Sequestration and histopathology in Plasmodium chabaudi malaria are influenced by the immune response in an organ-specific manner

Abstract

Infection with the malaria parasite, Plasmodium, is associated with a strong inflammatory response and parasite cytoadhesion (sequestration) in several organs. Here, we have carried out a systematic study of sequestration and histopathology during infection of C57Bl/6 mice with Plasmodium chabaudi AS and determined the influence of the immune response. This parasite sequesters predominantly in liver and lung, but not in the brain, kidney or gut. Histopathological changes occur in multiple organs during the acute infection, but are not restricted to the organs where sequestration takes place. Adaptive immunity, and signalling through the IFNγ receptor increased sequestration and histopathology in the liver, but not in the lung, suggesting that there are differences in the adhesion molecules and/or parasite ligands utilized and mechanisms of pathogenesis in these two organs. Exacerbation of pro-inflammatory responses during infection by deletion of the il10 gene resultsin the aggravation of damage to lung and kidney irrespective of the degree of sequestration. The immune response therefore affected both sequestration and histopathology in an organ-specific manner. P. chabaudi AS provides a good model to investigate the influence of the host response on the sequestration and specific organ pathology, which is applicable to human malaria.

Figure 1

Mature stages of Plasmodium chabaudi leave the peripheral circulation and accumulate in organs. C57BL/6 mice were maintained in a reverse 12 h light–dark cycle and infected with 10⁵ wild type P. chabaudi iRBC. Tail blood was taken hourly between 9 h and 17 h, on day 5 of infection.A. Stages of the parasite life cycle observed in peripheral blood.B. parasitemia in peripheral blood. Parasitemias at each time point were compared with parasitemia at 9.00 h.C. Percentage of microvessels in different organs containing P. chabaudi iRBC. 200 vessels were counted for each organ on H&E stained sections (see Experimental procedures).D. percentage of RBC infected within the microvessels of the different organs. 400 RBC were counted on each section for each organ on every sample. The red dotted line indicates the average peripheral parasitemia at 9.00 h.E. Representative tissue sections made at 12.00 h, 8 days after infection and stained by H&E. Infected red blood cells are indicated by arrows. Scale bars represent 10 μm. Each dot or bar represents the average for at least 8 mice (± SEM) (*P < 0.05; **P < 0.01; ***P < 0.001, Mann–Whitney test).

Figure 2

Plasmodium chabaudi sequester preferentially in lungs, liver and spleen in a time-dependent manner.A. Parasitemia in mice infected with 10⁵ wild-type C57BL/6 mice and luciferase-expressing P. chabaudi (PccASluc) infected RBC. Luciferase activity in organs was measured between 12.00 h and 14.00 h after whole body perfusion.B. Representative images of PccASluc accumulation in extracted organs.C. The luciferase activity (p/s) measured in each organ was normalized as described in Experimental procedures. Each bar represents the average for ten mice (± SEM). (*P < 0.05; **P < 0.01; ***P < 0.001, Mann–Whitney test).

Figure 3

Adaptive immune responses and IFNγ response increase sequestration in the liver during Plasmodium chabaudi infection. Wild type (wt) C57BL/6 mice and mice lacking an adaptive immune system (rag1^−/−) IFNγ receptor (ifnγr^−/−) or IL-10 (il10^−/−) were infected with 10⁵ luciferase-expressing P. chabaudi iRBC (PccASluc).A. Comparison of peripheral parasitemia in the different strains of mice on days 5 and 9 of infection measured at 9.00 h.B. Luciferase activity in liver, lungs and spleen of infected rag1^−/−, ifngr^−/− and il10^−/− mice expressed as fold change relative to luciferase activity in wt C57BL/6 mice measured after whole body perfusion at days 5 and 9 post infection. Each bar represents the average for at least six mice (± SEM). (*P < 0.05; **P < 0.01; ***P < 0.001, Mann–Whitney test).

Figure 4

Histopathological changes in organs during Plasmodium chabaudi infection.C57BL/6 mice were infected with 10⁵ wild-type P. chabaudi iRBC. Representative H&E sections from liver, lung and kidney of uninfected (control) or infected mice (day 8 p.i.) are shown.A. Liver: upper panel shows extramedullary haematopoiesis (EMH) (1) and pigmented kupfer cells (2); lower panel shows a region of focal necrosis (indicated within the marked area). Scale bars represent 100 μm. The upper right graph is a semi-quantitative measure of EMH, Kupffer cells and necrosis using the scoring system described in Experimental procedures. The lower right graph shows the increase in alanine transaminase (ALT) in plasma during infection as an indication of liver damage.B. Lung: upper left panel shows increased alveolar septae cellularity; upper right graph shows the enumeration of this cellularity using the scoring system described in Experimental procedures; lower left graph shows the number of IFNγ-producing lymphocytes in the lung of uninfected mice and mice infected with P. chabaudi for 5, 8 and 13 days. Lower right graph shows the increase in IgM in the BAL of P. chabaudi infected mice, as measured by ELISA.C. Kidney: left panels show tubular dilatation; right upper graph show a semi-quantitative measure of tubular dilatation using the scoring system described in Experimental procedures; lower graphs shows the amounts of creatinine and urea in plasma of uninfected and P. chabaudi infected mice at days 5, 8, 9 and 13. Each bar represents the average for at least five mice (± SEM). (*P < 0.05; **P < 0.01; ***P < 0.001, Mann–Whitney test).

Figure 5

Pro-inflammatory response increases damage in multiple organs during Plasmodium chabaudi infection. Wild type (wt) C57BL/6 mice and mice lacking an adaptive immune system (rag1^−/−) IFNγ receptor (ifnγr^−/−) or IL-10 (il10^−/−) were infected with 10⁵ wild-type P. chabaudi iRBC.A. Liver: increase in alanine transaminase (ALT) in plasma during infection as an indication of liver damage.B. Lung: upper panels show the number of IFNγ-producing lymphocytes in the lung of uninfected mice and mice infected with P. chabaudi for 9 days. Lower graph shows the increase in IgM in the BAL of P. chabaudi infected mice (measured by ELISA) expressed as fold change relative to uninfected mice of the same genetic background (indicated by the dotted line).C. Kidney: left panel show a semi-quantitative measure of tubular dilatation using the scoring system described in Experimental procedures; middle and right graphs shows the amounts of creatinine and urea in plasma of P. chabaudi infected mice at days 9. Each bar represents the average for at least five mice (± SEM). (*P < 0.05; **P < 0.01; ***P < 0.001, Mann–Whitney test).