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1.
Clin. transl. oncol. (Print) ; 24(10): 1881–1889, octubre 2022.
Artigo em Inglês | IBECS | ID: ibc-207944

RESUMO

All phases of carcinogenesis are affected by inflammation. Activation of the inflammasome is a crucial signaling mechanism that leads to acute and chronic inflammation. When specific nucleotide-binding domains, leucine-rich repeat-containing proteins (NLRs) are activated, inflammasomes are formed. The NLRP3 is one of the NLR family members with the most functional characterization. NLRP3 can modulate the immune systems, apoptosis, growth, and/or the gut microbiome to impact cancer development. Colorectal cancer (CRC) is one of the most common cancers, and it begins as a tissue overgrowth on the internal part of the rectum or colon. In vivo and in vitro studies showed that the NLRP3 inflammasome has a role in CRC development due to its broad activity in shaping immune responses. Here, onwards, we focus on the NLRP3 inflammasome role in CRC development, as well as the therapeutic prospective of modifying NLRP3 inflammasome in the context of anti-cancer therapy. (AU)


Assuntos
Humanos , Neoplasias Colorretais , Inflamassomos , Inflamação , Fagocitose
2.
Biol Pharm Bull ; 45(9): 1378-1384, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36047208

RESUMO

Pyridoxine (VB6) is a vitamin that is essential to maintain the homeostasis of the human body by contributing to various metabolic reactions. In the skin, although some studies have shown that VB6 is involved in regulating homeostasis through the attenuation of intracellular oxidative stress, there are few reports regarding the effects of VB6 on the prevention or improvement of skin aging. Thus, we conducted this study to determine the potential anti-skin pigmentation effect of VB6 focusing on the phagocytosis of melanosomes (MSs) by keratinocytes. The phagocytosis of MSs by keratinocytes is activated by oxidative stress and is an important factor of skin pigmentation and the eventual appearance of pigmented spots. First, we confirmed the antioxidant property of VB6 that enhanced the expression of several intracellular antioxidants via nuclear erythroid factor 2-related factor 2 (Nrf2). Although the incorporation of fluorescent beads (FBs), which are used as pseudo-MSs, into keratinocytes was increased under higher oxidation conditions caused by UVB and by the depletion of intracellular glutathione, treatment with VB6 suppressed the increased incorporation of FBs into those keratinocytes via Nrf2 activation. Furthermore, VB6 restored the decreased expression of differentiation marker proteins in keratinocytes caused by FB incorporation. Taken together, the results show that VB6 has the potential to prevent the appearance of pigmented spots by suppressing the activation of phagocytosis in keratinocytes caused by oxidative stress, and by restoring the differentiation of keratinocytes disrupted by FB incorporation.


Assuntos
Fator 2 Relacionado a NF-E2 , Piridoxina , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Humanos , Queratinócitos , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Fagocitose , Piridoxina/metabolismo , Piridoxina/farmacologia , Pigmentação da Pele , Raios Ultravioleta
3.
Front Immunol ; 13: 945485, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36105813

RESUMO

Microglia are phagocytosis-competent CNS cells comprising a spectrum of subtypes with beneficial and/or detrimental functions in acute and chronic neurodegenerative disorders. The heterogeneity of microglia suggests differences in phagocytic activity and phenotype plasticity between microglia subtypes. To study these issues, primary murine glial cultures were cultivated in the presence of serum, different growth factors and cytokines to obtain M0-like, M1-like, and M2-like microglia as confirmed by morphology, M1/M2 gene marker expression, and nitric oxide assay. Single-cell analysis after 3 hours of phagocytosis of E.coli particles or IgG-opsonized beads showed equal internalization by M0-like microglia, whereas M1-like microglia preferably internalized E.coli particles and M2-like microglia preferably internalized IgG beads, suggesting subtype-specific preferences for different phagocytosis substrates. Time-lapse live-cells imaging over 16 hours revealed further differences between microglia subtypes in phagocytosis preference and internalization dynamics. M0- and, more efficiently, M1-like microglia continuously internalized E.coli particles for 16 hours, whereas M2-like microglia discontinued internalization after approximately 8 hours. IgG beads were continuously internalized by M0- and M1-like microglia but strikingly less by M2-like microglia. M2-like microglia initially showed continuous internalization similar to M0-like microglia but again discontinuation of internalization after 8 hours suggesting that the time of substrate exposure differently affect microglia subtypes. After prolonged exposure to E.coli particles or IgG beads for 5 days all microglia subtypes showed increased internalization of E.coli particles compared to IgG beads, increased nitric oxide release and up-regulation of M1 gene markers, irrespectively of the phagocytosis substrate, suggesting phenotype plasticity. In summary, microglia subtypes show substrate- and time-dependent phagocytosis preferences and phenotype plasticity. The results suggest that prolonged phagocytosis substrate exposure enhances M1-like profiles and M2-M1 repolarization of microglia. Similar processes may also take place in conditions of acute and chronic brain insults when microglia encounter different types of phagocytic substrates.


Assuntos
Microglia , Óxido Nítrico , Animais , Imunoglobulina G/metabolismo , Camundongos , Microglia/metabolismo , Óxido Nítrico/metabolismo , Fagocitose , Fenótipo
4.
Methods Mol Biol ; 2543: 45-55, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36087258

RESUMO

Apoptotic cells are cleared from the body principally through recognition and engulfment by neighboring phagocytes, a process known as efferocytosis. During efferocytosis, phagocytes are recruited to the site/activated by "find me" signals released from apoptotic cells, precisely identify apoptotic cells by the recognition of "eat me" signals on the apoptotic cell surface, and engulf the apoptotic cells to prevent secondary necrosis and inflammation. Thus, efferocytosis is critical for tissue homeostasis in normal physiology. However, efferocytosis of apoptotic tumor cells-performed by tumor-associated macrophages-suppresses immunity within the tumor microenvironment and limits the antitumor response. This phenomenon is further exacerbated in tumor residual disease because of the high apoptotic cell burden generated by cytotoxic therapies. Blocking efferocytosis could be a powerful approach to boost tumor immunogenicity, particularly as a combination approach with cytotoxic therapies that produce many apoptotic cells, but little is currently known about the immune response to efferocytosis. Moreover, there is a dearth of in vivo models available to study the immunologic and therapeutic consequences of blocking efferocytosis in tumor residual disease.Here, we describe a model that enables in vivo studies of tumor immunology in the aftermath of cytotoxic therapy with an emphasis on the impact of efferocytosis. Orthotopic HER2+ mammary tumors are established in immune-competent mice, followed by a single administration of lapatinib, a receptor tyrosine kinase inhibitor of HER2, to the mice that induces widespread, transient apoptosis in the tumor microenvironment. In the days following lapatinib treatment, agents that block efferocytosis such as BMS-777607 are administered. Tissue is collected from cohorts of mice at day 2 (after lapatinib treatment only) to assess apoptosis, day 8 (after lapatinib treatment followed by blockade of efferocytosis) to assess the immune response to apoptosis and efferocytosis, and day 28 (after 4 consecutive weeks of treatment) to assess therapeutic efficacy. This model enables mechanistic studies of tumor immunology in residual disease as well as therapeutic efficacy studies of targeted agents that disrupt efferocytosis.


Assuntos
Macrófagos , Neoplasias , Animais , Apoptose/fisiologia , Lapatinib/farmacologia , Macrófagos/metabolismo , Camundongos , Necrose/patologia , Neoplasias/patologia , Fagocitose , Microambiente Tumoral
5.
Molecules ; 27(17)2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-36080360

RESUMO

Background: Targeting the CD47/SIRPα signaling pathway represents a novel approach to enhance anti-tumor immunity. However, the crystal structure of the CD47/SIRPα has not been fully studied. This study aims to analyze the structure interface of the complex of CD47 and IMM01, a novel recombinant SIRPα-Fc fusion protein. Methods: IMM01-Fab/CD47 complex was crystalized, and diffraction images were collected. The complex structure was determined by molecular replacement using the program PHASER with the CD47-SIRPαv2 structure (PDB code 2JJT) as a search model. The model was manually built using the COOT program and refined using TLS parameters in REFMAC from the CCP4 program suite. Results: Crystallization and structure determination analysis of the interface of IMM01/CD47 structure demonstrated CD47 surface buried by IMM01. Comparison with the literature structure (PDB ID 2JJT) showed that the interactions of IMM01/CD47 structure are the same. All the hydrogen bonds that appear in the literature structure are also present in the IMM01/CD47 structure. These common hydrogen bonds are stable under different crystal packing styles, suggesting that these hydrogen bonds are important for protein binding. In the structure of human CD47 in complex with human SIRPα, except SER66, the amino acids that form hydrogen bonds are all conserved. Furthermore, comparing with the structure of PDB ID 2JJT, the salt bridge interaction from IMM01/CD47 structure are very similar, except the salt bridge bond between LYS53 in IMM01 and GLU106 in CD47, which only occurs between the B and D chains. However, as the side chain conformation of LYS53 in chain A is slightly different, the salt bridge bond is absent between the A and C chains. At this site between chain A and chain C, there are a salt bridge bond between LYS53 (A) and GLU104 (C) and a salt bridge bond between HIS56 (A) and GLU106 (C) instead. According to the sequence alignment results of SIRPα, SIRPß and SIRPγ in the literature of PDB ID 2JJT, except ASP100, the amino acids that form common salt bridge bonds are all conserved. Conclusion: Our data demonstrated crystal structure of the IMM01/CD47 complex and provides a structural basis for the structural binding interface and future clinical applications.


Assuntos
Aminoácidos , Antígenos de Diferenciação , Antígeno CD47 , Receptores Imunológicos , Aminoácidos/química , Antígenos de Diferenciação/química , Antígeno CD47/química , Humanos , Fagocitose , Ligação Proteica , Receptores Imunológicos/química , Proteínas Recombinantes de Fusão/química
6.
Front Immunol ; 13: 757480, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36081498

RESUMO

CD47 is ubiquitously expressed on the surface of cells and plays a critical role in self-recognition. By interacting with SIRPα, TSP-1 and integrins, CD47 modulates cellular phagocytosis by macrophages, determines life span of individual erythrocytes, regulates activation of immune cells, and manipulates synaptic pruning during neuronal development. As such, CD47 has recently be regarded as one of novel innate checkpoint receptor targets for cancer immunotherapy. In this review, we will discuss increasing awareness about the diverse functions of CD47 and its role in immune system homeostasis. Then, we will discuss its potential therapeutic roles against cancer and outlines, the possible future research directions of CD47- based therapeutics against cancer.


Assuntos
Antígeno CD47 , Neoplasias , Humanos , Imunoterapia , Macrófagos , Fagocitose
7.
J Vis Exp ; (186)2022 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-36063018

RESUMO

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.


Assuntos
Células-Tronco Pluripotentes Induzidas , Microglia , Diferenciação Celular/fisiologia , Citofagocitose , Humanos , Fagocitose/fisiologia , Sinaptossomos
8.
J Neuroinflammation ; 19(1): 226, 2022 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-36104755

RESUMO

Evidence from experimental and clinical studies implicates immuno-inflammatory responses as playing an important role in epilepsy-induced brain injury. Captopril, an angiotensin-converting enzyme inhibitor (ACEi), has previously been shown to suppress immuno-inflammatory responses in a variety of neurological diseases. However, the therapeutic potential of captopril on epilepsy remains unclear. In the present study, Sprague Dawley (SD) rats were intraperitoneally subjected to kainic acid (KA) to establish a status epilepticus. Captopril (50 mg/kg, i.p.) was administered daily following the KA administration from day 3 to 49. We found that captopril efficiently suppressed the KA-induced epilepsy, as measured by electroencephalography. Moreover, captopril ameliorated the epilepsy-induced cognitive deficits, with improved performance in the Morris water maze, Y-maze and novel objective test. RNA sequencing (RNA-seq) analysis indicated that captopril reversed a wide range of epilepsy-related biological processes, particularly the glial activation, complement system-mediated phagocytosis and the production of inflammatory factors. Interestingly, captopril suppressed the epilepsy-induced activation and abnormal contact between astrocytes and microglia. Immunohistochemical experiments demonstrated that captopril attenuated microglia-dependent synaptic remodeling presumably through C3-C3ar-mediated phagocytosis in the hippocampus. Finally, the above effects of captopril were partially blocked by an intranasal application of recombinant C3a (1.3 µg/kg/day). Our findings demonstrated that captopril reduced the occurrence of epilepsy and cognitive impairment by attenuation of inflammation and C3-mediated synaptic phagocytosis. This approach can easily be adapted to long-term efficacy and safety in clinical practice.


Assuntos
Disfunção Cognitiva , Epilepsia , Animais , Captopril/farmacologia , Captopril/uso terapêutico , Disfunção Cognitiva/tratamento farmacológico , Disfunção Cognitiva/etiologia , Epilepsia/induzido quimicamente , Epilepsia/tratamento farmacológico , Inflamação/tratamento farmacológico , Ácido Caínico/toxicidade , Fagocitose , Ratos , Ratos Sprague-Dawley
9.
J Therm Biol ; 108: 103301, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36031222

RESUMO

Macrophages are considered to be key players in innate immunity and inflammatory responses. Domestic cattle with standard body size quickly reach their heat tolerance limit and are prone to heat stress. The combined effects of high temperature and endotoxemia on bovine monocyte-derived macrophages remain almost undisclosed. This study aims to unravel the molecular and functional responses of bovine monocyte-derived macrophages to thermal cum lipopolysaccharide induced stress challenge in vitro. The cells were incubated at 37 °C or 40 °C with lipopolysaccharide (1.0 µg/mL) for 24 h and 48 h. At the end of each treatment, cell viability, apoptotic rate, mitochondrial membrane potential, oxidative activity, phagocytosis, and autophagy functions were assessed and mRNA abundance of genes related to heat shock (HSP 70), inflammation (IL1ß, IL6, IL 12, TNF, INF γ), cell signalling (TLR4), cell viability (Bax, Bcl2), nitric oxide synthesis (NOS2) and natural resistant associated macrophage protein were quantified by quantitative polymerase chain reaction (qPCR). The results revealed the increased apoptosis, reduced mitochondrial membrane potential, and cell viability, decreased oxidative and phagocytosis ability in cells co-stimulated with LPS and thermal stimuli. Upregulation of HSP 70 gene and downregulation of natural resistant associated macrophage protein, cell signalling, and inflammation related genes mRNA expressions were also identified due to these stressors. In conclusion, the observed thermal cum LPS stress induced dysregulation in macrophage functionality may be one facet of the increased disease susceptibility in dairy cattle during thermal stress.


Assuntos
Lipopolissacarídeos , Macrófagos , Animais , Bovinos , Proteínas de Choque Térmico HSP70 , Inflamação , Fagocitose , RNA Mensageiro
10.
Front Immunol ; 13: 922377, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35967409

RESUMO

Neutrophils are the most abundant leukocytes in human peripheral blood. They form the first line of defense against invading foreign pathogens and might play a crucial role in malaria. According to World Health Organization (WHO), malaria is a globally significant disease caused by protozoan parasites from the Plasmodium genus, and it's responsible for 627,000 deaths in 2020. Neutrophils participate in the defense response against the malaria parasite via phagocytosis and reactive oxygen species (ROS) production. Neutrophils might also be involved in the pathogenesis of malaria by the release of toxic granules and the release of neutrophil extracellular traps (NETs). Intriguingly, malaria parasites inhibit the anti-microbial function of neutrophils, thus making malaria patients more susceptible to secondary opportunistic Salmonella infections. In this review, we will provide a summary of the role of neutrophils during malaria infection, some contradicting mouse model neutrophil data and neutrophil-related mechanisms involved in malaria patients' susceptibility to bacterial infection.


Assuntos
Armadilhas Extracelulares , Malária , Plasmodium , Animais , Humanos , Camundongos , Neutrófilos , Fagocitose
11.
Int Immunopharmacol ; 110: 109070, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35978514

RESUMO

Alzheimer's disease (AD) manifests as progressive deterioration in multiple cognitive and information processing domains, including memory and executive functions. Although AD's cause and cure remain elusive, increasing evidence supports a key role for microglial cells in AD pathogenesis via diverse mechanisms. ß-Amyloid (Aß) and tau triggered proteopathic and immunopathic processes are key contributors to AD pathology. These proteins aggregate into oligomers and fibrils, which eventually deposit in the central nervous system (CNS) as plaques and tangles. Aß and tau are directly synaptotoxic and neurotoxic, but also concomitantly induce neuroinflammation. As a central player in CNS immunity, microglia recognize different forms of misfolded proteins and initiate subsequent immune responses, mediating neuroinflammation and neuron-glia crosstalk. Microglia phagocytose debris and release cytokines to maintain brain homeostasis and synaptic integrity. However, microglia also exhibit harmful effects when subject to prolonged activation. This review describes the role of microglia in the proteopathic-immunopathic pathogeneses of AD. We summarize the microglial receptors involved in Aß recognition, and the role played by this interaction in explaining the interplay between Aß accumulation and AD progression through microglia-mediated neuroinflammation. Based on the dual proteopathic and immunopathic roles of microglia, we also review putative drug candidates targeting microglial receptors.


Assuntos
Doença de Alzheimer , Microglia , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Humanos , Microglia/metabolismo , Fagocitose
12.
Front Immunol ; 13: 933251, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35967335

RESUMO

Central line associated bloodstream infections (CLABSI) with Staphylococcus epidermidis are a major cause of morbidity in neonates, who have an increased risk of infection because of their immature immune system. As especially preterm neonates suffer from antibody deficiency, clinical studies into preventive therapies have thus far focused on antibody supplementation with pooled intravenous immunoglobulins from healthy donors (IVIG) but with little success. Here we study the potential of monoclonal antibodies (mAbs) against S. epidermidis to induce phagocytic killing by human neutrophils. Nine different mAbs recognizing Staphylococcal surface components were cloned and expressed as human IgG1s. In binding assays, clones rF1, CR5133 and CR6453 showed the strongest binding to S. epidermidis ATCC14990 and CR5133 and CR6453 bound the majority of clinical isolates from neonatal sepsis (19 out of 20). To study the immune-activating potential of rF1, CR5133 and CR6453, bacteria were opsonized with mAbs in the presence or absence of complement. We observed that activation of the complement system is essential to induce efficient phagocytosis of S. epidermidis. Complement activation and phagocytic killing could be enhanced by Fc-mutations that improve IgG1 hexamerization on cellular surfaces. Finally, we studied the ability of the mAbs to activate complement in r-Hirudin neonatal plasma conditions. We show that classical pathway complement activity in plasma isolated from neonatal cord blood is comparable to adult levels. Furthermore, mAbs could greatly enhance phagocytosis of S. epidermidis in neonatal plasma. Altogether, our findings provide insights that are crucial for optimizing anti-S. epidermidis mAbs as prophylactic agents for neonatal CLABSI.


Assuntos
Antineoplásicos Imunológicos , Staphylococcus epidermidis , Adulto , Anticorpos Monoclonais/farmacologia , Ativação do Complemento , Humanos , Imunoglobulinas Intravenosas , Recém-Nascido , Fagocitose
13.
ACS Synth Biol ; 11(8): 2820-2828, 2022 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-35930594

RESUMO

Histamine receptor 2 (HRH2) activation in the stomach results in gastric acid secretion, and HRH2 blockers are used for the treatment of peptidic ulcers and acid reflux. Over-the-counter HRH2 blockers carry a five-membered aromatic heterocycle, with two of them additionally carrying a tertiary amine that decomposes to N-nitrosodimethylamine, a human carcinogen. To discover a novel HRH2 blocker scaffold to serve in the development of next-generation HRH2 blockers, we developed an HRH2-based sensor in yeast by linking human HRH2 activation to cell luminescence. We used the HRH2-based sensor to screen a 403-member anti-infection chemical library and identified three HRH2 blockers, chlorquinaldol, chloroxine, and broxyquinoline, all sharing an 8-hydroxyquinoline scaffold, which is not found among known HRH2 antagonists. Critically, we validate their HRH2-blocking ability in mammalian cells. Molecular docking suggests that the HRH2 blockers bind the histamine binding pocket and structure-activity data point toward these blockers acting as competitive antagonists. Chloroxine and broxyquinoline are antimicrobials that can be found in the gastrointestinal tract at concentrations that would block HRH2, thus likely modulating gastric acid secretion. Taken together, this work demonstrates the utility of GPCR-based sensors for rapid drug discovery applications, identifies a novel HRH2 blocker scaffold, and provides further evidence that antimicrobials not only target the human microbiota but also the human host.


Assuntos
Fagocitose , Receptores Histamínicos , Animais , Humanos , Mamíferos , Simulação de Acoplamento Molecular , Oxiquinolina
14.
Front Immunol ; 13: 946857, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35911773

RESUMO

MicroRNA clusters are microRNAs (miRNAs) that are distributed in close proximity on chromosomes. In this study, we report a miRNA cluster identified from grass carp (Ctenopharyngodon idella), miR-462-731, which plays a positive role in host antibacterial immunity. The expression of miR-462-731 was disrupted after infection by Aeromonas hydrophila. Transcription factor ETS transcription factor ELK1 was identified to bind to the promoter of the miR-462-731 cluster and suppress its expression. In addition, miR-731 negatively regulates the expression of elk1, forms an elk1/miR-462-731 double negative feedback loop. In addition, we found that miR-731 directly targets ezrin a (ezra), participates in inducing PI3K/AKT signaling in macrophage, to induce macrophage polarization to the M1 phenotype with stronger phagocytosis. Our results demonstrate a novel elk1/miR-462-731 feedback loop. The data deepen our understanding of the relationship between macrophage polarization and phagocytosis in teleost fish.


Assuntos
Carpas , MicroRNAs , Animais , Carpas/genética , Carpas/metabolismo , Retroalimentação , Macrófagos , MicroRNAs/genética , MicroRNAs/metabolismo , Fagocitose , Fosfatidilinositol 3-Quinases
15.
Front Immunol ; 13: 952104, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36032131

RESUMO

Lymphatic filariasis (LF) is a mosquito-borne disease caused by filarial nematodes including Brugia malayi. Over 860 million people worldwide are infected or at risk of infection in 72 endemic countries. The absence of a protective vaccine means that current control strategies rely on mass drug administration programs that utilize inadequate drugs that cannot effectively kill adult parasites, thus established infections are incurable. Progress to address deficiencies in the approach to LF control is hindered by a poor mechanistic understanding of host-parasite interactions, including mechanisms of host immunomodulation by the parasite, a critical adaptation for establishing and maintaining infections. The canonical type 2 host response to helminth infection characterized by anti-inflammatory and regulatory immune phenotypes is modified by filarial nematodes during chronic LF. Current efforts at identifying parasite-derived factors driving this modification focus on parasite excretory-secretory products (ESP), including extracellular vesicles (EVs). We have previously profiled the cargo of B. malayi EVs and identified B. malayi galectin-1 and galectin-2 as among the most abundant EV proteins. In this study we further investigated the function of these proteins. Sequence analysis of the parasite galectins revealed highest homology to mammalian galectin-9 and functional characterization identified similar substrate affinities consistent with this designation. Immunological assays showed that Bma-LEC-2 is a bioactive protein that can polarize macrophages to an alternatively activated phenotype and selectively induce apoptosis in Th1 cells. Our data shows that an abundantly secreted parasite galectin is immunomodulatory and induces phenotypes consistent with the modified type 2 response characteristic of chronic LF infection.


Assuntos
Brugia Malayi , Filariose Linfática , Vesículas Extracelulares , Animais , Galectinas , Humanos , Mamíferos , Fagocitose
16.
Proc Natl Acad Sci U S A ; 119(32): e2122659119, 2022 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-35914149

RESUMO

Predatory protozoa play an essential role in shaping microbial populations. Among these protozoa, Acanthamoeba are ubiquitous in the soil and aqueous environments inhabited by Listeria monocytogenes. Observations of predator-prey interactions between these two microorganisms revealed a predation strategy in which Acanthamoeba castellanii assemble L. monocytogenes in aggregates, termed backpacks, on their posterior. The rapid formation and specific location of backpacks led to the assumption that A. castellanii may recruit L. monocytogenes by releasing an attractant. However, this hypothesis has not been validated, and the mechanisms driving this process remained unknown. Here, we combined video microscopy, microfluidics, single-cell image analyses, and theoretical modeling to characterize predator-prey interactions of A. castellanii and L. monocytogenes and determined whether bacterial chemotaxis contributes to the backpack formation. Our results indicate that L. monocytogenes captures are not driven by chemotaxis. Instead, random encounters of bacteria with amoebae initialize bacterial capture and aggregation. This is supported by the strong correlation between experimentally derived capture rates and theoretical encounter models at the single-cell level. Observations of the spatial rearrangement of L. monocytogenes trapped by A. castellanii revealed that bacterial aggregation into backpacks is mainly driven by amoeboid locomotion. Overall, we show that two nonspecific, independent mechanisms, namely random encounters enhanced by bacterial motility and predator surface-bound locomotion, drive backpack formation, resulting in a bacterial aggregate on the amoeba ready for phagocytosis. Due to the prevalence of these two processes in the environment, we expect this strategy to be widespread among amoebae, contributing to their effectiveness as predators.


Assuntos
Acanthamoeba castellanii , Listeria monocytogenes , Acanthamoeba castellanii/fisiologia , Quimiotaxia , Locomoção , Microfluídica , Microscopia de Vídeo , Fagocitose , Análise de Célula Única
17.
Int J Mol Sci ; 23(15)2022 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-35955687

RESUMO

Leishmanolysin, also known as major promastigote protease (PSP) or gp63, is the most abundant surface glycoprotein of Leishmania spp., and has been extensively studied and recognized as the main parasite virulence factor. Characterized as a metalloprotease, gp63 can be powerfully inactivated in the presence of a metal chelator. In this study, we first used the structural parameters of a 7-hydroxycoumarin derivative, L1 compound, to evaluate the theoretical-computational experiments against gp63, comparing it with an available metal chelator already described. The methodology followed was (i) analysis of the three-dimensional structure of gp63 as well as its active site, and searching the literature and molecular databases for possible inhibitors; (ii) molecular docking simulations and investigation of the interactions in the generated protein-ligand complexes; and (iii) the individual energy of the gp63 amino acids that interacted most with the ligands of interest was quantified by ab initio calculations using Molecular Fraction with Conjugated Caps (MFCC). MFCC still allowed the final quantum balance calculations of the protein interaction to be obtained with each inhibitor candidate binder. L1 obtained the best energy quantum balance result with -2 eV, followed by DETC (-1.4 eV), doxycycline (-1.3 eV), and 4-terpineol (-0.6 eV), and showed evidence of covalent binding in the enzyme active site. In vitro experiments confirmed L1 as highly effective against L. amazonensis parasites. The compound also exhibited a low cytotoxicity profile against mammalian RAW and 3T3 cells lines, presenting a selective index of 149.19 and 380.64 µM, respectively. L1 induced promastigote forms' death by necrosis and the ultrastructural analysis revealed disruption in membrane integrity. Furthermore, leakage of the contents and destruction of the parasite were confirmed by Spectroscopy Dispersion analysis. These results together suggested L1 has a potential effect against L. amazonensis, the etiologic agent of diffuse leishmaniasis, and the only one that currently does not have a satisfactory treatment.


Assuntos
Leishmania , Animais , Quelantes , Leishmania/metabolismo , Mamíferos/metabolismo , Metaloendopeptidases/metabolismo , Metaloproteases , Camundongos , Simulação de Acoplamento Molecular , Fagocitose
18.
Front Cell Infect Microbiol ; 12: 898796, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35909964

RESUMO

Calprotectin is a transition metal chelating protein of the innate immune response known to exert nutritional immunity upon microbial infection. It is abundantly released during inflammation and is therefore found at sites occupied by pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus. The metal limitation induced by this protein has previously been shown to mediate P. aeruginosa and S. aureus co-culture. In addition to the transition metal sequestration role of calprotectin, it has also been shown to have metal-independent antimicrobial activity via direct cell contact. Therefore, we sought to assess the impact of this protein on the biofilm architecture of P. aeruginosa and S. aureus in monomicrobial and polymicrobial culture. The experiments described in this report reveal novel aspects of calprotectin's interaction with biofilm communities of P. aeruginosa and S. aureus discovered using scanning electron microscopy and confocal laser scanning microscopy. Our results indicate that calprotectin can interact with microbial cells by stimulating encapsulation in mesh-like structures. This physical interaction leads to compositional changes in the biofilm extracellular polymeric substance (EPS) in both P. aeruginosa and S. aureus.


Assuntos
Biofilmes , Imunidade Inata , Complexo Antígeno L1 Leucocitário , Pseudomonas aeruginosa , Staphylococcus aureus , Antibacterianos/imunologia , Antibacterianos/farmacologia , Matriz Extracelular de Substâncias Poliméricas/genética , Matriz Extracelular de Substâncias Poliméricas/imunologia , Humanos , Imunidade Inata/genética , Imunidade Inata/imunologia , Complexo Antígeno L1 Leucocitário/genética , Complexo Antígeno L1 Leucocitário/imunologia , Fagocitose , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/imunologia , Staphylococcus aureus/genética , Staphylococcus aureus/imunologia
19.
Theranostics ; 12(12): 5488-5503, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35910792

RESUMO

Rationale: Cancer immunotherapy has demonstrated significant antitumor activity in a variety of tumors; however, extensive infiltration of immunosuppressive tumor-associated macrophages (TAMs) in the glioblastoma (GBM) tumor microenvironment (TME) and the existence of the blood-brain barrier (BBB) might lead to failure of the checkpoint blockade therapy. Methods: Herein, we have developed a smart "Trojan horse" BBB-permeable nanocapsule termed "NAcp@CD47" to deliver anti-CD47 antibodies and stimulator of interferon genes (STING) agonists into GBM tissues in a stealth-like manner to reshaped the immune microenvironment by switching the phenotype of microglia and macrophages. Results: Both in vitro and in vivo studies demonstrate that NAcp@CD47 could effectively penetrate the BBB, increase the polarization of M1-phenotype TAMs, help reduce tumor immunosuppression, and inhibit the orthotopic GBM growth by phagocytosis of macrophages and microglia. Conclusions: Our findings indicate that the well-designed NAcp@CD47 not only enhances the phagocytosis of cancer cells but also efficiently enhance antitumor immunogenicity and reverses immune suppression to convert uninflamed "cold" tumors into "hot" tumors.


Assuntos
Glioblastoma , Glioma , Nanocápsulas , Glioblastoma/tratamento farmacológico , Glioma/tratamento farmacológico , Humanos , Fatores Imunológicos , Imunoterapia , Fagocitose , Microambiente Tumoral
20.
PLoS Comput Biol ; 18(8): e1009937, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-36026476

RESUMO

The dynamic interplay between cell adhesion and protrusion is a critical determinant of many forms of cell motility. When modeling cell spreading on adhesive surfaces, traditional mathematical treatments often consider passive cell adhesion as the primary, if not exclusive, mechanistic driving force of this cellular motion. To better assess the contribution of active cytoskeletal protrusion to immune-cell spreading during phagocytosis, we here develop a computational framework that allows us to optionally investigate both purely adhesive spreading ("Brownian zipper hypothesis") as well as protrusion-dominated spreading ("protrusive zipper hypothesis"). We model the cell as an axisymmetric body of highly viscous fluid surrounded by a cortex with uniform surface tension and incorporate as potential driving forces of cell spreading an attractive stress due to receptor-ligand binding and an outward normal stress representing cytoskeletal protrusion, both acting on the cell boundary. We leverage various model predictions against the results of a directly related experimental companion study of human neutrophil phagocytic spreading on substrates coated with different densities of antibodies. We find that the concept of adhesion-driven spreading is incompatible with experimental results such as the independence of the cell-spreading speed on the density of immobilized antibodies. In contrast, the protrusive zipper model agrees well with experimental findings and, when adapted to simulate cell spreading on discrete adhesion sites, it also reproduces the observed positive correlation between antibody density and maximum cell-substrate contact area. Together, our integrative experimental/computational approach shows that phagocytic spreading is driven by cellular protrusion, and that the extent of spreading is limited by the density of adhesion sites.


Assuntos
Extensões da Superfície Celular , Citoesqueleto , Adesão Celular , Movimento Celular , Humanos , Fagocitose
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