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1.
Sci Adv ; 8(12): eabh4050, 2022 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-35319989

RESUMO

Radiotherapy is a mainstay cancer therapy whose antitumor effects partially depend on T cell responses. However, the role of Natural Killer (NK) cells in radiotherapy remains unclear. Here, using a reverse translational approach, we show a central role of NK cells in the radiation-induced immune response involving a CXCL8/IL-8-dependent mechanism. In a randomized controlled pancreatic cancer trial, CXCL8 increased under radiotherapy, and NK cell positively correlated with prolonged overall survival. Accordingly, NK cells preferentially infiltrated irradiated pancreatic tumors and exhibited CD56dim-like cytotoxic transcriptomic states. In experimental models, NF-κB and mTOR orchestrated radiation-induced CXCL8 secretion from tumor cells with senescence features causing directional migration of CD56dim NK cells, thus linking senescence-associated CXCL8 release to innate immune surveillance of human tumors. Moreover, combined high-dose radiotherapy and adoptive NK cell transfer improved tumor control over monotherapies in xenografted mice, suggesting NK cells combined with radiotherapy as a rational cancer treatment strategy.


Assuntos
Interleucina-8 , Células Matadoras Naturais , Neoplasias , Transferência Adotiva , Animais , Humanos , Imunidade , Interleucina-8/imunologia , Interleucina-8/metabolismo , Células Matadoras Naturais/imunologia , Camundongos , Neoplasias/imunologia , Neoplasias/radioterapia , Ensaios Antitumorais Modelo de Xenoenxerto
2.
Cytokine ; 151: 155804, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35063722

RESUMO

Coronavirus disease 2019 (COVID-19) is an infectious respiratory disorder caused by a new coronavirus called SARS-CoV-2. The pathophysiology of severe COVID-19 is associated with a "cytokine storm". IL-32 is a key modulator in the pathogenesis of various clinical conditions and is mostly induced by IL-8. IL-32 modulates important inflammatory pathways (including TNF-α, IL-6 and IL-1b), contributing to the pathogenesis of inflammatory diseases. Il-32 was never evaluated before in COVID-19 patients stratifying as mild-moderate and severe patients. A total of 64 COVID-19 patients, 27 healthy controls were consecutively enrolled in the study. Serum concentrations of biomarkers including IL-1ß, IL-10, IFN-γ, TNF-α and IL-6 were quantified by bead-based multiplex analysis and Serum concentration of IL-8 and IL-32 were determined by enzyme-linked immunosorbent assay (ELISA) kits. Interestingly, among the blood parameters, neutrophil and lymphocyte counts were significantly lower in severe COVID-19 patients than in the other, on the contrary, CRP was significantly higher in severe patients than in other groups. The cytokines that best distinguished controls from COVID-19 patients were IL-8 and IL-32, while IL-6 resulted the better variables for discriminate severe group. The best model performance for severe group was obtained by the combination of IL-32, IL-6, IFN-γ, and CRP serum concentration showing an AUC = 0.83. A cut off of 15 pg/ml of IL-6 greatly discriminate survivor from death patients. New insights related to the cytokine storm in COVID-19 patients, highlighting different severity of disease infection.


Assuntos
COVID-19/sangue , Citocinas/sangue , Interleucina-8/sangue , Interleucinas/sangue , Pulmão/imunologia , Idoso , Biomarcadores/sangue , COVID-19/imunologia , Síndrome da Liberação de Citocina/sangue , Síndrome da Liberação de Citocina/imunologia , Citocinas/imunologia , Feminino , Humanos , Interleucina-10/sangue , Interleucina-10/imunologia , Interleucina-8/imunologia , Interleucinas/imunologia , Contagem de Linfócitos/métodos , Linfócitos/imunologia , Masculino , Pessoa de Meia-Idade , Neutrófilos/imunologia , Estudos Prospectivos , SARS-CoV-2/imunologia
3.
Chembiochem ; 23(3): e202100552, 2022 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-34851004

RESUMO

Cytokines such as interleukin-8 activate the immune system during infection and interact with sulfated glycosaminoglycans with specific sulfation patterns. In some cases, these interactions are mediated by metal ion binding which can be used to tune surface-based glycan-protein interactions. We evaluated the effect of both hyaluronan sulfation degree and Fe3+ on interleukin-8 binding by electrochemical impedance spectroscopy and surface characterizations. Our results show that sulfation degree and metal ion interactions have a synergistic effect in tuning the electrochemical response of the glycated surfaces to the cytokine.


Assuntos
Compostos Férricos/química , Ácido Hialurônico/metabolismo , Interleucina-8/química , Polissacarídeos/química , Técnicas Eletroquímicas , Compostos Férricos/imunologia , Humanos , Ácido Hialurônico/química , Interleucina-8/imunologia , Modelos Moleculares , Estrutura Molecular , Polissacarídeos/imunologia
4.
mSphere ; 6(6): e0082021, 2021 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-34935443

RESUMO

The upper respiratory tract is the primary site of infection by porcine hemagglutinating encephalomyelitis virus (PHEV). In this study, primary porcine respiratory epithelial cells (PRECs) were cultured in an air-liquid interface (ALI) to differentiate into a pseudostratified columnar epithelium, proliferative basal cells, M cells, ciliated cells, and mucus-secreting goblet cells. ALI-PRECs recreates a cell culture environment morphologically and functionally more representative of the epithelial lining of the swine trachea than traditional culture systems. PHEV replicated actively in this environment, inducing cytopathic changes and progressive disruption of the mucociliary apparatus. The innate immunity against PHEV was comparatively evaluated in ALI-PREC cultures and tracheal tissue sections derived from the same cesarean-derived, colostrum-deprived (CDCD) neonatal donor pigs. Increased expression levels of TLR3 and/or TLR7, RIG1, and MyD88 genes were detected in response to infection, resulting in the transcriptional upregulation of IFN-λ1 in both ALI-PREC cultures and tracheal epithelia. IFN-λ1 triggered the upregulation of the transcription factor STAT1, which in turn induced the expression of the antiviral IFN-stimulated genes OAS1 and Mx1. No significant modulation of the major proinflammatory cytokines interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor alpha (TNF-α) was detected in response to PHEV infection. However, a significant upregulation of different chemokines was observed in ALI-PREC cultures (CCL2, CCL5, CXCL8, and CXCL10) and tracheal epithelium (CXCL8 and CXCL10). This study shed light on the molecular mechanisms driving the innate immune response to PHEV at the airway epithelium, underscoring the important role of respiratory epithelial cells in the maintenance of respiratory homeostasis and on the initiation, resolution, and outcome of the infectious process. IMPORTANCE The neurotropic betacoronavirus porcine hemagglutinating encephalomyelitis virus (PHEV) primarily infects and replicates in the swine upper respiratory tract, causing vomiting and wasting disease and/or encephalomyelitis in suckling pigs. This study investigated the modulation of key early innate immune genes at the respiratory epithelia in vivo, on tracheal tissue sections from experimentally infected pigs, and in vitro, on air-liquid interface porcine respiratory cell cultures. The results from the study underscore the important role of respiratory epithelial cells in maintaining respiratory homeostasis and on the initiation, resolution, and outcome of the PHEV infectious process.


Assuntos
Betacoronavirus 1/fisiologia , Interferons/genética , Interleucina-8/imunologia , Receptores de Reconhecimento de Padrão/genética , Mucosa Respiratória/imunologia , Mucosa Respiratória/virologia , Replicação Viral , Animais , Animais Recém-Nascidos , Betacoronavirus 1/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Imunidade Inata/genética , Imunidade Inata/imunologia , Interferons/imunologia , Interleucina-8/genética , Mucosa Respiratória/patologia , Suínos , Regulação para Cima , Replicação Viral/imunologia
5.
JCI Insight ; 6(24)2021 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-34784300

RESUMO

A substantial proportion of patients who have recovered from coronavirus disease-2019 (COVID-19) experience COVID-19-related symptoms even months after hospital discharge. We extensively immunologically characterized patients who recovered from COVID-19. In these patients, T cells were exhausted, with increased PD-1+ T cells, as compared with healthy controls. Plasma levels of IL-1ß, IL-1RA, and IL-8, among others, were also increased in patients who recovered from COVID-19. This altered immunophenotype was mirrored by a reduced ex vivo T cell response to both nonspecific and specific stimulation, revealing a dysfunctional status of T cells, including a poor response to SARS-CoV-2 antigens. Altered levels of plasma soluble PD-L1, as well as of PD1 promoter methylation and PD1-targeting miR-15-5p, in CD8+ T cells were also observed, suggesting abnormal function of the PD-1/PD-L1 immune checkpoint axis. Notably, ex vivo blockade of PD-1 nearly normalized the aforementioned immunophenotype and restored T cell function, reverting the observed post-COVID-19 immune abnormalities; indeed, we also noted an increased T cell-mediated response to SARS-CoV-2 peptides. Finally, in a neutralization assay, PD-1 blockade did not alter the ability of T cells to neutralize SARS-CoV-2 spike pseudotyped lentivirus infection. Immune checkpoint blockade ameliorates post-COVID-19 immune abnormalities and stimulates an anti-SARS-CoV-2 immune response.


Assuntos
COVID-19/complicações , Citocinas/imunologia , Inibidores de Checkpoint Imunológico/farmacologia , Receptor de Morte Celular Programada 1/imunologia , SARS-CoV-2/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Antígeno B7-H1/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Estudos de Casos e Controles , Citocinas/efeitos dos fármacos , Metilação de DNA , Feminino , Humanos , Imunofenotipagem , Técnicas In Vitro , Proteína Antagonista do Receptor de Interleucina 1/efeitos dos fármacos , Proteína Antagonista do Receptor de Interleucina 1/imunologia , Interleucina-1beta/efeitos dos fármacos , Interleucina-1beta/imunologia , Interleucina-8/efeitos dos fármacos , Interleucina-8/imunologia , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Regiões Promotoras Genéticas
6.
Gut Microbes ; 13(1): 1993583, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34747333

RESUMO

Gut microbiota have myriad roles in host physiology, development, and immunity. Though confined to the intestinal lumen by the epithelia, microbes influence distal systems via poorly characterized mechanisms. Recent work has considered the role of extracellular vesicles in interspecies communication, but whether they are involved in systemic microbe-host interaction is unclear. Here, we show that distinctive nanoparticles can be isolated from mouse blood within 2.5 h of consuming Lacticaseibacillus rhamnosus JB-1. In contrast to blood nanoparticles from saline-fed mice, they reproduced lipoteichoic acid-mediated immune functions of the original bacteria, including activation of TLR2 and increased IL-10 expression by dendritic cells. Like the fed bacteria, they also reduced IL-8 induced by TNF in an intestinal epithelial cell line. Though enriched for host neuronal proteins, these isolated nanoparticles also contained proteins and viral (phage) DNA of fed bacterial origin. Our data strongly suggest that oral consumption of live bacteria rapidly leads to circulation of their membrane vesicles and phages and demonstrate a nanoparticulate pathway whereby beneficial bacteria and probiotics may systemically affect their hosts.


Assuntos
Bacteriófagos/isolamento & purificação , Sangue/microbiologia , Sangue/virologia , Células Dendríticas/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Lactobacillus rhamnosus/metabolismo , Probióticos/farmacologia , Animais , Bacteriófagos/genética , Células Dendríticas/imunologia , Vesículas Extracelulares/química , Interleucina-8/genética , Interleucina-8/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Lactobacillus rhamnosus/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C/genética
7.
Int J Mol Sci ; 22(21)2021 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-34768826

RESUMO

Obstructive sleep apnea (OSA) is a disease with great cardiovascular risk. Interleukin-8 (IL-8), an important chemokine for monocyte chemotactic migration, was studied under intermittent hypoxia condition and in OSA patients. Monocytic THP-1 cells were used to investigate the effect of intermittent hypoxia on the regulation of IL-8 by an intermittent hypoxic culture system. The secreted protein and mRNA levels were studied by means of enzyme-linked immunosorbent assay and RT/real-time PCR. The chemotactic migration of monocytes toward a conditioned medium containing IL-8 was performed by means of the transwell filter migration assay. Peripheral venous blood was collected from 31 adult OSA patients and RNA was extracted from the monocytes for the analysis of IL-8 expression. The result revealed that intermittent hypoxia enhanced the monocytic THP-1 cells to actively express IL-8 at both the secreted protein and mRNA levels, which subsequently increased the migration ability of monocytes toward IL-8. The ERK, PI3K and PKC pathways were demonstrated to contribute to the activation of IL-8 expression by intermittent hypoxia. In addition, increased monocytic IL-8 expression was found in OSA patients, with disease severity dependence and diurnal changes. This study concluded the monocytic IL-8 gene expression can be activated by intermittent hypoxia and increased in OSA patients.


Assuntos
Hipóxia/metabolismo , Interleucina-8/biossíntese , Apneia Obstrutiva do Sono/metabolismo , Adulto , Feminino , Expressão Gênica , Humanos , Hipóxia/genética , Hipóxia/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Interleucina-8/metabolismo , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/metabolismo , RNA Mensageiro/genética , Apneia Obstrutiva do Sono/genética , Apneia Obstrutiva do Sono/imunologia , Células THP-1
8.
Front Immunol ; 12: 752612, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34616409

RESUMO

Background: Lymphopenia and the neutrophil/lymphocyte ratio may have prognostic value in COVID-19 severity. Objective: We investigated neutrophil subsets and functions in blood and bronchoalveolar lavage (BAL) of COVID-19 patients on the basis of patients' clinical characteristics. Methods: We used a multiparametric cytometry profiling based to mature and immature neutrophil markers in 146 critical or severe COVID-19 patients. Results: The Discovery study (38 patients, first pandemic wave) showed that 80% of Intensive Care Unit (ICU) patients develop strong myelemia with CD10-CD64+ immature neutrophils (ImNs). Cellular profiling revealed three distinct neutrophil subsets expressing either the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), the interleukin-3 receptor alpha (CD123), or programmed death-ligand 1 (PD-L1) overrepresented in ICU patients compared to non-ICU patients. The proportion of LOX-1- or CD123-expressing ImNs is positively correlated with clinical severity, cytokine storm (IL-1ß, IL-6, IL-8, TNFα), acute respiratory distress syndrome (ARDS), and thrombosis. BALs of patients with ARDS were highly enriched in LOX-1-expressing ImN subsets and in antimicrobial neutrophil factors. A validation study (118 patients, second pandemic wave) confirmed and strengthened the association of the proportion of ImN subsets with disease severity, invasive ventilation, and death. Only high proportions of LOX-1-expressing ImNs remained strongly associated with a high risk of severe thrombosis independently of the plasma antimicrobial neutrophil factors, suggesting an independent association of ImN markers with their functions. Conclusion: LOX-1-expressing ImNs may help identifying COVID-19 patients at high risk of severity and thrombosis complications.


Assuntos
COVID-19/complicações , Neutrófilos/imunologia , Receptores Depuradores Classe E/genética , Trombose/etiologia , Adulto , Idoso , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , COVID-19/genética , COVID-19/imunologia , COVID-19/virologia , Estado Terminal , Feminino , Humanos , Subunidade alfa de Receptor de Interleucina-3/genética , Subunidade alfa de Receptor de Interleucina-3/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Masculino , Pessoa de Meia-Idade , Síndrome do Desconforto Respiratório/etiologia , Síndrome do Desconforto Respiratório/genética , Síndrome do Desconforto Respiratório/imunologia , SARS-CoV-2/fisiologia , Receptores Depuradores Classe E/imunologia , Trombose/genética , Trombose/imunologia
9.
Int Immunopharmacol ; 101(Pt B): 108201, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34653729

RESUMO

One of the major clinical features of COVID-19 is a hyperinflammatory state, which is characterized by high expression of cytokines (such as IL-6 and TNF-α), chemokines (such as IL-8) and growth factors and is associated with severe forms of COVID-19. For this reason, the control of the "cytokine storm" represents a key issue in the management of COVID-19 patients. In this study we report evidence that the release of key proteins of the COVID-19 "cytokine storm" can be inhibited by mimicking the biological activity of microRNAs. The major focus of this report is on IL-8, whose expression can be modified by the employment of a molecule mimicking miR-93-5p, which is able to target the IL-8 RNA transcript and modulate its activity. The results obtained demonstrate that the production of IL-8 protein is enhanced in bronchial epithelial IB3-1 cells by treatment with the SARS-CoV-2 Spike protein and that IL-8 synthesis and extracellular release can be strongly reduced using an agomiR molecule mimicking miR-93-5p.


Assuntos
Células Epiteliais/imunologia , Interleucina-8/imunologia , MicroRNAs , Glicoproteína da Espícula de Coronavírus/imunologia , Brônquios/citologia , Linhagem Celular , Humanos , Interleucina-8/genética
10.
FASEB J ; 35(10): e21946, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34555226

RESUMO

Acute respiratory distress syndrome (ARDS) is a life-threatening illness characterized by decreased alveolar-capillary barrier function, pulmonary edema consisting of proteinaceous fluid, and inhibition of net alveolar fluid transport responsible for resolution of pulmonary edema. There is currently no pharmacotherapy that has proven useful to prevent or treat ARDS, and two trials using beta-agonist therapy to treat ARDS demonstrated no effect. Prior studies indicated that IL-8-induced heterologous desensitization of the beta2-adrenergic receptor (ß2 -AR) led to decreased beta-agonist-induced mobilization of cyclic adenosine monophosphate (cAMP). Interestingly, phosphodiesterase (PDE) 4 inhibitors have been used in human airway diseases characterized by low intracellular cAMP levels and increases in specific cAMP hydrolyzing activity. Therefore, we hypothesized that PDE4 would mediate IL-8-induced heterologous internalization of the ß2 -AR and that PDE4 inhibition would restore beta-agonist-induced functions. We determined that CINC-1 (a functional IL-8 analog in rats) induces internalization of ß2 -AR from the cell surface, and arrestin-2, PDE4, and ß2 -AR form a complex during this process. Furthermore, we determined that cAMP associated with the plasma membrane was adversely affected by ß2 -AR heterologous desensitization. Additionally, we determined that rolipram, a PDE4 inhibitor, reversed CINC-1-induced derangements of cAMP and also caused ß2 -AR to successfully recycle back to the cell surface. Finally, we demonstrated that rolipram could reverse CINC-1-mediated inhibition of beta-agonist-induced alveolar fluid clearance in a murine model of trauma-shock. These results indicate that PDE4 plays a role in CINC-1-induced heterologous internalization of the ß2 -AR; PDE4 inhibition reverses these effects and may be a useful adjunct in particular ARDS patients.


Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Interleucina-8/imunologia , Receptores Adrenérgicos beta 2/metabolismo , Animais , Líquido da Lavagem Broncoalveolar , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Quimiocina CXCL1/metabolismo , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/farmacologia , Regulação para Baixo/efeitos dos fármacos , Masculino , Camundongos , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores da Fosfodiesterase 4/farmacologia , beta-Arrestina 1/metabolismo
11.
Sci Rep ; 11(1): 18012, 2021 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-34504191

RESUMO

DNAM-1 is reportedly expressed on cytotoxic T and NK cells and, upon interaction with its ligands CD112 and CD155, plays an important role in tumor immunosurveillance. It has also been reported to be functionally expressed by myeloid cells, but expression and function on malignant cells of the myeloid lineage have not been studied so far. Here we analyzed expression of DNAM-1 in leukemic cells of acute myeloid leukemia (AML) patients. We found substantial levels of DNAM-1 to be expressed on leukemic blasts in 48 of 62 (> 75%) patients. Interaction of DNAM-1 with its ligands CD112 and CD155 induced release of the immunomodulatory cytokines IL-6, IL-8 IL-10 and TNF-α by AML cells and DNAM-1 expression correlated with a more differentiated phenotype. Multivariate analysis did not show any association of DNAM-1 positivity with established risk factors, but expression was significantly associated with clinical disease course: patients with high DNAM-1 surface levels had significantly longer progression-free and overall survival compared to DNAM-1low patients, independently whether patients had undergone allogenic stem cell transplantation or not. Together, our findings unravel a functional role of DNAM-1 in AML pathophysiology and identify DNAM-1 as a potential novel prognostic maker in AML.


Assuntos
Antígenos de Diferenciação de Linfócitos T/genética , Leucemia Mieloide Aguda/genética , Células Mieloides/metabolismo , Receptores de Superfície Celular/genética , Receptores Virais/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Diferenciação de Linfócitos T/imunologia , Feminino , Regulação Leucêmica da Expressão Gênica , Células HL-60 , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Células K562 , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/mortalidade , Leucemia Mieloide Aguda/patologia , Masculino , Pessoa de Meia-Idade , Células Mieloides/imunologia , Células Mieloides/patologia , Cultura Primária de Células , Prognóstico , Receptores de Superfície Celular/imunologia , Receptores Virais/imunologia , Transdução de Sinais , Análise de Sobrevida , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Células U937
12.
mSphere ; 6(4): e0059921, 2021 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-34346702

RESUMO

Vibrio parahaemolyticus is a marine Gram-negative bacterium that is a leading cause of seafood-borne gastroenteritis. Pandemic strains of V. parahaemolyticus rely on a specialized protein secretion machinery known as the type III secretion system 2 (T3SS2) to cause disease. The T3SS2 mediates the delivery of effector proteins into the cytosol of infected cells, where they subvert multiple cellular pathways. Here, we identify a new T3SS2 effector protein encoded by VPA1328 (VP_RS21530) in V. parahaemolyticus RIMD2210633. Bioinformatic analysis revealed that VPA1328 is part of a larger family of uncharacterized T3SS effector proteins with homology to the VopG effector protein in Vibrio cholerae AM-19226. These VopG-like proteins are found in many but not all T3SS2 gene clusters and are distributed among diverse Vibrio species, including V. parahaemolyticus, V. cholerae, V. mimicus, and V. diabolicus and also in Shewanella baltica. Structure-based prediction analyses uncovered the presence of a conserved C-terminal kinase domain in VopG orthologs, similar to the serine/threonine kinase domain found in the NleH family of T3SS effector proteins. However, in contrast to NleH effector proteins, in tissue culture-based infections, VopG did not impede host cell death or suppress interleukin 8 (IL-8) secretion, suggesting a yet undefined role for VopG during V. parahaemolyticus infection. Collectively, our work reveals that VopG effector proteins, a new family of likely serine/threonine kinases, is widely distributed in the T3SS2 effector armamentarium among marine bacteria. IMPORTANCE Vibrio parahaemolyticus is the leading bacterial cause of seafood-borne gastroenteritis worldwide. The pathogen relies on a type III secretion system to deliver a variety of effector proteins into the cytosol of infected cells to subvert cellular function. In this study, we identified a novel Vibrio parahaemolyticus effector protein that is similar to the VopG effector of Vibrio cholerae. VopG-like effectors were found in diverse Vibrio species and contain a conserved serine/threonine kinase domain that bears similarity to the kinase domain in the enterohemorrhagic Escherichia coli (EHEC) and Shigella NleH effectors that manipulate host cell survival pathways and host immune responses. Together our findings identify a new family of Vibrio effector proteins and highlight the role of horizontal gene transfer events among marine bacteria in shaping T3SS gene clusters.


Assuntos
Proteínas de Bactérias/genética , Sistemas de Secreção Tipo III/genética , Vibrio parahaemolyticus/enzimologia , Vibrio parahaemolyticus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Células CACO-2 , Biologia Computacional , Regulação Bacteriana da Expressão Gênica , Humanos , Interleucina-8/imunologia , Família Multigênica , Transporte Proteico , Serina/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Vibrio parahaemolyticus/metabolismo , Vibrio parahaemolyticus/patogenicidade
13.
J Infect Dis ; 224(4): 575-585, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34398243

RESUMO

Severe coronavirus disease 2019 (COVID-19) is associated with an overactive inflammatory response mediated by macrophages. Here, we analyzed the phenotype and function of neutrophils in patients with COVID-19. We found that neutrophils from patients with severe COVID-19 express high levels of CD11b and CD66b, spontaneously produce CXCL8 and CCL2, and show a strong association with platelets. Production of CXCL8 correlated with plasma concentrations of lactate dehydrogenase and D-dimer. Whole blood assays revealed that neutrophils from patients with severe COVID-19 show a clear association with immunoglobulin G (IgG) immune complexes. Moreover, we found that sera from patients with severe disease contain high levels of immune complexes and activate neutrophils through a mechanism partially dependent on FcγRII (CD32). Interestingly, when integrated in immune complexes, anti-severe acute respiratory syndrome coronavirus 2 IgG antibodies from patients with severe COVID-19 displayed a higher proinflammatory profile compared with antibodies from patients with mild disease. Our study suggests that IgG immune complexes might promote the acquisition of an inflammatory signature by neutrophils, worsening the course of COVID-19.


Assuntos
Anticorpos Antivirais/imunologia , Complexo Antígeno-Anticorpo/imunologia , COVID-19/imunologia , Imunoglobulina G/imunologia , Ativação de Neutrófilo/imunologia , Adulto , Idoso , Anticorpos Antivirais/sangue , Complexo Antígeno-Anticorpo/sangue , Antígenos CD/imunologia , Antígeno CD11b/imunologia , Moléculas de Adesão Celular/imunologia , Feminino , Proteínas Ligadas por GPI/imunologia , Humanos , Imunoglobulina G/sangue , Interleucina-8/imunologia , Masculino , Pessoa de Meia-Idade , Neutrófilos/imunologia , Receptores de IgG/imunologia , SARS-CoV-2/imunologia , Adulto Jovem
14.
Sci Rep ; 11(1): 17227, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34446770

RESUMO

Cattle vary in their susceptibility to infection and immunopathology, but our ability to measure and longitudinally profile immune response variation is limited by the lack of standardized immune phenotyping assays for high-throughput analysis. Here we report longitudinal innate immune response profiles in cattle using a low-blood volume, whole blood stimulation system-the ImmunoChek (IChek) assay. By minimizing cell manipulation, our standardized system minimizes the potential for artefactual results and enables repeatable temporal comparative analysis in cattle. IChek successfully captured biological variation in innate cytokine (IL-1ß and IL-6) and chemokine (IL-8) responses to 24-hr stimulation with either Gram-negative (LPS), Gram-positive (PamCSK4) bacterial or viral (R848) pathogen-associated molecular patterns (PAMPs) across a 4-month time window. Significant and repeatable patterns of inter-individual variation in cytokine and chemokine responses, as well as consistent high innate immune responder individuals were identified at both baseline and induced levels. Correlation coefficients between immune response read-outs (IL-1ß, IL-6 and IL-8) varied according to PAMP. Strong significant positive correlations were observed between circulating monocytes and IL-6 levels for null and induced responses (0.49-0.61) and between neutrophils and cytokine responses to R848 (0.38-0.47). The standardized assay facilitates high-throughput bovine innate immune response profiling to identify phenotypes associated with disease susceptibility and responses to vaccination.


Assuntos
Bovinos/imunologia , Imunidade Inata/imunologia , Testes Imunológicos/métodos , Padrões Moleculares Associados a Patógenos/imunologia , Imunidade Adaptativa/imunologia , Animais , Bovinos/sangue , Ensaio de Imunoadsorção Enzimática , Imidazóis/imunologia , Interleucina-1beta/sangue , Interleucina-1beta/imunologia , Interleucina-6/sangue , Interleucina-6/imunologia , Interleucina-8/sangue , Interleucina-8/imunologia , Lipopolissacarídeos/imunologia , Neutrófilos/imunologia , Padrões Moleculares Associados a Patógenos/sangue , Fatores de Tempo
15.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361797

RESUMO

Carpesium divaricatum Sieb. & Zucc., a traditional medicinal plant used as an inflammation-relieving remedy, is a rich source of terpenoids. At least 40 germacrane-type sesquiterpene lactones, representatives of four different structural groups, were isolated from the plant. Cytotoxicity against cancer cells in vitro is the most frequently described biological activity of the compounds. However, little is known about the selectivity of the cytotoxic effect. The anti-inflammatory activity of the germacranolides is also poorly documented. The objective of the present study was to assess the cytotoxic activity of selected C. divaricatum germacranolides-derivatives of 4,5,8,9-tetrahydroxy-3-oxo-germacran-6,12-olide towards cancer and normal cell lines (including cells of different p53 status). Moreover, to assess the anti-inflammatory effect of the compounds, the release of four proinflammatory cytokines/chemokines (IL-1ß, IL-8, TNF-α and CCL2) by lipopolysaccharide-stimulated human neutrophils was measured by ELISA. The investigated sesquiterpene lactones demonstrated nonselective activity towards prostate cancer (Du145 and PC3) and normal prostate epithelial cells (PNT2) as well as against melanoma cells (A375 and HTB140) and keratinocytes (HaCaT). Cytotoxic activity against osteosarcoma cells was independent of their p53 status. In sub-cytotoxic concentrations (0.5-2.5 µM) the studied compounds significantly decreased cytokine/chemokine release by lipopolysaccharide-stimulated human leukocytes.


Assuntos
Anti-Inflamatórios/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Asteraceae/química , Citotoxinas/farmacologia , Sesquiterpenos de Germacrano/farmacologia , Anti-Inflamatórios/química , Anti-Inflamatórios/classificação , Anti-Inflamatórios/isolamento & purificação , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/classificação , Antineoplásicos Fitogênicos/isolamento & purificação , Asteraceae/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Citotoxinas/química , Citotoxinas/classificação , Citotoxinas/isolamento & purificação , Doxorrubicina/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Concentração Inibidora 50 , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Componentes Aéreos da Planta/química , Componentes Aéreos da Planta/metabolismo , Extratos Vegetais/química , Plantas Medicinais , Polônia , Cultura Primária de Células , Sesquiterpenos de Germacrano/química , Sesquiterpenos de Germacrano/classificação , Sesquiterpenos de Germacrano/isolamento & purificação , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia
16.
Protein Expr Purif ; 187: 105950, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34363980

RESUMO

Interleukin-17A (IL-17A) produced by Th17 cells, contributes to the pathogenesis of various autoimmune diseases by stimulating the release of cytokines and chemokines and its regulation. Anti-IL-17A antibody which blocks the function of IL-17A has been proved to be an effective treatment of autoimmune disease. The aim of our study was to generate a potential humanized anti-IL-17A therapeutic monoclonal antibody (mAb) through a comprehensive panel of in vitro and in vivo biological activity studies, as well as physicochemical characterization. HZD37-5, a humanized monoclonal antibody specifically recognizing N78 loci of IL-17A, binds to human and rhesus monkeys, blocks IL-17 induced signal transduction and the release of IL-6, IL-8, CXCL-1 and G-GSF. In an in vivo efficacy mouse model, HZD37-5 significantly inhibited human IL-17A induced-keratinocyte chemoattractant (KC) secretion in a dose-dependent manner. The pharmacokinetics (PK) study result of HZD37-5 in rhesus monkeys indicated that HZD37-5 had favorable PK characteristics with limited distribution (78.0-78.8 ml/kg), slow elimination (5.00-6.45 ml/day/kg), long half-life (9.1-10.7 days) and high bioavailability (103%) following a single IV or SC dose at 1.5 mg/kg. These findings provided a comprehensive preclinical characterization of HZD37-5 and supported that it may be developed as a potential therapeutic for the treatment of autoimmune diseases, including psoriasis, psoriatic arthritis, axial spondyloarthritis, etc.


Assuntos
Anticorpos Monoclonais Humanizados/farmacocinética , Doenças Autoimunes/tratamento farmacológico , Interleucina-17/imunologia , Animais , Anticorpos Monoclonais Humanizados/imunologia , Quimiocina CXCL1/imunologia , Fatores Quimiotáticos/imunologia , Relação Dose-Resposta a Droga , Humanos , Interleucina-6/imunologia , Interleucina-8/imunologia , Queratinócitos/imunologia , Macaca mulatta , Camundongos , Coelhos , Transdução de Sinais
17.
Pharmacol Res Perspect ; 9(4): e00840, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34327862

RESUMO

Chronic obstructive pulmonary disease (COPD) often tends to respond poorly to glucocorticoid (GC) therapy. Reduced Histone deacetylase-2 (HDAC-2) activity is an important mechanism behind this GC insensitivity. In this study, we investigated the effects of three phosphodiesterase inhibitors (PDEIs), with an anti-inflammatory propensity, on cigarette smoke (CS)-induced pulmonary inflammation and HDAC-2 activity. Male C57BL/6 mice were exposed to cigarette smoke (CS) over the course of 30 weeks. Administration of the PDEIs commenced from the 29th week and followed a schedule of once daily treatments, 5 days a week, for 2 weeks. Roflumilast (ROF) was administered intragastrically (5 mg·kg-1 ), while pentoxifylline (PTX) (10 mg·kg-1 ) and theophylline (THEO) (10 mg·kg-1 ) were administered intraperitoneally, either alone or in combination with a GC (triamcinolone acetonide or TRI, 5 mg·kg-1 , i.m., single injection). Lung morphometry, as well as the activity of HDAC-2, pro-inflammatory cytokines and reactive oxygen species (ROS) were assessed at the end of the 30-week course. CS exposure was associated with a reduction in HDAC-2 activity and the up-regulation of ROS expression. PTX, ROF, and THEO administration led to the partial restoration of HDAC-2 activity, which was favorably associated with the reduction of ROS expression. However, combining TRI to any of these PDEIs did not synergistically augment HDAC-2 activity. Inactivation of HDAC-2 due to long-term CS exposure is closely related to exaggerated oxidative stress, and this reduced HDAC-2 activity could partially be restored through the use of PDEIs. This finding provides a potential novel approach for further clinical research.


Assuntos
Aminopiridinas/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Benzamidas/uso terapêutico , Pentoxifilina/uso terapêutico , Inibidores de Fosfodiesterase/uso terapêutico , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Teofilina/uso terapêutico , Aminopiridinas/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Benzamidas/farmacologia , Ciclopropanos/farmacologia , Ciclopropanos/uso terapêutico , Modelos Animais de Doenças , Histona Desacetilase 2/metabolismo , Interleucina-8/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos Endogâmicos C57BL , Pentoxifilina/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Espécies Reativas de Oxigênio/metabolismo , Fumaça/efeitos adversos , Fumar/efeitos adversos , Teofilina/farmacologia , Tabaco , Fator de Necrose Tumoral alfa/imunologia
18.
Rapid Commun Mass Spectrom ; 35(20): e9166, 2021 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-34270816

RESUMO

RATIONALE: Inflammation is a cascade of events mediated by a cytokine network triggering the cellular response. In order to monitor the modulation of the crucial inflammatory proteins, e.g., Tumour Necrosis Factor-α (TNF-α), Interferon-γ (INF-γ), Interleukin-8 (IL-8) and Interleukin-10 (IL-10), upon stimulation with endotoxins, differentiated and undifferentiated THP-1 cells were treated with lipopolysaccharides (LPSs) from E. coli, key cell wall components of Gram-negative bacteria. METHODS: The multiple reaction monitoring mass spectrometry (MRM-MS) method was optimized by using the standard proteins to be quantified, in order to construct external calibration curves and define the analytical parameters. The developed method was used to quantify the above-mentioned inflammatory proteins in THP-1 differentiated cells upon stimulation with LPSs with high accuracy, sensitivity, and robustness. RESULTS: The analysis of such proteins in MRM mode allowed the kinetics of stimulation along the time up to 24 h to be followed and the MS results were found to be comparable with those obtained by Western-blotting. A significant increase in TNF-α release triggered a cascade mechanism leading to the production of INF-γ and IL-8. IL-10, instead, was found to be constant throughout the process. CONCLUSIONS: The developed MRM-MS method allowed the quantification of TNF-α, INF-γ, IL-8 and IL-10 along a time-course from 2 to 24 h. Hence, a trace of the kinetics of the inflammatory response in THP-1 cells upon stimulation with E. coli LPSs was obtained. Finally, the extensibility of the developed MRM method to serum samples and other matrices demonstrated the versatility of the approach and the possibility to quantify multiple target proteins in different biological samples by using a few microliters in a single analysis.


Assuntos
Inflamação/imunologia , Lipopolissacarídeos/imunologia , Espectrometria de Massas/métodos , Monócitos/química , Monócitos/imunologia , Escherichia coli/imunologia , Escherichia coli/fisiologia , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Humanos , Inflamação/microbiologia , Interferon gama/química , Interferon gama/imunologia , Interleucina-10/química , Interleucina-10/imunologia , Interleucina-8/química , Interleucina-8/imunologia , Cinética , Lipopolissacarídeos/efeitos adversos , Células THP-1 , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/imunologia
19.
Invest Ophthalmol Vis Sci ; 62(7): 11, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34100890

RESUMO

Purpose: The insulin-like growth factor binding protein-3 (IGFBP-3) is a multifunctional secretory protein with well-known roles in cell growth and survival. Data in our laboratory suggest that IGFBP-3 may be functioning as a stress response protein in the corneal epithelium. The purpose of this study is to determine the role of IGFBP-3 in mediating the corneal epithelial cell stress response to hyperosmolarity, a well-known pathophysiological event in the development of dry eye disease. Methods: Telomerase-immortalized human corneal epithelial (hTCEpi) cells were used in this study. Cells were cultured in serum-free media with (growth) or without (basal) supplements. Hyperosmolarity was achieved by increasing salt concentrations to 450 and 500 mOsM. Metabolic and mitochondrial changes were assessed using Seahorse metabolic flux analysis and assays for mitochondrial calcium, polarization and mtDNA. Levels of IGFBP-3 and inflammatory mediators were quantified using ELISA. Cytotoxicity was evaluated using a lactate dehydrogenase assay. In select experiments, cells were cotreated with 500 ng/mL recombinant human (rh)IGFBP-3. Results: Hyperosmolar stress altered metabolic activity, shifting cells towards a respiratory phenotype. Hyperosmolar stress further altered mitochondrial calcium levels, depolarized mitochondria, decreased levels of ATP, mtDNA, and expression of IGFBP-3. In contrast, hyperosmolar stress increased production of the proinflammatory cytokines IL-6 and IL-8. Supplementation with rhIGFBP-3 abrogated metabolic and mitochondrial changes with only marginal effects on IL-8. Conclusions: These findings indicate that IGFBP-3 is a critical protein involved in hyperosmolar stress responses in the corneal epithelium. These data further support a new role for IGFBP-3 in the control of cellular metabolism.


Assuntos
Síndromes do Olho Seco , Epitélio Corneano/metabolismo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Pressão Osmótica/fisiologia , Estresse Fisiológico , Células Cultivadas , Síndromes do Olho Seco/imunologia , Síndromes do Olho Seco/metabolismo , Homeostase , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-6/imunologia , Interleucina-8/imunologia , Mitocôndrias/fisiologia , Concentração Osmolar , Estresse Fisiológico/imunologia , Estresse Fisiológico/fisiologia
20.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064458

RESUMO

Vitamin D and beta-glucans are both immunostimulants. Vitamin D exerts its beneficial effects on many components of the immune system. In macrophages, the hormone modulates both phagocytic activity and cytokine production; therefore, it plays an important role in mediating the innate immune response to infection. The immunomodulatory properties of beta-glucans are attributed to the ability of these fungal cell wall polysaccharides to bind to different receptors expressed on the cell surface of phagocytic and cytotoxic innate immune cells, including monocytes and macrophages. The intracellular signaling pathways activated by beta-glucans lead to enhanced phagocytosis and cytokine response. In this study we investigated the possible potentiation of immunomodulatory properties of the combined treatment with vitamin D and beta-glucans. The effects of 100 nM 1,25-dihydroxyvitamin D3 or 100 µg/mL beta-glucans were evaluated in human macrophages in terms of cytokine production, intracellular vesicle acidification and changes in energy metabolism, three hallmarks of macrophage antimicrobial activation. We found that all the analyzed parameters were enhanced by the co-treatment compared to the response to single molecules. The results of this study support the validity of a novel therapeutic approach that could boost the immune response, taking advantage of the synergy between two natural compounds.


Assuntos
Adjuvantes Imunológicos/farmacologia , Calcitriol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , beta-Glucanas/farmacologia , Diferenciação Celular , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Sinergismo Farmacológico , Regulação da Expressão Gênica/imunologia , Humanos , Interleucina-8/genética , Interleucina-8/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/imunologia , Transdução de Sinais , Células THP-1 , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/imunologia
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