RESUMO
The interleukin-6 (IL-6) membrane receptor and its circulating soluble form, sIL-6R, can be targeted by antibody therapy to reduce deleterious immune signaling caused by chronic overexpression of the pro-inflammatory cytokine IL-6. This strategy may also hold promise for treating acute hyperinflammation, such as observed in coronavirus disease 2019 (COVID-19), highlighting a need to define regulators of IL-6 homeostasis. We found that conventional dendritic cells (cDCs), defined in mice via expression of the transcription factor Zbtb46, were a major source of circulating sIL-6R and, thus, systemically regulated IL-6 signaling. This was uncovered through identification of a cDC-dependent but T cell-independent modality that naturally adjuvants plasma cell differentiation and antibody responses to protein antigens. This pathway was then revealed as part of a broader biological buffer system in which cDC-derived sIL-6R set the in-solution persistence of IL-6. This control axis may further inform the development of therapeutic agents to modulate pro-inflammatory immune reactions.
Assuntos
Células Dendríticas/imunologia , Interleucina-6/sangue , Interleucina-6/imunologia , Proteína ADAM17 , Animais , Diferenciação Celular , Imunidade Humoral , Imunoglobulina M/imunologia , Inflamação , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/imunologia , Interleucina-6/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Plasmócitos/imunologia , Receptores de Interleucina-6/sangue , Receptores de Interleucina-6/imunologia , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Receptor 7 Toll-Like/imunologiaRESUMO
Detailed understanding of the signaling intermediates that confer the sensing of intracellular viral nucleic acids for induction of type I interferons is critical for strategies to curtail viral mechanisms that impede innate immune defenses. Here we show that the activation of the microtubule-associated guanine nucleotide exchange factor GEF-H1, encoded by Arhgef2, is essential for sensing of foreign RNA by RIG-I-like receptors. Activation of GEF-H1 controls RIG-I-dependent and Mda5-dependent phosphorylation of IRF3 and induction of IFN-ß expression in macrophages. Generation of Arhgef2(-/-) mice revealed a pronounced signaling defect that prevented antiviral host responses to encephalomyocarditis virus and influenza A virus. Microtubule networks sequester GEF-H1 that upon activation is released to enable antiviral signaling by intracellular nucleic acid detection pathways.
Assuntos
Imunidade Inata/imunologia , Microtúbulos/imunologia , RNA Viral/imunologia , Fatores de Troca de Nucleotídeo Guanina Rho/imunologia , Transdução de Sinais/imunologia , Animais , Células COS , Chlorocebus aethiops , Proteína DEAD-box 58 , RNA Helicases DEAD-box/imunologia , RNA Helicases DEAD-box/metabolismo , Expressão Gênica/imunologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Imunidade Inata/genética , Immunoblotting , Vírus da Influenza A/genética , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Helicase IFIH1 Induzida por Interferon , Interferon beta/genética , Interferon beta/imunologia , Interferon beta/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microtúbulos/metabolismo , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais/genéticaRESUMO
Alcohol-related liver disease (ALD) accounts for the majority of cirrhosis and liver-related deaths worldwide. Activation of IFN-regulatory factor (IRF3) initiates alcohol-induced hepatocyte apoptosis, which fuels a robust secondary inflammatory response that drives ALD. The dominant molecular mechanism by which alcohol activates IRF3 and the pathways that amplify inflammatory signals in ALD remains unknown. Here we show that cytoplasmic sensor cyclic guanosine monophosphate-adenosine monophosphate (AMP) synthase (cGAS) drives IRF3 activation in both alcohol-injured hepatocytes and the neighboring parenchyma via a gap junction intercellular communication pathway. Hepatic RNA-seq analysis of patients with a wide spectrum of ALD revealed that expression of the cGAS-IRF3 pathway correlated positively with disease severity. Alcohol-fed mice demonstrated increased hepatic expression of the cGAS-IRF3 pathway. Mice genetically deficient in cGAS and IRF3 were protected against ALD. Ablation of cGAS in hepatocytes only phenocopied this hepatoprotection, highlighting the critical role of hepatocytes in fueling the cGAS-IRF3 response to alcohol. We identified connexin 32 (Cx32), the predominant hepatic gap junction, as a critical regulator of spreading cGAS-driven IRF3 activation through the liver parenchyma. Disruption of Cx32 in ALD impaired IRF3-stimulated gene expression, resulting in decreased hepatic injury despite an increase in hepatic steatosis. Taken together, these results identify cGAS and Cx32 as key factors in ALD pathogenesis and as potential therapeutic targets for hepatoprotection.
Assuntos
Junções Comunicantes/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Hepatopatias Alcoólicas/metabolismo , Nucleotidiltransferases/metabolismo , Adulto , Animais , Apoptose , Feminino , Hepatócitos/metabolismo , Humanos , Fígado/citologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Nucleotidiltransferases/genética , Transdução de SinaisRESUMO
Phagocytosis is a pivotal process by which macrophages eliminate microorganisms after recognition by pathogen sensors. Here we unexpectedly found that the self ligand and cell surface receptor SLAM functioned not only as a costimulatory molecule but also as a microbial sensor that controlled the killing of gram-negative bacteria by macrophages. SLAM regulated activity of the NADPH oxidase NOX2 complex and phagolysosomal maturation after entering the phagosome, following interaction with the bacterial outer membrane proteins OmpC and OmpF. SLAM recruited a complex containing the intracellular class III phosphatidylinositol kinase Vps34, its regulatory protein kinase Vps15 and the autophagy-associated molecule beclin-1 to the phagosome, which was responsible for inducing the accumulation of phosphatidylinositol-3-phosphate, a regulator of both NOX2 function and phagosomal or endosomal fusion. Thus, SLAM connects the gram-negative bacterial phagosome to ubiquitous cellular machinery responsible for the control of bacterial killing.
Assuntos
Antígenos CD/metabolismo , Infecções por Escherichia coli/imunologia , Escherichia coli/imunologia , Macrófagos/imunologia , Fagossomos/imunologia , Receptores de Superfície Celular/metabolismo , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Animais , Antígenos CD/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Bactérias/genética , Proteína Beclina-1 , Células Cultivadas , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Macrófagos/microbiologia , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Chaperonas Moleculares/genética , NADPH Oxidase 2 , NADPH Oxidases/metabolismo , Fagocitose , Fagossomos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Porinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Superfície Celular/genética , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Proteína VPS15 de Distribuição VacuolarRESUMO
Circulatory antigens transit through the small intestine via the fenestrated capillaries in the lamina propria prior to entering into the draining lymphatics. But whether or how this process controls mucosal immune responses remains unknown. Here we demonstrate that dendritic cells (DCs) of the lamina propria can sample and process both circulatory and luminal antigens. Surprisingly, antigen cross-presentation by resident CX3CR1(+) DCs induced differentiation of precursor cells into CD8(+) T cells that expressed interleukin-10 (IL-10), IL-13, and IL-9 and could migrate into adjacent compartments. We conclude that lamina propria CX3CR1(+) DCs facilitate the surveillance of circulatory antigens and act as a conduit for the processing of self- and intestinally absorbed antigens, leading to the induction of CD8(+) T cells, that partake in the control of T cell activation during mucosal immune responses.
Assuntos
Apresentação de Antígeno/imunologia , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Mucosa Intestinal/imunologia , Ativação Linfocitária/imunologia , Animais , Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Receptor 1 de Quimiocina CX3C , Diferenciação Celular/imunologia , Apresentação Cruzada/imunologia , Células Dendríticas/metabolismo , Enterite/imunologia , Enterite/prevenção & controle , Epitopos de Linfócito T/imunologia , Mucosa Intestinal/citologia , Intestino Delgado/imunologia , Camundongos , Receptores de Quimiocinas/imunologia , Receptores de Quimiocinas/metabolismoRESUMO
The molecular signaling cascades that regulate angiogenesis and microvascular remodeling are fundamental to normal development, healthy physiology, and pathologies such as inflammation and cancer. Yet quantifying such complex, fractally branching vascular patterns remains difficult. We review application of NASA's globally available, freely downloadable VESsel GENeration (VESGEN) Analysis software to numerous examples of 2D vascular trees, networks, and tree-network composites. Upon input of a binary vascular image, automated output includes informative vascular maps and quantification of parameters such as tortuosity, fractal dimension, vessel diameter, area, length, number, and branch point. Previous research has demonstrated that cytokines and therapeutics such as vascular endothelial growth factor, basic fibroblast growth factor (fibroblast growth factor-2), transforming growth factor-beta-1, and steroid triamcinolone acetonide specify unique "fingerprint" or "biomarker" vascular patterns that integrate dominant signaling with physiological response. In vivo experimental examples described here include vascular response to keratinocyte growth factor, a novel vessel tortuosity factor; angiogenic inhibition in humanized tumor xenografts by the anti-angiogenesis drug leronlimab; intestinal vascular inflammation with probiotic protection by Saccharomyces boulardii, and a workflow programming of vascular architecture for 3D bioprinting of regenerative tissues from 2D images. Microvascular remodeling in the human retina is described for astronaut risks in microgravity, vessel tortuosity in diabetic retinopathy, and venous occlusive disease.
Assuntos
Proteínas Angiogênicas/metabolismo , Artérias/anatomia & histologia , Artérias/metabolismo , Modelos Anatômicos , Modelos Cardiovasculares , Neovascularização Fisiológica , Transdução de Sinais , Remodelação Vascular , Proteínas Angiogênicas/genética , Animais , Astronautas , Bioimpressão , Simulação por Computador , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Fractais , Regulação da Expressão Gênica , Humanos , Neovascularização Patológica , Neovascularização Fisiológica/genética , Impressão Tridimensional , Oclusão da Veia Retiniana/metabolismo , Oclusão da Veia Retiniana/patologia , Vasos Retinianos/metabolismo , Vasos Retinianos/patologia , Transdução de Sinais/genética , Software , Remodelação Vascular/genética , Ausência de PesoRESUMO
Secretory IgA is a key host defense mechanism that controls the intestinal microbiota. We investigated the role of CD11c+CX3CR1+CD64+ macrophages in IgA production in the intestine. Intestinal CX3CR1+ macrophages directly induced IgA secretion by B cells. Ag delivery to lamina propria (LP) CX3CR1+ macrophages specifically induced intestinal IgA production. The induction of IgA by CX3CR1+ macrophages required BAFF, a proliferation-inducing ligand, and TNF-α, but was surprisingly independent of TLR-mediated microbial recognition and retinoic acid signaling. IgA secretion by CX3CR1+ macrophages was enhanced by LP CD8+ T cells through the secretion of IL-9 and IL-13. CX3CR1+ macrophages and CD8+ T cells induced IgA production by B cells independently of mesenteric lymph nodes and Peyer patches. Our data reveal a previously unrecognized cellular circuitry in which LP CX3CR1+ macrophages, B cells, and CD8+ T cells coordinate the protective Ig secretion in the small intestine upon peripheral Ag delivery.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunoglobulina A Secretora/biossíntese , Mucosa Intestinal/imunologia , Macrófagos/imunologia , Animais , Formação de Anticorpos/imunologia , Linfócitos B/imunologia , Receptor 1 de Quimiocina CX3C/imunologia , Imunidade nas Mucosas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Mid-hindbrain malformations can occur during embryogenesis through a disturbance of transient and localized gene expression patterns within these distinct brain structures. Rho guanine nucleotide exchange factor (ARHGEF) family members are key for controlling the spatiotemporal activation of Rho GTPase, to modulate cytoskeleton dynamics, cell division, and cell migration. We identified, by means of whole exome sequencing, a homozygous frameshift mutation in the ARHGEF2 as a cause of intellectual disability, a midbrain-hindbrain malformation, and mild microcephaly in a consanguineous pedigree of Kurdish-Turkish descent. We show that loss of ARHGEF2 perturbs progenitor cell differentiation and that this is associated with a shift of mitotic spindle plane orientation, putatively favoring more symmetric divisions. The ARHGEF2 mutation leads to reduction in the activation of the RhoA/ROCK/MLC pathway crucial for cell migration. We demonstrate that the human brain malformation is recapitulated in Arhgef2 mutant mice and identify an aberrant migration of distinct components of the precerebellar system as a pathomechanism underlying the midbrain-hindbrain phenotype. Our results highlight the crucial function of ARHGEF2 in human brain development and identify a mutation in ARHGEF2 as novel cause of a neurodevelopmental disorder.
Assuntos
Movimento Celular/genética , Mutação da Fase de Leitura/genética , Deficiência Intelectual/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Animais , Citoesqueleto/genética , Exoma/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Deficiência Intelectual/diagnóstico por imagem , Deficiência Intelectual/patologia , Imageamento por Ressonância Magnética , Masculino , Mesencéfalo/diagnóstico por imagem , Mesencéfalo/patologia , Camundongos , Linhagem , Rombencéfalo/diagnóstico por imagem , Rombencéfalo/patologia , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Genetic alterations that reduce the function of the immunoregulatory cytokine IL-10 contribute to colitis in mouse and man. Myeloid cells such as macrophages (MΦs) and dendritic cells (DCs) play an essential role in determining the relative abundance of IL-10 versus inflammatory cytokines in the gut. As such, using small molecules to boost IL-10 production by DCs-MΦs represents a promising approach to increase levels of this cytokine specifically in gut tissues. Toward this end, we screened a library of well-annotated kinase inhibitors for compounds that enhance production of IL-10 by murine bone-marrow-derived DCs stimulated with the yeast cell wall preparation zymosan. This approach identified a number of kinase inhibitors that robustly up-regulate IL-10 production including the Food and Drug Administration (FDA)-approved drugs dasatinib, bosutinib, and saracatinib that target ABL, SRC-family, and numerous other kinases. Correlating the kinase selectivity profiles of the active compounds with their effect on IL-10 production suggests that inhibition of salt-inducible kinases (SIKs) mediates the observed IL-10 increase. This was confirmed using the SIK-targeting inhibitor HG-9-91-01 and a series of structural analogs. The stimulatory effect of SIK inhibition on IL-10 is also associated with decreased production of the proinflammatory cytokines IL-1ß, IL-6, IL-12, and TNF-α, and these coordinated effects are observed in human DCs-MΦs and anti-inflammatory CD11c(+) CX3CR1(hi) cells isolated from murine gut tissue. Collectively, these studies demonstrate that SIK inhibition promotes an anti-inflammatory phenotype in activated myeloid cells marked by robust IL-10 production and establish these effects as a previously unidentified activity associated with several FDA-approved multikinase inhibitors.
Assuntos
Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Interleucina-10/biossíntese , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Compostos de Anilina/farmacologia , Animais , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Citocinas/biossíntese , Dasatinibe , Células Dendríticas/enzimologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Mediadores da Inflamação/metabolismo , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/enzimologia , Doenças Inflamatórias Intestinais/imunologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/enzimologia , Intestino Delgado/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Mieloides/efeitos dos fármacos , Células Mieloides/enzimologia , Células Mieloides/imunologia , Nitrilas/farmacologia , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/química , Pirimidinas/química , Pirimidinas/farmacologia , Quinolinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/enzimologia , Linfócitos T Reguladores/imunologia , Tiazóis/farmacologia , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND & AIMS: Intraepithelial T lymphocyte cells (IEL) are the first immune cells to respond to pathogens; they help maintain the integrity of the epithelial barrier. We studied the function of the mouse glycoprotein Signaling Lymphocyte Activation Molecule Family receptor (SLAMF) 4 (encoded by Slamf4) on the surface of CD8αß αß T-cell receptor (TCR)(+) IELs, and the roles of these cells in homeostasis of the small intestine in mice. METHODS: SLAMF4(-) CD8(+) αßTCR(+) cells isolated from spleens of OT-I Rag1(-/-) mice were induced to express gut-homing receptors and transferred to C57BL/6J mice; levels of SLAMF4(+) cells were measured in small intestine tissues. After administration of anti-CD3 or antigen, with or without anti-SLAM4, to C57BL/6J and Slamf4(-/-) mice, CD8αß αßTCR(+) IELs were collected; cytokine production and cytotoxicity were measured. Depletion of CX3CR1(+) phagocytes was assessed in mice by live-cell confocal imaging or by cytofluorometry; small intestine tissues were analyzed by histology and inflammation was quantified. RESULTS: Splenic CD8(+) αßTCR(+) cells began to express SLAMF4 only after migrating to the small intestine. Injection of C57BL/6J mice with anti-SLAMF4 and anti-CD3 increased levels of interleukin 10 and interferon gamma secretion by IEL, compared with injection of anti-CD3 only. Similarly, the number of granzyme B(+) cytotoxic CD8(+) αßTCR(+) IELs increased in Slamf4(-/-) mice after injection of anti-CD3 and anti-SLAMF4, administration of antigen, or injection of anti-CD3. Surprisingly, in vivo activation of CD8αß(+) IELs with anti-CD3 or antigen caused transient depletion of CX3CR1(+) phagocytes, which was prolonged by co-injection with anti-SLAMF4 or in Slamf4(-/-) mice. Anti-CD3 aggravated inflammation in the small intestines of Slamf4(-/-) mice and Eat2a(-/-)Eat2b(-/-) mice, indicated by flattened villi and crypt hyperplasia. CONCLUSIONS: In mice, the intestinal environment induces SLAMF4 expression and localization to the surface of CD8(+) αßTCR(+) IELs. Signaling via SLAMF4 controls expansion of cytotoxic CD8αß(+) IELs, which regulate the reversible depletion of lamina propria phagocytes and inflammation in the small intestine.
Assuntos
Antígenos CD/metabolismo , Proliferação de Células , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Ativação Linfocitária , Receptores Imunológicos/metabolismo , Linfócitos T Citotóxicos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antígenos CD/genética , Receptor 1 de Quimiocina CX3C , Movimento Celular , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Homeostase , Hiperplasia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Intestino Delgado/imunologia , Intestino Delgado/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagócitos/imunologia , Fagócitos/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Receptores Imunológicos/deficiência , Receptores Imunológicos/genética , Transdução de Sinais , Família de Moléculas de Sinalização da Ativação Linfocitária , Baço/imunologia , Baço/metabolismo , Linfócitos T Citotóxicos/imunologiaRESUMO
BACKGROUND & AIMS: Inducible chitinase 3-like-1 is expressed by intestinal epithelial cells (IECs) and adheres to bacteria under conditions of inflammation. We performed a structure-function analysis of the chitin-binding domains encoded by the chiA gene, which mediates the pathogenic effects of adherent invasive Escherichia coli (AIEC). METHODS: We created AIEC (strain LF82) with deletion of chiA (LF82-ΔchiA) or that expressed chiA with specific mutations. We investigated the effects of infecting different IEC lines with these bacteria compared with nonpathogenic E coli; chitinase activities were measured using the colloidal chitin-azure method. Colitis was induced in C57/Bl6 mice by administration of dextran sodium sulfate, and mice were given 10(8) bacteria for 15 consecutive days by gavage. Stool/tissue samples were collected and analyzed. RESULTS: LF82-ΔchiA had significantly less adhesion to IEC lines than LF82. Complementation of LF82-ΔchiA with the LF82 chiA gene, but not chiA from nonpathogenic (K12) E coli, increased adhesion. We identified 5 specific polymorphisms in the chitin-binding domain of LF82 chiA (at amino acids 362, 370, 378, 388, and 548) that differ from chiA of K12 and were required for LF82 to interact directly with IECs. This interaction was mediated by an N-glycosylated asparagine in chitinase 3-like-1 (amino acid 68) on IECs. Mice infected with LF82, or LF82-ΔchiA complemented with LF82 chiA, developed more severe colitis after administration of dextran sodium sulfate than mice infected with LF82-ΔchiA or LF82 that expressed mutant forms of chiA. CONCLUSIONS: AIEC adheres to an N-glycosylated chitinase 3-like-1 on IECs via the chitin-binding domain of chiA. This mechanism promotes the pathogenic effects of AIEC in mice with colitis.
Assuntos
Adesinas de Escherichia coli/metabolismo , Aderência Bacteriana/fisiologia , Quitinases/metabolismo , Colite/microbiologia , Células Epiteliais/microbiologia , Escherichia coli/patogenicidade , Mucosa Intestinal/microbiologia , Adesinas de Escherichia coli/química , Adesinas de Escherichia coli/genética , Animais , Aderência Bacteriana/genética , Biomarcadores/metabolismo , Linhagem Celular , Proteína 1 Semelhante à Quitinase-3 , Quitinases/química , Quitinases/genética , Colite/induzido quimicamente , Colite/enzimologia , Sulfato de Dextrana , Células Epiteliais/enzimologia , Escherichia coli/enzimologia , Escherichia coli/genética , Infecções por Escherichia coli/enzimologia , Infecções por Escherichia coli/microbiologia , Glicoproteínas/metabolismo , Glicosilação , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Polimorfismo GenéticoRESUMO
BACKGROUND & AIMS: Intestinal epithelial cells aid in mucosal defense by providing a physical barrier against entry of pathogenic bacteria and secreting antimicrobial peptides (AMPs). Autophagy is an important component of immune homeostasis. However, little is known about its role in specific cell types during bacterial infection in vivo. We investigated the role of autophagy in the response of intestinal epithelial and antigen-presenting cells to Salmonella infection in mice. METHODS: We generated mice deficient in Atg16l1 in epithelial cells (Atg16l1(f/f) × Villin-cre) or CD11c(+) cells (Atg16l1(f/f) × CD11c-cre); these mice were used to assess cell type-specific antibacterial autophagy. All responses were compared with Atg16l1(f/f) mice (controls). Mice were infected with Salmonella enterica serovar typhimurium; cecum and small-intestine tissues were collected for immunofluorescence, histology, and quantitative reverse-transcription polymerase chain reaction analyses of cytokines and AMPs. Modulators of autophagy were screened to evaluate their effects on antibacterial responses in human epithelial cells. RESULTS: Autophagy was induced in small intestine and cecum after infection with S typhimurium, and required Atg16l1. S typhimurium colocalized with microtubule-associated protein 1 light chain 3ß (Map1lc3b or LC3) in the intestinal epithelium of control mice but not in Atg16l1(f/f) × Villin-cre mice. Atg16l1(f/f) × Villin-cre mice also had fewer Paneth cells and abnormal granule morphology, leading to reduced expression of AMPs. Consistent with these defective immune responses, Atg16l1(f/f) × Villin-cre mice had increased inflammation and systemic translocation of bacteria compared with control mice. In contrast, we observed few differences between Atg16l1(f/f) × CD11c-cre and control mice. Trifluoperazine promoted autophagy and bacterial clearance in HeLa cells; these effects were reduced upon knockdown of ATG16L1. CONCLUSIONS: Atg16l1 regulates autophagy in intestinal epithelial cells and is required for bacterial clearance. It also is required to prevent systemic infection of mice with enteric bacteria.
Assuntos
Autofagia/fisiologia , Proteínas de Transporte/fisiologia , Mucosa Intestinal/fisiologia , Salmonelose Animal/prevenção & controle , Animais , Proteínas Relacionadas à Autofagia , Antígeno CD11c/fisiologia , Proteínas de Transporte/genética , Modelos Animais de Doenças , Células HeLa , Humanos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Salmonelose Animal/patologia , Salmonelose Animal/fisiopatologia , Salmonella typhimurium/isolamento & purificaçãoRESUMO
BACKGROUND & AIMS: Immunodeficiency and autoimmune sequelae, including colitis, develop in patients and mice deficient in Wiskott-Aldrich syndrome protein (WASP), a hematopoietic cell-specific intracellular signaling molecule that regulates the actin cytoskeleton. Development of colitis in WASP-deficient mice requires lymphocytes; transfer of T cells is sufficient to induce colitis in immunodeficient mice. We investigated the interactions between innate and adaptive immune cells in mucosal regulation during development of T cell-mediated colitis in mice with WASP-deficient cells of the innate immune system. METHODS: Naïve and/or regulatory CD4(+) T cells were transferred from 129 SvEv mice into RAG-2-deficient (RAG-2 KO) mice or mice lacking WASP and RAG-2 (WRDKO). Animals were observed for the development of colitis; effector and regulatory functions of innate immune and T cells were analyzed with in vivo and in vitro assays. RESULTS: Transfer of unfractionated CD4(+) T cells induced severe colitis in WRDKO, but not RAG-2 KO, mice. Naïve wild-type T cells had higher levels of effector activity and regulatory T cells had reduced suppressive function when transferred into WRDKO mice compared with RAG-2 KO mice. Regulatory T-cell proliferation, generation, and maintenance of FoxP3 expression were reduced in WRDKO recipients and associated with reduced numbers of CD103(+) tolerogenic dendritic cells and levels of interleukin-10. Administration of interleukin-10 prevented induction of colitis following transfer of T cells into WRDKO mice. CONCLUSIONS: Defective interactions between WASP-deficient innate immune cells and normal T cells disrupt mucosal regulation, potentially by altering the functions of tolerogenic dendritic cells, production of interleukin-10, and homeostasis of regulatory T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Colite/imunologia , Colo/imunologia , Imunidade Inata , Imunidade nas Mucosas , Mucosa Intestinal/imunologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Transferência Adotiva , Animais , Antígenos CD/metabolismo , Antígeno CD11b/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/transplante , Proliferação de Células , Células Cultivadas , Colite/genética , Colite/metabolismo , Colite/patologia , Colo/metabolismo , Colo/patologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismo , Tolerância Imunológica , Cadeias alfa de Integrinas/metabolismo , Interleucina-10/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Ativação Linfocitária , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Linfócitos T Reguladores/imunologia , Fatores de Tempo , Quimeras de Transplante , Proteína da Síndrome de Wiskott-Aldrich/genéticaRESUMO
BACKGROUND & AIMS: Signaling lymphocyte activation molecule (Slamf)1 is a co-stimulatory receptor on T cells and regulates cytokine production by macrophages and dendritic cells. Slamf1 regulates microbicidal mechanisms in macrophages, therefore we investigated whether the receptor affects development of colitis in mice. METHODS: We transferred CD45RB(hi) CD4(+) T cells into Rag(-/-) or Slamf1(-/-)Rag(-/-) mice to induce colitis. We also induced colitis by injecting mice with an antibody that activates CD40. We determined the severity of enterocolitis based on disease activity index, histology scores, and levels of cytokine production, and assessed the effects of antibodies against Slamf1 on colitis induction. We quantified migration of monocytes and macrophage to inflamed tissues upon induction of colitis or thioglycollate-induced peritonitis and in response to tumor necrosis factor-α in an air-pouch model of leukocyte migration. RESULTS: Colitis was reduced in Slamf1(-/-)Rag(-/-) mice, compared with Rag(-/-) mice, after transfer of CD45RB(hi) CD4(+) T cells or administration of the CD40 agonist. The numbers of monocytes and macrophages were reduced in inflamed tissues of Slamf1(-/-)Rag(-/-) mice, compared with Rag(-/-) mice, after induction of colitis and other inflammatory disorders. An antibody that inhibited Slamf1 reduced the level of enterocolitis in Rag(-/-) mice. CONCLUSIONS: Slamf1 contributes to the development of colitis in mice. It appears to indirectly regulate the appearance of monocytes and macrophages in inflamed intestinal tissues. Antibodies that inhibit Slamf1 reduce colitis in mice, so human SLAMF1 might be a therapeutic target for inflammatory bowel disease.
Assuntos
Antígenos CD/fisiologia , Colite/fisiopatologia , Receptores de Superfície Celular/fisiologia , Animais , Antígenos CD/genética , Antígenos CD40/efeitos adversos , Movimento Celular , Quimiocina CCL2/sangue , Quimiocina CCL7/sangue , Colite/sangue , Colite/induzido quimicamente , Modelos Animais de Doenças , Intestinos/patologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Membro 1 da Família de Moléculas de Sinalização da Ativação LinfocitáriaRESUMO
Gut-associated dendritic cells (DC) synthesize all-trans retinoic acid, which is required for inducing gut-tropic lymphocytes. Gut-associated DC from MyD88(-/-) mice, which lack most TLR signals, expressed low levels of retinal dehydrogenases (critical enzymes for all-trans retinoic acid biosynthesis) and were significantly impaired in their ability to induce gut-homing T cells. Pretreatment of extraintestinal DC with a TLR1/2 agonist was sufficient to induce retinal dehydrogenases and to confer these DC with the capacity to induce gut-homing lymphocytes via a mechanism dependent on MyD88 and JNK/MAPK. Moreover, gut-associated DC from TLR2(-/-) mice, or from mice in which JNK was pharmacologically blocked, were impaired in their education to imprint gut-homing T cells, which correlated with a decreased induction of gut-tropic T cells in TLR2(-/-) mice upon immunization. Thus, MyD88-dependent TLR2 signals are necessary and sufficient to educate DC with gut-specific imprinting properties and contribute in vivo to the generation of gut-tropic T cells.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Impressão Genômica/imunologia , Mucosa Intestinal/imunologia , Fator 88 de Diferenciação Mieloide/fisiologia , Transdução de Sinais/imunologia , Receptor 1 Toll-Like/fisiologia , Receptor 2 Toll-Like/fisiologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Células Dendríticas/citologia , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , Quimera por Radiação , Receptores de Retorno de Linfócitos/deficiência , Receptores de Retorno de Linfócitos/genética , Receptores de Retorno de Linfócitos/fisiologia , Transdução de Sinais/genéticaRESUMO
Less developed countries have a low incidence of immunological diseases like inflammatory bowel disease (IBD), perhaps prevented by the high prevalence of helminth infections in their populations. In the Rag IL-10(-/-) T cell transfer model of colitis, Heligmosomoides polygyrus, an intestinal helminth, prevents and reverses intestinal inflammation. This model of colitis was used to explore the importance of innate immunity in H. polygyrus protection from IBD. Rag mice briefly exposed to H. polygyrus before reconstitution with IL-10(-/-) colitogenic T cells are protected from colitis. Exposure to H. polygyrus before introduction of IL-10(-/-) and OT2 T cells reduced the capacity of the intestinal mucosa to make IFN-gamma and IL-17 after either anti-CD3 mAb or OVA stimulation. This depressed cytokine response was evident even in the absence of colitis, suggesting that the downmodulation in proinflammatory cytokine secretion was not just secondary to improvement in intestinal inflammation. Following H. polygyrus infection, dendritic cells (DCs) from the lamina propria of Rag mice displayed decreased expression of CD80 and CD86, and heightened expression of plasmacytoid dendritic cell Ag-1 and CD40. They were also less responsive to lamina proprias, producing less IL-12p40 and IL-10. Also diminished was their capacity to present OVA to OT2 T cells. These experiments infer that H. polygyrus does not require direct interactions with T or B cells to render animals resistant to colitis. DCs have an important role in driving both murine and human IBD. Data suggest that phenotypic alternations in mucosal DC function are part of the regulatory process.
Assuntos
Colite/imunologia , Colite/prevenção & controle , Imunidade Inata , Nematospiroides dubius/imunologia , Infecções por Strongylida/imunologia , Animais , Células Cultivadas , Colite/parasitologia , Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Modelos Animais de Doenças , Imunidade Inata/genética , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/parasitologia , Doenças Inflamatórias Intestinais/prevenção & controle , Interleucina-10/deficiência , Interleucina-10/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mucosa/imunologia , Mucosa/parasitologia , Mucosa/patologia , Ovalbumina/imunologia , Infecções por Strongylida/genética , Infecções por Strongylida/parasitologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/parasitologia , Subpopulações de Linfócitos T/transplanteRESUMO
Challenges to long-duration space exploration and colonization in microgravity and cosmic radiation environments by humans include poorly understood risks for gastrointestinal function and cancer. Nonetheless, constant remodeling of the intestinal microvasculature is critical for tissue viability, healthy wound healing, and successful prevention or recovery from vascular-mediated inflammatory or ischemic diseases such as cancer. Currently no automated image analysis programs provide quantitative assessments of the complex structure of the mucosal vascular system that are necessary for tracking disease development and tissue recovery. Increasing abnormalities to the microvascular network geometry were therefore mapped with VESsel GENeration Analysis (VESGEN) software from 3D tissue reconstructions of developing intestinal inflammation in a dextran sulfate sodium (DSS) mouse model. By several VESGEN parameters and a novel vascular network linking analysis, inflammation strongly disrupted the regular, lattice-like geometry that defines the normal microvascular network, correlating positively with the increased recruitment of dendritic cells during mucosal defense responses.
RESUMO
One of the most intriguing aspects of male reproductive physiology is the ability to generate spermatogenic cells - which are 'foreign' to the host - without triggering immune activation. After leaving the testis, spermatozoa enter the epididymis where they mature and are stored. In this study, we report a previously unrecognized dense network of dendritic cells (DCs) located at the base of the epididymal epithelium. This network was detected in transgenic mice expressing CD11c-EYFP and CX3CR1-GFP reporters. Epididymal DCs (eDCs) establish intimate interactions with the epithelium and project long dendrites between epithelial cells toward the lumen. We show that isolated eDCs express numerous leukocyte markers described previously in other organs that are in contact with the external environment, and present and cross-present ovalbumin to T cells in vitro. eDCs are, therefore, strategically positioned to regulate the complex interplay between immune tolerance and activation, a balance that is fundamental to male fertility.
Assuntos
Células Dendríticas/imunologia , Epididimo/imunologia , Tolerância Imunológica , Animais , Apresentação de Antígeno , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Biomarcadores/metabolismo , Antígeno CD11c/biossíntese , Antígeno CD11c/genética , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/metabolismo , Epididimo/citologia , Epididimo/metabolismo , Fertilidade , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Imunofenotipagem , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Ovalbumina/imunologia , Fenótipo , Receptores de Quimiocinas/biossíntese , Receptores de Quimiocinas/genética , Linfócitos T/imunologiaRESUMO
The human innate immune system responds to both pathogen and commensal bacteria at the molecular level using bacterial peptidoglycan (PG) recognition elements. Traditionally, synthetic and commercially accessible PG monosaccharide units known as muramyl dipeptide (MDP) and N-glycolyl MDP (ng-MDP) have been used to probe the mechanism of innate immune activation of pattern recognition receptors, such as NOD-like receptors. However, bacterial PG is a dynamic and complex structure, with various chemical modifications and trimming mechanisms that result in the production of disaccharide-containing elements. These molecules pose as attractive targets for immunostimulatory screening; however, studies are limited because of their synthetic accessibility. Inspired by disaccharide-containing compounds produced from the gut microbe Lactobacillus acidophilus, a robust and scalable chemical synthesis of PG-based disaccharide ligands was implemented. Together with a monosaccharide PG library, compounds were screened for their ability to stimulate proinflammatory genes in bone-marrow-derived macrophages. The data reveal distinct gene induction patterns for monosaccharide and disaccharide PG units, suggesting that PG innate immune signaling is more complex than a one activator-one pathway program, as biologically relevant fragments induce transcriptional programs to different degrees. These disaccharide molecules will serve as critical immunostimulatory tools to more precisely define specialized innate immune regulatory mechanisms that distinguish between commensal and pathogenic bacteria residing in the microbiome.
RESUMO
BACKGROUND & AIMS: The commensal microbiota is believed to have an important role in regulating immune responsiveness and preventing intestinal inflammation. Intestinal microbes produce signals that regulate inflammation via Toll-like receptor (TLR) signaling, but the mechanisms of this process are poorly understood. We investigated the role of the anti-inflammatory cytokine interleukin (IL)-10 in this signaling pathway using a mouse model of colitis. METHODS: Clinical, histopathologic, and functional parameters of intestinal inflammation were evaluated in TLR4(-/-), IL-10(-/-), and TLR4(-/-) x IL-10(-/-) mice that were free of specific pathogens and in TLR4(-/-) x IL-10(-/-) mice following eradication and reintroduction of Helicobacter hepaticus. Regulatory T-cell (Treg) function was evaluated by crossing each of the lines with transgenic mice that express green fluorescent protein under control of the endogenous regulatory elements of Foxp3. Apoptotic cells in the colonic lamina propria were detected by a TUNEL assay. RESULTS: TLR4-mediated signals have 2 interrelated roles in promoting inflammation in TLR4(-/-) x IL-10(-/-) mice. In the absence of TLR4-mediated signals, secretion of proinflammatory and immunoregulatory cytokines is dysregulated. Tregs (Foxp3(+)) that secrete interferon-gamma and IL-17 accumulate in the colonic lamina propria of TLR4(-/-) x IL-10(-/-) mice and do not prevent inflammation. Aberrant control of epithelial cell turnover results in the persistence of antigen-presenting cells that contain apoptotic epithelial fragments in the colonic lamina propria of Helicobacter-infected TLR4(-/-) mice. CONCLUSIONS: In mice that lack both IL-10- and TLR4-mediated signals, aberrant regulatory T-cell function and dysregulated control of epithelial homeostasis combine to exacerbate intestinal inflammation.