Exploring the relationship between plasmodium parasite density, immune response, and intestinal pathology during murine malaria

Abstract

Plasmodium spp., cause a wide range of symptoms that classically include fever and anaemia. One of the less investigated symptoms is malaria-associated diarrhoea. Consequences of this intestinal inflammation may be linked to observed dysbiosis and a breakdown of the gut barrier, leading to the increased risk of bacterial sepsis from enteropathogens. The mechanism for malaria caused intestinal inflammation remains unclear. Thus, the impact of parasitaemia on intestinal pathology during infection was investigated using a murine model of acute malaria. C57BL/6J mice were divided into two sets; one set was inoculated with serial blood passaged (SBP) Plasmodium chabaudi AS, and other with mosquito transmitted (MT) Plasmodium chabaudi AS. The P. chabaudi AS species was chosen specifically as it mirrors the P. falciparum infection, to compare two distinct infection models – high (SBP) vs low (MT) density of infection. Along with mock-treated animals, infected mice were monitored for 9 days with parasitaemia, weight and haemoglobin levels recorded. At the end parasite replicative of cycle 8, during schizogony, animals were culled. Plasma and faeces were also collected to measure inflammatory proteins such as IFNγ, IL-10, lactoferrin and albumin by ELISA. Intestinal length was measured and the tissue processed making swiss rolls. After formalin-fixation and processing, intestinal tissue sections were stained for histological analysis using haematoxylin and eosin stain (H&E), Alcian Blue, and fluorescently labelled with proliferation marker Ki-67. Results showed that during infection mice did not lose weight, whilst anaemia was significant in both the SBP and MT models. Parasitaemia for SBP mice ranged between 5-10% whereas the MT mice stayed below 5% at day 9. Infection showed increased serum IFNγ and IL-10 tracked with the higher SBP infection, as expected. In contrast to previous observations, faecal inflammatory proteins did not reveal any significant differences from controls. However, shortening of the intestinal length was observed only in MT mice. Further, histological analysis of H&E-stained small intestine showed a change in villous height and crypt depth ratio, indicating either crypt cell hyperplasia and/or villus atrophy. This data was supported by an increase in Ki-67 staining, particularly those with the higher parasitaemia (SBP). Finally, Alcian blue stained small intestine tissue revealed goblet cell hyperplasia in SBP mice. Altogether, this work demonstrates that intestinal pathology during malaria may be linked to parasite density and the magnitude of the systemic immune response.