NADPH phagocyte oxidase knockout mice control Trypanosoma cruzi proliferation, but develop circulatory collapse and succumb to infection

Abstract

(•)NO is considered to be a key macrophage-derived cytotoxic effector during Trypanosoma cruzi infection. On the other hand, the microbicidal properties of reactive oxygen species (ROS) are well recognized, but little importance has been attributed to them during in vivo infection with T. cruzi. In order to investigate the role of ROS in T. cruzi infection, mice deficient in NADPH phagocyte oxidase (gp91(phox) (-/-) or phox KO) were infected with Y strain of T. cruzi and the course of infection was followed. phox KO mice had similar parasitemia, similar tissue parasitism and similar levels of IFN-γ and TNF in serum and spleen cell culture supernatants, when compared to wild-type controls. However, all phox KO mice succumbed to infection between day 15 and 21 after inoculation with the parasite, while 60% of wild-type mice were alive 50 days after infection. Further investigation demonstrated increased serum levels of nitrite and nitrate (NOx) at day 15 of infection in phox KO animals, associated with a drop in blood pressure. Treatment with a NOS2 inhibitor corrected the blood pressure, implicating NOS2 in this phenomenon. We postulate that superoxide reacts with (•)NO in vivo, preventing blood pressure drops in wild type mice. Hence, whilst superoxide from phagocytes did not play a critical role in parasite control in the phox KO animals, its production would have an important protective effect against blood pressure decline during infection with T. cruzi.

Figure 1. NADPH oxidase deficient-mice control parasitemia, but succumb to infection with T. cruzi.

WT, phox KO and inf-γ KO mice were infected with 1000 blood-born trypomastigotes of Y strain of T. cruzi. Parasitemia (A) and mortality (B) were accessed daily. (A) Points represent mean ± SE of 5 animals per group of one from three different experiments performed with similar results. Asterisks represent P<0.05 by Student's t test. (B) Mortality curve is pooled from three different experiments and P<0.05 among all groups in the graph.

Figure 2. phox KO mice control parasite proliferation in target organs.

WT, phox KO and inf-γ KO mice infected with T. cruzi were sacrificed on days 10 and 15 post-infection and tissue parasitism in spleen, heart and liver evaluated by real-time PCR as described in material and methods. Bars represent mean ± SE of four animals per group. Arrows indicate P<0.05 between WT and phox KO animals. The parasitism of ifn-γ KO group is statistically different from WT and phox groups in all organs and times analyzed, except for the heart at day 10 post-infection.

Figure 3. WT and phox KO mice produce similar levels of IFN-γ and TNF.

(A) WT and phox KO animals infected with T. cruzi were bled at days 10 and 15 post-infection for cytokine measurements. (B) Infected mice were sacrificed at 10 days post-infection and spleen cells isolated and cultured for 72 hours, when supernatants were harvested. IFN-γ and TNF were measured by ELISA as described in material and methods. Bars represent mean ± SE of at least 4 animals per group. Experiment was repeated once with similar results.

Figure 4. Augmented NOx levels in phox KO mice infected with T. cruzi, when compared to WT.

(A) T. cruzi-infected mice were bled at 10 and 15 days post-infection and levels of nitrate and nitrite evaluated. Bars represent mean ± SE of 4 animals per group. Asterisks indicate P<0.05 by Student's t test. (B) Spleens from infected animals were harvested at 10 and 15 days post-infection and used for RNA extraction and real-time RT-PCR as described in material and methods. NOS2 expression was evaluated after normalization with HPRT constitutive gene.

Figure 5. T. cruzi- infected phox KO mice display dramatic blood pressure variations.

WT and phox KO animals were infected with T. cruzi and blood pressure evaluated in the tail (TBP) (A) or in the carotid artery (MAP) (B). Values represent mean ± SE of 6 mice of one from two performed (A) or 2–6 mice per time point pooled from 3 independent experiments (B). Asterisks represent P<0.05. (C) Drop in blood pressure is reverted by iNOS-specific inhibitor 1400SW. phox KO animals were infected with T. cruzi and blood pressure evaluated in the carotid artery (MAP) 16 days post-infection. Mice treated with 1400W received 15 mg/kg 24 and 1 hour before measurements. Values represent mean ± SE of 4 mice of one from two performed. Asterisks represent P<0.05. (D) 1400W did not revert mortality in phox KO mice. Mice were treated with 20 mg/kg of 1400W ip daily in single dose or divided in 2 doses starting on day 13 post-infection for 14 days. Mortality was accessed daily. Points represent mean cumulative mortality of 18–20 animals per group. Pool from 3 experiments performed with similar results (two experiments using one dose per day regime and one using two doses per day regime).