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1.
Eur J Immunol ; : e2350716, 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38837757

RESUMO

Immune mediators affect multiple biological functions of intestinal epithelial cells (IECs) and, like Paneth and Paneth-like cells, play an important role in intestinal epithelial homeostasis. IFN-γ a prototypical proinflammatory cytokine disrupts intestinal epithelial homeostasis. However, the mechanism underlying the process remains unknown. In this study, using in vivo and in vitro models we demonstrate that IFN-γ is spontaneously secreted in the small intestine. Furthermore, we observed that this cytokine stimulates mitochondrial activity, ROS production, and Paneth and Paneth-like cell secretion. Paneth and Paneth-like secretion downstream of IFN-γ, as identified here, is mTORC1 and necroptosis-dependent. Thus, our findings revealed that the pleiotropic function of IFN-γ also includes the regulation of Paneth cell function in the homeostatic gut.

2.
J Leukoc Biol ; 115(3): 565-572, 2024 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-38128116

RESUMO

The chemokine Cxcl1 plays a crucial role in recruiting neutrophils in response to infection. The early events in chemokine-mediated neutrophil extravasation involve a sequence of highly orchestrated steps including rolling, adhesion, arrest, and diapedesis. Cxcl1 function is determined by its properties of reversible monomer-dimer equilibrium and binding to Cxcr2 and glycosaminoglycans. Here, we characterized how these properties orchestrate extravasation using intravital microscopy of the cremaster. Compared to WT Cxcl1, which exists as both a monomer and a dimer, the trapped dimer caused faster rolling, less adhesion, and less extravasation. Whole-mount immunofluorescence of the cremaster and arrest assays confirmed these data. Moreover, the Cxcl1 dimer showed impaired LFA-1-mediated neutrophil arrest that could be attributed to impaired Cxcr2-mediated ERK signaling. We conclude that Cxcl1 monomer-dimer equilibrium and potent Cxcr2 activity of the monomer together coordinate the early events in neutrophil recruitment.


Assuntos
Glicosaminoglicanos , Neutrófilos , Quimiocina CXCL1/metabolismo , Neutrófilos/metabolismo , Movimento Celular , Glicosaminoglicanos/metabolismo , Quimiocinas/metabolismo , Infiltração de Neutrófilos , Receptores de Interleucina-8B/metabolismo
3.
Life Sci Alliance ; 6(12)2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37813486

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis among all human cancers as it is highly resistant to chemotherapy. K-Ras mutations usually trigger the development and progression of PDAC. We hypothesized that compounds stabilizing the KRas4B/PDE6δ complex could serve as PDAC treatments. Using in silico approaches, we identified the small molecules C14 and P8 that reduced K-Ras activation in primary PDAC cells. Importantly, C14 and P8 significantly prevented tumor growth in patient-derived xenotransplants. Combined treatment with C14 and P8 strongly increased cytotoxicity in PDAC cell lines and primary cultures and showed strong synergistic antineoplastic effects in preclinical murine PDAC models that were superior to conventional therapeutics without causing side effects. Mechanistically, C14 and P8 reduced tumor growth by inhibiting AKT and ERK signaling downstream of K-RAS leading to apoptosis, specifically in PDAC cells. Thus, combined treatment with C14 and P8 may be a superior pharmaceutical strategy to improve the outcome of PDAC.


Assuntos
Antineoplásicos , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Camundongos , Animais , Linhagem Celular Tumoral , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Antineoplásicos/farmacologia , Neoplasias Pancreáticas
4.
J Leukoc Biol ; 114(6): 672-683, 2023 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-37820030

RESUMO

Neutrophils infiltrate several types of cancer; however, whether their presence is associated with disease progression remains controversial. Here, we show that colon tumors overexpress neutrophil chemoattractants compared to healthy tissues, leading to their recruitment to the invasive margin and the central part of colon tumors. Of note, tumor-associated neutrophils expressing tumor necrosis factor α, which usually represents an antitumoral phenotype, were predominantly located in the invasive margin. Tumor-associated neutrophils from the invasive margin displayed an antitumoral phenotype with higher ICAM-1 and CD95 expression than neutrophils from healthy adjacent tissues. A higher neutrophil/lymphocyte ratio was found at later stages compared to the early phases of colon cancer. A neutrophil/lymphocyte ratio ≤3.5 predicted tumor samples had significantly more neutrophils at the invasive margin and the central part. Moreover, tumor-associated neutrophils at the invasive margin of early-stage tumors showed higher ICAM-1 and CD95 expression. Coculture of colon cancer cell lines with primary neutrophils induced ICAM-1 and CD95 expression, confirming our in situ findings. Thus, our data demonstrate that tumor-associated neutrophils with an antitumoral phenotype characterized by high ICAM-1 and CD95 expression infiltrate the invasive margin of early-stage colon tumors, suggesting that these cells can combat the disease at its early courses. The presence of tumor-associated neutrophils with antitumoral phenotype could help predict outcomes of patients with colon cancer.


Assuntos
Neoplasias do Colo , Neutrófilos , Humanos , Neutrófilos/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Neoplasias do Colo/patologia , Fenótipo
5.
Pharmacol Ther ; 244: 108374, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36889441

RESUMO

Metallothionein-2 (MT-2) was originally discovered as a mediator of zinc homeostasis and cadmium detoxification. However, MT-2 has recently received increased attention because altered expression of MT-2 is closely related to various diseases such as asthma and cancers. Several pharmacological strategies have been developed to inhibit or modify MT-2, revealing its potential as drug target in diseases. Therefore, a better understanding of the mechanisms of MT-2 action is warranted to improve drug development for potential clinical applications. In this review, we highlight recent advances in determining the protein structure, regulation, binding partners, and new functions of MT-2 in inflammatory diseases and cancers.


Assuntos
Neoplasias , Zinco , Humanos , Zinco/metabolismo , Metalotioneína/metabolismo , Neoplasias/tratamento farmacológico , Cádmio/metabolismo
6.
J Leukoc Biol ; 113(3): 315-325, 2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36808495

RESUMO

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that is still fatal in many cases. T cell blasts are characterized by hyperactivation and strong proliferative and migratory capacities. The chemokine receptor CXCR4 is involved in mediating malignant T cell properties, and cortactin has been shown to control CXCR4 surface localization in T-ALL cells. We have previously shown that cortactin overexpression is correlated with organ infiltration and relapse in B-ALL. However, the role of cortactin in T cell biology and T-ALL remains elusive. Here, we analyzed the functional relevance of cortactin for T cell activation and migration and the implications for T-ALL development. We found that cortactin is upregulated in response to T cell receptor engagement and recruited to the immune synapse in normal T cells. Loss of cortactin caused reduced IL-2 production and proliferation. Cortactin-depleted T cells showed defects in immune synapse formation and migrated less due to impaired actin polymerization in response to T cell receptor and CXCR4 stimulation. Leukemic T cells expressed much higher levels of cortactin compared to normal T cells that correlated with greater migratory capacity. Xenotransplantation assays in NSG mice revealed that cortactin-depleted human leukemic T cells colonized the bone marrow significantly less and failed to infiltrate the central nervous system, suggesting that cortactin overexpression drives organ infiltration, which is a major complication of T-ALL relapse. Thus, cortactin could serve as a potential therapeutic target for T-ALL and other pathologies involving aberrant T cell responses.


Assuntos
Cortactina , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Humanos , Animais , Camundongos , Linfócitos T/metabolismo , Leucócitos , Recidiva , Movimento Celular/fisiologia
7.
J Leukoc Biol ; 113(3): 229-230, 2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36806852
8.
Nat Commun ; 13(1): 7029, 2022 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-36396641

RESUMO

Neutrophil diapedesis is an immediate step following infections and injury and is driven by complex interactions between leukocytes and various components of the blood vessel wall. Here, we show that perivascular mast cells (MC) are key regulators of neutrophil behaviour within the sub-endothelial space of inflamed venules. Using confocal intravital microscopy, we observe directed abluminal neutrophil motility along pericyte processes towards perivascular MCs, a response that created neutrophil extravasation hotspots. Conversely, MC-deficiency and pharmacological or genetic blockade of IL-17A leads to impaired neutrophil sub-endothelial migration and breaching of the pericyte layer. Mechanistically, identifying MCs as a significant cellular source of IL-17A, we establish that MC-derived IL-17A regulates the enrichment of key effector molecules ICAM-1 and CXCL1 in nearby pericytes. Collectively, we identify a novel MC-IL-17A-pericyte axis as modulator of the final steps of neutrophil diapedesis, with potential translational implications for inflammatory disorders driven by increased neutrophil diapedesis.


Assuntos
Neutrófilos , Migração Transendotelial e Transepitelial , Neutrófilos/fisiologia , Pericitos , Interleucina-17 , Mastócitos
9.
Immunol Lett ; 248: 99-108, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35841974

RESUMO

Inflammatory bowel diseases (IBD) including Crohn's disease (CD) and ulcerative colitis (UC) are complex inflammatory disorders of the digestive tract. Dysfunctional intestinal epithelial barrier, uncontrolled neutrophil recruitment into the colon, and oxidative stress are major features of IBD. IBD cannot be cured, but symptoms can be alleviated with anti-inflammatory drugs, which often show adverse effects. Thus, safer alternative treatment options are needed. Given the known anti-inflammatory properties of Annickia polycarpa extract (APE), we hypothesized that APE improves the outcome of the inflammatory response during colitis. We assessed APE effects on colon histology, epithelial barrier function and neutrophil recruitment during DSS-induced colitis in mice treated with APE. APE treatment significantly reduced the disease activity index and prevented DSS-induced colon damage as evidenced by reduced colon shortening, ulcerations, crypt dysplasia, edema formation, and leukocyte infiltration. Expression of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1ß were significantly diminished in APE-treated mice. Importantly, APE administration reduced neutrophil infiltration into the lamina propria leading to reduced oxidative stress, tight junction disruption and epithelial permeability in the colon. Thus, we propose APE as additional treatment strategy to attenuate colitis symptoms and enhance life quality of individuals with IBD.


Assuntos
Colite Ulcerativa , Colite , Doenças Inflamatórias Intestinais , Animais , Anti-Inflamatórios/efeitos adversos , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/patologia , Colite Ulcerativa/metabolismo , Colo/patologia , Sulfato de Dextrana/efeitos adversos , Sulfato de Dextrana/metabolismo , Modelos Animais de Doenças , Inflamação/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos
10.
Front Cell Infect Microbiol ; 12: 885191, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35706909

RESUMO

Typical enteroaggregative Escherichia coli (tEAEC) is a diarrheagenic E. coli pathotype associated with pediatric and traveler's diarrhea. Even without diarrhea, EAEC infections in children also lead to increased gut inflammation and growth shortfalls. EAEC strain's defining phenotype is the aggregative adherence pattern on epithelial cells attributable to the aggregative adherence fimbriae (AAF). EAEC only causes diarrhea in humans; therefore, not much is known of the exact intestinal region of infection and damage or its interactions with intestinal enterocytes in vivo and in situ. This study aimed to develop a new tEAEC mouse model of infection, characterize the microbiota of infected mice, and evaluate in situ the expression of host adherence and surface molecules triggering EAEC infection and the role of the EAEC AAF-II in adherence. Six-week-old C57BL/6 mice, without previous antibiotic treatment, were orally challenged with EAEC 042 strain or EAEC 042 AAF-II mutant (ΔAAF/II) strain, or DAEC-MXR strain (diffusely adherent E. coli clinical isolate), and with saline solution (control group). Paraffin sections of the colon and ileum were stained with H&E and periodic acid-Schiff. ZO-1, ß-catenin, MUC1, and bacteria were analyzed by immunofluorescence. EAEC-infected mice, in comparison with DAEC-MXR-infected and control mice, significantly lost weight during the first 3 days. After 7 days post-infection, mucus production was increased in the colon and ileum, ZO-1 localization remained unaltered, and morphological alterations were more pronounced in the ileum since increased expression and apical localization of ß-catenin in ileal enterocytes were observed. EAEC-infected mice developed dysbiosis 21 days post-infection. At 4 days post-infection, EAEC strain 042 formed a biofilm on ileal villi and increased the expression and apical localization of ß-catenin in ileal enterocytes; these effects were not seen in animals infected with the 042 ΔAAF/II strain. At 3 days post-infection, MUC1 expression on ileal enterocytes was mainly detectable among infected mice and colocalized with 042 strains on the enterocyte surface. We developed a novel mouse model of EAEC infection, which mimics human infection, not an illness, revealing that EAEC 042 exerts its pathogenic effects in the mouse ileum and causes dysbiosis. This model is a unique tool to unveil early molecular mechanisms of EAEC infection in vivo and in situ.


Assuntos
Infecções por Escherichia coli , Íleo , Microbiota , Mucina-1 , beta Catenina , Adesinas de Escherichia coli/genética , Animais , Aderência Bacteriana/genética , Diarreia/microbiologia , Modelos Animais de Doenças , Disbiose , Escherichia coli/genética , Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mucina-1/genética , Muco/metabolismo , Viagem , beta Catenina/genética
11.
Biomedicines ; 10(5)2022 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-35625756

RESUMO

Sepsis is a systemic infection that can lead to multi-organ failure. It is characterised by an uncontrolled immune response with massive neutrophil influx into peripheral organs. Neutrophil extravasation into tissues depends on actin remodeling and actin-binding proteins such as cortactin, which is expressed ubiquitously, except for neutrophils. Endothelial cortactin is necessary for proper regulation of neutrophil transendothelial migration and recruitment to sites of infection. We therefore hypothesised that cortactin plays a crucial role in sepsis development by regulating neutrophil trafficking. Using a murine model of sepsis induced by cecal ligation and puncture (CLP), we showed that cortactin-deficient (KO) mice survive better due to reduced lung injury. Histopathological analysis of lungs from septic KO mice revealed absence of oedema, reduced vascular congestion and mucus deposition, and better-preserved alveoli compared to septic wild-type (WT) mice. Additionally, sepsis-induced cytokine storm, excessive neutrophil infiltration into the lung and oxidative stress were significantly reduced in KO mice. Neutrophil depletion 12 h after sepsis improved survival in WT mice by averting lung injury, similar to both neutrophil-depleted and non-depleted KO mice. Our findings highlight a critical role of cortactin for lung neutrophil infiltration and sepsis severity.

12.
Eur J Cell Biol ; 101(2): 151214, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35286924

RESUMO

Sepsis remains an important health problem worldwide due to inefficient treatments often resulting in multi-organ failure. Neutrophil recruitment is critical during sepsis. While neutrophils are required to combat invading bacteria, excessive neutrophil recruitment contributes to tissue damage due to their arsenal of molecular weapons that do not distinguish between host and pathogen. Thus, neutrophil recruitment needs to be fine-tuned to ensure bacterial killing, while avoiding neutrophil-inflicted tissue damage. We recently showed that the actin-binding protein HS1 promotes neutrophil extravasation; and hypothesized that HS1 is also a critical regulator of sepsis progression. We evaluated the role of HS1 in a model of lethal sepsis induced by cecal-ligation and puncture. We found that septic HS1-deficient mice had a better survival rate compared to WT mice due to absence of lung damage. Lungs of septic HS1-deficient mice showed less inflammation, fibrosis, and vascular congestion. Importantly, systemic CLP-induced neutrophil recruitment was attenuated in the lungs, the peritoneum and the cremaster in the absence of HS1. Lungs of HS1-deficient mice produced significantly more interleukin-10. Compared to WT neutrophils, those HS1-deficient neutrophils that reached the lungs had increased surface levels of Gr-1, ICAM-1, and L-selectin. Interestingly, HS1-deficient neutrophils had similar F-actin content and phagocytic activity, but they failed to polymerize actin and deform in response to CXCL-1 likely explaining the reduced systemic neutrophil recruitment in HS1-deficient mice. Our data show that HS1 deficiency protects against sepsis by attenuating neutrophil recruitment to amounts sufficient to combat bacterial infection, but insufficient to induce tissue damage.


Assuntos
Neutrófilos , Sepse , Animais , Modelos Animais de Doenças , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos/fisiologia , Neutrófilos/metabolismo
13.
Trends Cell Biol ; 32(2): 94-97, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34625363

RESUMO

Membrane-cytoskeleton interactions (MCIs) are mediated by actin-binding proteins (ABPs). Ezrin is a crucial ABP that links membranes to actin filaments during lamellipodia formation, cell polarization, and migration. We discuss the concept of MCI and the potential of ezrin as a druggable target for treating inflammatory diseases and cancers.


Assuntos
Proteínas do Citoesqueleto , Citoesqueleto , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Humanos , Proteínas dos Microfilamentos/metabolismo
14.
J Leukoc Biol ; 111(6): 1147-1158, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-34826347

RESUMO

Severe coronavirus disease 2019 (COVID-19) is characterized by lung injury, cytokine storm, and increased neutrophil-to-lymphocyte ratio (NLR). Current therapies focus on reducing viral replication and inflammatory responses, but no specific treatment exists to prevent the development of severe COVID-19 in infected individuals. Angiotensin-converting enzyme-2 (ACE2) is the receptor for SARS-CoV-2, the virus causing COVID-19, but it is also critical for maintaining the correct functionality of lung epithelium and endothelium. Coronaviruses induce activation of a disintegrin and metalloprotease 17 (ADAM17) and shedding of ACE2 from the cell surface resulting in exacerbated inflammatory responses. Thus, we hypothesized that ADAM17 inhibition ameliorates COVID-19-related lung inflammation. We employed a preclinical mouse model using intratracheal instillation of a combination of polyinosinic:polycytidylic acid (poly(I:C)) and the receptor-binding domain of the SARS-CoV-2 spike protein (RBD-S) to mimic lung damage associated with COVID-19. Histologic analysis of inflamed mice confirmed the expected signs of lung injury including edema, fibrosis, vascular congestion, and leukocyte infiltration. Moreover, inflamed mice also showed an increased NLR as observed in critically ill COVID-19 patients. Administration of the ADAM17/MMP inhibitors apratastat and TMI-1 significantly improved lung histology and prevented leukocyte infiltration. Reduced leukocyte recruitment could be explained by reduced production of proinflammatory cytokines and lower levels of the endothelial adhesion molecules ICAM-1 and VCAM-1. Additionally, the NLR was significantly reduced by ADAM17/MMP inhibition. Thus, we propose inhibition of ADAM17/MMP as a novel promising treatment strategy in SARS-CoV-2-infected individuals to prevent the progression toward severe COVID-19.


Assuntos
Tratamento Farmacológico da COVID-19 , Lesão Pulmonar , Proteína ADAM17 , Enzima de Conversão de Angiotensina 2 , Animais , Modelos Animais de Doenças , Humanos , Lesão Pulmonar/etiologia , Lesão Pulmonar/prevenção & controle , Metaloproteinases da Matriz , Camundongos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus
16.
J Vis Exp ; (172)2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34180884

RESUMO

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier impairment has been associated with the development of various pathologies such as inflammatory bowel diseases (IBD). In the inflamed mucosa, superficial erosions or micro-erosions that corrupt epithelial monolayers correspond to sites of high permeability. Several mechanisms have been implicated in the formation of micro-erosions including cell shedding and apoptosis. These micro-erosions often represent microscopic epithelial gaps randomly distributed in the colon. Visualization and quantification of those epithelial gaps has emerged as an important tool to investigate intestinal epithelial barrier function. Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa. Additional use of cell death markers revealed that HPR encompass apoptotic foci where epithelial extrusion/shedding occurs. The protocol described here provides a simple but effective approach to visualize and quantify micro-erosions in the intestine, which is a very useful tool in disease models, in which the intestinal epithelial barrier is compromised.


Assuntos
Doenças Inflamatórias Intestinais , Mucosa Intestinal , Colo , Células Epiteliais , Imunofluorescência , Humanos , Permeabilidade , Coloração e Rotulagem
17.
Am J Pathol ; 191(9): 1537-1549, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34139193

RESUMO

Epithelial barrier impairment is a hallmark of several pathologic processes in the gut, including inflammatory bowel diseases. Several intracellular signals prevent apoptosis in intestinal epithelial cells. Herein, we show that in colonocytes, rictor/mammalian target of rapamycin complex 2 (mTORC2) signaling is a prosurvival stimulus. Mechanistically, mTORC2 activates Akt, which, in turn, inhibits apoptosis by phosphorylating B-cell lymphoma 2 (BCL2) associated agonist of cell death (Bad) and preventing caspase-3 activation. Nevertheless, during inflammation, rictor/mTORC2 signaling declines and Akt activity is reduced. Consequently, active caspase-3 increases in surface colonocytes undergoing apoptosis/anoikis and causes epithelial barrier breakdown. Likewise, Rictor ablation in intestinal epithelial cells interrupts mTORC2/Akt signaling and increases apoptosis/anoikis of surface colonocytes without affecting the crypt architecture. The increase in epithelial permeability induced by Rictor ablation produces a mild inflammatory response in the colonic mucosa, but minimally affects the development/establishment of colitis. The data identify a previously unknown mechanism by which rictor/mTORC2 signaling regulates apoptosis/anoikis in intestinal epithelial cells during colitis and clarify its role in the maintenance of the intestinal epithelial barrier.


Assuntos
Apoptose/fisiologia , Colite/patologia , Células Epiteliais/metabolismo , Mucosa Intestinal/patologia , Proteína Companheira de mTOR Insensível à Rapamicina/metabolismo , Animais , Colite/metabolismo , Células Epiteliais/patologia , Mucosa Intestinal/metabolismo , Camundongos , Transdução de Sinais/fisiologia
18.
Front Cell Dev Biol ; 9: 625719, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34012961

RESUMO

The intestinal epithelial barrier (IEB) depends on stable interepithelial protein complexes such as tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton. During inflammation, the IEB is compromised due to TJ protein internalization and actin remodeling. An important actin regulator is the actin-related protein 2/3 (Arp2/3) complex, which induces actin branching. Activation of Arp2/3 by nucleation-promoting factors is required for the formation of epithelial monolayers, but little is known about the relevance of Arp2/3 inhibition and endogenous Arp2/3 inhibitory proteins for IEB regulation. We found that the recently identified Arp2/3 inhibitory protein arpin was strongly expressed in intestinal epithelial cells. Arpin expression decreased in response to tumor necrosis factor (TNF)α and interferon (IFN)γ treatment, whereas the expression of gadkin and protein interacting with protein C-kinase α-subunit 1 (PICK1), other Arp2/3 inhibitors, remained unchanged. Of note, arpin coprecipitated with the TJ proteins occludin and claudin-1 and the AJ protein E-cadherin. Arpin depletion altered the architecture of both AJ and TJ, increased actin filament content and actomyosin contractility, and significantly increased epithelial permeability, demonstrating that arpin is indeed required for maintaining IEB integrity. During experimental colitis in mice, arpin expression was also decreased. Analyzing colon tissues from ulcerative colitis patients by Western blot, we found different arpin levels with overall no significant changes. However, in acutely inflamed areas, arpin was significantly reduced compared to non-inflamed areas. Importantly, patients receiving mesalazine had significantly higher arpin levels than untreated patients. As arpin depletion (theoretically meaning more active Arp2/3) increased permeability, we wanted to know whether Arp2/3 inhibition would show the opposite. Indeed, the specific Arp2/3 inhibitor CK666 ameliorated TNFα/IFNγ-induced permeability in established Caco-2 monolayers by preventing TJ disruption. CK666 treatment also attenuated colitis development, colon tissue damage, TJ disruption, and permeability in dextran sulphate sodium (DSS)-treated mice. Our results demonstrate that loss of arpin triggers IEB dysfunction during inflammation and that low arpin levels can be considered a novel hallmark of acute inflammation.

19.
Curr Biol ; 31(10): 2051-2064.e8, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-33711252

RESUMO

Hematopoietic-specific protein 1 (Hem1) is an essential subunit of the WAVE regulatory complex (WRC) in immune cells. WRC is crucial for Arp2/3 complex activation and the protrusion of branched actin filament networks. Moreover, Hem1 loss of function in immune cells causes autoimmune diseases in humans. Here, we show that genetic removal of Hem1 in macrophages diminishes frequency and efficacy of phagocytosis as well as phagocytic cup formation in addition to defects in lamellipodial protrusion and migration. Moreover, Hem1-null macrophages displayed strong defects in cell adhesion despite unaltered podosome formation and concomitant extracellular matrix degradation. Specifically, dynamics of both adhesion and de-adhesion as well as concomitant phosphorylation of paxillin and focal adhesion kinase (FAK) were significantly compromised. Accordingly, disruption of WRC function in non-hematopoietic cells coincided with both defects in adhesion turnover and altered FAK and paxillin phosphorylation. Consistently, platelets exhibited reduced adhesion and diminished integrin αIIbß3 activation upon WRC removal. Interestingly, adhesion phenotypes, but not lamellipodia formation, were partially rescued by small molecule activation of FAK. A full rescue of the phenotype, including lamellipodia formation, required not only the presence of WRCs but also their binding to and activation by Rac. Collectively, our results uncover that WRC impacts on integrin-dependent processes in a FAK-dependent manner, controlling formation and dismantling of adhesions, relevant for properly grabbing onto extracellular surfaces and particles during cell edge expansion, like in migration or phagocytosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Adesão Celular , Movimento Celular , Integrinas/metabolismo , Macrófagos/metabolismo , Fagocitose , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Quinase 1 de Adesão Focal/metabolismo , Masculino , Camundongos , Paxilina/metabolismo , Fosforilação , Pseudópodes
20.
Trends Immunol ; 42(3): 182-185, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33485795

RESUMO

A considerable portion of our knowledge on T and B cell biology is acquired from research using acute lymphoblastic leukemia (ALL) cell lines, which are invaluable tools used in many immunology and leukemia studies. Here, we discuss the advantages and limitations of ALL cell lines and provide guidelines on their proper usage.


Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Linfócitos B , Linhagem Celular , Humanos
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