Neutrophils are immune cells specialized in producing large amounts of reactive oxygen species (ROS) to kill microbes. However, the mechanisms by which these cells regulate the balance of different ROS species and mitigate oxidative stress remain unclear. Here, we demonstrate that superoxide dismutase 1 (SOD1) plays a crucial role in ROS formation and antimicrobial activity in neutrophils. Our findings reveal that SOD1 modulates the ratio of superoxide (O2-) to hydrogen peroxide (H2O2) during the ROS burst, thereby supporting myeloperoxidase (MPO) enzymatic activity. By employing biochemical, cell biological, and genetic approaches, we show that SOD1 is crucial for ROS formation during NETosis and microbial infections, as it reduces oxidative stress and enables complete neutrophil activation. Impairment of SOD1 activity increases cysteine oxidation and lipid peroxidation. Neutrophils isolated from a patient with a SOD1 mutation exhibit decreased ROS production and impaired neutrophil extracellular trap (NET) formation. Our findings suggest that SOD1 is a novel regulatory factor in the oxidative burst that enables the full immunological response of neutrophils.

Anti-neutrophil cytoplasmic antibodies directed to myeloperoxidase (MPO-ANCA) are key molecules in the pathogenesis of ANCA-associated vasculitis (AAV), however, the mechanisms of autoantibody production have not been elucidated. We hypothesized that an aberrant PTM occurs in the MPO of MPO-ANCA-positive AAV (MPO-AAV), which induces immune responses to self MPO. To test this, we purified MPO proteins from neutrophils of 8 patients with MPO-AAV and 8 healthy subjects, digested them with trypsin, and comprehensively quantified PTMs of the MPO peptides using the sequential window acquisition of all theoretical fragment ion spectra (SWATH) method of LC-MS. Among the 1034 detected MPO peptides, 38 peptides were increased in the patients with MPO-AAV relative to the healthy subjects, whereas 10 peptides were decreased in the patients (p < 0.05). Interestingly, oxidative modifications were found in 11 of the 38 increased peptides (1.14- to 3.29-fold), but not in the decreased peptides. These included oxidation of Met577, Phe686, Met688 and Met719, dioxidation of Met409, Phe605, Trp679 and Met719, and kynurenylation of Trp255. Conversely, glycosylation was detected in 4 of the 10 decreased peptides (-1.32- to -2.32-fold), but not in the increased peptides. They were O-type glycans at Ser357 and Ser731, and N-type glycans at Asn355 and Asn729. In animal experiments, immunization of mice with in vitro oxidized or unoxidized mouse MPO (mMPO) showed that not only anti-oxidized mMPO antibodies but also anti-unoxidized mMPO antibodies were preferentially produced in the oxidized mMPO-immunized mice relative to the unoxidized mMPO-immunized mice (anti-oxidized mMPO antibodies, 6/8 vs 1/9, p < 0.05; anti-unoxidized mMPO antibodies, 4/8 vs 0/9, p < 0.05). Our results suggest that the increased oxidative modifications of MPO in MPO-AAV may break immune tolerance and trigger the MPO-ANCA production. SIGNIFICANCE: ANCA-associated vasculitis (AAV) is a systemic and refractory disease that causes life-threatening multi-organ involvement such as necrotizing glomerulonephritis and lung hemorrhage. MPO-ANCA is an autoantibody that plays a key role in the pathogenesis of AAV. Therefore, elucidation of the mechanism of MPO-ANCA production is crucial to overcoming this disease. In this study, we applied a SWATH-MS analysis to the detection of aberrant PTMs, and found increased oxidative modifications of neutrophil MPO in patients with MPO-ANCA-positive AAV (MPO-AAV) for the first time. Immunization of in vitro oxidized MPO induced autoantibodies to the intact unoxidized MPO, indicating that the increased oxidative modifications of MPO may break the immune tolerance in MPO-AAV. This study suggests a novel trigger mechanism for MPO-ANCA production, which may lead to a curative treatment for AAV.

The polymorphonuclear granulocyte (PMN) kills ingested bacteria by mechanisms that include myeloperoxidase (MPO) and a sudden increase in oxygen consumption (the oxidative burst), both of which are iron dependent. The magnitude of the oxidative burst and activity of MPO were determined in PMNs during the progression of iron deficiency (ID) and following its treatment in rats. As ID developed, the oxidative burst after zymosan activation was less depressed than the activity of MPO. There was no change in the oxidative burst after activation with phorbol myristate acetate (PMA) or in the generation of superoxide (O2- ) by NADPH oxidase-containing particles from PMNs. Following iron treatment, impairment of the oxidative burst after zymosan activation was corrected after 1 day. In contrast, the deficit in MPO activity was not corrected until 7 days after initiation of iron treatment. The pattern of recovery in MPO activity after iron treatment corresponded to the prolonged period of maturation of the PMN primary granule since the formation of primary granules, which contain MPO, takes place only in the early, mitotic stages of maturation. The tendency of the PMN to maintain the oxidative burst allows the cell to preserve its capacity for bacterial killing during the progression of iron deficiency.

The NADPH oxidase of neutrophils, essential for innate immunity, passes electrons across the phagocytic membrane to form superoxide in the phagocytic vacuole. Activity of the oxidase requires that charge movements across the vacuolar membrane are balanced. Using the pH indicator SNARF, we measured changes in pH in the phagocytic vacuole and cytosol of neutrophils. In human cells, the vacuolar pH rose to ~9, and the cytosol acidified slightly. By contrast, in Hvcn1 knock out mouse neutrophils, the vacuolar pH rose above 11, vacuoles swelled, and the cytosol acidified excessively, demonstrating that ordinarily this channel plays an important role in charge compensation. Proton extrusion was not diminished in Hvcn1-/- mouse neutrophils arguing against its role in maintaining pH homeostasis across the plasma membrane. Conditions in the vacuole are optimal for bacterial killing by the neutral proteases, cathepsin G and elastase, and not by myeloperoxidase, activity of which was unphysiologically low at alkaline pH

Hypochlorous acid (HOCl), produced in inflammatory conditions by the enzyme myeloperoxidase, and its anion hypochlorite (OCl(-)) exist in vivo at almost equal concentrations. Their reactions with hyaluronan and heparin (as a model for sulfated glycosaminoglycans in the extracellular matrix) have been studied as a function of pH. The major product in these reactions is the chloramide derivative of the glycosaminoglycans. Spectral, chloramide yield, and kinetic measurements show sharply contrasting behavior of heparin and hyaluronan and the data allow the calculation of second-order rate constants for the reactions of both HOCl and OCl(-) for all reaction pathways leading to the formation of chloramides and also oxidation products. By comparison with hyaluronan, it can be demonstrated that both N-sulfate and O-sulfate groups in heparin influence the proportions of these pathways in this glycosaminoglycan. Evidence is also given for further oxidation pathways involving a reaction of HOCl with the chloramide product of hyaluronan but not with heparin. The significance of these results for the mechanisms of inflammation, particularly for fragmentation of extracellular matrix glycosaminoglycans, is discussed

The susceptibility of a number of human neutrophil granule enzymes to oxidative inactivation was investigated. Addition of H2O2 to the cell-free medium from stimulated neutrophils resulted in inactivation of all enzymes tested. This was inhibited by azide and methionine, indicating that inactivation was due to myeloperoxidase-derived oxidants. Lysozyme was more than 50% inactivated by one addition of 100 nmol of H2O2/ml, whereas myeloperoxidase, beta-glucuronidase, gelatinase and collagenase were almost completely inactivated by three additions. Cathepsin G was slightly less susceptible, whereas elastase was extremely resistant to oxidative attack. Myeloperoxidase-dependent enzyme inactivation may be a means whereby the neutrophil can terminate the activity of its granule enzymes and control the release of degradative enzymes into the tissues.

Neutrophils are among the most important components of the innate immune response, which provides the first line of host defense. The antimicrobial potential of neutrophils has been traditionally divided into either non-oxidative or oxidative mechanisms. Two of the most important antimicrobial systems of these mechansims are granule-associated antimicrobial proteases and peptides and the nicotinamid adenine dinucleotide phosphate (NADPH) oxidase generating reactive oxygen species (ROS). In the past, studies often focused on the effects of either non-oxidative or oxidative mechanisms and decades of research have provided a detailed understanding of the regulation, generation and actions of these processes alone. Recent evidence challenged the established view of two independent mechanisms and proposed the cooperation of the NADPH oxidase with granule proteases in the killing of microorganisms. Furthermore, a novel phagocytosis-independent antimicrobial mechanism was found by the discovery of neutrophil extracellular traps (NETs). The formation of NETs was found dependent on NADPH oxidase activation and the production of ROS but NETs are believed to kill entrapped pathogens by NETsassociated granule proteases and peptides. Consequently, NETs present additional evidence for the interaction of non-oxidative and oxidative killing mechanisms. Together, these findings opened a new field of investigation with many controversies to be elucidated and underscored that we need further insight into the mechanisms by which neutrophils specifically recognize and overcome pathogens. In this thesis, we followed the question whether the murine antimicrobial peptide cathelin-related antimicrobial peptide (CRAMP) is an important component of the non-oxidative arm of neutrophil defense against S. aureus. This was motivated because little is known about cathelicidin function and activity in neutrophils and seems of crucial interest since mice lack the major constituent of human neutrophils - the -defensins. We further aimed to specify the relationship between the NADPH oxidase and CRAMP with focus on the antimicrobial activity of CRAMP in association with NETs and in NADPH oxidase-deficient mice (gp91phox-/-). As a result, we could demonstrate a previously unknown intracellular antimicrobial activity of CRAMP against S. aureus. Specifically, CRAMP colocalized with S. aureus in phagolysosomes and we showed first evidence for the presence of intracellular active CRAMP. Most interestingly, phagolysosomal localization and intracellular activity of CRAMP was found independent of a functional NADPH oxidase controversially to our expectations. Investigation of NET-dependent killing of S. aureus revealed a negligible role for CRAMP due to inactivation of the peptide in association with NETs. This point is of particular relevance and should be considered in the current opinion of NETs-mediated antimicrobial activity. In summary, our data provided deeper knowledge about one specific member of the non-oxidative killing mechanism and gives reason to reconsider the controversial results about interaction of NADPH oxidase in activating non-oxidative mechanisms. In addition, we followed the question whether recognition of S. aureus by TLR2 regulates the induction of non-oxidative and oxidative killing responses as well as the induction of NETs. The background of this study is based on several previous reports. First, results of our group evidenced a relationship between TLR2-mediated staphylococcal killing by neutrophils and the susceptibility of S. aureus to cationic antimicrobial peptides. Additionally, TLR2 activation has been shown to up-regulate 7 cathelicidin expression. Second, TLR2 was demonstrated to induce phosphorylation of p47phox and up-regulation of p47phox mRNA in macrophages. However, little attention has been paid to similar studies in neutrophils. Third, recognition of pathogens by TLRs was hypothesized to induce formation of NETs but there is as yet no evidence. First results unraveled a role for TLR2 in rapid induction of the NADPH oxidase, whereas TLR2 signaling had no influence on CRAMP activity. Interestingly, pathogen sensing for the induction of NETs formation did not depend on TLR2- MyD88 signaling. Taken together, the results demonstrate a role for TLR2 in mediating rapid killing of S. aureus by accelerating the activation of the NADPH oxidase complex possibly by influencing assembly. Further studies of the mechanisms underlying the relationship between pathogen sensing and non-oxidative and oxidative killing mechanisms would contribute greatly to our understanding of how the innate immune system resolves bacterial infections and will help in the development of therapeutic strategies to assist in clearance of pathogenic bacteria

ABSTRACT During infections, Staphylococcus aureus is exposed to hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN), which are produced by the neutrophil myeloperoxidase as potent antimicrobial killing agents. In this work, we applied RNAseq transcriptomics, Brx-roGFP2 biosensor measurements, and phenotype analyses to investigate the stress responses and defense mechanisms of S. aureus COL toward HOSCN stress. Based on the RNAseq transcriptome profile, HOSCN exerts strong thiol-specific oxidative, electrophile, and metal stress responses as well as protein damage in S. aureus, which is indicated by the strong induction of the HypR, TetR1, PerR, QsrR, MhqR, CstR, CsoR, CzrA, AgrA, HrcA, and CtsR regulons. Phenotype analyses of various mutants in HOSCN-responsive genes revealed that the HOSCN reductase MerA conferred the highest resistance toward HOSCN stress in S. aureus COL, whereas the QsrR and MhqR electrophile stress regulons do not contribute to protection. Brx-roGFP2 biosensor measurements and bacillithiol (BSH)-specific Western blot analyses revealed a strong oxidative shift of the bacillithiol redox potential (E BSH) and increased S-bacillithiolations in S. aureus, indicating that BSH is oxidized to bacillithiol disulfide (BSSB) under HOSCN stress. While the ΔmerA mutant was delayed in recovery of the reduced E BSH, overproduction of MerA in the ΔhypR mutant enabled faster recovery of E BSH due to efficient HOSCN detoxification. Moreover, both MerA and BSH were shown to contribute to HOSCN resistance in growth assays. In summary, HOSCN provokes a thiol-specific oxidative, electrophile, and metal stress response, an oxidative shift in E BSH and increased S-bacillithiolation in S. aureus. IMPORTANCE Staphylococcus aureus colonizes the skin and the airways but can also lead to life-threatening systemic and chronic infections. During colonization and phagocytosis by immune cells, S. aureus encounters the thiol-reactive oxidant HOSCN. The understanding of the adaptation mechanisms of S. aureus toward HOSCN stress is important to identify novel drug targets to combat multi-resistant S. aureus isolates. As a defense mechanism, S. aureus uses the flavin disulfide reductase MerA, which functions as HOSCN reductase and protects against HOSCN stress. Moreover, MerA homologs have conserved functions in HOSCN detoxification in other bacteria, including intestinal and respiratory pathogens. In this work, we studied the comprehensive thiol-reactive mode of action of HOSCN and its effect on the reversible shift of the E BSH to discover new defense mechanisms against the neutrophil oxidant. These findings provide new leads for future drug design to fight the pathogen at the sites of colonization and infections. Staphylococcus aureus colonizes the skin and the airways but can also lead to life-threatening systemic and chronic infections. During colonization and phagocytosis by immune cells, S. aureus encounters the thiol-reactive oxidant HOSCN. The understanding of the adaptation mechanisms of S. aureus toward HOSCN stress is important to identify novel drug targets to combat multi-resistant S. aureus isolates. As a defense mechanism, S. aureus uses the flavin disulfide reductase MerA, which functions as HOSCN reductase and protects against HOSCN stress. Moreover, MerA homologs have conserved functions in HOSCN detoxification in other bacteria, including intestinal and respiratory pathogens. In this work, we studied the comprehensive thiol-reactive mode of action of HOSCN and its effect on the reversible shift of the E BSH to discover new defense mechanisms against the neutrophil oxidant. These findings provide new leads for future drug design to fight the pathogen at the sites of colonization and infections.

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pós-Graduação em Farmácia.Introdução: O gênero Esenbeckia (Rutaceae) compreende cerca de 30 espécies, sendo característico de regiões tropicais. A espécie Esenbeckia leiocarpa Engl. é conhecida popularmente no Brasil como guarantã, pau-duro, goiabeira ou guarataia. Espécies pertencentes ao gênero Esenbeckia têm sido utilizadas no tratamento de febre e/ou da malária por indivíduos da região amazônica. Estudos in vitro e in vivo já demonstraram que espécies do gênero Esenbeckia possuem importantes ações farmacológicas, como atividade anticolinesterásica, antimalárica, anti-helmíntica e antimicrobiana. Objetivos: Estudar o efeito e o mecanismo de ação anti-inflamatória do extrato bruto hidroalcoólico (EBH), frações, subfrações e compostos isolados da Esenbeckia leiocarpa Engl. utilizando modelos experimentais in vivo e in vitro. Materiais e Métodos: O efeito do EBH, frações n-hexano (Hex), acetato de etila (AcOEt) ou alcaloide (Alc), subfrações polar (Pol) e apolar (Apol), ou dos compostos di-hidrocorinanteol (DHC) e beta-sitosterol ( -Sit) isolados do EBH foram investigados sobre migração de leucócitos, exsudação, mieloperoxidase (MPO), adenosina-desaminase (ADA), nitrato/nitrito (NOx), interleucina-1 beta (IL-1 ), fator de necrose tumoral-alfa (TNF- ) e proteína inibitória kappa B-alfa (I B- ) na inflamação induzida pela carragenina (Cg) no modelo da bolsa de ar, em camundongos. Ainda, o mecanismo de ação anti-inflamatória exercido pelo EBH, fração Alc e composto DHC foram avaliados sobre a apoptose de neutrófilos humanos, bem como sobre a adesão celular e diferentes funções neutrofílicas, como: fagocitose, degranulação, produção de espécies reativas de oxigênio (EROs) e citocinas/quimiocinas. Ainda, foi avaliado o efeito do -Sit sobre a captação de cálcio (Ca2+). Resultados: EBH, Alc, Pol, Apol, DHC e -Sit, foram efetivos em inibir a migração de leucócitos, a exsudação e as concentrações de MPO, ADA, NOx, IL-1 e TNF- (P < 0,05). As frações Hex e AcOEt inibiram todos os parâmetros inflamatórios estudados, exceto a exsudação. O composto DHC inibiu a degradação da proteína inibitória I B- . Ainda, EBH, Alc e DHC induziram a apoptose de neutrófilos humanos, quando comparados à apoptose espontânea (AE) (P < 0,05). EBH e Alc, mas não o DHC promoveram aumento significativo, tanto da fagocitose como do processo de adesão celular dos neutrófilos a células A549 (P < 0,05). EBH e Alc promoveram a degranulação, tanto de vesículas secretórias como de grânulos específicos/de gelatinase dos neutrófilos (P < 0,05). EBH, Alc e DHC promoveram também a degranulação de grânulos azurófilos de neutrófilos (P < 0,01). EBH, Alc e DHC inibiram significativamente a produção de EROs, assim como a produção de citocinas/quimiocinas, como interleucina-6 (IL-6), proteína inflamatória de macrófagos-1 alfa (MIP-1 ), proteína inflamatória de macrófagos-1 beta (MIP-1 ) e oncogene-alfa relacionado ao crescimento (GRO- ) por neutrófilos ativados. -Sit promoveu aumento significativo na captação de Ca2+ de forma tempo- e concentração-dependente em neutrófilos ativados, que foi prontamente inibido pela nifedipina, BAPTA-AM, LY294002 e colchicina (P < 0,05). Conclusão: Este estudo demonstrou que a Esenbeckia leiocarpa Engl. possui potente efeito anti-inflamatório. Este efeito parece estar relacionado à inibição da migração de leucócitos, essencialmente de neutrófilos ativados, uma vez que a planta promoveu a degranulação destas células, inibiu as concentrações de enzimas relacionadas à atividade neutrofílica (MPO e ADA), assim como a produção de EROs, aumentou a adesão e a fagocitose, além de induzir a apoptose de neutrófilos. Ainda, o material vegetal inibiu a exsudação, efeito este relacionado também à inibição das concentrações de .NO e de citocinas/quimiocinas de caráter pró-inflamatório: IL-1 , TNF- , IL-6, MIP-1 e MIP-1 e GRO- . O DHC parece ser um dos responsáveis pelo efeito observado, uma vez que também demonstrou efeito anti-inflamatório por meio da inibição da migração de leucócitos, enzimas e citocinas/quimiocinas pró-inflamatórias, além de inibir a ativação do NF- B. Ainda, o -Sit foi efetivo em inibir a resposta inflamatória de forma dependente da captação de Ca2+.Introduction: The genus Esenbeckia (Rutaceae) includes about 30 species, and is characteristic from tropical regions. Esenbeckia leiocarpa Engl. is popularly known in Brazil as "guarantã", "pau-duro", "goiabeira" or "guarataia". Esenbeckia species have been used for treatment of fever and/or malaria by inhabitants of Brazilian amazon region. In vitro and in vivo studies have already demonstrated that Esenbeckia species have important pharmacological actions, such as anticholinesterasic, antimalaric, anthelmintic and antimicrobial activities. Objectives: The aim of this work was to evaluate the effect, as well as the mechanism of anti-inflammatory action of the crude hydroalcoholic extract (CHE), fractions, subfractions, and isolated compounds from Esenbeckia leiocarpa Engl. using in vivo and in vitro models. Materials and Methods: The effect of CHE, fractions n-hexane (Hex), ethyl acetate (AcOEt), and alkaloid (Alk), subfractions polar (Pol) and nonpolar (Nonpol), or isolated compounds dihydrocorynantheol (DHC) and beta-sitosterol (?-Sit) isolated from CHE upon leukocyte migration, exudation, myeloperoxidase (MPO) adenosine-deaminase (ADA), nitrate/nitrite (NOx), interleukin-1 beta (IL-1?), tumour necrosis factor-alpha (TNF-?), and inhibitory kappa-B-alpha protein (I B- ) degradation in the inflammation induced by carrageenan (Cg) in the mouse air pouch model were investigated. Also, the mechanism of anti-inflammatory action of CHE, Alk and DHC on apoptosis of human neutrophil, as well as cell adhesion, and different neutrophil functions, such as: phagocytosis, degranulation, reactive oxygen species (ROS) and cytokines/chemokines production were evaluated. Also, the effect of -Sit on the calcium (Ca2+) uptake was evaluated. Results: The CHE, Alk, Pol, Nonpol, DHC and ?-Sit, inhibited leukocytes, exudation, MPO, ADA, NOx, IL-1?, and TNF-? (P < 0.05). The Hex and AcOEt fractions inhibited all of the proinflammatory parameters, except for the exudation. The compound DHC prevented the I B- degradation. Also, CHE, Alk, and DHC accelerated the spontaneous apoptosis of human neutrophils (P < 0.05). CHE and Alk, but not DHC also increased both the phagocytosis and the adhesion exerted by neutrophils onto A549 cells (P < 0.05). CHE and Alk also promoted the degranulation of secretory vesicles and specific/gelatinase granules (P < 0.05). Also, CHE, Alk and DHC induced the degranulation of azurophilic granules (P < 0.01). Moreover, CHE, Alk and DHC significantly inhibited the ROS production, as well as the production of cytokines/chemokines, such as interleukin-6 (IL-6), macrophage inflammatory protein-1 alpha (MIP-1 ), macrophage inflammatory protein-1 beta (MIP-1 ), and growth-related oncogene-alpha (GRO- ) by activated neutrophils. -Sit promoted a time- and dose-dependent increase of the calcium uptake in activated neutrophils that was promptly reversed by nifedipine, BAPTA-AM, LY294002, and colchicine (P < 0.05). Conclusion: This study showed that Esenbeckia leiocarpa Engl. has a potential anti-inflammatory effect. Such effect seems to be related to the inhibition of leukocyte migration, essentially activated neutrophils, since this herb promoted the degranulation on these cells, and inhibited the concentration of enzymes related to neutrophil activity (MPO and ADA), as well as the ROS production. It also increased both the phagocytosis and adhesion, besides inducing neutrophil apoptosis. Esenbeckia leiocarpa Engl. also inhibited the exudation, in a process related to the inhibition of .NO concentrations, as well as concentrations of proinflammatory cytokines/chemokines: IL-1 , TNF- , IL-6, MIP-1 e MIP-1 e GRO- . DHC seems to be one of the most responsible for this effect, since it also showed an anti-inflammatory effect by inhibiting leukocyte migration, enzymes and pro-inflammatory cytokines/chemokines, besides inhibiting NF- B activation. Also, -Sit was effective in inhibiting the inflammatory response in a Ca2+ uptake-dependent manner

The leukotoxin of Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of inflammatory periodontal disease. We examined a potential mechanism for detoxification of this microbial product by the neutrophil myeloperoxidase system. Exposure to myeloperoxidase, H2O2, and a halide resulted in marked inactivation of leukotoxin, an effect which required each component of the myeloperoxidase system. Toxin inactivation was blocked by agents which inhibit heme enzymes (azide, cyanide) or degrade H2O2 (catalase). Reagent H2O2 could be replaced by the peroxide-generating enzyme system glucose oxidase plus glucose. The latter system, in fact, was more potent than reagent H2O2 in terms of the capacity to inactivate high concentrations of toxin. Toxin inactivation was complete within 1 to 2 min at 37 degrees C. These observations suggest a possible role for oxidative inactivation of leukotoxin by secretory products of neutrophils.

A novel feature of polymorphonuclear granulocyte (PMN) biology is their ability to generate neutrophil extracellular traps (NETs) via a distinct process of cell death termed NETosis. NETs consist of chromosomal extruded DNA decorated with granular components that include antimicrobial peptides and proteases. The molecular pathways leading to NETosis involve generation of reactive oxygen species (ROS), calcium mobilization, nuclear delobulation involving the enzymatic activities of myeloperoxidase (MPO) and neutrophil elastase (NE), and chromatin modification via the citrullination of histones by peptidyl arginine deiminase 4 (PAD4). A number of studies have implicated NETs in the etiology of auto-inflammatory or autoimmune conditions such as preeclampsia (PE), Felty’s syndrome, systemic lupus erythematosus (SLE), multiple sclerosis (MS), and, most recently, rheumatoid arthritis (RA). In the context of RA these findings are especially interesting, as NETs have been proposed to contribute to the generation of anti-citrullinated protein antibody (ACPA) auto-antigens, and may also be a target for autoantibodies. As we have previously detected significantly increased concentrations of cell-free DNA in the sera of RA patients compared with healthy controls, we were intrigued whether the elevation in this material involved NETosis. The premise for this investigation was that a link between circulating cell-free DNA levels and NETs has previously been made in a number of conditions including preeclampsia, sepsis, cancer, thrombosis or even storage of blood transfusion products. In view of these findings and reports on the complex involvement of neutrophil NETs in autoimmunity, we sought to investigate the NETotic response of PMN in RA, with particular regard to the underlying signal transduction cascade, and whether the products of overt NETosis could be diagnostically useful. Our data confirm and extend upon these observations in that we examined for changes in the underlying signal transduction cascade required for the induction of NETosis. These findings indicate that the propensity of circulatory PMN in RA patients to undergo NETosis is associated with elevations in members of this cascade including increased intracellular ROS production, enhanced expression of NE and MPO, and increased nuclear translocation of PAD4 and citrullination of histones, notably H3. Furthermore, by examining kinetic changes during extended in vitro culture it was observed that PMN from RA cases exhibited different nuclear morphometric characteristics, having a lower proportion of the classical lobulated phenotype. In the presence of a secondary stimuli, RA PMN also progressed more rapidly and extensively to a NETotic spread phenotype than controls, a finding confirmed by analysis of culture supernatants for the products of NETosis. Akin to what has been observed in an array of other pathological conditions ranging from SLE to cancer, PMN from RA patients exhibited an increased response to further stimulation. This response is in part mediated via the action of PAD4, as the effect of PMA could be significantly reduced by treatment with Cl-amidine, an inhibitor of PAD4. In addition, PMA treatment lead to an increased nuclear presence of this enzyme, where it presumably could be able to carry out a more extensive citrullination of histone proteins, thereby speeding up the induction of NETosis. A very intriguing and novel feature of our data is the detection of the extracellular presence of PAD4 on extruded NETs. The concomitant extracellular presence of this enzyme on NETs may increase the spectrum of citrullinated proteins and their local concentration significantly, a feature which could be important in the generation of ACPA. It is furthermore possible that the extracellular localisation of PAD4 may play a role in the development of anti-PAD4 antibodies observed in cases with RA. Hence, our data support and extend upon recent reports indicating that NETs can be a source for citrullinated autoantigens, or react with ACPA. Further presence of NETs in the intervillous space of preeclamptic placenta was previously reported by our lab. Using in vitro experiments, we have shown that trophoblast micro-debris could stimulate neutrophils to form NETs. However the effect of such phenomena remained elusive. Recent data suggests, NETs could act as a lattice, to stimulate platelet adhesion, promotes coagulation and thrombotic events. Given that PE, intrauterine growth restriction (IUGR) and even fetal loss are broadly related to elevated thrombotic events causing dysfunctions at the interface between innate immunity and haemostasis, it is of cardinal importance to investigate whether NETs could elucidate such a response. In this study, we adopted mice sFLT-1 overexpression model, to elucidate the impact of NETosis in murine pregnancy outcome. Preliminary results indicates significant drop in pregnancies after sFLT-1 overexpression although there was only a partial induction of PE phenotype characterized by minor elevation in blood pressure and proteinuria. Staining of placentas from mice that retained pregnancy after sFLT-1 overexpression indicates heavy neutrophil infiltration in the feto-placental junction, with zones positive for NET specific markers. Plasma isolated from sFLT-1 treated mouse also indicates elevation of NET specific markers compared to healthy pregnant controls. Minor increase in plasma TAT complexes (although not significant), along with a drop in platelet count, indicates elevated thrombotic events. Further ongoing experiments will compare the impact of sFLT-1 overexpression in normal WT mice to those that are unable to form NETs

Abstract Pimenta pseudocaryophyllus (Gomes) Landrum is a Brazilian native plant. The mechanisms by which it promotes analgesia are unknown. We demonstrated the analgesic effect of P. pseudocaryophyllus dried extract (3 mg/kg; i.p.) in the following models of inflammatory pain (maximal inhibition): phenyl-p-benzoquinone (89%), formalin (72% - 1st phase and 96% - 2nd phase for flinches, and 50% - 1st phase and 71% - 2nd phase for licking behavior), complete Freund’s adjuvant (95% - flinches and 33% - licking behavior), and carrageenin (56% - mechanical and 85% - thermal hyperalgesia) without motor impairment. Its analgesic effect depends on inhibiting neutrophil recruitment (95% - histopathology, 83% - myeloperoxidase activity, and 80% - LysM-eGFP mice), oxidative stress (86% - GSH and 98% - superoxide anion), and cytokine production (35% - IL-33, 80% - TNF-α, and 95% - IL-1β). The present study advances in understanding the analgesic mechanisms of P. pseudocaryophyllus. Graphical Abstract

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2010A Rosmarinus officinalis L. (Laminaceae), conhecida como alecrim, é uma planta nativa da região mediterrânea, que atualmente é cultivada em várias partes do mundo, inclusive no Brasil. Esta planta é utilizada na medicina popular para o tratamento de doenças inflamatórias como a asma brônquica. Além disso, estudos já demonstraram que seus constituintes possuem atividade anti-inflamatória. Objetivo: O objetivo deste trabalho foi avaliar o efeito anti-inflamatório do extrato bruto (CE) da Rosmarinus officinalis L., bem como de suas frações: hexano (HEX), acetato de etila (AcOEt) e etanólica (ET), e dos compostos isolados: carnosol (CA), ácido betulínico (BA) e ácido ursólico (UA), no modelo da pleurisia induzida pela carragenina, em camundongos. Metodologia: As partes aéreas da Rosmarinus officinalis L. foram secas à temperatura ambiente e submetidas à maceração com etanol 96% por 15 dias para a obtenção do CE. A seguir, o CE foi submetido a uma filtração em sílica utilizando-se solventes em ordem crescente de polaridade resultando nas frações HEX, AcOEt e ET. A partir das frações AcOEt e HEX foram isolados três compostos: CA, BA e UA. Para a avaliação da atividade anti-inflamatória foi utilizado o modelo da pleurisia induzida pela carragenina, em camundongos, e foram avaliados os seguintes parâmetros inflamatórios: influxo de leucócitos, atividade da mieloperoxidase (MPO), além das concentrações de nitrito/nitrato (NOx), exsudação, interleucina-1 beta (IL-1?) e fator de necrose tumoral-alfa (TNF-?). Nesse protocolo experimental, foram utilizados camundongos albinos suíços, os quais foram inicialmente distribuídos em diferentes grupos e tratados com CE (25 - 100 mg/kg), HEX (10 - 50 mg/kg), AcOEt (5 - 50 mg/kg), ET (10 - 50 mg/kg), CA (1 - 10 mg/kg), BA (1 - 10 mg/kg) ou UA (2,5 - 25 mg/kg), administrados por via intraperitoneal (i.p.), 0,5 h antes da administração da carragenina (1%, i.pl.). Os parâmetros inflamatórios (leucócitos e exsudação) foram avaliados 4 h após. Para avaliar a exsudação, os animais foram tratados previamente (10 min.) com solução Azul de Evans (25 mg/kg, i.v.). Para avaliar a atividade da MPO e as concentrações de NOx, IL-1? e TNF-?, diferentes grupos de animais foram tratados com CE (50 mg/kg, i.p.), HEX (25 mg/kg, i.p.), AcOEt (10 mg/kg, i.p.), ET (50 mg/kg, i.p.), CA (2,5 mg/kg, i.p.), BA (2,5 mg/kg, i.p.) ou UA (25 mg/kg, i.p.) administrados 0,5 h antes da carragenina. A dexametasona e indometacina foram utilizadas como fármacos anti-inflamatórios de referência. Diferenças estatísticas entre os grupos foram determinadas pela análise dos testes de variância (ANOVA) e/ou t de Student. Valores de P < 0,05 foram considerados significativos. Resultados: O extrato bruto da Rosmarinus officinalis L. (50 - 100 mg/kg), as frações (10 - 50 mg/kg), assim como os compostos isolados (2,5 - 25 mg/kg) inibiram: influxo de leucócitos, neutrófilos, atividade da MPO, além das concentrações de NOx, exsudação, IL-1? e TNF-? (P < 0,05). A indometacina e a dexametasona inibiram todos os parâmetros inflamatórios estudados (P < 0,05). Conclusão: O extrato bruto, frações e compostos isolados da Rosmarinus officinalis L. demonstraram importante atividade anti-inflamatória. Os compostos isolados: carnosol, ácido betulínico e ácido ursólico podem ser os responsáveis por este efeito anti-inflamatório

Degenerative-dystrophic processes in the articular tissues are accompanied by the changes in the values of congenital immunity, being a result of immune responses to aseptic inflammation. The purpose of this study - to investigate the oxygen-dependent mechanisms of neutrophil phagocytic activity (NBT-test, myeloperoxidase activity), the biochemical values of lipid peroxidation, the oxidative modification of proteins and the activity of enzyme antioxidant protection in children (at the age of 12-16 years) with osteochondropathy of the femoral head.The increase in spontaneous NBT-test and myeloperoxidase content in neutrophils, the decrease in NBT stimulation index, as well as the statistically significant increase in the activity of superoxide dismutase, the main producer of hydroperoxide, was revealed in the group examined. At the same time, no increase in catalase activity and in the content of lipoperoxidation products was observed, however, redistribution of the products of oxidative modification of proteins with ketone predominance was found. In case of osteochondropathy development in children the activation of the oxygendependent mechanisms of neutrophil phagocytic activity is observed, as well as the increase in the number of the secondary products of protein peroxidation - ketones. An increase in SOD activity, with a simultaneous and significant decrease in catalase activity evidence of accumulation of hydroperoxide causing neutrophil activation and chemotaxis. The study data can be used for predicting osteonecrosis development as additional criteria when decision-making of the advisability of performing reconstructive surgeries of the joint.

Dissertação (mestrado) - Universidade Federal de Santa catarina, Centro de Ciências Biológicas. Programa de Pós-graduação em Biotecnologia

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pos-Graduação em Nutrição.As espécies reativas de oxigênio (ERO) estão continuamente relacionadas com o consumo de oxigênio nos organismos aeróbicos, incluindo os seres humanos. Sua produção é aumentada em eventos patológicos, como no processo inflamatório, dentre eles a doença periodontal. O principal objetivo do presente estudo foi avaliar a incidência do estresse oxidativo no tecido gengival em indivíduos com doença periodontal crônica. Dezoito indivíduos participaram do estudo e foram divididos em dois grupos (n=9 em cada grupo): controle (pacientes saudáveis # média de idade 46,77 ± 12,94 ), com profundidade de sondagem de até 3mm; e grupo experimental (pacientes com periodontite # média de idade 55,5 ± 11,32), com profundidade de sondagem entre 6-9mm. A avaliação nutricional baseou-se no consumo alimentar e em medidas antropométricas. Fragmentos de tecido gengival foram coletados para análise enzimática de antioxidantes: catalase (CAT), glutationa peroxidase (GPx), glutationa S-transferase (GST), glutationa redutase (GR); antioxidantes não enzimáticos: glutationa total (GT), glutationa reduzida (GSH); e biomarcadores do estresse oxidativo, tal como, glutationa oxidada (GSSG) e lipoperoxidação tecidual, avaliado como TBARS (substâncias reativas ao ácido tiobarbitúrico). A atividade da mieloperoxidase (MPO) também foi analisada. Em relação ao estado nutricional, foram obtidos os seguintes resultados no grupo controle: índice de massa corporal (IMC) = 24,88 ± 3,72; circunferência braquial (CB) = 30,30 ± 3,86; prega cutânea triciptal (PCT) = 14,55 ± 5,56; circunferência muscular do braço (CBM) = 24,23 ± 3,86. No grupo experimental índice de massa corporal (IMC) = 27,65 ± 4,32; circunferência braquial (CB) = 33,56 ± 2,66; prega cutânea triciptal (PCT) = 32,26 ± 10,76; circunferência muscular do braço (CBM) = 23,34 ± 3,12. O consumo alimentar do grupo controle (indivíduos considerados normais em relação ao estado nutricional) mostrou uma presença elevada de antioxidantes comparado ao grupo experimental (considerados indivíduos pré-obesos). Os indivíduos com doença periodontal mostraram um aumento da GSSG e concentrações de TBARS no tecido gengival, comparados ao pacientes saudáveis. Além disso, o grupo experimental, mostrou também um aumento da atividade da GPx, GST e MPO e concentrações de GT nos fragmentos gengivais analisados, quando comparado ao grupo controle. As atividades da CAT e da GR não apresentaram significância estatística entre os grupos. A análise das amostras obtidas em relação estado dos antioxidantes, atividade da MPO e o estado nutricional, sugere uma forte correlação entre biomarcadores do estresse oxidativo e doença periodontal, a qual provavelmente estava associada com o estado nutricional dos indivíduos pesquisados

Nowadays, severe acute pancreatitis (SAP) remains a critical clinical disease with a high mortality rate. Current treatments are mainly supportive, lacking specific therapies targeting the core pathological mechanisms including neutrophil extracellular traps (NETs)-mediated systemic inflammation and progressive acinar cell necrosis, which may lead to many complications. Therefore, in this study, we developed a novel dual-targeting nanodrug delivery system named PBs@CCR2-ApopEVs, combining Prussian blue nanoparticles (PBs) and CCR2-overexpressing mesenchymal stem cell-derived apoptotic extracellular vesicles (ApopEVs). Through the specific binding of CCR2 with numerous CCL2 released by neutrophils in SAP, the PBs@CCR2-ApopEVs can be actively recruited to the pancreatic inflammation site. Then PBs effectively inhibit neutrophil extracellular trap (NETs) formation by suppressing myeloperoxidase and elastase expression, as well as Gasdermin D pathway activation, thereby reducing oxidative stress and inflammatory responses. Simultaneously, we confirmed that MSCs-ApopEVs could activate the p-STAT3/SOX4 pathway to promote acinar-to-ductal metaplasia (ADM) process, enhancing the self-protection ability of acinar cells and reducing necrosis. This dual-approach strategy—targeting anti-inflammatory effects externally while reinforcing cytoprotective mechanisms internally—stands out as a novel therapeutic avenue for SAP management, providing a new approach for SAP treatment and significantly improving therapeutic efficacy.

Oxidative stress plays an important role in the progression of vascular endothelial dysfunction. The two major systems generating vascular oxidative stress are the NADPH oxidase and the xanthine oxidase pathways. Allopurinol, a xanthine oxidase inhibitor, has been in clinical use for over 40 years in the treatment of chronic gout. Allopurinol has also been shown to improve endothelial dysfunction, reduce oxidative stress burden and improve myocardial efficiency by reducing oxygen consumption in smaller mechanistic studies involving various cohorts at risk of cardiovascular events. This article aims to explain the role of xanthine oxidase in vascular oxidative stress and to explore the mechanisms by which allopurinol is thought to improve vascular and myocardial indices

Tumor-infiltrating neutrophils (TINs) have been observed to be linked with poor prognosis in patients with pancreatic ductal adenocarcinoma (PDAC). In the inflammation and oxidative stress-rich tumor microenvironment, neutrophils are likely to undergo neutrophil extracellular traps (NETs) formation which have unique capability of shielding the tumor cells from anti-tumor immune response and facilitate tumor progression. However, the extent of NET infiltration in PDAC and the specific mechanisms by which NETs contribute to tumorigenesis remain unclear. This study aims to investigate NETs infiltration in PDAC and their impact on PDAC progression. Tumor-infiltrating neutrophils (CD15) and NETs specific markers (citrullinated histone H3, Myeloperoxidase) in the tumor tissue and blood of 15 PDAC patients and 15 healthy volunteers were determined by flow cytometry, gene expression and immunofluorescence. NETs were cocultured with Panc1 cells to assess their effect on tumor growth and behavior through various tumorigenic assays (MTT, colony formation assay, scratch assay). The gene expression analysis was done for epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) modulation, stemness and dormant cell markers through qRT-PCR and further confirmation at protein level by western blot. In both blood and tissue samples of PDAC patients, relative percentage of NETs were markedly increased and the levels of citrullinated histone is positively correlated with higher tumor stage & grade. Moreover, circulating NETs shows a significant linear positive correlation with local pancreatic tumor NETs, indicates an association between NETs infiltration and disease severity. In vitro, NETs increased tumor migration and induced a more aggressive mesenchymal phenotype. They promoted tumor progression and invasion by downregulating epithelial markers (E-cadherin, ZO-1) and upregulating mesenchymal markers (N-cadherin, Vimentin, Snail, Twist, Zeb1, IL8, TGFβ) as well ECM-modulating markers (αSMA, Desmin, MMP, TIMP). Additionally, NETs contributed to tumor relapse by awakening dormant cancer cells (Dec2, p21, p27, p38) and increasing the expression of proliferative markers (Ki67, ERK, Bcl2) along with stemness markers (Sox2, Oct4, Nanog). NETs contribute to a pro-metastatic cancer phenotype by activating EMT and reactivating dormant cancer cells. Therefore, NETs may serve as independent diagnostic biomarker and a potential therapeutic target in PDAC. Sakshi Bansal, Anjali Aggarwal, Vinit Sharma, Rajesh Gupta, Harjeet Singh, Naresh Sachdeva, Alka Bhatia. Neutrophil extracellular traps drive the tumorigenic potential in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3447.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Ciências Médicas, Florianópolis, 2013Introdução: A Croton antisyphiliticus Mart., conhecida popularmente como canela-de-perdiz ou curraleira, é uma planta arbustiva do cerrado brasileiro, encontrada principalmente no estado de Minas Gerais. A medicina popular descreve o uso desta espécie no tratamento da sífilis, inflamação, lesões ulcerativas, eczemas e reumatismos. Objetivo: Avaliar o efeito anti-inflamatório do extrato bruto, frações e compostos isolados obtidos da espécie Croton antisyphiliticus Mart. (Euphorbiaceae), no modelo da pleurisia induzida pela carragenina (Cg), em camundongos. Metodologia: Partes aéreas da Croton antisyphiliticus foram secas, maceradas e submetidas o processo de extração com etanol 96%, para obtenção do extrato bruto hidroalcoólico (EBH). Através de uma extração líquido-líquido, o EBH foi fracionado com o uso de diferentes solventes de polaridade crescente em: hexano (Hex), acetato de etila (AcOEt) e aquosa (Aq). A fração Aq foi cromatografada em coluna de sílica gel, fornecendo 2 compostos: vitexina (VT) e ácido quínico (AcQ). Nesse estudo, foram utilizados camundongos albinos Swiss, fêmeas (18-22 g), os quais foram submetidos ao modelo experimental da pleurisia induzida pela Cg. No protocolo experimental, os animais receberam injeção endovenosa do corante Azul de Evans (25 mg/kg), para análise indireta da exsudação. Em seguida, diferentes grupos de animais foram tratados com o material vegetal (EBH, frações ou compostos isolados), 0,5 h antes da indução da pleurisia com uma injeção intrapleural de Cg (1%). Após 4 h de pleurisia, avaliou-se o efeito do EBH, das suas frações: Hex, AcOEt e Aq, bem como dos composto isolados: VT e AcQ, obtidos das partes aéreas da Croton antisyphiliticus, sobre a migração de leucócitos, exsudação, mieloperoxidase (MPO), adenosina-deaminase (ADA), metabólitos de óxido nítrico (NOx), fator de necrose tumoral-alfa (TNF-?) e interleucina-17 (IL-17). A análise estatística utilizada foi o teste de variância ANOVA, complementado pelo teste de Newman-Keuls. Valores de P 0,05). Conclusão: Este estudo demonstrou que a Croton antisyphiliticus Mart. apresentou importante efeito anti-inflamatório, sendo a sua fração Aq, entre as frações avaliadas, a mais efetiva em diminuir a inflamação, o qual parece estar relacionado com a inibição da migração de leucócitos, principalmente os neutrófilos ativados, haja vista a capacidade da planta em reduzir a atividade das enzimas MPO e ADA. O material vegetal estudado também foi capaz de reduzir a concentração de mediadores pró-inflamatórios como: NOx, TNF-? e IL-17. Essa redução pode estar relacionada com a diminuição do recrutamento celular e extravazamento vascular para o sítio inflamatório, promovidos pelos compostos isolados, a VT e o AcQ.Abstract : Introduction: Croton antisyphiliticus Mart. is popularly named as canela-de-perdiz or curraleira. It is a shrub plant found in the Brazilian savanna, mainly in the state of Minas Gerais. The folk medicine describes the use of this species in the treatment of syphilis, inflammation, ulcer, eczemas and rheumatisms. Objective: To evaluate the anti-inflammatory effect of the crude extract, its fractions and isolated compounds obtained from the aerial parts of Croton antisiphyliticus Mart., in the carrageenan-induced pleurisy model. Methodology: Aerial parts of the plant were dried, macerated and extracted with ethanol 96% to obtain the crude hydroalcoholic extract (CHE), which was partitioned with solvents of increasing polarity to obtain 3 fractions: hexane (Hex), ethyl acetate (EA) and aqueous (Aq). The Aq fraction was chromatographed on silica gel column and resulted in 3 isolated compounds: vitexin (VT) and quinic acid (QA). Swiss mice, female (18-22 g), were used in the murine carrageenan-induced pleurisy model. In this experimental protocol, animals received a previous injection of Evans Blue Dye (25 mg/kg) intravenous, in order to evaluate the exudate concentration. Then, different groups of animals were pre-treated, by intraperitoneal route (i.p.), with the vegetable products obtained from Croton antisyphiliticus (CHE, its fractions and isolated compounds), 0.5 h before pleurisy induction with an intrapleural (i.pl.) injection of carrageenan (Cg: 1%). The anti-inflammatory effect of CHE, its fractions (Hex, EA and Aq) and isolated compounds (VT and QA) was investigated upon: leukocyte migration (neutrophyls), exudation, myeloperoxidase (MPO), adenosine-deaminase (ADA), nitric oxide metabolities (NOx), tumoral necrosis factor- alpha (TNF-á) and interlekin-17 (IL-17). It was used to statistical analysis the variance test of ANOVA, which was complemented by Newman-Keuls test. Values of P 0.05). Conclusion: This study demonstrated the anti-inflammatory effect of Croton antisyphiliticus Mart. may be related to the inhibition of leukocyte migration, mainly activated neutrophils, since this plant is capable to reduce the activity of enzymes that is related to neutrophil activation (MPO and ADA). The extracted vegetable products obtained from this plant also decreased the levels of pro-inflammatory mediators, such as: NOx, TNF-á and IL-17. Among the analyzed fractions, Aq fraction performed the best anti-inflammatory effect, which may be associated with its isolated compounds, VT and AcQ, in the inhibition of leukocyte recruitment and vascular fluid leakage in the inflammatory site