Claudia N. Paiva

Are Reactive Oxygen Species Always Detrimental to Pathogens

Reactive oxygen species (ROS) are deadly weapons used by phagocytes and other cell types, such as lung epithelial cells, against pathogens. ROS can kill pathogens directly by causing oxidative damage to biocompounds or indirectly by stimulating pathogen elimination by various nonoxidative mechanisms, including pattern recognition receptors signaling, autophagy, neutrophil extracellular trap formation, and T-lymphocyte responses. Thus, one should expect that the inhibition of ROS production promote infection. Increasing evidences support that in certain particular infections, antioxidants decrease and prooxidants increase pathogen burden. In this study, we review the classic infections that are controlled by ROS and the cases in which ROS appear as promoters of infection, challenging the paradigm. We discuss the possible mechanisms by which ROS could promote particular infections. These mechanisms are still not completely clear but include the metabolic effects of ROS on pathogen physiology, ROS-induced damage to the immune system, and ROS-induced activation of immune defense mechanisms that are subsequently hijacked by particular pathogens to act against more effective microbicidal mechanisms of the immune system. The effective use of antioxidants as therapeutic agents against certain infections is a realistic possibility that is beginning to be applied against viruses.

Oxidative stress fuels Trypanosoma cruzi infection in mice

Oxidative damage contributes to microbe elimination during macrophage respiratory burst. Nuclear factor, erythroid-derived 2, like 2 (NRF2) orchestrates antioxidant defenses, including the expression of heme-oxygenase-1 (HO-1). Unexpectedly, the activation of NRF2 and HO-1 reduces infection by a number of pathogens, although the mechanism responsible for this effect is largely unknown. We studied Trypanosoma cruzi infection in mice in which NRF2/HO-1 was induced with cobalt protoporphyrin (CoPP). CoPP reduced parasitemia and tissue parasitism, while an inhibitor of HO-1 activity increased T. cruzi parasitemia in blood. CoPP-induced effects did not depend on the adaptive immunity, nor were parasites directly targeted. We also found that CoPP reduced macrophage parasitism, which depended on NRF2 expression but not on classical mechanisms such as apoptosis of infected cells, induction of type I IFN, or NO. We found that exogenous expression of NRF2 or HO-1 also reduced macrophage parasitism. Several antioxidants, including NRF2 activators, reduced macrophage parasite burden, while pro-oxidants promoted it. Reducing the intracellular labile iron pool decreased parasitism, and antioxidants increased the expression of ferritin and ferroportin in infected macrophages. Ferrous sulfate reversed the CoPP-induced decrease in macrophage parasite burden and, given in vivo, reversed their protective effects. Our results indicate that oxidative stress contributes to parasite persistence in host tissues and open a new avenue for the development of anti-T. cruzi drugs.

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.

CCL2/MCP-1 controls parasite burden, cell infiltration, and mononuclear activation during acute Trypanosoma cruzi infection

CCL2/MCP‐1 has emerged recently as a critical factor in infectious and autoimmune myocarditis. In fact, this chemokine is produced in great amounts in hearts from Trypanosoma cruzi‐infected mice and is known to enhance parasite uptake and destruction by macrophages. Herein, we studied the involvement of CCL2 in tissue inflammation and resistance to T. cruzi. Infected CCL2−/− mice developed higher parasitemias and died earlier than WT mice. Close to their death, T. cruzi‐infected CCL2−/− presented greater amounts of TNF, IFN‐γ, and IL‐10 in plasma than WTs and clinical signs of systemic inflammatory response. Amastigote nests were more frequent in hearts and livers from infected CCL2−/− tissues than in WTs, and reduced numbers of leukocytes infiltrated their tissues. Leukocytes formed diffuse but not focal infiltrates in hearts from infected CCL2−/− mice, and perivascular cuffs could still be found in their livers. Infected CCL2−/− mice had smaller percentages of activated CD11b (Mac‐1)+CD107b (Mac‐3)+ macrophages and CD8+CD69hi cells among heart and liver infiltrates than WTs (flow cytometry), indicating that CCL2 controls subset migration/activation. CCL2 accumulated among focal heart infiltrates, suggesting that this chemokine is involved in retention of mononuclear cells in particular spots. Peritoneal macrophages from CCL2−/− mice displayed decreased trypanocidal activity. Our results demonstrate that CCL2 contributes to reduce parasite growth and indicate that it does so by controlling the distribution, cellular composition, and state of activation of inflammatory infiltrates in acute T. cruzi infection.

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 wildtype 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.—Magalhaes, E. S., Paiva, C. N., Souza, H. S. P., Pyrrho, A. S., Mourao‐Sa, D., Figueiredo, R. T., Vieira‐de‐Abreu, A., Dutra, H. S., Silveira, M. S., Gaspar‐Elsas, M. I. C., Xavier‐Elsas, P., Bozza, P. T., Bozza, M. T. Macrophage migration inhibitory factor is critical to interleukin‐5‐driven eosinophilopoiesis and tissue eosinophilia triggered by Schistosoma mansoni infection. FASEB J. 23, 1262–1271 (2009)

Macrophage-dependent IL-1β production induces cardiac arrhythmias in diabetic mice

Diabetes mellitus (DM) encompasses a multitude of secondary disorders, including heart disease. One of the most frequent and potentially life threatening disorders of DM-induced heart disease is ventricular tachycardia (VT). Here we show that toll-like receptor 2 (TLR2) and NLRP3 inflammasome activation in cardiac macrophages mediate the production of IL-1β in DM mice. IL-1β causes prolongation of the action potential duration, induces a decrease in potassium current and an increase in calcium sparks in cardiomyocytes, which are changes that underlie arrhythmia propensity. IL-1β-induced spontaneous contractile events are associated with CaMKII oxidation and phosphorylation. We further show that DM-induced arrhythmias can be successfully treated by inhibiting the IL-1β axis with either IL-1 receptor antagonist or by inhibiting the NLRP3 inflammasome. Our results establish IL-1β as an inflammatory connection between metabolic dysfunction and arrhythmias in DM.

Chronic Trypanosoma cruzi-elicited cardiomyopathy: from the discovery to the proposal of rational therapeutic interventions targeting cell adhesion molecules and chemokine receptors - how to make a dream come true

One hundred years ago, Carlos Chagas discovered a new disease, the American trypanosomiasis. Chagas and co-workers later characterised the diseases common manifestation, chronic cardiomyopathy, and suggested that parasitic persistence coupled with inflammation was the key underlying pathogenic mechanism. Better comprehension of the molecular mechanisms leading to clinical heart afflictions is a prerequisite to developing new therapies that ameliorate inflammation and improve heart function without hampering parasite control. Here, we review recent data showing that distinct cell adhesion molecules, chemokines and chemokine receptors participate in anti-parasite immunity and/or detrimental leukocyte trafficking to the heart. Moreover, we offer evidence that CC-chemokine receptors may be attractive therapeutic targets aiming to regain homeostatic balance in parasite/host interaction thereby improving prognosis, supporting that it is becoming a non-phantasious proposal.

Migration inhibitory factor (MIF) released by macrophages upon recognition of immune complexes is critical to inflammation in Arthus reaction

Deposition of immune complexes (IC) triggers FcγR‐dependent inflammation, leading to tissue damage in rheumatoid arthritis, systemic lupus erythematous, immune glomerulonephritis, and several immune vasculitides. Evidences support a role for macrophage migration inhibitory factor (MIF) in a number of inflammatory diseases, but the triggering of its secretion and its physiopathological role upon IC deposition remain elusive. Herein, we show that human macrophages secreted MIF after IC recognition, which in turn controlled the secretion of TNF. Macrophages from Mif−/− mice produced smaller amounts of TNF when stimulated with IgG‐opsonized erythrocytes than wild‐type (WT) cells. Using passive reverse Arthus reaction in the peritoneum and lungs as a model for IC‐induced inflammation, we demonstrated that Mif−/− mice had a milder response, observed by reduced neutrophil recruitment, vascular leakage, and secretion of TNF, MIP‐2, and keratinocyte‐derived chemokine compared with WT controls. Adoptive transfer of alveolar macrophages from WT to Mif−/− mice rescued pulmonary neutrophil recruitment and TNF production upon passive reverse Arthus reaction. Our study indicates that Arthus inflammatory reaction is largely dependent on MIF and poses macrophages as a source of the MIF released upon IC recognition. These results give experimental support to the proposition that blockade of MIF might constitute an adjunctive, therapeutic approach to IC disease.

Unraveling the lethal synergism between Trypanosoma cruzi infection and LPS: a role for increased macrophage reactivity.

Various infections sensitize to lethal shock by promoting hyperactivation of macrophages to LPS stimulation. Although macrophages are thought to be deactivated upon contact with apoptotic cells during Trypanosoma cruzi infection, T. cruzi infection also sensitizes mice to endotoxemia. Herein, we studied the mechanisms of sensitization to endotoxemia in T. cruzi‐infected mice in order to solve the paradox. Live (but not fixed) trypomastigotes from various stocks sensitized mice to endotoxemia. Mice deficient in glycolipid recognition (TLR2–/– and CD1d–/–) were sensitized by infection to challenge with LPS. Infected mice hyperproduced TNF and IL‐10 upon LPS challenge. Infected TNF‐R1–/–, macrophage migration inhibitory factor (MIF)–/– and IFN‐γ–/– mice were lethally sensitized, but infected TNF‐R1–/– mice administered anti‐MIF survived shock with LPS. Macrophages from infected mice hyperproduced TNF in response to LPS stimulation and displayed increased expression of TLR4 compared to non‐infected controls. Treatment with the PGE2 synthesis inhibitor acetylsalicylic acid (AAS) in vivo reduced parasitemia and enhanced LPS‐stimulated production of TNF by macrophages, but the effect was less in infected mice than in normal mice. Nevertheless, AAS treatment did not increase the susceptibility of infected mice to sublethal shock with LPS. Our results point to independent MIF and TNF/TNF‐R1 lethal pathways and suggest a role for hyperactivated macrophages in T. cruzi‐sensitized LPS‐induced shock.

Macrophage migration inhibitory factor in protozoan infections.

Macrophage migration inhibitory factor (MIF) is a cytokine that plays a central role in immune and inflammatory responses. In the present paper, we discussed the participation of MIF in the immune response to protozoan parasite infections. As a general trend, MIF participates in the control of parasite burden at the expense of promoting tissue damage due to increased inflammation.