Browsing by Author "Soares, Miguel P."
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- Atherogenesis May Involve the Prooxidant and Proinflammatory Effects of Ferryl HemoglobinPublication . Potor, László; Bányai, Emese; Becs, Gergely; Soares, Miguel P.; Balla, György; Balla, József; Jeney, ViktóriaOxidized cell-free hemoglobin (Hb), including covalently cross-linked Hb multimers, is present in advanced atherosclerotic lesions. Oxidation of Hb produces methemoglobin (Fe(3+)) and ferryl hemoglobin (Fe(4+) = O(2-)). Ferryl iron is unstable and can return to the Fe(3+) state by reacting with specific amino acids of the globin chains. In these reactions globin radicals are produced followed by termination reactions yielding covalently cross-linked Hb multimers. Despite the evanescent nature of the ferryl state, herein we refer to this oxidized Hb as "ferryl Hb." Our aim in this work was to study formation and biological effects of ferrylHb. We demonstrate that ferrylHb, like metHb, can release its heme group, leading to sensitization of endothelial cells (ECs) to oxidant-mediated killing and to oxidation of low-density lipoprotein (LDL). Furthermore, we observed that both oxidized LDL and lipids derived from human atherosclerotic lesions trigger Hb oxidation and subsequent production of covalently cross-linked ferrylHb multimers. Previously we showed that ferrylHb disrupts EC monolayer integrity and induces expression of inflammatory cell adhesion molecules. Here we show that when exposed to ferrylHb, EC monolayers exhibit increased permeability and enhanced monocyte adhesion. Taken together, interactions between cell-free Hb and atheroma lipids engage in a vicious cycle, amplifying oxidation of plaque lipids and Hb. These processes trigger EC activation and cytotoxicity.
- Beyond killing: Can we find new ways to manage infection?Publication . Vale, Pedro F.; McNally, Luke; Doeschl-Wilson, Andrea; King, Kayla C.; Popat, Roman; Domingo-Sananes, Maria R.; Allen, Judith E.; Soares, Miguel P.; Kümmerli, RolfThe antibiotic pipeline is running dry and infectious disease remains a major threat to public health. An efficient strategy to stay ahead of rapidly adapting pathogens should include approaches that replace, complement or enhance the effect of both current and novel antimicrobial compounds. In recent years, a number of innovative approaches to manage disease without the aid of traditional antibiotics and without eliminating the pathogens directly have emerged. These include disabling pathogen virulence-factors, increasing host tissue damage control or altering the microbiota to provide colonization resistance, immune resistance or disease tolerance against pathogens. We discuss the therapeutic potential of these approaches and examine their possible consequences for pathogen evolution. To guarantee a longer half-life of these alternatives to directly killing pathogens, and to gain a full understanding of their population-level consequences, we encourage future work to incorporate evolutionary perspectives into the development of these treatments.
- Characterization of Plasma Labile Heme in Hemolytic ConditionsPublication . Gouveia, Zélia; Carlos, Ana Rita; Yuan, Xiaojing; Aires-da-Silva, Frederico; Stocker, Roland; J. Maghzal, Ghassan; Leal, Sónia S.; Gomes, Cláudio M.; Todorovic, Smilja; Iranzo, Olga; Ramos, Susana; Santos, Ana Catarina; Hamza, Iqbal; Gonçalves, João; Soares, Miguel P.Extracellular hemoglobin (Hb), a byproduct of hemolysis, can release its prosthetic heme groups upon oxidation. This produces metabolically active heme that is exchangeable between acceptor proteins, macromolecules and low molecular weight ligands, termed here labile heme. As it accumulates in plasma labile heme acts in a pro-oxidant manner and regulates cellular metabolism while exerting pro-inflammatory and cytotoxic effects that foster the pathogenesis of hemolytic diseases. Here we developed and characterized a panel of heme-specific single domain antibodies (sdAbs) that together with a cellular-based heme reporter assay, allow for quantification and characterization of labile heme in plasma during hemolytic conditions. Using these approaches we demonstrate that labile heme generated during hemolytic conditions is bound to plasma molecules with an affinity higher than 10(-7) M and that 2-8% (~2-5 μM) of the total amount of heme detected in plasma can be internalized by bystander cells, i.e. bioavailable heme. Acute, but not chronic, hemolysis is associated with transient reduction of plasma heme binding capacity (HBC1/2 ), that is, the ability of plasma molecules to bind labile heme with an affinity higher than 10(-7) M. The heme-specific sdAbs neutralize the pro-oxidant activity of soluble heme in vitro, suggesting that these maybe used to counter the pathologic effects of labile heme during hemolytic conditions. Finally, we show that heme-specific sdAbs can be used to visualize cellular heme. In conclusion, we describe a panel of heme-specific sdAbs that when used with other approaches provide novel insights to the pathophysiology of heme. This article is protected by copyright. All rights reserved.
- Disease tolerance and immunity in host protection against infectionPublication . Soares, Miguel P.; Teixeira, Luis; Moita, Luis F.The immune system probably evolved to limit the negative effects exerted by pathogens on host homeostasis. This defence strategy relies on the concerted action of innate and adaptive components of the immune system, which sense and target pathogens for containment, destruction or expulsion. Resistance to infection refers to these immune functions, which reduce the pathogen load of an infected host as the means to preserve homeostasis. Immune-driven resistance to infection is coupled to an additional, and arguably as important, defence strategy that limits the extent of dysfunction imposed on host parenchymal tissues during infection, without exerting a direct negative effect on pathogens. This defence strategy, known as disease tolerance, relies on tissue damage control mechanisms that prevent the deleterious effects of pathogens and that uncouples immune-driven resistance mechanisms from immunopathology and disease. In this Review, we provide a unifying view of resistance and disease tolerance in the framework of immunity to infection.
- Ferritin H Deficiency in Myeloid Compartments Dysregulates Host Energy Metabolism and Increases Susceptibility to Mycobacterium tuberculosis InfectionPublication . Reddy, Vineel P.; Chinta, Krishna C.; Saini, Vikram; Glasgow, Joel N.; Hull, Travis D.; Traylor, Amie; Rey-Stolle, Fernanda; Soares, Miguel P.; Madansein, Rajhmun; Rahman, Md Aejazur; Barbas, Coral; Nargan, Kievershen; Naidoo, Threnesan; Ramdial, Pratistadevi K.; George, James F.; Agarwal, Anupam; Steyn, Adrie J. C.Iron is an essential factor for the growth and virulence of Mycobacterium tuberculosis (Mtb). However, little is known about the mechanisms by which the host controls iron availability during infection. Since ferritin heavy chain (FtH) is a major intracellular source of reserve iron in the host, we hypothesized that the lack of FtH would cause dysregulated iron homeostasis to exacerbate TB disease. Therefore, we used knockout mice lacking FtH in myeloid-derived cell populations to study Mtb disease progression. We found that FtH plays a critical role in protecting mice against Mtb, as evidenced by increased organ burden, extrapulmonary dissemination, and decreased survival in Fth−/− mice. Flow cytometry analysis showed that reduced levels of FtH contribute to an excessive inflammatory response to exacerbate disease. Extracellular flux analysis showed that FtH is essential for maintaining bioenergetic homeostasis through oxidative phosphorylation. In support of these findings, RNAseq and mass spectrometry analyses demonstrated an essential role for FtH in mitochondrial function and maintenance of central intermediary metabolism in vivo. Further, we show that FtH deficiency leads to iron dysregulation through the hepcidin–ferroportin axis during infection. To assess the clinical significance of our animal studies, we performed a clinicopathological analysis of iron distribution within human TB lung tissue and showed that Mtb severely disrupts iron homeostasis in distinct microanatomic locations of the human lung. We identified hemorrhage as a major source of metabolically inert iron deposition. Importantly, we observed increased iron levels in human TB lung tissue compared to healthy tissue. Overall, these findings advance our understanding of the link between iron-dependent energy metabolism and immunity and provide new insight into iron distribution within the spectrum of human pulmonary TB. These metabolic mechanisms could serve as the foundation for novel host-directed strategies.
- Heme Cytotoxicity and the Pathogenesis of Immune-Mediated Inflammatory DiseasesPublication . Larsen, Rasmus; Gouveia, Zélia; Soares, Miguel P.; Gozzelino, RaffaellaHeme, iron (Fe) protoporphyrin IX, functions as a prosthetic group in a range of hemoproteins essential to support life under aerobic conditions. The Fe contained within the prosthetic heme groups of these hemoproteins can catalyze the production of reactive oxygen species. Presumably for this reason, heme must be sequestered within those hemoproteins, thereby shielding the reactivity of its Fe-heme. However, under pathologic conditions associated with oxidative stress, some hemoproteins can release their prosthetic heme groups. While this heme is not necessarily damaging per se, it becomes highly cytotoxic in the presence of a range of inflammatory mediators such as tumor necrosis factor. This can lead to tissue damage and, as such, exacerbate the pathologic outcome of several immune-mediated inflammatory conditions. Presumably, targeting "free heme" may be used as a therapeutic intervention against these diseases.
- Identification of cyclins A1, E1 and vimentin as downstream targets of heme oxygenase-1 in vascular endothelial growth factor-mediated angiogenesisPublication . Bauer, Andrea; Mylroie, Hayley; Thornton, C. Clare; Calay, Damien; Birdsey, Graeme M.; Kiprianos, Allan P.; Wilson, Garrick K.; Soares, Miguel P.; Yin, Xiaoke; Mayr, Manuel; Randi, Anna M.; Mason, Justin C.Angiogenesis is an essential physiological process and an important factor in disease pathogenesis. However, its exploitation as a clinical target has achieved limited success and novel molecular targets are required. Although heme oxygenase-1 (HO-1) acts downstream of vascular endothelial growth factor (VEGF) to modulate angiogenesis, knowledge of the mechanisms involved remains limited. We set out identify novel HO-1 targets involved in angiogenesis. HO-1 depletion attenuated VEGF-induced human endothelial cell (EC) proliferation and tube formation. The latter response suggested a role for HO-1 in EC migration, and indeed HO-1 siRNA negatively affected directional migration of EC towards VEGF; a phenotype reversed by HO-1 over-expression. EC from Hmox1(-/-) mice behaved similarly. Microarray analysis of HO-1-depleted and control EC exposed to VEGF identified cyclins A1 and E1 as HO-1 targets. Migrating HO-1-deficient EC showed increased p27, reduced cyclin A1 and attenuated cyclin-dependent kinase 2 activity. In vivo, cyclin A1 siRNA inhibited VEGF-driven angiogenesis, a response reversed by Ad-HO-1. Proteomics identified structural protein vimentin as an additional VEGF-HO-1 target. HO-1 depletion inhibited VEGF-induced calpain activity and vimentin cleavage, while vimentin silencing attenuated HO-1-driven proliferation. Thus, vimentin and cyclins A1 and E1 represent VEGF-activated HO-1-dependent targets important for VEGF-driven angiogenesis.
- IL-22 controls iron-dependent nutritional immunity against systemic bacterial infectionsPublication . Sakamoto, Kei; Kim, Yun-Gi; Hara, Hideki; Kamada, Nobuhiko; Caballero-Flores, Gustavo; Tolosano, Emanuela; Soares, Miguel P.; Puente, José L.; Inohara, Naohiro; Núñez, GabrielHost immunity limits iron availability to pathogenic bacteria, but whether immunity limits pathogenic bacteria from accessing host heme, the major source of iron in the body, remains unclear. Using Citrobacter rodentium, a mouse enteric pathogen and Escherichia coli, a major cause of sepsis in humans as models, we find that interleukin-22, a cytokine best known for its ability to promote epithelial barrier function, also suppresses the systemic growth of bacteria by limiting iron availability to the pathogen. Using an unbiased proteomic approach to understand the mechanistic basis of IL-22 dependent iron retention in the host, we have identified that IL-22 induces the production of the plasma hemoglobin scavenger haptoglobin and heme scavenger hemopexin. Moreover, the anti-microbial effect of IL-22 depends on the induction of hemopexin expression, while haptogloblin is dispensable. Impaired pathogen clearance in infected Il22(-/-) mice was restored by administration and hemopexin-deficient mice had increased pathogen loads after infection. These studies reveal a previously unrecognized host defense mechanism regulated by IL-22 that relies on the induction of hemopexin to limit heme availability to bacteria leading to suppression of bacterial growth during systemic infections.
- Involvement of the p62/NRF2 signal transduction pathway on erythrophagocytosisPublication . Santarino, Inês B.; Viegas, Michelle S.; Domingues, Neuza S.; Ribeiro, Ana M.; Soares, Miguel P.; Vieira, Otília V.Erythrophagocytosis, the phagocytic removal of damaged red blood cells (RBC), and subsequent phagolysosome biogenesis are important processes in iron/heme metabolism and homeostasis. Phagolysosome biogenesis implies the interaction of nascent phagosomes with endocytic compartments and also autophagy effectors. Here, we report that besides recruitment of microtubule-associated protein-1-light chain 3 (LC3), additional autophagy machinery such as sequestosome 1 (p62) is also acquired by single-membrane phagosomes at very early stages of the phagocytic process and that its acquisition is very important to the outcome of the process. In bone marrow-derived macrophages (BMDM) silenced for p62, RBC degradation is inhibited. P62, is also required for nuclear translocation and activation of the transcription factor Nuclear factor E2-related Factor 2 (NRF2) during erythrophagocytosis. Deletion of the Nrf2 allele reduces p62 expression and compromises RBC degradation. In conclusion, we reveal that erythrophagocytosis relies on an interplay between p62 and NRF2, potentially acting as protective mechanism to maintain reactive oxygen species at basal levels and preserve macrophage homeostasis.
- Macrophage and epithelial cell H-ferritin expression regulates renal inflammationPublication . Bolisetty, Subhashini; Zarjou, Abolfazl; Hull, Travis D.; Traylor, Amie M.; Perianayagam, Anjana; Joseph, Reny; Kamal, Ahmed I.; Arosio, Paolo; Soares, Miguel P.; Jeney, Viktoria; Balla, Jozsef; George, James F.; Agarwal, AnupamInflammation culminating in fibrosis contributes to progressive kidney disease. Cross-talk between the tubular epithelium and interstitial cells regulates inflammation by a coordinated release of cytokines and chemokines. Here we studied the role of heme oxygenase-1 (HO-1) and the heavy subunit of ferritin (FtH) in macrophage polarization and renal inflammation. Deficiency in HO-1 was associated with increased FtH expression, accumulation of macrophages with a dysregulated polarization profile, and increased fibrosis following unilateral ureteral obstruction in mice: a model of renal inflammation and fibrosis. Macrophage polarization in vitro was predominantly dependent on FtH expression in isolated bone marrow-derived mouse monocytes. Using transgenic mice with conditional deletion of FtH in the proximal tubules (FtH(PT-/-)) or myeloid cells (FtH(LysM-/-)), we found that myeloid FtH deficiency did not affect polarization or accumulation of macrophages in the injured kidney compared with wild-type (FtH(+/+)) controls. However, tubular FtH deletion led to a marked increase in proinflammatory macrophages. Furthermore, injured kidneys from FtH(PT-/-) mice expressed significantly higher levels of inflammatory chemokines and fibrosis compared with kidneys from FtH(+/+) and FtH(LysM-/-) mice. Thus, there are differential effects of FtH in macrophages and epithelial cells, which underscore the critical role of FtH in tubular-macrophage cross-talk during kidney injury.