Lipopolysaccharide Induces Immune Activation and SIV Replication in Rhesus Macaques of Chinese Origin

<div><p>Background</p><p>Chronic immune activation is a hallmark of progressive HIV infection and a key determinant of immunodeficiency in HIV-infected individuals. Bacterial lipopolysaccharide (LPS) in the circulation has been implicated as a key factor in HIV infection-related systemic immune activation. We thus investigate the impact of LPS on systemic immune activation in simian immunodeficiency virus (SIV)-infected rhesus macaques of Chinese origin.</p><p>Methods</p><p>The animals were inoculated intravenously with SIVmac239. The levels of plasma viral load and host inflammatory cytokines in PBMC were measured by real-time RT-PCR. CD4/CD8 ratio and systemic immune activation markers were examined by flow cytometric analysis of PBMCs. White blood cell and neutrophil counts and C Reactive Protein levels were determined using biochemistry analyzer. The plasma levels of LPS were determined by Tachypleus Amebocyte Lysate (TAL) test.</p><p>Results</p><p>The animals inoculated with SIVmac239 became infected as evidenced by the increased plasma levels of SIV RNA and decreased CD4/CD8 ratio. LPS administration of SIV-infected animals induced a transient increase of plasma SIV RNA and immune activation, which was indicated by the elevated expression of the inflammatory cytokines and CD4+HLA-DR+ T cells in PBMCs.</p><p>Conclusions</p><p>These data support the concept that LPS is a driving factor in systemic immune activation of HIV disease.</p></div

Effect of LPS on inflammatory cytokines in PBMCs.

<p>SIVmac239-infected animals were intravenously injected with a single dose of LPS (50 µg/kg, solid circles and lines, n = 3) or saline (open circle and dashed lines, n = 3) at 45 weeks postinfection. Blood samples were collected at indicated time points post-LPS administration and PBMCs were isolated. The levels of cytokines (IL-6, IL-8, IFN-α, and TNF-α) in PBMCs were determined by real-time PCR and normalized to GAPDH mRNA. Data are expressed as fold of control (before LPS administration, cytokine mRNA/GAPDH mRNA, which was defined as 1).</p

SIV infection of Chinese rhesus monkeys.

<p>Six animals were intravenously inoculated with SIVmac239 (10³ TCID50). Blood samples were collected from the animals at the indicated time points postinfection. A: Plasma levels of SIV GAG gene RNA were measured by real-time PCR. B: CD4/CD8 ratios were measured by flow cytometry. C: Average SIV loads and CD4/CD8 ratios (mean ± SEM) of SIV-infected animals.</p

Effect of LPS on SIV replication.

<p>SIV-infected animals were intravenously injected with either a single dose of LPS (50 µg/kg; solid circles and lines, n = 3) or saline (open circle and dashed lines, n = 3) at 45 weeks postinfection. The plasma samples were collected at the indicated time points after LPS treatment. A: The plasma levels of LPS were determined by Tachypleus Amebocyte Lysate (TAL) test. B: SIV loads were measured by real-time PCR for SIV GAG gene expression.</p

Effect of LPS on CD4/CD8 ratio, counts of CD4+ T cells and frequency of CD4+HLA-DR+ T cells.

<p>SIVmac239-infected animals were intravenously injected with either LPS (50 µg/kg; solid circles and lines, n = 3) or saline (open circle and dashed lines, n = 3) at 45 weeks postinfection. At the indicated time points after LPS administration, the CD4/CD8 ratios (A), total CD4+ T cell counts (B), and CD4+HLA-DR+ T cell percentage in PBMCs (C) were determined by flow cytometry.</p

Primer Pairs for Real-Time RT-PCR.

<p>Primer Pairs for Real-Time RT-PCR.</p

Animals Used for the Study.

<p>*I.V. = intravenous;</p><p>**analyzed at day 315 (45 weeks) post SIV inoculation.</p

Effect of LPS on WBC, neutrophils and CRP.

<p>SIVmac239-infected animals were intravenously injected with either LPS (50 µg/kg; solid circles and lines, n = 3) or saline (open circle and dashed lines, n = 3) at 45 weeks postinfection. At the indicated time points after LPS administration, white blood cell (WBC) counts (A), neutrophil counts (B), and C Reactive Protein (CRP) levels (C) were determined by a biochemistry analyzer.</p

Changes in neutralization sensitivity of R5 viruses evolving over time in macaque BR24.

<p>Susceptibility of BR24 R5 pseudoviruses to neutralization with b12, 447-52D and T20 was determined, with sensitivity of variants from the inoculating virus SHIV_SF162P3N (P3N) shown for reference. The vertical dashed line indicates the time of coreceptor switching, and the dotted area designates the period of marked envelope conformational changes. Data are representative of at least two independent experiments (error bars, s.d.). * above the bars indicate IC₅₀ values that are statistically different between the acute (w2) and the evolving R5 viruses.</p

Entry efficiency, PSC-RANTES and sCD4 sensitivity of R5 viruses evolving over time in CA28.

<p>Entry of luciferase reporter viruses expressing CCR5-using envelopes into TZM-bl cells (A), and susceptibility of the reporter viruses to neutralization with PSC-RANTES (B) and sCD4 (C) were determined. The solid and dashed vertical lines indicate the two switch events in CA28 leading to the emergence of distinct dual-tropic and X4 viruses, respectively. The numbers in the brackets denote the number of envelope clones analyzed at each time point. Absolute CD4+ T-cell count in the animal over the course of infection is shown in (C), and values above the bars indicate fold increase in sCD4 sensitivity of CA28 viruses compared to viruses in the SHIV_SF162P3N inoculum (P3N). *<i>P</i><0.05 (Mann-Whitney <i>U</i> test). Data are representative of at least three independent experiments (error bars, s.d.).</p