RESUMO
The Aedes aegypti mosquito is a vector of many infectious agents, including flaviviruses such as Zika virus. Components of mosquito saliva have pleomorphic effects on the vertebrate host to enhance blood feeding, and these changes also create a favorable niche for pathogen replication and dissemination. Here, we demonstrate that human CD47, which is known to be involved in various immune processes, interacts with a 34-kilodalton mosquito salivary protein named Nest1. Nest1 is up-regulated in blood-fed female A. aegypti and facilitates Zika virus dissemination in human skin explants. Nest1 has a stronger affinity for CD47 than its natural ligand, signal regulatory protein α, competing for binding at the same interface. The interaction between Nest1 with CD47 suppresses phagocytosis by human macrophages and inhibits proinflammatory responses by white blood cells, thereby suppressing antiviral responses in the skin. This interaction elucidates how an arthropod protein alters the human response to promote arbovirus infectivity.
Assuntos
Aedes , Pele , Zika virus , Aedes/imunologia , Aedes/virologia , Animais , Humanos , Pele/imunologia , Pele/virologia , Zika virus/imunologia , Zika virus/fisiologia , Feminino , Proteínas de Insetos/imunologia , Infecção por Zika virus/imunologia , Proteínas e Peptídeos Salivares/imunologia , Mosquitos Vetores/imunologia , Mosquitos Vetores/virologia , Antígeno CD47RESUMO
BACKGROUND: Incidences of inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, are escalating worldwide and can be considered a global public health problem. Given that the gold standard approach to IBD therapeutics focuses on reducing the severity of symptoms, there is an urgent unmet need to develop alternative therapies that halt not only inflammatory processes but also promote mucosal repair. Previous studies have identified increased stem cell factor (SCF) expression in inflamed intestinal mucosal tissues. However, the role that SCF plays in mediating intestinal inflammation and repair has not been explored. METHODS: Changes in the expression of SCF were evaluated in the colonic tissue of healthy mice and during dextran sodium sulfate (DSS)-induced colitis. Furthermore, mucosal wound healing and colitis severity were analyzed in mice subjected to either mechanical biopsy or DSS treatment, respectively, following intestinal epithelial cell-specific deletion of SCF or anti-SCF antibody administration. RESULTS: We report robust expression of SCF by intestinal epithelial cells during intestinal homeostasis with a switch to immune cell-produced SCF during colitis. Data from mice with intestinal epithelial cell-specific deletion of SCF highlight the importance of immune cell-produced SCF in driving the pathogenesis of colitis. Importantly, antibody-mediated neutralization of total SCF or the specific SCF248 isoform decreased immune cell infiltration and enhanced mucosal wound repair following biopsy-induced colonic injury or DSS-induced colitis. CONCLUSIONS: These data demonstrate that SCF functions as a pro-inflammatory mediator in mucosal tissues and that specific neutralization of SCF248 could be a viable therapeutic option to reduce intestinal inflammation and promote mucosal wound repair in individuals with IBD.
Our investigation demonstrates that blocking cleavable SCF248 isoform by administration of specific stem cell factor antibodies enhances healing of the intestinal mucosa and restores critical barrier function, suggesting an alternative therapeutic option to treat individuals with active IBD.
Assuntos
Colite Ulcerativa , Colite , Doenças Inflamatórias Intestinais , Animais , Camundongos , Colite/tratamento farmacológico , Colite/patologia , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/patologia , Sulfato de Dextrana , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Inflamação/patologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Fator de Células-Tronco/antagonistas & inibidores , Fator de Células-Tronco/metabolismoRESUMO
Polymorphonuclear neutrophils (PMNs) play a critical role in clearing invading microbes and promoting tissue repair following infection/injury. However, dysregulated PMN trafficking and associated tissue damage is pathognomonic of numerous inflammatory mucosal diseases. The final step in PMN influx into mucosal lined organs (including the lungs, kidneys, skin, and gut) involves transepithelial migration (TEpM). The ß2-integrin CD11b/CD18 plays an important role in mediating PMN intestinal trafficking, with recent studies highlighting that terminal fucose and GlcNAc glycans on CD11b/CD18 can be targeted to reduce TEpM. However, the role of the most abundant terminal glycan, sialic acid (Sia), in regulating PMN epithelial influx and mucosal inflammatory function is not well understood. Here we demonstrate that inhibiting sialidase-mediated removal of α2-3-linked Sia from CD11b/CD18 inhibits PMN migration across intestinal epithelium in vitro and in vivo. Sialylation was also found to regulate critical PMN inflammatory effector functions, including degranulation and superoxide release. Finally, we demonstrate that sialidase inhibition reduces bacterial peptide-mediated CD11b/CD18 activation in PMN and blocks downstream intracellular signaling mediated by spleen tyrosine kinase (Syk) and p38 MAPK. These findings suggest that sialylated glycans on CD11b/CD18 represent potentially novel targets for ameliorating PMN-mediated tissue destruction in inflammatory mucosal diseases.
Assuntos
Neutrófilos , Migração Transendotelial e Transepitelial , Mucosa Intestinal , Neuraminidase , Neutrófilos/fisiologia , Polissacarídeos , Antígeno CD11b/imunologia , Antígenos CD18/imunologiaRESUMO
Clinical symptoms in many inflammatory diseases of the intestine are directly related to neutrophil (PMN) migration across colonic mucosa and into the intestinal lumen, yet in-vivo studies detailing this process are lacking. Using real-time intravital microscopy and a new distal colon loop model, we report distinct PMN migratory dynamics in response to several models of acute colonic injury. PMNs exhibited rapid swarming responses after mechanically induced intestinal wounds. Similar numbers of PMNs infiltrated colonic mucosa after wounding in germ-free mice, suggesting microbiota-independent mechanisms. By contrast, acute mucosal injury secondary to either a treatment of mice with dextran sodium sulfate or an IL-10 receptor blockade model of colitis resulted in lamina propria infiltration with PMNs that were largely immotile. Biopsy wounding of colonic mucosa in DSS-treated mice did not result in enhanced PMN swarming however, intraluminal application of the neutrophil chemoattractant LTB4 under such conditions resulted in enhanced transepithelial migration of PMNs. Analyses of PMNs that had migrated into the colonic lumen revealed that the majority of PMNs were directly recruited from the circulation and not from the immotile pool in the mucosa. Decreased PMN motility parallels upregulation of the receptor CXCR4 and apoptosis. Similarly, increased expression of CXCR4 on human PMNs was observed in colonic biopsies from people with active ulcerative colitis. This new approach adds an important tool to investigate mechanisms regulating PMN migration across mucosa within the distal intestine and will provide new insights for developing future anti-inflammatory and pro-repair therapies.
RESUMO
Dysregulated neutrophil (PMN) transmigration across epithelial surfaces (TEpM) significantly contributes to chronic inflammatory diseases, yet mechanisms defining this process remain poorly understood. In the intestine, uncontrolled PMN TEpM is a hallmark of disease flares in ulcerative colitis. Previous in vitro studies directed at identifying molecular determinants that mediate TEpM have shown that plasma membrane proteins including CD47 and CD11b/CD18 play key roles in regulating PMN TEpM across monolayers of intestinal epithelial cells. Here, we show that CD47 modulates PMN TEpM in vivo using an ileal loop assay. Importantly, using novel tissue-specific CD47 knockout mice and in vitro approaches, we report that PMN-expressed, but not epithelial-expressed CD47 plays a major role in regulating PMN TEpM. We show that CD47 associates with CD11b/CD18 in the plasma membrane of PMN, and that loss of CD47 results in impaired CD11b/CD18 activation. In addition, in vitro and in vivo studies using function blocking antibodies support a role of CD47 in regulating CD11b-dependent PMN TEpM and chemotaxis. Taken together, these findings provide new insights for developing approaches to target dysregulated PMN infiltration in the intestine. Moreover, tissue-specific CD47 knockout mice constitute an important new tool to study contributions of cells expressing CD47 to inflammation in vivo.
Assuntos
Antígeno CD47/metabolismo , Inflamação/imunologia , Intestinos/imunologia , Neutrófilos/imunologia , Animais , Antígeno CD11b/metabolismo , Antígenos CD18/metabolismo , Antígeno CD47/genética , Células Cultivadas , Quimiotaxia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ativação de Neutrófilo , Especificidade de Órgãos , Migração Transendotelial e TransepitelialRESUMO
Dysregulated healing of injured mucosa is a hallmark of many pathological conditions, including inflammatory bowel disease. Mucosal injury and chronic intestinal inflammation are also associated with alterations in epithelial glycosylation. Previous studies have revealed that inflammation-induced glycan sialyl Lewis A on epithelial CD44v6 acts as a ligand for transmigrating PMNs. Here we report that robust sialylated Lewis glycan expression was induced in colonic mucosa from individuals with ulcerative colitis and Crohn disease as well as in the colonic epithelium of mice with colitis induced by dextran sodium sulfate (DSS). Targeting of sialylated epithelial Lewis glycans with mAb GM35 reduced disease activity and improved mucosal integrity during DSS-induced colitis in mice. Wound healing studies revealed increased epithelial proliferation and migration responses as well as improved mucosal repair after ligation of epithelial sialyl Lewis glycans. Finally, we showed that GM35-mediated increases in epithelial proliferation and migration were mediated through activation of kinases that signal downstream of CD44v6 (Src, FAK, Akt). These findings suggest that sialylated Lewis glycans on CD44v6 represent epithelial targets for improved recovery of intestinal barrier function and restitution of mucosal homeostasis after inflammation or injury.
Assuntos
Colite/metabolismo , Colo/patologia , Células Epiteliais/metabolismo , Mucosa Intestinal/patologia , Cicatrização/fisiologia , Animais , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colo/metabolismo , Modelos Animais de Doenças , Células Epiteliais/patologia , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Polymorphonuclear neutrophils (PMNs) play a critical role in the innate immune response to invading pathogens. However, dysregulated mucosal trafficking of PMNs and associated epithelial tissue damage is a pathological hallmark of numerous inflammatory conditions including inflammatory bowel disease. The glycoprotein CD11b/CD18 plays a well-described role in regulating PMN transepithelial migration and PMN inflammatory functions. Previous studies have demonstrated that targeting of the N-linked glycan Lewis X on CD11b blocks PMN transepithelial migration (TEpM). Given evidence of glycosylation-dependent regulation of CD11b/CD18 function, we performed MALDI TOF Mass Spectrometry (MS) analyses on CD11b/CD18 purified from human PMNs. Unusual glycan epitopes identified on CD11b/CD18 included high Mannose oligosaccharides recognized by the Galanthus Nivalis lectin and biantennary galactosylated N-glycans recognized by the Phaseolus Vulgaris erythroagglutinin lectin. Importantly, we show that selective targeting of glycans on CD11b with such lectins results in altered intracellular signaling events that inhibit TEpM and differentially affect key PMN inflammatory functions including phagocytosis, superoxide release and apoptosis. Taken together, these data demonstrate that discrete glycan motifs expressed on CD11b/CD18 such as biantennary galactose could represent novel targets for selective manipulation of CD11b function and reduction of PMN-associated tissue damage in chronic inflammatory diseases.
Assuntos
Antígeno CD11b/imunologia , Antígenos CD18/imunologia , Epitopos/imunologia , Neutrófilos/imunologia , Antígeno CD11b/química , Antígenos CD18/química , Epitopos/química , Humanos , Lectinas de Ligação a Manose/química , Lectinas de Ligação a Manose/farmacologia , Neutrófilos/química , Fagocitose , Fito-Hemaglutininas/química , Fito-Hemaglutininas/farmacologia , Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Superóxidos/química , Superóxidos/imunologia , Migração Transendotelial e TransepitelialRESUMO
CD47 is a ubiquitously expressed transmembrane glycoprotein that regulates inflammatory responses and tissue repair. Here, we show that normal mice treated with anti-CD47 antibodies, and Cd47-null mice have impaired intestinal mucosal wound healing. Furthermore, intestinal epithelial cell (IEC)-specific loss of CD47 does not induce spontaneous immune-mediated intestinal barrier disruption but results in defective mucosal repair after biopsy-induced colonic wounding or Dextran Sulfate Sodium (DSS)-induced mucosal damage. In vitro analyses using primary cultures of CD47-deficient murine colonic IEC or human colonoid-derived IEC treated with CD47-blocking antibodies demonstrate impaired epithelial cell migration in wound healing assays. Defective wound repair after CD47 loss is linked to decreased epithelial ß1 integrin and focal adhesion signaling, as well as reduced thrombospondin-1 and TGF-ß1. These results demonstrate a critical role for IEC-expressed CD47 in regulating mucosal repair and raise important considerations for possible alterations in wound healing secondary to therapeutic targeting of CD47.
Assuntos
Antígeno CD47/metabolismo , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/fisiopatologia , Cicatrização/fisiologia , Animais , Antígeno CD47/genética , Colite/induzido quimicamente , Colite/genética , Colite/metabolismo , Sulfato de Dextrana , Modelos Animais de Doenças , Humanos , Mucosa Intestinal/patologia , Intestinos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/genética , Trombospondina 1/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Cicatrização/genéticaRESUMO
Mucosal wound repair is coordinated by dynamic crosstalk between endogenous and exogenous mediators and specific receptors on epithelial cells and infiltrating immune cells. One class of such receptor-ligand pairs involves formyl peptide receptors (FPRs) that have been shown to influence inflammatory response and repair. Here we explored the role of murine Fpr2/3, an ortholog of human FPR2/receptor for lipoxin A4 (ALX), in orchestrating intestinal mucosal repair. Compared with wild-type (WT) mice, Fpr2/3-/- mice exhibited delayed recovery from acute experimental colitis and perturbed repair after biopsy-induced colonic mucosal injury. Decreased numbers of infiltrating monocytes were observed in healing wounds from Fpr2/3-/- mice compared with WT animals. Bone marrow transplant experiments revealed that Fpr2/3-/- monocytes showed a competitive disadvantage when infiltrating colonic wounds. Moreover, Fpr2/3-/- monocytes were defective in chemotactic responses to the chemokine CC chemokine ligand (CCL)20, which is up-regulated during early phases of inflammation. Analysis of Fpr2/3-/- monocytes revealed altered expression of the CCL20 receptor CC chemokine receptor (CCR)6, suggesting that Fpr2/3 regulates CCL20-CCR6-mediated monocyte chemotaxis to sites of mucosal injury in the gut. These findings demonstrate an important contribution of Fpr2/3 in facilitating monocyte recruitment to sites of mucosal injury to influence wound repair.-Birkl, D., O'Leary, M. N., Quiros, M., Azcutia, V., Schaller, M., Reed, M., Nishio, H., Keeney, J., Neish, A. S., Lukacs, N. W., Parkos, C. A., Nusrat, A. Formyl peptide receptor 2 regulates monocyte recruitment to promote intestinal mucosal wound repair.
Assuntos
Movimento Celular , Inflamação/terapia , Mucosa Intestinal/fisiologia , Monócitos/metabolismo , Receptores de Formil Peptídeo/fisiologia , Cicatrização , Animais , Transplante de Medula Óssea , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Colite/induzido quimicamente , Colite/metabolismo , Colite/patologia , Sulfato de Dextrana/toxicidade , Inflamação/etiologia , Inflamação/patologia , Mucosa Intestinal/lesões , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/citologia , Receptores CCR6/genética , Receptores CCR6/metabolismoRESUMO
Junctional adhesion molecule-A (JAM-A) is a transmembrane glycoprotein expressed on leukocytes, endothelia, and epithelia that regulates biological processes including barrier function and immune responses. While JAM-A has been reported to facilitate tissue infiltration of leukocytes under inflammatory conditions, the contributions of leukocyte-expressed JAM-A in vivo remain unresolved. We investigated the role of leukocyte-expressed JAM-A in acute peritonitis induced by zymosan, lipopolysaccharide (LPS), or TNFα using mice with selective loss of JAM-A in myelomonocytic cells (LysM-Cre;Jam-afl/fl). Surprisingly, in LysM-Cre;Jam-afl/fl mice, loss of JAM-A did not affect neutrophil (PMN) recruitment into the peritoneum in response to zymosan, LPS, or TNFα although it was significantly reduced in Jam-aKO mice. In parallel, Jam-aKO peritoneal macrophages exhibited diminished CXCL1 chemokine production and decreased activation of NF-kB, whereas those from LysM-Cre;Jam-afl/fl mice were unaffected. Using Villin-Cre;Jam-afl/fl mice, targeted loss of JAM-A on intestinal epithelial cells resulted in increased intestinal permeability along with reduced peritoneal PMN migration as well as lower levels of CXCL1 and active NF-kB similar to that observed in Jam-aKO animals. Interestingly, in germ-free Villin-Cre;Jam-afl/fl mice, PMN recruitment was unaffected suggesting dependence on gut microbiota. Such observations highlight the functional link between a leaky gut and regulation of innate immune responses.
Assuntos
Moléculas de Adesão Celular/metabolismo , Mucosa Intestinal/imunologia , Macrófagos/imunologia , Neutrófilos/imunologia , Peritonite/imunologia , Receptores de Superfície Celular/metabolismo , Junções Íntimas/patologia , Animais , Moléculas de Adesão Celular/genética , Células Cultivadas , Quimiocina CXCL1/metabolismo , Modelos Animais de Doenças , Microbioma Gastrointestinal , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , NF-kappa B/metabolismo , Infiltração de Neutrófilos , Peritonite/induzido quimicamente , Permeabilidade , Receptores de Superfície Celular/genética , ZimosanRESUMO
In response to injury, epithelial cells migrate and proliferate to cover denuded mucosal surfaces and repair the barrier defect. This process is orchestrated by dynamic crosstalk between immune cells and the epithelium; however, the mechanisms involved remain incompletely understood. Here, we report that IL-10 was rapidly induced following intestinal mucosal injury and was required for optimal intestinal mucosal wound closure. Conditional deletion of IL-10 specifically in CD11c-expressing cells in vivo implicated macrophages as a critical innate immune contributor to IL-10-induced wound closure. Consistent with these findings, wound closure in T cell- and B cell-deficient Rag1-/- mice was unimpaired, demonstrating that adaptive immune cells are not absolutely required for this process. Further, following mucosal injury, macrophage-derived IL-10 resulted in epithelial cAMP response element-binding protein (CREB) activation and subsequent synthesis and secretion of the pro-repair WNT1-inducible signaling protein 1 (WISP-1). WISP-1 induced epithelial cell proliferation and wound closure by activating epithelial pro-proliferative pathways. These findings define the involvement of macrophages in regulating an IL-10/CREB/WISP-1 signaling axis, with broad implications in linking innate immune activation to mucosal wound repair.
Assuntos
Proteínas de Sinalização Intercelular CCN/metabolismo , Interleucina-10/metabolismo , Macrófagos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Antígenos CD11/metabolismo , Proliferação de Células , Colo/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Humanos , Inflamação , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , CicatrizaçãoRESUMO
CD47 is known to play an important role in CD4+ T cell homeostasis. We recently reported a reduction in mice deficient in the Cd47 gene (Cd47-/-) CD4+ T cell adhesion and transendothelial migration (TEM) in vivo and in vitro as a result of impaired expression of high-affinity forms of LFA-1 and VLA-4 integrins. A prior study concluded that Cd47-/- mice were resistant to experimental autoimmune encephalomyelitis (EAE) as a result of complete failure in CD4+ T cell activation after myelin oligodendrocyte glycoprotein peptide 35-55 aa (MOG35-55) immunization. As the prior EAE study was published before our report, authors could not have accounted for defects in T cell integrin function as a mechanism to protect Cd47-/- in EAE. Thus, we hypothesized that failure of T cell activation involved defects in LFA-1 and VLA-4 integrins. We confirmed that Cd47-/- mice were resistant to MOG35-55-induced EAE. Our data, however, supported a different mechanism that was not a result of failure of CD4+ T cell activation. Instead, we found that CD4+ T cells in MOG35-55-immunized Cd47-/- mice were activated, but clonal expansion contracted within 72 h after immunization. We used TCR crosslinking and mitogen activation in vitro to investigate the underlying mechanism. We found that naïve Cd47-/- CD4+ T cells exhibited a premature block in proliferation and survival because of impaired activation of LFA-1, despite effective TCR-induced activation. These results identify CD47 as an important regulator of LFA-1 and VLA-4 integrin-adhesive functions in T cell proliferation, as well as recruitment, and clarify the roles played by CD47 in MOG35-55-induced EAE.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Antígeno CD47/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/prevenção & controle , Antígeno-1 Associado à Função Linfocitária/metabolismo , Transferência Adotiva , Animais , Anticorpos Monoclonais/farmacologia , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/imunologia , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quimiocinas/farmacologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Humanos , Imunização , Integrina alfa4beta1/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Linfonodos/efeitos dos fármacos , Linfonodos/patologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Polymorphonuclear neutrophils (PMNs) play a critical role in host defense against infection and in the resolution of inflammation. However, immune responses mediated by PMN must be tightly regulated to facilitate elimination of invading pathogens without inducing detrimental inflammation and host tissue damage. Specific engagement of cell surface immunoreceptors by a diverse range of extracellular signals regulates PMN effector functions through differential activation of intracellular signaling cascades. Although mechanisms of PMN activation mediated via cell signaling pathways have been well described, less is known about negative regulation of PMN function by immune signaling cascades. Here, we provide an overview of immunoreceptor-mediated negative regulation of key PMN effector functions including maturation, migration, phagocytosis, reactive oxygen species release, degranulation, apoptosis, and NET formation. Increased understanding of mechanisms of suppression of PMN effector functions may point to possible future therapeutic targets for the amelioration of PMN-mediated autoimmune and inflammatory diseases.
RESUMO
Wiskott-Aldrich syndrome (WAS) is associated with mutations in the WAS protein (WASp), which plays a critical role in the initiation of T cell receptor-driven (TCR-driven) actin polymerization. The clinical phenotype of WAS includes susceptibility to infection, allergy, autoimmunity, and malignancy and overlaps with the symptoms of dedicator of cytokinesis 8 (DOCK8) deficiency, suggesting that the 2 syndromes share common pathogenic mechanisms. Here, we demonstrated that the WASp-interacting protein (WIP) bridges DOCK8 to WASp and actin in T cells. We determined that the guanine nucleotide exchange factor activity of DOCK8 is essential for the integrity of the subcortical actin cytoskeleton as well as for TCR-driven WASp activation, F-actin assembly, immune synapse formation, actin foci formation, mechanotransduction, T cell transendothelial migration, and homing to lymph nodes, all of which also depend on WASp. These results indicate that DOCK8 and WASp are in the same signaling pathway that links TCRs to the actin cytoskeleton in TCR-driven actin assembly. Further, they provide an explanation for similarities in the clinical phenotypes of WAS and DOCK8 deficiency.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Transporte/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Movimento Celular , Proteínas do Citoesqueleto , Células HEK293 , Humanos , Sinapses Imunológicas/metabolismo , Linfonodos/citologia , Mecanotransdução Celular , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Mapas de Interação de Proteínas , Multimerização Proteica , Transporte Proteico , Linfócitos T/fisiologiaRESUMO
BACKGROUND: Hyperglycemia is acknowledged as a pro-inflammatory condition and a major cause of vascular damage. Nevertheless, we have previously described that high glucose only promotes inflammation in human vascular cells previously primed with pro-inflammatory stimuli, such as the cytokine interleukin (IL)1ß. Here, we aimed to identify the cellular mechanisms by which high glucose exacerbates the vascular inflammation induced by IL1ß. METHODS: Cultured human aortic smooth muscle cells (HASMC) and isolated rat mesenteric microvessels were treated with IL1ß in medium containing 5.5-22 mmol/L glucose. Glucose uptake and consumption, lactate production, GLUT1 levels, NADPH oxidase activity and inflammatory signalling (nuclear factor-κB activation and inducible nitric oxide synthase expression) were measured in HASMC, while endothelium-dependent relaxations to acetylcholine were determined in rat microvessels. Pharmacological inhibition of IL1 receptors, NADPH oxidase and glucose-6-phosphate dehydrogenase (G6PD), as well as silencing of G6PD, were also performed. Moreover, the pentose phosphate pathway (PPP) activity and the levels of reduced glutathione were determined. RESULTS: We found that excess glucose uptake in HASMC cultured in 22 mM glucose only occurred following activation with IL1ß. However, the simple entry of glucose was not enough to be deleterious since over-expression of the glucose transporter GLUT1 or increased glucose uptake following inhibition of mitochondrial respiration by sodium azide was not sufficient to trigger inflammatory mechanisms. In fact, besides allowing glucose entry, IL1ß activated the PPP, thus permitting some of the excess glucose to be metabolized via this route. This in turn led to an over-activation NADPH oxidase, resulting in increased generation of free radicals and the subsequent downstream pro-inflammatory signalling. Moreover, in rat mesenteric microvessels high glucose incubation enhanced the endothelial dysfunction induced by IL1ß by a mechanism which was abrogated by the inhibition of the PPP. CONCLUSIONS: A pro-inflammatory stimulus like IL1ß transforms excess glucose into a vascular deleterious agent by causing an increase in glucose uptake and its subsequent diversion into the PPP, promoting the pro-oxidant conditions required for the exacerbation of pro-oxidant and pro-inflammatory pathways. We propose that over-activation of the PPP is a crucial mechanism for the vascular damage associated to hyperglycemia.
Assuntos
Glucose/metabolismo , Inflamação/metabolismo , Miócitos de Músculo Liso/metabolismo , Via de Pentose Fosfato , Animais , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Glutationa , Humanos , Hiperglicemia/metabolismo , Interleucina-1beta/farmacologia , Masculino , Miócitos de Músculo Liso/efeitos dos fármacos , NADPH Oxidases/metabolismo , Oxirredução/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismoRESUMO
The mechanisms driving T cell homing to lymph nodes and migration to tissue are well described but little is known about factors that affect T cell egress from tissues. Here, we generate mice with a T cell-specific deletion of the scaffold protein A kinase anchoring protein 9 (AKAP9) and use models of inflammatory disease to demonstrate that AKAP9 is dispensable for T cell priming and migration into tissues and lymph nodes, but is required for T cell retention in tissues. AKAP9 deficiency results in increased T cell egress to draining lymph nodes, which is associated with impaired T cell re-activation in tissues and protection from organ damage. AKAP9-deficient T cells exhibit reduced microtubule-dependent recycling of TCRs back to the cell surface and this affects antigen-dependent activation, primarily by non-classical antigen-presenting cells. Thus, AKAP9-dependent TCR trafficking drives efficient T cell re-activation and extends their retention at sites of inflammation with implications for disease pathogenesis.
Assuntos
Proteínas de Ancoragem à Quinase A/genética , Movimento Celular/imunologia , Encefalomielite Autoimune Experimental/imunologia , Linfonodos/imunologia , Ativação Linfocitária/imunologia , Proteínas Associadas aos Microtúbulos/genética , Nefrite/imunologia , Traumatismo por Reperfusão/imunologia , Linfócitos T/imunologia , Proteínas de Ancoragem à Quinase A/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Adesão Celular/imunologia , Ensaios de Migração de Leucócitos , Células Cultivadas , Endossomos , Técnicas de Inativação de Genes , Membrana Basal Glomerular/imunologia , Técnicas In Vitro , Inflamação , Rim/irrigação sanguínea , Rim/imunologia , Camundongos , Proteínas Associadas aos Microtúbulos/imunologia , Receptores de Antígenos de Linfócitos T , Migração Transendotelial e Transepitelial/imunologiaRESUMO
Leukocyte CD18 integrins increase their affinity for ligand by transmitting allosteric signals to and from their ligand-binding αI domain. Mechanical forces induce allosteric changes that paradoxically slow dissociation by increasing the integrin/ligand bond lifetimes, referred to as catch bonds. Mac-1 formed catch bonds with its ligands. However, a Mac-1 gene (ITGAM) coding variant (rs1143679, R77H), which is located in the ß-propeller domain and is significantly associated with systemic lupus erythematosus risk, exhibits a marked impairment in 2D ligand affinity and affinity maturation under mechanical force. Targeted mutations and activating antibodies reveal that the failure in Mac-1 R77H allostery is rescued by induction of cytoplasmic tail separation and full integrin extension. These findings demonstrate roles for R77, and the ß-propeller in which it resides, in force-induced allostery relay and integrin bond stabilization. Defects in these processes may have pathological consequences, as the Mac-1 R77H variant is associated with increased susceptibility to lupus.
RESUMO
Previous studies have demonstrated that the cell surface receptor Slamf1 (CD150) is requisite for optimal NADPH-oxidase (Nox2) dependent reactive oxygen species (ROS) production by phagocytes in response to Gram- bacteria. By contrast, Slamf8 (CD353) is a negative regulator of ROS in response to Gram+ and Gram- bacteria. Employing in vivo migration after skin sensitization, induction of peritonitis, and repopulation of the small intestine demonstrates that in vivo migration of Slamf1-/- dendritic cells and macrophages is reduced, as compared to wt mice. By contrast, in vivo migration of Slamf8-/- dendritic cells, macrophages and neutrophils is accelerated. These opposing effects of Slamf1 and Slamf8 are cell-intrinsic as judged by in vitro migration in transwell chambers in response to CCL19, CCL21 or CSF-1. Importantly, inhibiting ROS production of Slamf8-/- macrophages by diphenyleneiodonium chloride blocks this in vitro migration. We conclude that Slamf1 and Slamf8 govern ROS-dependent innate immune responses of myeloid cells, thus modulating migration of these cells during inflammation in an opposing manner.
Assuntos
Antígenos CD/fisiologia , Células Mieloides/metabolismo , Receptores de Superfície Celular/fisiologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Movimento Celular/genética , Movimento Celular/fisiologia , Quimiotaxia/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Células Mieloides/citologia , Neutrófilos/citologia , Neutrófilos/metabolismo , Peritonite/metabolismo , Peritonite/patologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Membro 1 da Família de Moléculas de Sinalização da Ativação LinfocitáriaRESUMO
The immune cell system is a critical component of host defense. Recruitment of immune cells to sites of infection, immune reaction, or injury is complex and involves coordinated adhesive interactions between the leukocyte and the endothelial cell monolayer that lines blood vessels. This article reviews basic mechanisms in the recruitment of leukocytes to tissues and then selectively reviews new concepts that are emerging based on advances in live cell imaging microscopy and mouse strains. These emerging concepts are altering the conventional paradigms of inflammatory leukocyte recruitment established in the early 1990s. Indeed, recent publications have identified previously unrecognized contributions from pericytes and interstitial leukocytes and their secreted products that guide leukocytes to their targets. Investigators have also begun to design organs on a chip. Recent reports indicate that this avenue of research holds much promise.