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Elevated plasma lipopolysaccharide is not sufficient to drive natural killer cell activation in HIV-1-infected individuals

Gregson, John NSa; Steel, Alana,b; Bower, Markc; Gazzard, Brian Gb; Gotch, Frances Ma; Goodier, Martin Ra

doi: 10.1097/QAD.0b013e3283199780
Basic Science: Concise Communications

Background: Lipopolysaccharide (LPS) is elevated in the plasma of individuals chronically infected with HIV-1 and is thought to contribute to chronic immune activation of myeloid cells and T-cells. Natural killer (NK) cells can also be stimulated by LPS in vitro.

Objectives: To measure plasma LPS levels in individuals with HIV-1 infection, with or without suppressed plasma viral load, and in individuals with or without inflammatory bowel diseases (IBD). To compare the expression of NK cell receptors and activation markers in individuals with HIV-1 infection and in HIV-1-negative individuals with active IBD.

Methods: NK cells were studied by flow cytometry in treatment-naïve viraemic HIV-1-positive individuals (n = 14), aviraemic HIV-1-positive individuals (n = 19), HIV-1-negative individuals with inflammatory bowel disease (n = 10) and HIV-1-negative healthy control individuals (n = 17). Plasma endotoxin (LPS) was measured using the limulus amoebocyte assay.

Results: Viraemic and aviraemic HIV-1-positive individuals and patients with IBD have elevated levels of plasma LPS compared with HIV-1-negative individuals.

HIV-1-positive individuals had significant changes in activation marker or NK cell receptor expression, whereas NK cells from IBD patients had similar levels to HIV-1-negative controls. NK cells from HIV-1-positive individuals are refractory to further stimulation by LPS in vitro.

Conclusion: Elevated plasma LPS alone does not account for the chronic activation and receptor loss in NK cells from HIV-1-infected individuals.

aDepartment of Immunology, Imperial College London, UK

bDivision of HIV-GUM, UK

cClinical Oncology Unit, Chelsea and Westminster Hospital, London, UK.

Received 5 June, 2008

Revised 10 September, 2008

Accepted 16 September, 2008

Correspondence to Dr Martin R. Goodier, PhD, Imperial College London, London, UK.

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HIV-1 infection results in chronic immune activation, contributing to immune dysfunction. There is evidence that chronic activation of T-cells not only results from chronic antigen-specific activation, but also as a bystander effect of myeloid cell activation by toll-like receptor (TLR) ligands [1]. Increased levels of the TLR4-ligand lipopolysaccharide (LPS) and components of the LPS- activating complex are detected in the plasma of chronically infected HIV-1-positive individuals [1]. These changes may result from damage to the intestinal epithelia and microbial translocation from the gastrointestinal tract in HIV-1-positive individuals, as observed previously in patients with inflammatory bowel diseases (IBD) [2,3]. Monocytes from HIV-1-infected individuals make poor tumour necrosis factor and IL-1 responses to LPS, indicating that chronic over-stimulation renders these cells refractory to further stimulation in vitro [1]. Sequences derived from HIV-1 mRNA also promote myeloid cell activation via TLR-7/8 [4].

Natural killer (NK) cells undergo a chronic partial activation during HIV-1 infection resulting in elevated expression of activation markers and contributing to the generation of a dysfunctional CD56CD16+ NK cell population in viraemic HIV-1-positive individuals [5,6]. Chronic activation of NK cells is also caused by opportunistic viral infections during HIV-1 infection as demonstrated by the expansion of an NK cell population bearing NKG2C, a receptor for cytomegalovirus antigens [7,8].

NK cells can also be activated in vitro by microbial products, including LPS, in the presence of macrophages or dendritic cells [9,10]. We, therefore, investigated NK cell activation and receptor expression in groups of individuals with elevated levels of plasma LPS, including HIV-1-positive individuals and HIV-1-negative individuals with IBD.

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Materials and methods

Study population

HIV-1-positive patients (n = 33) were recruited from local HIV-GUM clinics. Comparisons were made between treatment-naive individuals with detectable plasma viraemia (viraemic, n = 14, median HIV-1 RNA copies/ml blood = 4836 (range = 133–83140); median CD4+ T-cells/μl blood = 379 (range 123–674); median age = 40 (range = 33–53); and HAART-treated individuals with plasma viraemia below the limit of detection (aviraemic, n = 19, <50 copies HIV-1 RNA/ml blood; CD4 T cells/μl 506 (68–896); age = 40 (23–58). HIV-1-negative individuals with active Crohn's disease (n = 5) and ulcerative colitis (n = 5); CD4+ T cells/μl blood = 695 (581–1113); age = 60 (24–71) were recruited from the local gastroenterology clinic. HIV-1-negative healthy control individuals [n = 17, age = 37 (22–64)] were recruited locally. All samples were obtained with informed consent and full ethical approval.

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Lipopolysaccharide measurements in plasma

LPS measurements in plasma were determined using a commercially available limulus amoebocyte lysate assay (LAL) (Lonza, Basel, Switzerland) according to manufacturer's instructions and after heating plasma to 70°C according to a previously published protocol [1].

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NK cell receptor and activation marker expression

Analysis of NK and CD8+ T cells was performed within ficoll-hypaque isolated peripheral blood mononuclear cells (PBMC) using a FacsCalibur instrument (Becton Dickinson, Oxford, UK). NK cell and CD8+ T-cell subsets were detected using a cocktail of fluorochrome-conjugated monoclonal antibodies such as anti-CD56 allophycocyanin, anti-CD8 Fluorescein isothiocyanate (Beckmann Coulter, Marseille, France) and anti-CD3 PerCP (Becton Dickinson). Receptors and activation markers were detected using phycoerythrin-conjugated monoclonal antibodies such as anti-NKp30, anti-NKp44, anti-NKp46, anti-human leucocyte antigen DR-1 (HLA-DR) and anti-CD25 (Beckmann Coulter), anti-CD38 and anti-CD69 and mouse isotype control reagents (Beckton Dickinson). NK cell and CD8+ T-cell specific receptor expression was determined after gating on CD3CD56+ and CD3+CD8+ cells, respectively. For in-vitro stimulation, NK cells were cultured for up to 5 days in the presence of LPS from Escherichia coli 026:B6 (1 ng/ml, 100 ng/ml, Sigma, Poole UK) or IL-15 (50 ng/ml; Peprotech, London, UK) and activation marker and natural cytotoxicity receptor (NCR) expression assessed.

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Statistical analysis

Statistical analyses were performed using Mann–Whitney U-test for comparison between groups and Wilcoxon signed rank test for comparing of NK cell responses induced by LPS or IL-15 with those observed in cells cultured in medium alone. StatView 5.01 software was used (Abacus, Berkley, California, USA) and P values less than 0.05 were considered as significant.

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Elevated plasma lipopolysaccharide in HIV-1-infected individuals

As plasma viraemia has previously been shown to influence the proportion of NK cells expressing activation markers and activating receptors, plasma LPS was measured in viraemic and aviraemic HIV-1-positive individuals compared with HIV-1-negative healthy control individuals and IBD patients (Fig. 1). Plasma LPS concentration was significantly elevated in HIV-1-positive viraemic (median n = 0.333 U/ml, range = 0.181–0.902) and aviraemic individuals (median n = 0.349 EU/ml/ml, range = 0.108–0.870) and in IBD patients (median n = 0.349, range = 0.124–0.710) compared with healthy HIV-1-negative individuals (median n = 0.172, range = 0.117–0.310) (Fig. 1a).

Fig. 1

Fig. 1

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Decreased natural cytotoxicity receptors and elevated activation markers in HIV-1-infected individuals but not in individuals with inflammatory bowel disease

As NK cells are chronically partially activated during HIV-1 infection, we investigated the relationship between increased levels of plasma LPS and the expression of activation markers (CD25, CD69 and HLA-DR) and NCRs (NKp30, NKp44 and NKp46).

Viraemic HIV-1-positive individuals had a significantly elevated proportion of CD69+ (median n = 14.5%, range = 0.9–43.5%) and HLA-DR+ NK cells (20.4%, 7.1–67.5%) compared with HIV-1-negative individuals (4.9%, 0.4–20.9%, P = 0.0321 and 4.1%, 0.6–13%, P < 0.0001, respectively) (Fig. 1b). No increase was observed in the proportion of NK cells expressing CD69 in aviraemic HIV-1-positive individuals (7%, 0.5–28.9%), and a reduced proportion of HLA-DR expressing NK cells (10.7, 2.9–57.5%) was detected compared with viraemic individuals, despite both groups of patients having elevated levels of LPS in plasma. This indicates that plasma viral load, rather than LPS, had a role in driving NK cell activation in these patients (Fig. 1b). There was no significant difference in the proportion of NK cells from HIV-1-positive individuals expressing CD25 compared with HIV-1-negative individuals (Fig. 1b), and also no change was observed in CD38 expression (data not shown). Despite HIV-1-negative individuals with IBD having elevated plasma LPS, no difference was observed in the proportion of NK cells expressing activation markers CD69 and HLA-DR [7%, (4.5–9.1%) and 3.4%, (0.3–9.4%), respectively] compared with HIV-1-negative individuals (Fig. 1b).

A decreased proportion of NK cells expressing NCRs, NKp30 and NKp46, was observed in both viraemic (median n = 42.7%, range = 4.4–82.4% and 79.1, 25.9–97.8%, respectively) and aviraemic HIV-1-positive individuals (44.8%, 6.4–91.3% and 64.1%, 14.2–97.7%) compared with HIV-1-negative individuals (77.5%, 31.9–90.6% and 93.4%, 50.4–96.9%), but not in IBD patients (72.8%, 21.8–89.9% and 89.3%, 55.3–96.5%), again indicating that elevated plasma LPS alone is not sufficient to drive these changes (Fig. 1c).

A higher proportion of CD8+ T cells from viraemic HIV-1-positive individuals expressed HLA-DR (median n = 31.9%, range = 10.9–58.2%) and CD38 (53.8%, 10.5–95%) compared with HIV-1-negative individuals (10.5%, 2.2–17.5% and 19.3%, 1.8–57.2%), and the expression of these activation markers was reduced in CD8+ T-cells from aviraemic HIV-1-positive individuals receiving HAART (15.4%, 2.1–55.3% and 28.7%, 7.8–94%) (Fig. 1d). However, despite having elevated levels of plasma LPS, no difference was observed in the proportion of CD8+HLA-DR+ and CD8+CD38+ T cells in IBD patients (5.9%, 2.7–20.7% and 15.4%, 5.1–47.9%, respectively) compared with HIV-1-negative healthy individuals (Fig. 1d).

These data indicate that elevated plasma LPS alone, as observed in aviraemic HIV-1-positive individuals and IBD patients, does not drive chronic NK cell activation and that plasma viraemia plays an important role.

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Stimulation of NK cells by lipopolysaccharide in vitro

LPS stimulates NK cells to produce cytokines and proliferate in vitro in the presence of myeloid cells [9,10]. PBMC were, therefore, stimulated with LPS to investigate which phenotypic changes in NK cells could be potentially attributable to LPS, and whether the in-vitro activation of NK cells was refractory in HIV-1-positive individuals.

Low and high concentrations of LPS stimulated an increase in the proportion of NK cells expressing CD25 and CD69 from HIV-1-negative individuals compared with cells cultured in medium alone. LPS, 1 ng/ml: CD25 median increase = 4.9%, range = −1.2 to 14%; CD69 38.3%, 19.9–58.8% and LPS, 100 ng/ml: CD25 median increase = 6.9%, range = 0.9–24.1%; CD69 42.2%, 19.4–63.5% (Fig. 2a,b). Little upregulation of HLA-DR was observed in NK cells from HIV-1-negative individuals (LPS, 1 ng/ml: median increase = 3.4%, range = 0.4–18.3%; LPS, 100 ng/ml: median increase = 5.2%, range = −1.1 to 27.1%), suggesting that LPS alone is unlikely to account for the chronic upregulation of HLA-DR expression on NK cells in HIV-1-positive individuals (Fig. 2c).

Fig. 2

Fig. 2

Ongoing activation of NK cells, from both viraemic and aviraemic HIV-1-positive individuals, was observed in cells cultured in medium compared with freshly isolated NK cells. In-vitro stimulation with LPS did not result in further significant stimulation of CD25 or CD69 expression in HIV-1-positive individuals. In contrast, IL-15 stimulated significant CD25 and CD69 expression in both HIV-1-positive viraemic and aviraemic individuals and in HIV-1-negative individuals (Fig. 2a,b).

NCR, NKp30 and NKp46, are lost in in-vitro culture, and LPS stimulation had no significant impact on the proportion of NK cells expressing these NCR either in HIV-1-negative controls or HIV-1-positive individuals, indicating that changes to NCR expression during HIV-1 infection are unlikely to be driven by LPS. IL-15, however, promoted the maintenance of NKp30 expression in HIV-1-negative individuals and in both HIV-1-positive viraemic and aviraemic individuals (Fig. 2d). LPS stimulated a modest, but significant, induction of NKp44 in NK cells from HIV-1-negative individuals, but this response was not significant in HIV-1-positive individuals (LPS, 1 ng/ml: HIV-1-negative, median increase = 4.8%, range = 0.3–26.6%; HIV-1-positive, viraemic = 1.7%, −1.7 to 13.9%; HIV-1-positive, aviraemic = 2.4%, −0.1 to 8.4%; LPS, 100 ng/ml: HIV-1-negative = 2.1%, −0.1 to 23.1%; viraemic = 1.1%, −3.2 to 13.7%; aviraemic = 1.1%, −0.7 to 11.9%) (Fig. 2e). The induction of NKp44 in response to IL-15 was also reduced in HIV-1-positive compared with HIV-1-negative individuals (HIV-1-negative median increase = 34.3%, range = 1.4–50.3%; viraemic = 4.3%, −0.2 to 47.2; aviraemic = 4.9%, −0.2 to 62.8%), suggesting that intrinsic IL-15 signalling pathways that induce NKp44 may be defective in HIV-1-positive individuals (Fig. 2e).

These data suggest that multiple pathways, including those initiated by LPS, induce the expression of different NK cell activation markers, and that pathways leading to the expression of NKp44, CD25 and CD69 in NK cells from HIV-1-positive individuals are not stimulated further by LPS in vitro.

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There is evidence for a role of HIV-1 envelope proteins, and HIV-1 viral RNA in driving nonspecific activation of lymphocytes [4,11,12]. Translocation of microbial products, including bacterial LPS, from the gastrointestinal tract may also contribute to chronic immune activation in HIV-1-positive individuals [1].

A dominant role for HIV-1 plasma viraemia in chronic NK cell activation is supported here by an activated phenotype in viraemic HIV-1-positive individuals compared with aviraemic individuals, and a lack of activation in NK cells from IBD patients, despite all groups having elevated plasma LPS. Detection of elevated plasma LPS concentrations in aviraemic individuals receiving antiretroviral therapy in this study are consistent with original studies from Brenchley et al. [1] in which, although partially reduced, levels remained significantly elevated in HIV-1-positive patients receiving HAART for 48 weeks compared with HIV-1-negative controls, and were most dramatically reduced in those individuals with the greatest recovery in CD4+ T cells/μl. Similar levels of plasma LPS could, therefore, reflect the similar ranges of CD4+ T cells/μl in viraemic and aviraemic individuals in our study.

Overall, whilst our studies cannot exclude the possibility that LPS contributes to chronic immune activation in HIV-1-positive individuals, it is evident that the potential role of LPS is diminished in the absence of HIV-1 plasma viraemia.

Weak induction of NKp44, CD25 and CD69 is observed on in-vitro stimulation of NK cells from HIV-1-negative individuals with LPS compared with those mediated by IL-15. Poor induction of HLA-DR by LPS in vitro is consistent with low expression in IBD patients and aviraemic HIV-1-positive individuals who have elevated plasma LPS compared with healthy controls, and indicates that other factors contribute to chronic NK cell activation during HIV-1 infection. We observed significant ongoing activation of CD25 and CD69 in NK cells from HIV-1-positive individuals in the absence of LPS and, in contrast to IL-15 stimulation, no additional response was observed on in-vitro culture with LPS. Furthermore, LPS stimulated little or no induction of NKp44 in HIV-1-positive individuals. Plasmacytoid dendritic cells, although reduced in number, have been recently shown to chronically produce IFN-α in viraemic HIV-1-positive individuals and to be refractory for further IFN-α production on in-vitro stimulation [13]. Ongoing IFN-α production from plasmacytoid dendritic cells, in response to viral RNA, would drive both the elevated expression of NK cell activation markers ex-vivo and the ongoing partial activation of NK cells in viraemic HIV-1-positive individuals [13–15]. Such a mechanism would account for both the dominance of HIV-1 plasma viraemia over elevated plasma LPS levels and the lack of in-vitro stimulation by LPS.

As in other studies, a reduction in the proportion of total blood dendritic cell subsets in viraemic HIV-1-positive individuals is observed here (proportion of PBMC: HIV-1-negative control: median n = 1.12%, range = 0.32–2.93%, HIV-1 viraemic: median n = 0.54%, range = 0.19–1.54%, P = 0.0215) [16,17]. A reduced impact of LPS on NK cell activation in HIV-1-positive individuals in vitro could relate to changes in numbers of dendritic cell subsets. We observed no impact of HIV-1 infection on the proportion of CD14+HLA-DR+ monocytes (data not shown), which should respond to LPS in vitro [10]. However, peripheral blood monocytes have inhibitory effects on NK cell responses to LPS and a shift in the balance of dendritic and monocytic cell populations and the factors they produce could have an impact on the outcome of such responses in HIV-1-positive individuals [10].

Sequences derived from HIV-1 RNA stimulate monocytes, plasmacytoid and myeloid dendritic cells via TLR-7/8 [18,19]. Signalling pathways for LPS stimulation via TLR4 and HIV-1 RNA stimulation via TLR 7/8 converge, so that in-vivo stimulation by HIV-1 viral RNA could affect subsequent in-vitro responsiveness to LPS [20].

Further analysis will reveal the importance of different accessory cell populations, responsiveness to different TLR ligands and the role of stimulatory cytokines in chronic NK cell activation during HIV-1 infection.

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J.N.G. and M.R.G. designed and performed the experiments and wrote the manuscript and M.R.G. was a grant holder. A.S., M.B. and B.G.G. identified and recruited patients to the study and were involved in study design and review of the manuscript. F.M.G. was a grant holder and reviewed and provided revision to the final manuscript. These studies were funded by grants from the Joint Research Committee of the Trustees of Chelsea and Westminster Hospital, London, by the St Stephen's AIDS Trust and by the European Commission Europrise Consortium.

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activation; HIV-1; NK cells; plasma endotoxin

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