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. 2010 Apr 29;6(4):e1000867.
doi: 10.1371/journal.ppat.1000867.

Inadequate clearance of translocated bacterial products in HIV-infected humanized mice

Ursula Hofer  1 , Erika Schlaepfer, Stefan Baenziger, Marc Nischang, Stephan Regenass, Reto Schwendener, Werner Kempf, David Nadal, Roberto F Speck
Affiliations

Inadequate clearance of translocated bacterial products in HIV-infected humanized mice

Ursula Hofer et al. PLoS Pathog. .

Abstract

Bacterial translocation from the gut and subsequent immune activation are hallmarks of HIV infection and are thought to determine disease progression. Intestinal barrier integrity is impaired early in acute retroviral infection, but levels of plasma lipopolysaccharide (LPS), a marker of bacterial translocation, increase only later. We examined humanized mice infected with HIV to determine if disruption of the intestinal barrier alone is responsible for elevated levels of LPS and if bacterial translocation increases immune activation. Treating uninfected mice with dextran sodium sulfate (DSS) induced bacterial translocation, but did not result in elevated plasma LPS levels. DSS-induced translocation provoked LPS elevation only when phagocytic cells were depleted with clodronate liposomes (clodrolip). Macrophages of DSS-treated, HIV-negative mice phagocytosed more LPS ex vivo than those of control mice. In HIV-infected mice, however, LPS phagocytosis was insufficient to clear the translocated LPS. These conditions allowed higher levels of plasma LPS and CD8+ cell activation, which were associated with lower CD4+/CD8+ cell ratios and higher viral loads. LPS levels reflect both intestinal barrier and LPS clearance. Macrophages are essential in controlling systemic bacterial translocation, and this function might be hindered in chronic HIV infection.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Gastrointestinal barrier dysfunction did not completely explain HIV-associated plasma LPS elevation in humanized mice.
4 to 6 weeks after HIV or mock infection, humanized mice received DSS 0.75% or normal drinking water for 2 weeks. (A) In cultures of organ suspensions, bacterial colony forming units from MLN (white bar) and spleens (black bar, mean, SD) were quantified; percentages of positive organs in the different groups are indicated at the bottom of the respective bars (n = 50, pooled data from two independent experiments). (B) The mice showed a trend towards higher levels of bacterial translocation (assessed by an index that includes number, species, and location of bacteria detected, P = 0.1, 0.08 and 0.13, respectively) after HIV infection or DSS treatment. (C) DSS+/HIV− mice (black square) had plasma LPS levels similar to those of control mice (black circle). Only HIV+ mice without (white circle, *, P = 0.015) or with DSS treatment (white square, **, P = 0.005) showed significant increases of plasma LPS (n = 59, pooled data from two independent experiments). (D and E) Both groups of HIV+ mice showed a trend towards higher plasma sC14 (P = 0.06 for HIV+/DSS− and P = 0.1 for HIV+/DSS+ mice) and had significantly higher LBP values (***, P = 0.0006 for HIV+/DSS− and **, P = 0.008 for HIV+/DSS+ mice).
Figure 2
Figure 2. The combination of bacterial translocation and disturbed LPS clearance induced plasma LPS elevation.
(A) Humanized mice were injected intraperitoneally with clodrolip (1 mg/20 g body weight) to deplete phagocytic CD11b intermediate cells (spleens of representative mock PBS or clodrolip treated mice 48 h after injection). (B) After 1 week of DSS 0.75% (square, block arrow down) treatment and a second injection of clodrolip (0.5 mg/20 g body weight) (block arrow up, block arrow down), plasma LPS was only increased in mice that received both treatments (*, P = 0.006, n = 37, pooled data from two independent experiments). (C) Liver macrophages, isolated from HIV− or HIV+ mice that received either normal drinking water or 0.75% DSS for 2 weeks, were incubated ex vivo with FITC-LPS at 37°C or 4°C (shaded or open histogram), and mean fluorescence intensity of phagocytic cells (values upper right corner) was measured. (D) Values were normalized to the mean FITC-LPS signal of cells from HIV−/DSS− mice (black circle). DSS-induced bacterial translocation increased FITC-LPS phagocytosis (black square, ***, P<0.0001), but HIV infection abrogated this effect (white circle, P = 0.2), independent of DSS treatment (white square, P = 0.19) (n = 59, pooled data from two independent experiments).
Figure 3
Figure 3. HIV-infected humanized mice had high levels of CD8+ T-cell activation.
Control (black symbols) or HIV-infected mice (white symbols) received normal drinking water (circles) or 0.75% DSS (squares) for 2 weeks. (A) Activation levels in the spleen were determined by flow cytometry of human HLA-DR and CD38 staining of CD45+CD8+ (one representative animal per group) and CD45+CD4+ splenocytes. (B) DSS treatment alone slightly increased activation levels of CD4+ (*, P = 0.004) and CD8+ cells (*, P = 0.007), over those in uninfected, untreated control mice. HIV infection drastically increased CD8+ cell activation in animals that received normal (***, P<0.0001) or DSS (***, P<0.0001) water (n = 50, pooled data from two independent experiments).
Figure 4
Figure 4. Activation of CD8+ cells was associated with lower CD4+/CD8+ cell ratios and higher viral loads.
Human CD8+ splenocyte activation levels defined by HLA−DR+ and CD38+ co-staining from HIV−/DSS− (black circle), HIV−/DSS+ (black square), HIV+/DSS− (white circle), and HIV+/DSS+ animals (white square) were correlated with CD4+ cell-activation levels, CD4+/CD8+ cell ratio, and viral load. (A) In uninfected mice, activation levels of CD8+ and CD4+ cells were tightly correlated (n = 23, pooled data from two independent experiments). (B) But in HIV+ mice, this relationship was not as clear (n = 27, pooled data from two independent experiments). (C) In HIV+ mice, higher percentages of activated CD8+ cells correlated with lower CD4+/CD8+ cell ratios. (D) When activation levels were adjusted for overall engraftment by calculating percentages of HLA-DR+CD38+CD8+ cells of total cells, including murine cells, CD8+ cell activation correlated also with higher viral loads.
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