Heme oxygenase-1 in protozoan infections: A tale of resistance and disease tolerance.

Heme oxygenase (HO-1) mediates the enzymatic cleavage of heme, a molecule with proinflammatory and prooxidant properties. HO-1 activity deeply impacts host capacity to tolerate infection through reduction of tissue damage or affecting resistance, the ability of the host to control pathogen loads. In this Review, we will discuss the contribution of HO-1 in different and complex protozoan infections, such as malaria, leishmaniasis, Chagas disease, and toxoplasmosis. The complexity of these infections and the pleiotropic effects of HO-1 constitute an interesting area of study and an opportunity for drug development.

Mitochondrial Reactive Oxygen Species Participate in Signaling Triggered by Heme in Macrophages and upon Hemolysis.

Hemolysis causes an increase of intravascular heme, oxidative damage, and inflammation in which macrophages play a critical role. In these cells, heme can act as a prototypical damage-associated molecular pattern, inducing TLR4-dependent cytokine production through the MyD88 pathway, independently of TRIF. Heme promotes reactive oxygen species (ROS) generation independently of TLR4. ROS and TNF production contribute to heme-induced necroptosis and inflammasome activation; however, the role of ROS in proinflammatory signaling and cytokine production remains unknown. In this study, we demonstrate that heme activates at least three signaling pathways that contribute to a robust MAPK phosphorylation and cytokine expression in mouse macrophages. Although heme did not induce a detectable Myddosome formation, the TLR4/MyD88 axis was important for phosphorylation of p38 and secretion of cytokines. ROS generation and spleen tyrosine kinase (Syk) activation induced by heme were critical for most proinflammatory signaling pathways, as the antioxidant N-acetyl-l-cysteine and a Syk inhibitor differentially blocked heme-induced ROS, MAPK phosphorylation, and cytokine production in macrophages. Early generated mitochondrial ROS induced by heme was Syk dependent, selectively promoted the phosphorylation of ERK1/2 without affecting JNK or p38, and contributed to CXCL1 and TNF production. Finally, lethality caused by sterile hemolysis in mice required TLR4, TNFR1, and mitochondrial ROS, supporting the rationale to target these pathways to mitigate tissue damage of hemolytic disorders.

ROS and Trypanosoma cruzi: Fuel to infection, poison to the heart.

The activation of macrophage respiratory burst in response to infection with Trypanosoma cruzi inflicts oxidative damage to the host's tissues. For decades, the role of reactive oxygen species (ROS) in the elimination of T. cruzi was taken for granted, but recent evidence suggests parasite growth is stimulated in oxidative environments. It is still a matter of debate whether indeed oxidative environments provide ideal conditions (e.g., iron availability in macrophages) for T. cruzi growth and whether indeed ROS signals directly to stimulate growth. Nitric oxide (NO) and ROS combine to form peroxynitrite, participating in the killing of phagocytosed parasites by activated macrophages. In response to infection, mitochondrial ROS are produced by cardiomyocytes. They contribute to oxidative damage that persists at the chronic stage of infection and is involved in functional impairment of the heart. In this review, we discuss how oxidative stress helps parasite growth during the acute stage and how it participates in the development of cardiomyopathy at the chronic stage.

Cardiac arrhythmias after renal I/R depend on IL-1ß.

AIMS: Cardiac arrhythmias are one of the most important remote complications after kidney injury. Renal ischemia reperfusion (I/R) is a major cause of acute renal injury predisposing to several remote dysfunctions, including cardiac electrical disturbance. Since IL-1ß production dependent on NLRP3 represents a link between tissue malfunctioning and cardiac arrhythmias, here we tested the hypothesis that longer ventricular repolarization and arrhythmias after renal I/R depend on this innate immunity sensor. METHODS AND RESULTS: Nlrp3-/- and Casp1-/- mice reacted to renal I/R with no increase in plasma IL-1ß, different from WT (wild-type) I/R. A prolonged QJ interval and an increased susceptibility to ventricular arrhythmias were found after I/R compared to Sham controls in wild-type mice at 15 days post-perfusion, but not in Nlrp3-/- or CASP1-/- I/R, indicating that the absence of NLRP3 or CASP1 totally prevented longer QJ interval after renal I/R. In contrast with WT mice, we found no renal atrophy and no renal dysfunction in Nlrp3-/- and Casp1-/- mice after renal I/R. Depletion of macrophages in vivo after I/R and a day before IL-1ß peak (at 7 days post-perfusion) totally prevented prolongation of QJ interval, suggesting that macrophages might participate as sensors of tissue injury. Moreover, treatment of I/R-WT mice with IL-1r antagonist (IL-1ra) from 8 to 15 days post perfusion did not interfere with renal function, but reversed QJ prolongation, prevented the increase in susceptibility to ventricular arrhythmias and rescued a close to normal duration and amplitude of calcium transient. CONCLUSION: Taken together, these results corroborate the hypothesis that IL-1ß is produced after sensing renal injury through NRLP3-CASP1, and IL-1ß on its turn triggers longer ventricular repolarization and increase susceptibility to cardiac arrhythmias. Still, they offer a therapeutic approach to treat cardiac arrhythmias that arise after renal I/R.

Resveratrol Reverses Functional Chagas Heart Disease in Mice.

Chronic chagasic cardiomyopathy (CCC) develops years after acute infection by Trypanosoma cruzi and does not improve after trypanocidal therapy, despite reduction of parasite burden. During disease, the heart undergoes oxidative stress, a potential causative factor for arrhythmias and contractile dysfunction. Here we tested whether antioxidants/ cardioprotective drugs could improve cardiac function in established Chagas heart disease. We chose a model that resembles B1-B2 stage of human CCC, treated mice with resveratrol and performed electrocardiography and echocardiography studies. Resveratrol reduced the prolonged PR and QTc intervals, increased heart rates and reversed sinus arrhythmia, atrial and atrioventricular conduction disorders; restored a normal left ventricular ejection fraction, improved stroke volume and cardiac output. Resveratrol activated the AMPK-pathway and reduced both ROS production and heart parasite burden, without interfering with vascularization or myocarditis intensity. Resveratrol was even capable of improving heart function of infected mice when treatment was started late after infection, while trypanocidal drug benznidazole failed. We attempted to mimic resveratrol's actions using metformin (AMPK-activator) or tempol (SOD-mimetic). Metformin and tempol mimicked the beneficial effects of resveratrol on heart function and decreased lipid peroxidation, but did not alter parasite burden. These results indicate that AMPK activation and ROS neutralization are key strategies to induce tolerance to Chagas heart disease. Despite all tissue damage observed in established Chagas heart disease, we found that a physiological dysfunction can still be reversed by treatment with resveratrol, metformin and tempol, resulting in improved heart function and representing a starting point to develop innovative therapies in CCC.

Silymarin reduces profibrogenic cytokines and reverses hepatic fibrosis in chronic murine schistosomiasis.

In chronic schistosomiasis, hepatic fibrosis is linked to the portal hypertension that causes morbidity in Schistosoma mansoni infection. Silymarin (SIL) is a hepatoprotective and antioxidant medicament largely prescribed against liver diseases that has previously been shown to prevent fibrosis during acute murine schistosomiasis. Here we employed silymarin to try to reverse established hepatic fibrosis in chronic schistosomiasis. Silymarin or vehicle was administered to BALB/c mice every 48 h, starting on the 40th (80 days of treatment), 70th (50 days), or 110th (10 days) day postinfection (dpi). All mice were sacrificed and analyzed at 120 dpi. Treatment with silymarin reduced liver weight and granuloma sizes, reduced the increase in alanine aminotransferase and aspartate aminotransferase levels, and reduced the established hepatic fibrosis (assessed by hydroxyproline contents and picrosirius staining). Treatment with silymarin also reduced the levels of interleukin-13 (IL-13) in serum and increased the gamma interferon (IFN-γ)/IL-13 ratio. There was a linear correlation between IL-13 levels in serum and hydroxyproline hepatic content in both infected untreated and SIL-treated mice, with decreased IL-13 levels corresponding to decreased hydroxyproline hepatic contents. Treatment with either SIL or N-acetylcysteine reduced both proliferation of fibroblast cell lines and basal/IL-13-induced production of collagen I, indicating that besides inhibiting IL-13 production during infection, SIL antioxidant properties most likely contribute to inhibition of collagen production downstream of IL-13. These results show that silymarin interferes with fibrogenic cytokines, reduces established fibrosis, and inhibits downstream effects of IL-13 on fibrogenesis, indicating the drug as a safe and cheap treatment to liver fibrotic disease in schistosomiasis.

Heme amplifies the innate immune response to microbial molecules through spleen tyrosine kinase (Syk)-dependent reactive oxygen species generation.

Infectious diseases that cause hemolysis are among the most threatening human diseases, because of severity and/or global distribution. In these conditions, hemeproteins and heme are released, but whether heme affects the inflammatory response to microorganism molecules remains to be characterized. Here, we show that heme increased the lethality and cytokine secretion induced by LPS in vivo and enhanced the secretion of cytokines by macrophages stimulated with various agonists of innate immune receptors. Activation of nuclear factor κB (NF-κB) and MAPKs and the generation of reactive oxygen species were essential to the increase in cytokine production induced by heme plus LPS. This synergistic effect of heme and LPS was blocked by a selective inhibitor of spleen tyrosine kinase (Syk) and was abrogated in dendritic cells deficient in Syk. Moreover, inhibition of Syk and the downstream molecules PKC and PI3K reduced the reactive oxygen species generation by heme. Our results highlight a mechanism by which heme amplifies the secretion of cytokines triggered by microbial molecule activation and indicates possible pathways for therapeutic intervention during hemolytic infectious diseases.

Macrophage migration inhibitory factor is critical to interleukin-5-driven eosinophilopoiesis and tissue eosinophilia triggered by Schistosoma mansoni infection.

Macrophage migration inhibitory factor (MIF) participates in the pathogenesis of inflammatory diseases, including asthma, in which it enhances airway hypersensitivity and tissue eosinophilia. Herein, we investigated the role of MIF in eosinophilopoiesis and tissue eosinophilia using Schistosoma mansoni infection. MIF-deficient (Mif(-/-)) mice had similar numbers of adult worms, eggs, and granulomas compared to wild-type mice, but the size of granulomas was strikingly reduced due to smaller numbers of eosinophils. MIF did not affect the acquired response to infection, as Mif(-/-) mice produced normal amounts of Th2 cytokines and IgE. Nevertheless, recombinant MIF (rMIF) behaved as a chemoattractant for eosinophils, what could partially explain the reduced eosinophilia in infected Mif(-/-) mice. Moreover, the percentage of eosinophils was reduced in bone marrows of Mif(-/-) mice chronically infected with S. mansoni compared to wild type. Mif(-/-) had impaired eosinophilopoiesis in response to interleukin (IL)-5 and addition of rMIF to bone marrow cultures from IL-5 transgenic mice enhanced the generation of eosinophils. In the absence of MIF, eosinophil precursors were unable to survive the IL-5-supplemented cell culture, and were ingested by macrophages. Treatment with pancaspase inhibitor z-VAD or rMIF promoted the survival of eosinophil progenitors. Together, these results indicate that MIF participates in IL-5-driven maturation of eosinophils and in tissue eosinophilia associated with S. mansoni infection.

The Role of MIF on Eosinophil Biology and Eosinophilic Inflammation.

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that participates in innate and adaptive immune responses. MIF contributes to the resistance against infection agents, but also to the cellular and tissue damage in infectious, autoimmune, and allergic diseases. In the past years, several studies demonstrated a critical role for MIF in the pathogenesis of type-2-mediated inflammation, including allergy and helminth infection. Atopic patients have increased MIF amounts in affected tissues, mainly produced by immune cells such as macrophages, Th2 cells, and eosinophils. Increased MIF mRNA and protein are found in activated Th2 cells, while eosinophils stock pre-formed MIF protein and secrete high amounts of MIF upon stimulation. In mouse models of allergic asthma, the lack of MIF causes an almost complete abrogation of the cardinal signs of the disease including mucus secretion, eosinophilic inflammation, and airway hyper-responsiveness. Additionally, blocking the expression of MIF in animal models leads to significant reduction of pathological signs of eosinophilic inflammation such as rhinitis, atopic dermatitis, eosinophilic esophagitis and helminth infection. A number of studies indicate that MIF is important in the effector phase of type-2 immune responses, while its contribution to Th2 differentiation and IgE production is not consensual. MIF has been found to intervene in different aspects of eosinophil physiology including differentiation, survival, activation, and migration. CD4+ T cells and eosinophils express CD74 and CXCR4, receptors able to signal upon MIF binding. Blockage of these receptors with neutralizing antibodies or small molecule antagonists also succeeds in reducing the signals of inflammation in experimental allergic models. Together, these studies demonstrate an important contribution of MIF on eosinophil biology and in the pathogenesis of allergic diseases and helminth infection.