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1.
Front Immunol ; 15: 1493946, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39687618

RESUMO

During intestinal and liver invasion by the protozoan parasite Entamoeba histolytica, extensive tissue destruction linked to large neutrophil infiltrates is observed. It has been proposed that microbicidal components of neutrophils are responsible for the damage, however, the mechanism by which they are released and act in the extracellular space remains unknown. In previous studies, we have shown that E. histolytica trophozoites induce NET formation, leading to the release of neutrophil granule content into extruded DNA. In this work, we evaluate the possible participation of NETs in the development of amoeba-associated pathology and analyze the contribution of anti-microbial components of the associated granules. E. histolytica-induced NETs were isolated and their effect on the viability and integrity of HCT 116 colonic and Hep G2 liver cultures were evaluated. The results showed that simple incubation of cell monolayers with purified NETs for 24 h resulted in cell detachment and death in a dose-dependent manner. The effect was thermolabile and correlated with the amount of DNA and protein present in NETs. Pretreatment of NETs with specific inhibitors of some microbicidal components suggested that serine proteases, are mostly responsible for the damage caused by NETs on HCT 116 cells, while the MPO activity was the most related to Hep G2 cells damage. Our study also points to a very important role of DNA as a scaffold for the activity of these proteins. We show evidence of the development of NETs in amoebic liver abscesses in hamsters as a preamble to evaluate their participation in tissue damage. In conclusion, these studies demonstrate that amoebic-induced NETs have potent cytotoxic effects on target cells and, therefore, may be responsible for the intense damage associated with tissue invasion by this parasite.


Assuntos
Entamoeba histolytica , Armadilhas Extracelulares , Peroxidase , Serina Proteases , Entamoeba histolytica/enzimologia , Humanos , Animais , Serina Proteases/metabolismo , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Peroxidase/metabolismo , Células Hep G2 , Colo/parasitologia , Colo/patologia , Fígado/parasitologia , Fígado/patologia , Células HCT116 , Neutrófilos/imunologia , Entamebíase/parasitologia , Entamebíase/imunologia , Hepatócitos/parasitologia , Mesocricetus , Cricetinae
2.
Int J Biol Macromol ; 283(Pt 2): 137423, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-39537074

RESUMO

Human Respiratory syncytial virus (hRSV) mainly affects immunosuppressed patients requiring hospitalization. No specific treatment is financially accessible, and available vaccines do not cover all risk groups. During hRSV infection, there is a robust neutrophilic influx into the airways. hRSV-activated neutrophils release substantial neutrophil extracellular traps (NETs) in lung tissue, comprising DNA, histones, cytosolic, and granular proteins. NETs form mucus buildup in the lungs, compromising respiratory capacity and neutralizing viral particles. Understanding responsible NETs molecules requires improvement. We evaluated NETs interacting with hRSV particles and their contribution to anti-hRSV NET effects. Immunoblotting, immunoprecipitation, and peptide sequencing assays confirmed hRSV binding to a 50-75 kDa NET protein, Myeloperoxidase (MPO). MPO, a microbicide enzyme in NETs, interacts with hRSV, likely at F0 protein (site IV) on the viral surface. Additionally, MPO (32 µM) and NETs (0.4 µg/mL) reduced in vitro replication of clinical (hRSV A and B) and laboratory (Long) hRSV isolates by approximately 30 %, reversible by selective MPO inhibitor (PF-06281355; 48 µM). Thus, MPO contributes to virucidal NET effects on diverse hRSV strains, enhancing comprehension of NETs' role in infection and aiding treatment strategies for respiratory diseases.


Assuntos
Armadilhas Extracelulares , Peroxidase , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Humanos , Vírus Sincicial Respiratório Humano/efeitos dos fármacos , Armadilhas Extracelulares/metabolismo , Armadilhas Extracelulares/efeitos dos fármacos , Peroxidase/metabolismo , Infecções por Vírus Respiratório Sincicial/virologia , Vírion/metabolismo , Vírion/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , Neutrófilos/metabolismo , Neutrófilos/efeitos dos fármacos
3.
PLoS Pathog ; 20(10): e1012592, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39378227

RESUMO

Neutrophils rapidly infiltrate sites of infection and possess several microbicidal strategies, such as neutrophil extracellular traps release and phagocytosis. Enhanced neutrophil infiltration is associated with higher susceptibility to Leishmania infection, but neutrophil effector response contribution to this phenotype is uncertain. Here, we show that neutrophils from susceptible BALB/c mice (B/c) produce more NETs in response to Leishmania major than those from resistant C57BL/6 mice (B6), which are more phagocytic. The absence of neutrophil elastase contributes to phagocytosis regulation. Microarray analysis shows enrichment of genes involved in NET formation (mpo, pi3kcg, il1b) in B/c, while B6 shows upregulation of genes involved in phagocytosis and cell death (Arhgap12, casp9, mlkl, FasL). scRNA-seq in L. major-infected B6 showed heterogeneity in the pool of intralesional neutrophils, and we identified the N1 subset as the putative subpopulation involved with phagocytosis. In vivo, imaging validates NET formation in infected B/c ears where NETing neutrophils were mainly uninfected cells. NET digestion in vivo augmented parasite lymphatic drainage. Hence, a balance between NET formation and phagocytosis in neutrophils may contribute to the divergent phenotype observed in these mice.


Assuntos
Leishmania major , Leishmaniose Cutânea , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neutrófilos , Fagocitose , Animais , Leishmania major/imunologia , Neutrófilos/imunologia , Camundongos , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/parasitologia , Armadilhas Extracelulares/imunologia , Suscetibilidade a Doenças , Feminino
4.
J Leukoc Biol ; 117(1)2024 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-39241110

RESUMO

Neutrophils interact with Leishmania when the sandfly vector inoculates these parasites in the host with saliva and promastigotes-derived extracellular vesicles (EVs). It has been shown that this co-injection induces inflammation and exacerbates leishmaniasis lesions. EVs are a heterogeneous group of vesicles released by cells that play a crucial role in intercellular communication. Neutrophils are among the first cells to interact with the parasites and release neutrophil extracellular traps (NETs) that ensnare and kill the promastigotes. Here, we show that Leishmania amazonensis EVs induce NET formation and identify molecular mechanisms involved. We showed the requirement of neutrophils' toll-like receptors for EVs-induced NET. EVs carrying the virulence factors lipophosphoglycan and the zinc metalloproteases were endocytosed by some neutrophils and snared by NETs. EVs-induced NET formation required reactive oxygen species, myeloperoxidase, elastase, peptidyl arginine deiminase, and Ca++. The proteomic analysis of the EVs cargo revealed 1,189 proteins; the 100 most abundant identified comprised some known Leishmania virulent factors. Importantly, L. amazonensis EVs-induced NETs lead to the killing of promastigotes and could participate in the exacerbated inflammatory response induced by the EVs, which may play a role in the pathogenesis process.


Assuntos
Armadilhas Extracelulares , Vesículas Extracelulares , Neutrófilos , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Humanos , Animais , Leishmania/imunologia , Espécies Reativas de Oxigênio/metabolismo , Receptores Toll-Like/metabolismo , Leishmania mexicana/imunologia , Leishmaniose/imunologia , Leishmaniose/patologia
5.
Life Sci ; 352: 122895, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-38986896

RESUMO

AIMS: To investigate the SARS-CoV-2 Spike protein (Spk)-induced inflammatory response and its downmodulation by diminazene aceturate (DIZE). MATERIALS AND METHODS: Through inducing Spk inflammation in murine models, leukocyte migration to the peritoneum, levels of myeloperoxidase (MPO), malondialdehyde (MDA), rolling and adhesion of mesenteric leukocytes, and vascular permeability were investigated. Extracellular DNA traps (DETs) induced by Spk and the production of IL-6 and TNF-α were analyzed using human neutrophils, monocytes, and macrophages. In silico assays assessed the molecular interaction between DIZE and molecules related to leukocyte migration and DETs induction. KEY FINDINGS: Spk triggered acute inflammation, demonstrated by increasing leukocyte migration. Oxidative stress was evidenced by elevated levels of MPO and MDA in the peritoneal liquid. DIZE attenuated cell migration, rolling, and leukocyte adhesion, improved vascular barrier function, mitigated DETs, and reduced the production of Spk-induced pro-inflammatory cytokines. Computational studies supported our findings, showing the molecular interaction of DIZE with targets such as ß2 integrin, PI3K, and PAD2 due to its intermolecular coupling. SIGNIFICANCE: Our results outline a novel role of DIZE as a potential therapeutic agent for mitigating Spk-induced inflammation.


Assuntos
COVID-19 , Movimento Celular , Diminazena , Armadilhas Extracelulares , Inflamação , Leucócitos , SARS-CoV-2 , Diminazena/farmacologia , Diminazena/análogos & derivados , Animais , Camundongos , Humanos , Movimento Celular/efeitos dos fármacos , Armadilhas Extracelulares/metabolismo , Armadilhas Extracelulares/efeitos dos fármacos , Leucócitos/metabolismo , Leucócitos/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Inflamação/metabolismo , Inflamação/tratamento farmacológico , COVID-19/metabolismo , Masculino , Tratamento Farmacológico da COVID-19 , Adesão Celular/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus
6.
Vet Immunol Immunopathol ; 274: 110793, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38943998

RESUMO

Mastitis, an inflammation of the mammary gland affecting milk production and quality in dairy herds, is often associated with Staphylococcus spp. in goats. Neutrophils are crucial in combating infections by migrating into milk and deploying various defense strategies, including the release of neutrophil extracellular traps (NETs) composed of DNA, histones, and bactericidal proteins. This study investigated whether NETs are released by goat neutrophils stimulated in vitro by Staphylococcus aureus and Staphylococcus warneri, two common pathogens of goat mastitis. PMNs were isolated from blood from healthy adult goats. We evaluated goat NET formation by stimulating cells with: phorbol 12-myristate 13-acetate (PMA) as a positive control, cytochalasin for inhibition of actin polymerization, S. aureus, and S. warneri. NET formation was observed in response to chemical stimulation and bacterial presence, effectively trapping pathogens. Variations in NET formation between S. aureus and S. warneri suggest pathogen-specific responses. These findings suggest that the formation of NETs may be an important complementary mechanism in the defense against mastitis in goats. In conclusion, this study unveils a novel defense mechanism in goats, indicating the role of NETs against S. aureus and S. warneri in mastitis.


Assuntos
Armadilhas Extracelulares , Doenças das Cabras , Cabras , Mastite , Neutrófilos , Infecções Estafilocócicas , Staphylococcus aureus , Animais , Cabras/imunologia , Armadilhas Extracelulares/imunologia , Feminino , Mastite/veterinária , Mastite/imunologia , Mastite/microbiologia , Doenças das Cabras/imunologia , Doenças das Cabras/microbiologia , Neutrófilos/imunologia , Staphylococcus aureus/imunologia , Infecções Estafilocócicas/veterinária , Infecções Estafilocócicas/imunologia , Staphylococcus/imunologia
7.
Int J Mol Sci ; 25(11)2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38892404

RESUMO

Reproductive failure in dogs is often due to unknown causes, and correct diagnosis and treatment are not always achieved. This condition is associated with various congenital and acquired etiologies that develop inflammatory processes, causing an increase in the number of leukocytes within the female reproductive tract (FRT). An encounter between polymorphonuclear neutrophils (PMNs) and infectious agents or inflammation in the FRT could trigger neutrophil extracellular traps (NETs), which are associated with significantly decreased motility and damage to sperm functional parameters in other species, including humans. This study describes the interaction between canine PMNs and spermatozoa and characterizes the release of NETs, in addition to evaluating the consequences of these structures on canine sperm function. To identify and visualize NETs, May-Grünwald Giemsa staining and immunofluorescence for neutrophil elastase (NE) were performed on canine semen samples and sperm/PMN co-cultures. Sperm viability was assessed using SYBR/PI and acrosome integrity was assessed using PNA-FITC/PI by flow cytometry. The results demonstrate NETs release in native semen samples and PMN/sperm co-cultures. In addition, NETs negatively affect canine sperm function parameters. This is the first report on the ability of NETs to efficiently entrap canine spermatozoa, and to provide additional data on the adverse effects of NETs on male gametes. Therefore, NETs formation should be considered in future studies of canine reproductive failure, as these extracellular fibers and NET-derived pro-inflammatory capacities will impede proper oocyte fertilization and embryo implantation. These data will serve as a basis to explain certain reproductive failures of dogs and provide new information about triggers and molecules involved in adverse effects of NETosis for domestic pet animals.


Assuntos
Armadilhas Extracelulares , Neutrófilos , Espermatozoides , Animais , Cães , Armadilhas Extracelulares/metabolismo , Masculino , Espermatozoides/metabolismo , Neutrófilos/metabolismo , Motilidade dos Espermatozoides , Feminino , Elastase de Leucócito/metabolismo , Técnicas de Cocultura , Acrossomo/metabolismo
8.
Sci Rep ; 14(1): 9107, 2024 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-38643283

RESUMO

Neutrophil extracellular traps (NETs) are defense mechanisms that trap and kill microorganisms and degrade cytokines. However, excessive production, dysregulation of suppression mechanisms, or inefficient removal of NETs can contribute to increased inflammatory response and the development of pathological conditions. Therefore, research has focused on identifying drugs that inhibit or delay the NET release process. Since reactive oxygen species (ROS) play a significant role in NET release, we aimed to investigate whether resveratrol (RSV), with a wide range of biological and pharmacological properties, could modulate NET release in response to different stimuli. Thus, human neutrophils were pretreated with RSV and subsequently stimulated with PMA, LPS, IL-8, or Leishmania. Our findings revealed that RSV reduced the release of NETs in response to all tested stimuli. RSV decreased hydrogen peroxide levels in PMA- and LPS-stimulated neutrophils, inhibited myeloperoxidase activity, and altered the localization of neutrophil elastase. RSV inhibition of NET generation was not mediated through A2A or A2B adenosine receptors or PKA. Based on the observed effectiveness of RSV in inhibiting NET release, our study suggests that this flavonoid holds potential as a candidate for treating NETs involving pathologies.


Assuntos
Armadilhas Extracelulares , Humanos , Armadilhas Extracelulares/metabolismo , Resveratrol/farmacologia , Resveratrol/metabolismo , Peróxido de Hidrogênio/metabolismo , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo
9.
Immunobiology ; 229(3): 152803, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38640572

RESUMO

Neutrophil extracellular traps (NETs) are cell-extruded DNA strands coated with neutrophils' nuclear proteins and enzymes from cytotoxic granules, produced by NETosis, a cell death pathway. They perform an important defensive role in innate immunity, but their increased production and/or inefficient degradation expose new antigens, such as DNA or citrullinated histone peptides, triggering autoimmunity. This study aimed to access possible associations between serum NETs levels with epidemiological, clinical, and serological data from a well-characterized SLE Brazilian patients' cohort. NET levels were evaluated in one hundred seventy serum samples of patients with Systemic Lupus Erythematosus (SLE) using an Immunoassay. Univariate and multivariate binary logistic regression used clinical patients' data as independent variables. Parametric and non-parametric tests compared log10 base serum NET levels transformed between patients' groups. SLE patients were also dichotomized into "High serum NET levels" and "Low serum NET levels" groups. All analyses were performed in R language 4.1.2, and p < 0.05 were considered significant. Increased susceptibility for high serum NET levels was observed in SLE patients with Raynaud's phenomenon (OR = 2.30, 95 % CI = 1.06-5.21 and p = 0.039), independently of any other risk factor. Also, SLE patients with Raynaud's phenomenon presented higher mean NET serum levels (mean = -0.13 vs. -0.51, p = 0.01). In addition, higher mean NET serum levels were associated with glomerulonephritis (mean = -0.45 vs. -0.12, p = 0.03). Ultimately, the SLEDAI index scored higher in the high NETs serum levels group (median = 2.0 vs. 0.0, p = 6 × 10-3). The formation of NETs might be implicated in Raynaud's phenomenon, glomerulonephritis, and disease index score in SLE patients. Our results highlight the importance of serum NET levels as a possible therapeutical target to modulate the clinical course of SLE.


Assuntos
Armadilhas Extracelulares , Lúpus Eritematoso Sistêmico , Doença de Raynaud , Humanos , Armadilhas Extracelulares/metabolismo , Armadilhas Extracelulares/imunologia , Feminino , Masculino , Brasil/epidemiologia , Adulto , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/imunologia , Doença de Raynaud/etiologia , Doença de Raynaud/sangue , Doença de Raynaud/imunologia , Pessoa de Meia-Idade , Neutrófilos/imunologia , Índice de Gravidade de Doença , Glomerulonefrite/sangue , Glomerulonefrite/imunologia , Glomerulonefrite/diagnóstico , Adulto Jovem , Biomarcadores/sangue
10.
Andrology ; 12(8): 1799-1807, 2024 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-38469742

RESUMO

BACKGROUND: Severe acute syndrome coronavirus 2 can invade a variety of tissues, including the testis. Even though this virus is scarcely found in human semen polymerase chain reaction tests, autopsy studies confirm the viral presence in all testicular cell types, including spermatozoa and spermatids. OBJECTIVE: To investigate whether the severe acute syndrome coronavirus 2 is present inside the spermatozoa of negative polymerase chain reaction-infected men up to 3 months after hospital discharge. MATERIALS AND METHODS: This cross-sectional study included 13 confirmed moderate-to-severe COVID-19 patients enrolled 30-90 days after the diagnosis. Semen samples were obtained and examined with real-time polymerase chain reaction for RNA detection and by transmission electron microscopy. RESULTS: In moderate-to-severe clinical scenarios, we identified the severe acute syndrome coronavirus 2 inside spermatozoa in nine of 13 patients up to 90 days after discharge from the hospital. Moreover, some DNA-based extracellular traps were reported in all studied specimens. DISCUSSION AND CONCLUSION: Although severe acute syndrome coronavirus 2 was not present in the infected men's semen, it was intracellularly present in the spermatozoa till 3 months after hospital discharge. The Electron microscopy (EM) findings also suggest that spermatozoa produce nuclear DNA-based extracellular traps, probably in a cell-free DNA-dependent manner, similar to those previously described in the systemic inflammatory response to COVID-19. In moderate-to-severe cases, the blood-testes barrier grants little defence against different pathogenic viruses, including the severe acute syndrome coronavirus 2. The virus could also use the epididymis as a post-testicular route to bind and fuse to the mature spermatozoon and possibly accomplish the reverse transcription of the single-stranded viral RNA into proviral DNA. These mechanisms can elicit extracellular cell-free DNA formation. The potential implications of our findings for assisted conception must be addressed, and the evolutionary history of DNA-based extracellular traps as preserved ammunition in animals' innate defence might improve our understanding of the severe acute syndrome coronavirus 2 pathophysiology in the testis and spermatozoa.


Assuntos
COVID-19 , Microscopia Eletrônica de Transmissão , SARS-CoV-2 , Espermatozoides , Humanos , Masculino , COVID-19/virologia , COVID-19/patologia , Espermatozoides/virologia , Espermatozoides/ultraestrutura , Estudos Transversais , Adulto , Pessoa de Meia-Idade , Armadilhas Extracelulares/virologia
11.
Int J Mol Sci ; 25(4)2024 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-38396814

RESUMO

Methadone is an effective and long-lasting analgesic drug that is also used in medication-assisted treatment for people with opioid use disorders. Although there is evidence that methadone activates µ-opioid and Toll-like-4 receptors (TLR-4s), its effects on distinct immune cells, including mast cells (MCs), are not well characterized. MCs express µ-opioid and Toll-like receptors (TLRs) and constitute an important cell lineage involved in allergy and effective innate immunity responses. In the present study, murine bone-marrow-derived mast cells (BMMCs) were treated with methadone to evaluate cell viability by flow cytometry, cell morphology with immunofluorescence and scanning electron microscopy, reactive oxygen species (ROS) production, and intracellular calcium concentration ([Ca2+]i) increase. We found that exposure of BMMCs to 0.5 mM or 1 mM methadone rapidly induced cell death by forming extracellular DNA traps (ETosis). Methadone-induced cell death depended on ROS formation and [Ca2+]i. Using pharmacological approaches and TLR4-defective BMMC cultures, we found that µ-opioid receptors were necessary for both methadone-induced ROS production and intracellular calcium increase. Remarkably, TLR4 receptors were also involved in methadone-induced ROS production as it did not occur in BMMCs obtained from TLR4-deficient mice. Finally, confocal microscopy images showed a significant co-localization of µ-opioid and TLR4 receptors that increased after methadone treatment. Our results suggest that methadone produces MCETosis by a mechanism requiring a novel crosstalk pathway between µ-opioid and TLR4 receptors.


Assuntos
Analgésicos Opioides , Armadilhas Extracelulares , Humanos , Animais , Camundongos , Analgésicos Opioides/farmacologia , Receptor 4 Toll-Like/metabolismo , Metadona/farmacologia , Mastócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Medula Óssea/metabolismo , Cálcio/metabolismo , Armadilhas Extracelulares/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptores Toll-Like/metabolismo
12.
Dev Comp Immunol ; 155: 105151, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38423491

RESUMO

This study explores Neutrophil Extracellular Trap (NET) formation in equine neutrophils, which is crucial for eliminating infections and is implicated in various equine inflammatory diseases. We investigated the molecular pathways involved in NET release by equine neutrophils in response to stimuli. We use PMA, A23187, LPS, PAF, OZ, and cytokines, observing NET release in response to PMA, PAF, and A23187. In contrast, LPS, OZ, and the cytokines tested did not induce DNA release or did not consistently induce citrullination of histone 4. Peptidyl-arginine deiminase inhibition completely halted NET release, while NADPH oxidase and mitochondrial reactive oxygen species only played a role in PMA-induced NETs. Neutrophil elastase inhibition modestly affected PAF-induced NET liberation but not in PMA or A23187-induced NET, while myeloperoxidase did not contribute to NET release. We expect to provide a foundation for future investigations into the role of NETs in equine health and disease and the search for potential therapeutic targets.


Assuntos
Armadilhas Extracelulares , Neutrófilos , Animais , Cavalos , Neutrófilos/metabolismo , Armadilhas Extracelulares/metabolismo , Calcimicina/metabolismo , Lipopolissacarídeos/metabolismo , Citocinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo
13.
Oral Dis ; 30(7): 4751-4761, 2024 10.
Artigo em Inglês | MEDLINE | ID: mdl-38178616

RESUMO

OBJECTIVE: This study investigated the concentrations of neutrophil extracellular traps (NET) and salivary cytokines (IL-1ß, IL-6, IL-8/CXCL8, TNF, and TGF-ß1) in patients undergoing chemotherapy and their associations with oral mucositis (OM) and Candida infection. MATERIALS AND METHODS: This prospective longitudinal study performed at a Brazilian service included 60 adults diagnosed with hematolymphoid diseases. Saliva samples were collected on days D0, D3, D10, and D15. Cytokines were analyzed by ELISA and NET formation by identification of the myeloperoxidase-DNA complex. Oral Candida spp. was cultured. RESULTS: OM occurred in 43.3% of patients and oral candidiasis in 20%. However, 66% of individuals had positive cultures for C. albicans. Higher concentrations of IL-6, IL-8/CXCL8, and TNF and lower concentrations of TGF-ß1 were observed in patients with OM. C. albicans infection contributed to the increase in IL-8/CXCL8, TGF-ß1, and TNF. Individuals with OM or with oral candidiasis had significant reductions in NET formation. In contrast, individuals with C. albicans and with concomitant C. albicans and OM exhibited higher NET formation. CONCLUSION: The kinetics of cytokine levels and NET formation in chemotherapy-induced OM appears to be altered by Candida infection, even in the absence of clinical signs of oral candidiasis.


Assuntos
Candidíase Bucal , Citocinas , Armadilhas Extracelulares , Saliva , Estomatite , Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Citocinas/metabolismo , Candidíase Bucal/microbiologia , Estomatite/microbiologia , Estomatite/metabolismo , Estudos Prospectivos , Adulto , Armadilhas Extracelulares/metabolismo , Saliva/microbiologia , Saliva/metabolismo , Idoso , Estudos Longitudinais , Fator de Crescimento Transformador beta1/metabolismo , Interleucina-8/metabolismo , Interleucina-8/análise , Fator de Necrose Tumoral alfa/metabolismo , Candida albicans , Antineoplásicos , Interleucina-6/análise , Interleucina-6/metabolismo , Interleucina-1beta/metabolismo , Neoplasias Hematológicas/complicações
14.
J Clin Periodontol ; 51(4): 452-463, 2024 04.
Artigo em Inglês | MEDLINE | ID: mdl-38115803

RESUMO

AIM: We sought to investigate the release of neutrophil extracellular traps (NETs) in neutrophils from individuals with rheumatoid arthritis (RA) and controls and compare the presence of NETs in gingival tissues according to periodontal status. Also, the association between single nucleotide polymorphisms (SNPs) of the peptidyl arginine deaminase type 4 (PADI4) gene and the GTG haplotype with RA, periodontitis and NETs was evaluated in vitro. MATERIALS AND METHODS: Peripheral neutrophils were isolated by density gradient, and NET concentration was determined by the PicoGreen method. Immunofluorescence was studied to identify NETs by co-localization of myeloperoxidase (MPO)-citrullinated histone H3 (H3Cit). Genotyping for SNPs (PADI4_89; PADI4_90; PADI4_92; and PADI4_104) was performed in 87 individuals with RA and 111 controls. RESULTS: The release of NETs in vitro was significantly higher in individuals with RA and periodontitis and when stimulated with Porphyromonas gingivalis. Gingival tissues from subjects with RA and periodontitis revealed increased numbers of MPO-H3Cit-positive cells. Individuals with the GTG haplotype showed a higher release of NETs in vitro and worse periodontal parameters. CONCLUSIONS: The release of NETs by circulating neutrophils is associated with RA and periodontitis and is influenced by the presence of the GTG haplotype.


Assuntos
Artrite Reumatoide , Armadilhas Extracelulares , Periodontite , Humanos , Desiminases de Arginina em Proteínas/genética , Artrite Reumatoide/genética , Periodontite/genética , Neutrófilos , Polimorfismo de Nucleotídeo Único
15.
Clin Orthop Relat Res ; 482(4): 727-733, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-37882792

RESUMO

BACKGROUND: The diagnosis of periprosthetic joint infection (PJI) is a major challenge in clinical practice. The role of neutrophils in fighting infection has been increasingly understood, and one mechanism of action of these cells is neutrophil extracellular traps. However, little is known about this process in PJI. QUESTIONS/PURPOSES: (1) Are the biomarkers of neutrophil extracellular trap formation (citrullinated histone H3 [H3Cit], cell-free DNA [cf-DNA], and myeloperoxidase [MPO]) increased in the synovial fluid of patients with PJI? (2) What is the diagnostic accuracy of biomarkers of neutrophil extracellular trap formation for PJI? METHODS: Between May 2020 and March 2021, 43 patients who underwent revision THA or TKA were enrolled in this study. Eleven patients were excluded and 32 patients were categorized into the PJI group (n = 16) or non-PJI group (n = 16) according to the 2018 Second International Consensus Meeting on Musculoskeletal Infection criteria. There were 15 men and 17 women in this study, with a median (range) age of 70 years (60 to 80 years). Twenty-seven patients had TKA and five had THA. We measured cf-DNA, MPO, and H3Cit in synovial fluid. The sensitivity, specificity, and receiver operating characteristic curve were calculated for each biomarker using the Musculoskeletal Infection Society criteria as the gold standard for diagnosis and considering a clinical surveillance of 2 years for patients in the non-PJI group. RESULTS: Patients with PJI had higher levels of synovial fluid cf-DNA (median [range] 130 ng/µL [18 to 179] versus 2 ng/µL [0 to 6]; p < 0.001), MPO (1436 ng/µL [55 to 3996] versus 0 ng/µL [0 to 393]; p < 0.001), and H3Cit (2115 ng/µL [5 to 2885] versus 3 ng/µL [0 to 87]; p < 0.001) than those in the non-PJI group. In receiver operating characteristic curve analyses, we observed near-perfect performance for all biomarkers evaluated, with an area under the curve of 1 (95% CI 0.9 to 1), 0.98 (95% CI 0.9 to 1), and 0.94 (95% CI 0.8 to 0.99) for cf-DNA, MPO, and H3Cit, respectively. The sensitivity for detecting PJI using synovial fluid was 100% for cf-DNA, 94% for MPO, and 88% for H3Cit. The specificity was 100% for cf-DNA and MPO, and 88% for H3Cit. CONCLUSION: Our results show that neutrophils in the periprosthetic microenvironment release neutrophil extracellular traps as part of the bactericidal arsenal to fight infection. These results allow a better understanding of the cellular and molecular processes that occur in this microenvironment, enabling the design of more assertive strategies for identifying new biomarkers and improving the available ones. Novel studies are needed to define whether and how neutrophil extracellular trap-related biomarkers can be useful for diagnosing PJI. LEVEL OF EVIDENCE: Level II, diagnostic study.


Assuntos
Artrite Infecciosa , Artroplastia de Quadril , Artroplastia do Joelho , Armadilhas Extracelulares , Prótese do Joelho , Infecções Relacionadas à Prótese , Masculino , Humanos , Feminino , Idoso , Artroplastia do Joelho/efeitos adversos , Artroplastia do Joelho/métodos , Armadilhas Extracelulares/química , Sensibilidade e Especificidade , Artroplastia de Quadril/efeitos adversos , Artroplastia de Quadril/métodos , Prótese do Joelho/efeitos adversos , Líquido Sinovial/química , Biomarcadores/análise , Artrite Infecciosa/diagnóstico , DNA , Infecções Relacionadas à Prótese/diagnóstico , Infecções Relacionadas à Prótese/cirurgia
16.
AIDS Res Hum Retroviruses ; 40(5): 308-316, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-37772695

RESUMO

Human immunodeficiency virus (HIV) infection is still an important public health problem, which justifies the research of new therapies to combat it. Recent studies show that Extracellular Traps (ETs) are cellular mechanisms useful in the capture and destruction of some viruses, such as the HIV. Here, we show that neutrophils from peripheral blood, genital tissues, and placenta are activated when exposed to human immunodeficiency virus type 1 (HIV-1) and release Neutrophil Extracellular Traps (NETs). The NETs can capture, neutralize, and inactivate the virus and, also, protect other target cells from HIV infection, as long as the DNA and other constituents of the NETs remain intact. Further, the review indicates that the immunoprotective role of NETs in the context of HIV-1 infection is a promising finding for the development of new antiviral therapies. It is necessary, however, the development of studies that evaluate the tissue injury that NETs can cause and the biological relationships with other cells to improve them as therapeutic targets.


Assuntos
Armadilhas Extracelulares , Infecções por HIV , HIV-1 , Neutrófilos , Armadilhas Extracelulares/imunologia , Humanos , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/fisiologia , HIV-1/imunologia , Neutrófilos/imunologia , Feminino , Gravidez , Placenta/virologia , Placenta/imunologia
17.
Br J Pharmacol ; 181(3): 429-446, 2024 02.
Artigo em Inglês | MEDLINE | ID: mdl-37625900

RESUMO

BACKGROUND AND PURPOSE: Rheumatoid arthritis (RA) is a chronic autoimmune disease that can cause bone erosion due to increased osteoclastogenesis. Neutrophils involvement in osteoclastogenesis remains uncertain. Given that neutrophil extracellular traps (NETs) can act as inflammatory mediators in rheumatoid arthritis, we investigated the role of NETs in stimulating bone loss by potentiating osteoclastogenesis during arthritis. EXPERIMENTAL APPROACH: The level of NETs in synovial fluid from arthritis patients was assessed. Bone loss was evaluated by histology and micro-CT in antigen-induced arthritis (AIA)-induced WT mice treated with DNase or in Padi4-deficient mice (Padi4flox/flox LysMCRE ). The size and function of osteoclasts and the levels of RANKL and osteoprotegerin (OPG) released by osteoblasts that were incubated with NETs were measured. The expression of osteoclastogenic marker genes and protein levels were evaluated by qPCR and western blotting. To assess the participation of TLR4 and TLR9 in osteoclastogenesis, cells from Tlr4-/- and Tlr9-/- mice were cultured with NETs. KEY RESULTS: Rheumatoid arthritis patients had higher levels of NETs in synovial fluid than osteoarthritis patients, which correlated with increased levels of RANKL/OPG. Moreover, patients with bone erosion had higher levels of NETs. Inhibiting NETs with DNase or Padi4 deletion alleviated bone loss in arthritic mice. Consistently, NETs enhanced RANKL-induced osteoclastogenesis that was dependent on TLR4 and TLR9 and increased osteoclast resorptive functions in vitro. In addition, NETs stimulated the release of RANKL and inhibited osteoprotegerin in osteoblasts, favouring osteoclastogenesis. CONCLUSIONS AND IMPLICATIONS: Inhibiting NETs could be an alternative strategy to reduce bone erosion in arthritis patients.


Assuntos
Artrite Reumatoide , Armadilhas Extracelulares , Humanos , Animais , Camundongos , Osteoprotegerina/metabolismo , Osteoprotegerina/farmacologia , Osteogênese , Armadilhas Extracelulares/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Artrite Reumatoide/metabolismo , Osteoclastos/metabolismo , Desoxirribonucleases/metabolismo , Ligante RANK/metabolismo
18.
Braz. j. biol ; 84: e252936, 2024. graf, ilus
Artigo em Inglês | VETINDEX | ID: biblio-1374648

RESUMO

Neutrophil extracellular traps (NETs) were first reported as a microbicidal strategy for activated neutrophils. Through an immunologic response against several stimuli, neutrophils release their DNA together with proteins from granules, nucleus, and cytoplasm (e.g., elastase and myeloperoxidase). To date, NETs have been implicated in tissue damage during intense inflammatory processes, mainly when their release is dependent on oxygen radical generation. Flavonoids are antioxidant and anti-inflammatory agents; of these, quercetin is commonly found in our daily diet. Therefore, quercetin could exert some protective activity against tissue damage induced by NETs. In our in vitro assays, quercetin reduced NETs, myeloperoxidase (MPO), and elastase release from neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA). The activity of these enzymes also decreased in the presence of quercetin. Quercetin also reduced the cytotoxic effect of NETs on alveolar cells (A549 cell line). Further, in silico assays indicated favorable interactions between quercetin and NET proteins (MPO and elastase). Overall, our results demonstrate that quercetin decreases deleterious cellular effects of NETs by reducing their release from activated neutrophils, and diminishing the enzymatic activity of MPO and elastase, possibly through direct interaction.


As armadilhas extracelulares de neutrófilos (NETs) foram relatadas pela primeira vez como uma estratégia microbicida para neutrófilos ativados. Por meio de uma resposta imunológica contra vários estímulos, os neutrófilos liberam seu DNA ligado a proteínas de grânulos, núcleo e citoplasma (por exemplo, elastase e mieloperoxidase). Até o momento, os NETs têm sido implicadas em danos aos tecidos durante processos inflamatórios intensos, principalmente quando sua liberação depende da geração de radicais de oxigênio. Os flavonóides são agentes antioxidantes e anti-inflamatórios, e destes, a quercetina é comumente encontrada em nossa dieta diária. Portanto, a quercetina pode exercer alguma atividade protetora contra o dano tecidual induzido por NETs. Em nossos ensaios in vitro, a quercetina reduziu a liberação das NETs, mieloperoxidase (MPO) e elastase a partir de neutrófilos estimulados com forbol 12-miristato 13-acetato (PMA). A atividade dessas enzimas também foi diminuída na presença de quercetina. A quercetina também reduziu o efeito citotóxico dos NETs sobre células alveolares (linha celular A549). Além disso, os ensaios in silico indicaram interações favoráveis entre a quercetina e as proteínas da NET (MPO e elastase). No geral, nossos resultados demonstram que a quercetina diminui os efeitos deletérios das NETs, reduzindo sua liberação à partir de neutrófilos ativados e diminuindo a atividade enzimática de MPO e elastase, possivelmente por meio de interação direta.


Assuntos
Quercetina , Flavonoides , Armadilhas Extracelulares , Imunidade , Anti-Inflamatórios , Neutrófilos , Antioxidantes
19.
Front Immunol ; 14: 1282278, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38115994

RESUMO

Introduction: Toxoplasma gondii, responsible for causing toxoplasmosis, is a prevalent food and waterborne pathogen worldwide. It commonly infects warm-blooded animals and affects more than a third of the global human population. Once ingested, the parasite enters the host's small intestine and rapidly disseminates throughout the body via the bloodstream, infiltrating various tissues. Leukocyte-driven responses are vital against T. gondii, with neutrophils playing a dual role: swiftly recruited to infection sites, releasing inflammatory mediators, and serving as a replication hub and Trojan horses, aiding parasite spread. Neutrophils from various hosts release extracellular traps (NETs) against the protozoan. However, gaps persist regarding the mechanisms of NETs production to parasite and their significance in infection control. This study investigates the interplay between human neutrophils and T. gondii, exploring dynamics, key molecules, and signaling pathways involved in NETs production upon protozoan challenge. Methods and Results: Using confocal and electron microscopy, live cell imaging, pharmacological inhibitors, and DNA quantification assays, we find that human neutrophils promptly release both classical and rapid NETs upon pathogen stimulation. The NETs structure exhibits diverse phenotypes over time and is consistently associated with microorganisms. Mechanisms involve neutrophil elastase and peptidylarginine deiminase, along with intracellular calcium signaling and the PI3K pathway. Unexpectedly, human traps do not diminish viability or infectivity, but potentially aid in capturing parasites for subsequent neutrophil phagocytosis and elimination. Discussion: By revealing NETs formation mechanisms and their nuanced impact on T. gondii infection dynamics, our findings contribute to broader insights into host-pathogen relationships.


Assuntos
Armadilhas Extracelulares , Toxoplasma , Toxoplasmose , Animais , Humanos , Armadilhas Extracelulares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Toxoplasmose/metabolismo , Neutrófilos/metabolismo , Toxoplasma/fisiologia
20.
Vet Res ; 54(1): 105, 2023 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-37953317

RESUMO

Fasciola hepatica causes liver fluke disease, a worldwide neglected and re-emerging zoonotic disease, leading to hepatitis in humans and livestock. In the pathogenesis, flukes actively migrate through liver parenchyma provoking tissue damage. Here, parasites must confront leukocytes of the innate immune system in vivo. Polymorphonuclear neutrophils (PMN) are the most abundant granulocytes and first ones arriving at infection sites. PMN may display neutrophil extracellular traps (NETs), consisting of nuclear DNA, decorated with histones, enzymes, and antimicrobial peptides. We investigated for the first time whether F. hepatica soluble antigens (FhAg) can also trigger NETosis and innate immune reactions in exposed ovine PMN. Thus, isolated PMN were co-cultured with FhAg and NET formation was visualized by immunofluorescence and scanning electron microscopy analyses resulting in various phenotypes with spread NETs being the most detected in vitro. In line, NETs quantification via Picogreen®-fluorometric measurements revealed induction of anchored- and cell free NETs phenotypes. Live cell 3D-holotomographic microscopy revealed degranulation of stimulated PMN at 30 min exposure to FhAg. Functional PMN chemotaxis assays showed a significant increase of PMN migration (p = 0.010) and intracellular ROS production significantly increased throughout time (p = 0.028). Contrary, metabolic activities profiles of FhAg-exposed PMN did not significantly increase. Finally, in vivo histopathological analysis on F. hepatica-parasitized liver tissue sections of sheep showed multifocal infiltration of inflammatory cells within liver parenchyma, and further fluorescence microscopy analyses confirmed NETs formation in vivo. Overall, we hypothesized that NET-formation is a relevant host defence mechanism that might have a role in the pathogenesis of fasciolosis in vivo.


Assuntos
Armadilhas Extracelulares , Fasciola hepatica , Humanos , Animais , Ovinos , Neutrófilos , Imunidade Inata , Armadilhas Extracelulares/metabolismo , Técnicas de Cocultura/veterinária
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