Different behavior of myeloperoxidase in two rodent amoebic liver abscess models

Abstract

The protozoan Entamoeba histolytica is the etiological agent of amoebiasis, which can spread to the liver and form amoebic liver abscesses. Histological studies conducted with resistant and susceptible models of amoebic liver abscesses (ALAs) have established that neutrophils are the first cells to contact invasive amoebae at the lesion site. Myeloperoxidase is the most abundant enzyme secreted by neutrophils. It uses hydrogen peroxide secreted by the same cells to oxidize chloride ions and produce hypochlorous acid, which is the most efficient microbicidal system of neutrophils. In a previous report, our group demonstrated that myeloperoxidase presents amoebicidal activity in vitro. The aim of the current contribution was to analyze in vivo the role of myeloperoxidase in a susceptible (hamsters) and resistant (Balb/c mice) animal models of ALAs. In liver samples of hamsters and mice inoculated intraportally with Entamoeba histolytica trophozoites, the number of neutrophils in ALAs was determined by enzymatic activity. The presence of myeloperoxidase was observed by staining, and its expression and activity were quantified in situ. A significant difference existed between the two animal models in the number of neutrophils and the expression and activity of myeloperoxidase, which may explain the distinct evolution of amoebic liver abscesses. Hamsters and mice were treated with an MPO inhibitor (4-aminobenzoic acid hydrazide). Hamsters treated with ABAH showed no significant differences in the percentage of lesions or in the percentage of amoebae damaged compared with the untreated hamsters. ABAH treated mice versus untreated mice showed larger abscesses and a decreased percentage of damaged amoebae in these lesion at all stages of evolution. Further studies are needed to elucidate the host and amoebic mechanisms involved in the adequate or inadequate activation and modulation of myeloperoxidase.

Fig 1. Percentage of neutrophils stained by AS-D esterase in ALAs from hamsters and Balb/c mice.

Liver sections were stained with AS-D chloroacetate esterase. Neutrophils were identified by their well-known morphology and a positive stain. The total number of cells were counted in the inflammatory focus (8 infiltrates per slide/3 slides per animal), as were the number of cells positive to AS-D chloroacetate esterase (neutrophils) by using Image-Pro Plus 5.1 with 40x magnification. The percentage of neutrophils was calculated. During the evolution of hamster ALA, the percentage of neutrophils diminished progressively. In mice, the percentage of neutrophils was less compare with the hamsters but the changes showed similar behaviour, except at 24 hours. At the latter times post infection, the percentage of neutrophils was higher in mice than hamsters. Data represent the mean ± SD of the three independents experiments, (n = 5). P-values were determined by the Student’s t-test (*** p<0.001; ** p<0.01).

Fig 2. Neutrophils positive to esterase in hamster and mouse ALAs.

Liver tissue was processed by histochemistry. Neutrophils were positive to AS-D chloroacetate esterase in ALA from hamsters and mice at 3, 6, 12 and 24 h post inoculation. Amoebae are present inside the inflammatory infiltrate (arrows). Barr = 50μm.

Fig 3. Neutrophils positive to MPO in hamster and mice ALAs.

Liver tissue was processed by immunohistochemistry to detect the presence of MPO in ALA of hamsters (A, C, E, G, I) and mice (B, D, F, H, J) at 3, 6, 12 and 24 h post-inoculation. Negative controls was performed with an irrelevant, antibody no label was observed (A, B). (C) Hamster liver lesions, neutrophils were positive in small inflammatory foci at 3 h post-inoculation (arrowhead). (E) At 6 h post-inoculation, amoebae (arrows) were positive to MPO. (G) At 12 h, appears extensive inflammatory reaction composed by damaged neutrophils positive to MPO. (I) At 24 h, inflammatory cells lysed on the border of a necrotic area were also stained to MPO. (D) Mouse liver lesions; inflammatory cells showed MPO label at 3 h post-inoculation (arrowhead), amoeba is seen (arrow). (F) At 6 h post-inoculation, neutrophils appear surround the damaged amoeba (arrow). (H) At 12 h appear damaged amoeba (arrow); the inflammatory infiltrate was constituted by neutrophils positive to MPO (arrowhead). (J) At 24 h staining for MPO was evident, the amoeba present signs of damage (arrow). Barr = 50μm.

Fig 4. Different kinetics of cells positive to MPO in hamster and mouse ALAs model.

Hamsters showed an increase in neutrophils positive to MPO at 3 and 6 h, with a reduction in the number of cell at 12 and 24 h. In mice the MPO was positive at 6 and 12 h and decrease importantly at 24 h. Data represent the mean ± SD of three independent experiments (n = 5). P-values were determined by the Student’s t-test (*** p<0.001; ** p<0.01; * p<0.05).

Fig 5. Differential expression of the mpo gene in hamster and mice ALAs.

Mice tissue samples show a significant high expression of the mpo gene at all post-inoculation times compared with the control group. The difference between these two ALAs models were higher at 3 and 12 h (p<0.001). Hamsters ALA did not show expression of the mpo gene compared with the control group, the lowest expression was found at 6 and 12 h (p<0.001). There is a statistically significant difference between the species and post-inoculation time (p<0.001). Data represent the mean ± SD of three independent experiments, (n = 5). P-values were determined by the one-way ANOVA (*** p<0.001).

Fig 6. E. histolytica stimulate the MPO activity in ALA of mice and to decrease in hamsters.

Mice tissue samples from ALA showed a significant higher MPO activity from 6–24 h (p<0.001). Hamster liver samples from ALAs showed a significantly reduction of MPO activity from 6 to 24 h (p<0.01). There is a statistically significant difference between the species and post-inoculation time. Data represent the mean ± SD of the three independent experiments, (n = 5). P-values were determined by the one-way ANOVA (***p<0.001; ** p<0.01; *p<0.05).

Fig 7. The MPO inhibitor (ABAH) has effect in mouse ALA but not in hamster ALA.

Hamsters and mice were treated with 40 mg/Kg of ABAH every 12 h during 5 days, followed by E. histolytica inoculation and sacrifice at 3, 6, and 12 h post-inoculation. Abscess size is expressed as a percentage of the total liver weight. (A) Hamsters treated with ABAH (diagonal bars) did not present differences in the percentage of lesion with respect to the control group with ABAH (gray bars). (B) Mice treated with ABAH presented a larger size of ALAs at all times tested (diagonal bars). The DMSO control group did not present hepatic damage. Data represent the mean ± SD, (n = 6). P-values were determined by the Student’s t-test (***p<0.001; ** p<0.01).

Fig 8. The MPO inhibitor (ABAH) has affect in the percentage of damage amoebae in mouse ALAs but not in amoebae from hamster ALAs.

(A) Hamster treated with ABAH not showed significant differences in the percentage of damaged amoebae respect to the control group without ABAH. (B) Mice treated with ABAH showed less percentage of damaged amoebae around 50% at 6 h and 30% at 12 h (that correlates with atypical morphology). Untreated mice presented about 70% of trophozoites damaged at 6 and 12 h. Data represent the mean ± SD (n = 6). P-values were determined by Bonferroni t-test (***p < 0.001). (C) Undamaged and damaged amoebae in ALA samples stained with H&E. ALA in hamster (A and B) and mice (C and D) at six hours post-inoculation. A and B show undamaged amoebae (with their typical morphology) surrounded by inflammatory cells. C and D show damaged amoebae (arrows) in close contact with the inflammatory cells. Bar = 25 μm.