Figure 1.
Histological and immunohistochemical characterization of cell infiltrates during ALA.
(A) H&E staining of mouse liver abscesses (indicated by the square in the top row of images) at the indicated times post-infection with E. histolytica trophozoites. (B) PAS staining shows E. histolytica trophozoites (arrowheads) within the abscess. (C and D) Tissue sections were stained with anti-7/4 (C) and anti-F4/80 (D) antibodies followed by HRP-conjugated secondary antibody to detect neutrophils and macrophages, respectively (brown).
Figure 2.
Neutrophil recruitment during ALA and effects of selective neutrophil depletion.
(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); P-values were determined by the unpaired Student's t-test (*P<0.05).
Figure 3.
Characterization of Kupffer cell populations during ALA.
(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 CD11bloF4/80hi cells (subset 1); transient inflammatory monocyte-derived Kupffer cells were defined as CD11bhiF4/80lo (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); P-values were determined by the unpaired Student's t-test; *P<0.05.
Figure 4.
ALA formation following depletion of Kupffer cells by clodronate liposomes.
(A) Gating strategy to define resident CD11b+F4/80hi (subset 1) and transient inflammatory CD11bhiF4/80lo (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 E. histolytica trophozoites. Data represent the mean ± SEM; P-values were determined by the Mann-Whitney U and unpaired Student's t test (*P<0.05).
Figure 5.
Role of Ly6Chi inflammatory monocytes in abscess formation.
(A) CCL2 mRNA levels in liver tissue of mice infected with E. histolytica 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 Ly6ChiLy6G−CD11b+ cells (region 1); Ly6Clo monocytes were defined as Ly6CloLy6G−CD11b+ cells (region 2); and neutrophils were defined as Ly6G+Ly6CloCD11b+ 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); P-values were determined by the Mann-Whitney U test (*P<0.05; **P<0.01; ***P<0.001).
Figure 6.
Contribution of NO and TNFα to liver tissue damage during ALA.
iNOS (A) and TNFα (B) mRNA levels in liver tissue of mice infected with E. histolytica 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 (TNFαlo) and high (TNFαhi) 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+Ly6lo and CD11b+Ly6Chi 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); P-values were determined by the Mann-Whitney U test (*P<0.05, **P<0.01, ***P<0.001).