TNFα-Mediated Liver Destruction by Kupffer Cells and Ly6Chi Monocytes during Entamoeba histolytica Infection

Amebic liver abscess (ALA) is a focal destruction of liver tissue due to infection by the protozoan parasite Entamoeba histolytica (E. histolytica). Host tissue damage is attributed mainly to parasite pathogenicity factors, but massive early accumulation of mononuclear cells, including neutrophils, inflammatory monocytes and macrophages, at the site of infection raises the question of whether these cells also contribute to tissue damage. Using highly selective depletion strategies and cell-specific knockout mice, the relative contribution of innate immune cell populations to liver destruction during amebic infection was investigated. Neutrophils were not required for amebic infection nor did they appear to be substantially involved in tissue damage. In contrast, Kupffer cells and inflammatory monocytes contributed substantially to liver destruction during ALA, and tissue damage was mediated primarily by TNFα. These data indicate that besides direct antiparasitic drugs, modulating innate immune responses may potentially be beneficial in limiting ALA pathogenesis

Overexpression of Differentially Expressed Genes Identified in Non-pathogenic and Pathogenic Entamoeba histolytica Clones Allow Identification of New Pathogenicity Factors Involved in Amoebic Liver Abscess Formation

We here compared pathogenic (p) and non-pathogenic (np) isolates of Entamoeba histolytica to identify molecules involved in the ability of this parasite to induce amoebic liver abscess (ALA)-like lesions in two rodent models for the disease. We performed a comprehensive analysis of 12 clones (A1–A12) derived from a non-pathogenic isolate HM-1:IMSS-A and 12 clones (B1–B12) derived from a pathogenic isolate HM-1:IMSS-B. “Non-pathogenicity” included the induction of small and quickly resolved lesions while “pathogenicity” comprised larger abscess development that overstayed day 7 post infection. All A-clones were designated as non-pathogenic, whereas 4 out of 12 B-clones lost their ability to induce ALAs in gerbils. No correlation between ALA formation and cysteine peptidase (CP) activity, haemolytic activity, erythrophagocytosis, motility or cytopathic activity was found. To identify the molecular framework underlying different pathogenic phenotypes, three clones were selected for in-depth transcriptome analyses. Comparison of a non-pathogenic clone A1np with pathogenic clone B2p revealed 76 differentially expressed genes, whereas comparison of a non-pathogenic clone B8np with B2p revealed only 19 differentially expressed genes. Only six genes were found to be similarly regulated in the two non-pathogenic clones A1np and B8np in comparison with the pathogenic clone B2p. Based on these analyses, we chose 20 candidate genes and evaluated their roles in ALA formation using the respective gene-overexpressing transfectants. We conclude that different mechanisms lead to loss of pathogenicity. In total, we identified eight proteins, comprising a metallopeptidase, C2 domain proteins, alcohol dehydrogenases and hypothetical proteins, that affect the pathogenicity of E. histolytica.AUTHOR SUMMARY: The pathogen Entamoeba histolytica can live asymptomatically in the human gut, or it can disrupt the intestinal barrier and induce life-threatening abscesses in different organs, most often in the liver. The molecular framework that enables this invasive, highly pathogenic phenotype is still not well understood. In order to identify factors that are positively or negatively correlated for invasion and destruction of the liver, we used a unique tool, E. histolytica clones that differ dramatically in their pathogenicity, while sharing almost identical genetic background. Based on comprehensive transcriptome studies of these clones, we identified a set of candidate genes that are potentially involved in pathogenicity. Using ectopic overexpression of the most promising candidates, either in pathogenic or in non-pathogenic Entamoeba clones, we identified genes where high expression reduced pathogenicity and only one gene that increased pathogenicity to a certain extend. Taken together, the current study identifies novel pathogenicity factors of E. histolytica and highlights the observation that various different genes contribute to pathogenicity

ALA formation following depletion of Kupffer cells by clodronate liposomes.

<p>(A) Gating strategy to define resident CD11b⁺F4/80^hi (subset 1) and transient inflammatory CD11b^hiF4/80^lo (subset 2) Kupffer cells in the livers of mice five days after a single intravenous (i.v.) administration of clodronate liposomes (clod) or empty liposomes (ctrl) three days post-infection. Data represent the mean ± SEM of three independent experiments (n = 3 animals/group). (B) Gating strategy to define CD11b⁺CD68⁺ (region 1) and CD11b⁺CD68⁻ (region 2) Kupffer cells following treatment with clodronate liposomes (clod) or empty liposomes (ctrl) three days post-infection. (C) Immunohistochemical staining of liver tissue sections two days post i.v. administration of empty liposomes (ctrl) or clodronate liposomes (clod) using an anti-F4/80 antibody; Kupffer cells are indicated by the brown staining. (D) Gating strategy to define CD11b⁺Ly6G⁻Ly6C⁺ inflammatory monocytes derived from total liver and blood leukocytes five days post-clodronate treatment and three days post-infection. Data represent the mean ± SEM of three independent experiments (n = 3 animals/group). (E) Abscess size in wild-type (WT), clodronate-treated (clod), and control (ctrl) mice was monitored by MRI at the indicated times post-infection. Data represent the mean ± SEM of two experiments (3–4 mice/group). (F) PAS staining of abscessed liver tissue sections from control (ctrl) or clodronate-treated mice three days post-treatment and one day post-infection. Arrows indicate <i>E. histolytica</i> trophozoites. Data represent the mean ± SEM; <i>P</i>-values were determined by the Mann-Whitney U and unpaired Student's t test (^*<i>P</i><0.05).</p

Characterization of Kupffer cell populations during ALA.

<p>(A) Gating strategy to define liver macrophage subpopulations in the abscessed region of an infected liver (abscess), a healthy region from the same liver lobe (healthy tissue), and liver tissue from a naïve animal (naïve) at the indicated time points post-infection. Resident Kupffer cells were defined as CD11b^loF4/80^hi cells (subset 1); transient inflammatory monocyte-derived Kupffer cells were defined as CD11b^hiF4/80^lo (subset 2). (B) Representative histograms depict Ly6C expression levels. Data are shown in the bar graphs as mean ± SEM of two independent experiments at the indicated time points post infection (n = 6 animals/group); <i>P</i>-values were determined by the unpaired Student's t-test; ^*<i>P</i><0.05.</p

Neutrophil recruitment during ALA and effects of selective neutrophil depletion.

<p>(A) Levels of CCL3 mRNA were increased in liver tissue of infected mice (ALA) compared with sham-operated mice (sham) or naïve mice (naïve). (B) Gating strategy to define neutrophils isolated from the abscessed region of the infected liver (abscess), a healthy region from the same liver lobe (healthy tissue), and liver tissue of a naïve mouse (naïve) following intrahepatic amebic infection (n = 3–4 animals/group). Neutrophils were defined as CD11b⁺Ly6G⁺ cells. (C) FACS analysis of blood leukocytes at the indicated time points after neutrophil depletion with anti-Ly6G and anti-GR1 antibodies; control mice were subjected to depletion with a non-specific immunoglobulin (rat IgG). CD11b pre-gated cells were further defined as neutrophils by the expression of Ly6G (n = 5 animals/group) and as blood monocytes by the expression of Ly6C (n = 5 animals/group). Depletion efficacy was estimated on indicated time points after the first treatment. (D) Representative T₂ weighted MRI images of mouse liver tissue showing the size of the abscess (arrowheads) following depletion with anti-Ly6G or anti-GR1 antibodies compared to control mice at the indicated times post-infection. (E) Abscess volume in control mice and anti-Ly6G- and anti-GR1-treated mice. Data represent the mean ± SEM of three independent experiments (n = 9–13); <i>P</i>-values were determined by the unpaired Student's t-test (^*<i>P</i><0.05).</p

Contribution of NO and TNFα to liver tissue damage during ALA.

<p>iNOS (A) and TNFα (B) mRNA levels in liver tissue of mice infected with <i>E. histolytica</i> trophozoites (ALA), sham-operated (sham) and naïve (naïve) mice at the indicated times post-infection (n = 4). (C and D) MRI-based determination of abscess volume in wild-type (WT), iNOS^−/− as well as L-NMMA treated mice (C) and in wild-type mice treated with an anti-TNFα antibody 24 h before infection (D). Time post-infection is indicated (4–5 animals/group). (E) Gating strategy to define liver leukocytes producing TNFα; cells were defined as low (TNFα^lo) and high (TNFα^hi) producers of TNFα. Leukocytes were isolated from infected (ALA), sham-operated (sham) and naïve (naïve) mice at the indicated times post-infection. Cells were further characterized according to the expression of Ly6C as CD11b⁺Ly6^lo and CD11b⁺Ly6C^hi monocytes. (F) Gating strategy to define the numbers of TNFα-positive F4/80⁺ Kupffer cells in livers of naive, sham immunized and infected mice on indicated time points. Data represent the mean ± SEM of two independent experiments (2–5 animals/group); <i>P</i>-values were determined by the Mann-Whitney U test (^*<i>P</i><0.05, ^**<i>P</i><0.01, ^***<i>P</i><0.001).</p

Testosterone modulates murine NKT cell specific IFNγ production.

<p>A) Liver lymphocytes from placebo or testosterone treated naïve female mice were gradient purified, T cells were isolated by magnetic cell sorting and co-cultured for 48 h with αGalCer (2 µg/ml) or EhLPPG (4 µg/ml) pre-pulsed APCs. Medium control comprises NKT cells co-cultured with naive APC’s. IFN<b>γ</b> in the supernatant was quantified using ELISA. B) Liver lymphocytes were isolated from naïve male or orchiectomized male mice and co-cultured with pre-pulsed APCs as described above. IFN<b>γ</b> production was quantified using ELISA. C/D) Identical experiments as performed in A and B but cells were isolated from the livers of ameba-infected female and male mice- Three independent experiments were performed (data express mean +/− SD, statistics: student`s t test).</p

Role of Ly6C^hi inflammatory monocytes in abscess formation.

<p>(A) CCL2 mRNA levels in liver tissue of mice infected with <i>E. histolytica</i> trophozoites (ALA), sham-operated mice (sham) and naïve mice (naïve) at the indicated times (n = 4). (B) Abscess volume was determined by MRI at the indicated time points in wild-type (WT) and CCR2^−/− mice. Data represent the mean ± SEM of three independent experiments (n = 3–4 animals/group). (C) Gating strategy to define liver leukocytes from wild-type (WT) and CCR2^−/− mice five days post-infection. Inflammatory monocytes were defined as Ly6C^hiLy6G⁻CD11b⁺ cells (region 1); Ly6C^lo monocytes were defined as Ly6C^loLy6G⁻CD11b⁺ cells (region 2); and neutrophils were defined as Ly6G⁺Ly6C^loCD11b⁺ cells. (D) Representative MRI images of abscesses (arrows) in infected wild-type (WT) and CCR2^−/− mice that received an adoptive transfer of CD115⁺ WT monocytes 6 hours post-infection; time post-infection is indicated. (E) MRI-based determination of abscess volume at the indicated times. Shown are representative data (mean ± SEM) of one out of two independent experiments (each 4–5 animals/group); <i>P</i>-values were determined by the Mann-Whitney U test (^*<i>P</i><0.05; ^**<i>P</i><0.01; ^***<i>P</i><0.001).</p

Influence of gonadectomy on the size of ALA in the mouse model for the disease.

<p>Eight weeks old male and female mice were gonadectomized and intrahepatically challenged with 1×10⁵<i>E. histolytica</i> trophozoites seven weeks after castration. The animals were sacrificed 7 days post infection, the sizes of abscesses were determined and transformed into score values (score 0: no abscess; score 1: abscess <1 mm; score 2∶1 to 5 mm; score 3: >5 mm) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055694#pone-0055694-g001" target="_blank">Fig. 1A</a>) or the re-isolation rate of live <i>E. histolytica</i> trophozoites from abscessed liver tissue was determined (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055694#pone-0055694-g001" target="_blank">Fig. 1B</a>). Results were obtained from 3 independent trials each comprising 7 animals (statistics: Mann Whitney U test).</p

Characterization of NKT cells frequencies and NKT cell specific IFNγ production in female and male mice.

<p>A) NKT cell frequencies in the liver of female and male mice at different ages are shown. NKT cell numbers were determined as percentage of αGalCer -CD1d tetramer positive cells to CD3⁺ T cells. B) IFN<b>γ</b> production of NKT cells sorted (αGalCer -CD1d tetramer) from the liver of female or male mice after 48 h of co-incubation with αGalCer (4 µg/ml) or EhLLPG (8 µg/ml) pre-pulsed APCs. Medium control includes NKT cells co-cultured with naive APC’s. IFN<b>γ</b> production was quantified by ELISA (5 independent experiments were performed, a summary of data is expressed as mean +/− SD, statistics: student t test). C) IFN<b>γ</b> production of male or female NKT cells upon stimulation with αGalCer pre-pulsed male or female APCs.</p