Systems Inc., Branchburg, New Jersey, USA; detection limit 40 copies/ml) and T-cell counts (routine flow cytometry) were done as part of the clinical routine.
Quantification of high mobility group box-1 protein and lipopolysaccharide
Plasma samples were collected with venepuncture and stored at −80°C until analysis.
LPS was analyzed by Lamilus Amebocyte Lysate colometric assay (Lonza, Walkersville, Maryland, USA) according to the manufacturer's instructions, with the following modifications: samples were diluted 10-fold to avoid interference with background color and preheated to 70°C for 12 min prior to analyses to dissolve immune complexes, as this procedure yielded higher LPS levels.
HMGB1 was analyzed by ELISA (Shino-Test Corporation, Oonodai, Kanawaga, Japan) according to the manufacturer's instructions. Although HMGB1 is known to form complexes that might interfere with the detection , preheating did not affect the HMGB1 levels and was, therefore, not performed.
Serial samples from the same individuals were analyzed in the same run. The interassay coefficients of variation for both HMGB1 and LPS were less than 10%.
As levels of LPS and HMGB1 were skewly distributed, nonparametric statistics were applied. Differences between patient groups and controls at baseline were evaluated with Mann–Whitney U test, and intragroup changes from baseline to end of study were evaluated by Wilcoxon test. Correlation analyses were performed using the Spearman method. Trend analyses were performed with the Jonckheere–Terpstra test. A significance level of 0.05 was used. The statistical analyses were performed with SPSS software, version 15.0 (SPSS Inc., Chicago, Illinois, USA).
High mobility group box-1 protein and lipopolysaccharide in HIV-1 before and after 2-year antiretroviral therapy
Plasma levels of both HMGB1 and LPS were higher in naive HIV-1 patients as compared with controls (P < 0.001 for both) (Fig. 1). In patients with undetectable viral load after ART, plasma LPS levels were reduced by a median of 36% from baseline to the same median levels as the controls (P < 0.001), although not normalized in all patients. Patients with LPS above median after 2 years had a tendency to higher pretreatment viral load (P = 0.064), of whom 13 with available samples were also analyzed after 3 years: eight continued to decrease and five were normalized. Levels of HMGB1 were reduced by 35% from baseline (P = 0.001), however, not normalized. In ART nonresponders, there was no reduction in levels of LPS or HMGB1.
The impact of high mobility group box-1 protein and lipopolysaccharide on plasma HIV-1 RNA
There was no significant association between LPS and HMGB1 levels with age, sex or mode of transmission. However, there was a significant trend to higher LPS levels in HIV-1-positive individuals of African or Oriental origin compared with white patients (P = 0.007).
Levels of HMGB1 and LPS were not intercorrelated, and none of the substances were directly correlated with CD4+ T cells, CD8+ T cells or viral load, respectively. In order to evaluate a potentially interacting effect, the patients were divided according to the 50th percentile of HMGB1 and LPS, respectively. Notably, viral load was two-fold higher in patients with LPS and HMGB1, above median as compared with other patients (P = 0.005) (Fig. 2), whereas elevated levels of LPS and HMGB1 alone had no significant impact on viral load. This association was particularly strong in African patients, resulting in a five-fold higher viral load (P = 0.006), but not in whites. The combination of increased LPS and HMGB1 was associated with a 25% lower increase (not significant) in CD4+ cell count in patients with undetectable viral load after ART, but not in ART nonresponders.
The main findings of this study were: (i) that plasma levels of LPS and HMGB1 were elevated in patients with HIV-1 infection as compared with controls, (ii) that patients with LPS and HMGB1 above median had substantially higher viral loads than other patients, and (iii) that median levels of LPS were normalized and HMGB1 was reduced after 2 years of effective ART.
Our results confirm and extend the work by Brenchley et al.  reporting elevated plasma levels of LPS in HIV-1 patients, suggesting translocation of gut microbes. Also in the original report , LPS levels were reduced but not normalized after 1 year of ART. Our results imply that prolongation of ART might substantially further improve and potentially normalize the mucosal barrier, in line with a recent study  showing normalized intestinal permeability and expression of tight junction proteins in suppressively treated patients. Our patients with LPS above median after 2 years had a tendency to higher pretreatment viral load, and such patients might need suppressive ART for longer time to normalize the mucosal barrier, as suggested from some of our patients with available 3-year data (not shown). Notably, ART nonresponders had no reduction in LPS or HMGB1 levels after 2 years, suggesting that a virologic response is mandatory for restoration of the gut barrier, although persistence of HIV-1 has been reported in gut-associated lymphoid tissue despite 10-year effective ART .
This study confirms our previous observation that plasma HMGB1 levels are elevated in nontreated HIV-1-infected individuals . Here, we show, for the first time, a reduction of HMGB1 levels by ART. However, HMGB1 levels were not normalized, implying that low-grade tissue necrosis and immune activation might persist despite 2-year effective ART. A concern of the HMGB1 ELISA is the possibility of plasma components that might bind to the protein and result in decreased detectable levels . However, one would expect that such a masking factor is higher in HIV-1-infected persons with hypergammaglobulinemia, and the difference compared with controls, in this study, is thus probably a conservative estimate.
One limitation is the retrospective design, introducing the possibilities of selection bias and problems with sample handling, although very few patients were excluded due to poor quality of plasma samples. Another limitation is that the controls were not matched for ethnicity. However, when analyzing whites separately, there was still a significant difference in LPS and HMGB1 between HIV-1-infected patients and controls (P < 0.001 for both).
Interestingly, LPS levels were higher in African patients, in line with a recent study  from a Kenyan cohort. African patients in general have a higher burden of intestinal infections, including helminths [16,17], and with the exception of two individuals, our patients had arrived to Sweden only few months before inclusion in the study. Notably, although LPS levels decreased by ART in all ethnic groups, LPS levels remained higher after treatment in African and Oriental patients compared with whites. It can be noted that we have earlier reported higher levels of tumor necrosis factor-alpha in HIV-1-infected Africans who had recently immigrated to Sweden as compared with white patients from Sweden .
As HMGB1 and LPS have been shown to form immunologically active complexes [8,19], one aim of the study was to specifically evaluate the combined effect of these substances on viral load and disease progression. Of interest, HIV-1 patients with elevated levels of both HMGB1 and LPS had a two-fold higher viral load. This association was largely driven by patients of African origin, who had a five-fold increased viral load in the presence of elevated LPS and HMGB1. Additionally, there was a nonsignificant tendency to reduced restoration of CD4+ cell counts, but only in patients with undetectable viral load after 2-year ART.
Previous studies [6,11] have shown that both LPS and HMGB1 might be associated with HIV-1 disease progression, and taken together, our results suggest an interacting effect of these substances. HMGB1 tends to form complexes with LPS  and bacterial CpG-DNA  that might stimulate TLR4 and TLR9, respectively. Thus, we have also found that HMGB1 in combination with the TLR ligands, LPS, flagellin and CpG-DNA synergistically induce HIV-1 replication in vitro. Moreover, polymorphisms in the TLR4 and TLR9 genes are associated with HIV-1 viral load in vivo.
We show that HIV-1-positive patients with elevated plasma levels of HMGB1 and LPS had higher viral load than other patients, and that 2 years of effective ART reduced levels of HMGB1 and potentially normalized LPS levels. As LPS and HMGB1 tend to form complexes that stimulate TLRs in vitro, we propose that such complexes might be involved in the immune activation and pathogenesis of HIV-1 infection.
The study was supported by the Swedish Medical Research Council, Karolinska Institutet and the Swedish International Developing Agency.
All the authors contributed in planning the study and in critically reviewing the manuscript. M.T., P.N., J.N., A.L. and S.A. performed the laboratory analyses. P.N. provided linkage to the study cohort and coordinated the study. M.T. performed statistical analyses and drafted the manuscript. A.S. supervised and financially supported the study as research group leader.
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