Microbial translocation is associated with increased monocyte activation and dementia in AIDS patients

Abstract

Elevated plasma lipopolysaccharide (LPS), an indicator of microbial translocation from the gut, is a likely cause of systemic immune activation in chronic HIV infection. LPS induces monocyte activation and trafficking into brain, which are key mechanisms in the pathogenesis of HIV-associated dementia (HAD). To determine whether high LPS levels are associated with increased monocyte activation and HAD, we obtained peripheral blood samples from AIDS patients and examined plasma LPS by Limulus amebocyte lysate (LAL) assay, peripheral blood monocytes by FACS, and soluble markers of monocyte activation by ELISA. Purified monocytes were isolated by FACS sorting, and HIV DNA and RNA levels were quantified by real time PCR. Circulating monocytes expressed high levels of the activation markers CD69 and HLA-DR, and harbored low levels of HIV compared to CD4(+) T-cells. High plasma LPS levels were associated with increased plasma sCD14 and LPS-binding protein (LBP) levels, and low endotoxin core antibody levels. LPS levels were higher in HAD patients compared to control groups, and were associated with HAD independently of plasma viral load and CD4 counts. LPS levels were higher in AIDS patients using intravenous heroin and/or ethanol, or with Hepatitis C virus (HCV) co-infection, compared to control groups. These results suggest a role for elevated LPS levels in driving monocyte activation in AIDS, thereby contributing to the pathogenesis of HAD, and provide evidence that cofactors linked to substance abuse and HCV co-infection influence these processes.

Figure 1. Increased plasma LPS, sCD14, and CCL2 are associated with HAD.

(A) Levels of LPS, sCD14, CCL2, and IL-6 were quantified in plasma of AIDS patients (n = 119) and uninfected controls (n = 25). (B) Spearman correlation (r and p-values) was calculated to determine the relationship between the LPS levels and sCD14, and between sCD14 and plasma CCL2 or IL-6. (C) Levels of plasma VL, CD4 counts, LPS, sCD14, CCL2, and IL-6 were compared in AIDS patients classified in 5 groups based on the degree of neurocognitive impairment (NCI) (None, No NCI; HAD, HIV-associated dementia; MCMD, minor cognitive and motor disorder; NPI-O, neuropsychiatric impairment due to conditions other than HIV; and ANI, asymptomatic NCI). Kruskal-Wallis test determined significant differences between the 5 groups in 1C. Median values in 1A and 1C are indicated as horizontal lines and significant differences between the 4 NCI groups versus the no NCI group (None) were determined using the Mann-Whitney test.

Figure 2. Substance abuse and HCV co-infection are associated with high plasma LPS levels.

(A) CD4 counts and plasma levels of VL, LPS, and sCD14 were compared in AIDS patients with or without substance abuse as indicated. (B) CD4 counts and plasma levels of VL, LPS, and sCD14 were compared in AIDS patients with IVDU heroin (H), cocaine (C), and H+C. (C) CD4 counts and plasma levels of VL, LPS, and sCD14 were compared in AIDS patients with and without HCV co-infection. Median values are indicated as horizontal lines and statistical significance between groups was calculated using the Mann-Whitney test; significant differences (p<0.05) are indicated.

Figure 3. Increased plasma lipopolysaccharide binding protein (LBP) levels in AIDS patients with HAD.

(A) LBP levels were quantified in plasma from AIDS patients (n = 119) and uninfected controls (n = 14) by ELISA. (B) Spearman correlation (r and p-values) was calculated to determine relationships between LBP levels and plasma LPS. (C) LBP levels were compared between AIDS patients classified into 5 groups based on the level of NCI as in Figure 1C or (D–E) classified into groups based on patterns of substance abuse as in Figure 2A–B. Median values are indicated as horizontal lines and statistical significance between groups was calculated using the Mann-Whitney test; significant differences (p<0.05) are indicated.

Figure 4. Decreased plasma IgM endotoxin core antibody (EndoCAb) levels in AIDS patients with HAD.

(A) Levels of IgM EndoCAb were quantified in plasma from AIDS patients (n = 119) and uninfected controls (n = 14) by ELISA. (B) Spearman correlation (r and p-values) was calculated to determine relationships between IgM EndoCAb levels and plasma LPS. (C) IgM EndoCAb levels were compared between AIDS patients classified into 5 groups based on the level of NCI as in Figure 1C or (D–E) classified into groups based on patterns of substance abuse as in Figure 2A–B. Median values are indicated as horizontal lines and statistical significance between groups was calculated using the Mann-Whitney test; significant differences (p<0.05) are indicated.

Figure 5. Increased CD16⁺ monocyte frequency in AIDS patients with and without HAD.

(A) The frequency of CD16⁺ Mo within the total Mo population was analyzed in AIDS (n = 38) and uninfected subjects (n = 25). Total and CD16⁺ Mo were distinguished from granulocytes by HLA-DR and lack of CD16b/CD66b expression, and from NK cells by higher forward and side scatter characteristics (FSC and SSC), CD14, CD4, and CD33, and lack of CD56 expression . (B) The frequency of CD16⁺ Mo was compared in AIDS patients with HAD (n = 29) and no NCI (n = 9). (C) The frequency of CD16⁺ Mo, plasma VL, and plasma levels of LPS and sCD14 were compared in patients receiving HAART ≥8 weeks (wks) and untreated or on HAART <8 wks. Median values are indicated as horizontal lines and statistical significance between groups was calculated using the Mann-Whitney test; significant differences (p<0.05) are indicated.

Figure 6. Upregulation of CD69 and CCR5 expression on CD16⁺ and CD16⁻ monocyte subsets.

(A) PBMC from AIDS subjects were stained with fluorochrome-conjugated Abs. CD14 and CD16 expression identified three Mo subsets: CD14^highCD16⁻ (gate R2), CD14^highCD16⁺ (gate R3), and CD14^lowCD16⁺ (gate R4). (B) The frequency of each Mo subset was compared in AIDS (n = 11) and uninfected subjects (n = 8). The expression of CD69 (C) and CCR5 (D) was analyzed for each Mo subset from AIDS (n = 9–11) and uninfected subjects (n = 5–9). Median values are indicated as horizontal lines, and statistical significance in 6B–D was calculated using the Mann-Whitney test.

Figure 7. Monocytes are a minor reservoir for HIV replication compared to CD4+ T-cells in vivo.

Highly pure CD4⁺ T-cells and total or CD16⁺ and CD16⁻ Mo subsets were sorted from the same donor peripheral blood sample by FACS from HIV-infected patients with (n = 12) or without (n = 1) AIDS. 8 of the 12 AIDS subjects had HAD. Levels of cell-associated (A) HIV DNA and (B) RNA were quantified by real time PCR and RT-PCR, respectively. (C) CD4⁺ T-cells and CD16⁺ or CD16⁻ Mo (10⁵ cells/well) from patients with high plasma VL were co-cultured with PHA/IL-2 activated Mo∶CD4⁺ T-cell co-cultures from HIV-uninfected subjects (Mo∶T-cell ratio of 1∶2; 0.5×10⁶ cells/well). Co-cultures were maintained up to 32 days and supernatants were recovered every 4 days. Shown are levels of HIV-p24 in supernatants quantified by ELISA at days 24, 28, and 32.