NADPH Phagocyte Oxidase Knockout Mice Control Trypanosoma cruzi Proliferation, but Develop Circulatory Collapse and Succumb to Infection

•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 (gp91phox−/− 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

Oxidative Stress: Promoter of Allergic Sensitization to Protease Allergens?

Allergies arise from aberrant T helper type 2 responses to allergens. Several respiratory allergens possess proteolytic activity, which has been recognized to act as an adjuvant for the development of a Th2 response. Allergen source-derived proteases can activate the protease-activated receptor-2, have specific effects on immune cells by cleaving cell membrane-bound regulatory molecules, and can disrupt tight junctions. The protease activity can induce a non-allergen-specific inflammatory response in the airways, which will set the stage for an allergen-specific Th2 response. In this review, we will discuss the evidence for the induction of oxidative stress as an underlying mechanism in Th2 sensitization to proteolytic allergens. We will discuss recent data linking the proteolytic activity of an allergen to its potential to induce oxidative stress and how this can facilitate allergic sensitization. Based on experimental data, we propose that a less proficient anti-oxidant response to allergen-induced oxidative stress contributes to the susceptibility to allergic sensitization. Besides the effect of oxidative stress on the immune response, we will also discuss how oxidative stress can increase the immunogenicity of an allergen by chemical modification

<i>phox</i> KO mice control parasite proliferation in target organs.

<p>WT, <i>phox</i> KO and <i>inf-γ</i> KO mice infected with <i>T. cruzi</i> 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 <i>phox</i> KO animals. The parasitism of <i>ifn-γ</i> KO group is statistically different from WT and <i>phox</i> groups in all organs and times analyzed, except for the heart at day 10 post-infection.</p

Serum AST and amylase in WT and <i>phox</i> KO mice infected with <i>T. cruzi.</i>

<p>Values from AST and amylase are combined from 3 independent experiments with n = 3 for each independent experiment.</p>a<p>AST values are expressed in IU/L;</p>b<p>Amylase values are expressed in U/L.</p

NADPH oxidase deficient-mice control parasitemia, but succumb to infection with <i>T. cruzi</i>.

<p>WT, <i>phox</i> KO and <i>inf-γ</i> KO mice were infected with 1000 blood-born trypomastigotes of Y strain of <i>T. cruzi</i>. 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 <i>t</i> test. (B) Mortality curve is pooled from three different experiments and P<0.05 among all groups in the graph.</p

WT and <i>phox</i> KO mice produce similar levels of IFN-γ and TNF.

<p>(A) WT and <i>phox</i> KO animals infected with <i>T. cruzi</i> 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.</p

Augmented NOx levels in <i>phox</i> KO mice infected with <i>T. cruzi</i>, when compared to WT.

<p>(A) <i>T. cruzi</i>-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 <i>t</i> 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.</p