RESUMO
The integrin α4ß7 selectively regulates lymphocyte trafficking and adhesion in the gut and gut-associated lymphoid tissue (GALT). Here, we describe unexpected involvement of the tyrosine phosphatase Shp1 and the B cell lectin CD22 (Siglec-2) in the regulation of α4ß7 surface expression and gut immunity. Shp1 selectively inhibited ß7 endocytosis, enhancing surface α4ß7 display and lymphocyte homing to GALT. In B cells, CD22 associated in a sialic acid-dependent manner with integrin ß7 on the cell surface to target intracellular Shp1 to ß7. Shp1 restrained plasma membrane ß7 phosphorylation and inhibited ß7 endocytosis without affecting ß1 integrin. B cells with reduced Shp1 activity, lacking CD22 or expressing CD22 with mutated Shp1-binding or carbohydrate-binding domains displayed parallel reductions in surface α4ß7 and in homing to GALT. Consistent with the specialized role of α4ß7 in intestinal immunity, CD22 deficiency selectively inhibited intestinal antibody and pathogen responses.
Assuntos
Linfócitos B/enzimologia , Imunidade nas Mucosas , Cadeias beta de Integrinas/metabolismo , Integrinas/metabolismo , Mucosa Intestinal/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/virologia , Quimiotaxia de Leucócito , Modelos Animais de Doenças , Endocitose , Feminino , Cadeias beta de Integrinas/imunologia , Integrinas/imunologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/virologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 6/deficiência , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Rotavirus/imunologia , Rotavirus/patogenicidade , Infecções por Rotavirus/genética , Infecções por Rotavirus/imunologia , Infecções por Rotavirus/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/deficiência , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/genética , Transdução de Sinais , Técnicas de Cultura de TecidosRESUMO
C-type lectin receptors sense a diversity of endogenous and exogenous ligands that may trigger differential responses. Here, we have found that human and mouse Mincle bind to a ligand released by Leishmania, a eukaryote parasite that evades an effective immune response. Mincle-deficient mice had milder dermal pathology and a tenth of the parasite burden compared to wild-type mice after Leishmania major intradermal ear infection. Mincle deficiency enhanced adaptive immunity against the parasite, correlating with increased activation, migration, and priming by Mincle-deficient dendritic cells (DCs). Leishmania triggered a Mincle-dependent inhibitory axis characterized by SHP1 coupling to the FcRγ chain. Selective loss of SHP1 in CD11c+ cells phenocopies enhanced adaptive immunity to Leishmania. In conclusion, Leishmania shifts Mincle to an inhibitory ITAM (ITAMi) configuration that impairs DC activation. Thus, ITAMi can be exploited for immune evasion by a pathogen and may represent a paradigm for ITAM-coupled receptors sensing self and non-self.
Assuntos
Imunidade Adaptativa/imunologia , Células Dendríticas/imunologia , Motivo de Ativação do Imunorreceptor Baseado em Tirosina/imunologia , Lectinas Tipo C/imunologia , Leishmania major/imunologia , Proteínas de Membrana/imunologia , Transdução de Sinais/imunologia , Animais , Antígeno CD11c/imunologia , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Receptores Fc/imunologiaRESUMO
The motheaten mouse has long served as a paradigm for complex autoimmune and inflammatory disease. Null mutations in Ptpn6, which encodes the nonreceptor protein-tyrosine phosphatase Shp1, cause the motheaten phenotype. However, Shp1 regulates multiple signaling pathways in different hematopoietic cell types, so the cellular and molecular mechanism of autoimmunity and inflammation in the motheaten mouse has remained unclear. By using floxed Ptpn6 mice, we dissected the contribution of innate immune cells to the motheaten phenotype. Ptpn6 deletion in neutrophils resulted in cutaneous inflammation, but not autoimmunity, providing an animal model of human neutrophilic dermatoses. By contrast, dendritic cell deletion caused severe autoimmunity, without inflammation. Genetic and biochemical analysis showed that inflammation was caused by enhanced neutrophil integrin signaling through Src-family and Syk kinases, whereas autoimmunity resulted from exaggerated MyD88-dependent signaling in dendritic cells. Our data demonstrate that disruption of distinct Shp1-regulated pathways in different cell types combine to cause motheaten disease.
Assuntos
Autoimunidade/imunologia , Células Dendríticas/imunologia , Inflamação/imunologia , Neutrófilos/imunologia , Animais , Autoimunidade/genética , Antígeno CD11c/genética , Antígeno CD11c/imunologia , Antígeno CD11c/metabolismo , Calgranulina A/genética , Calgranulina A/imunologia , Calgranulina A/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Células Dendríticas/metabolismo , Derme/imunologia , Derme/metabolismo , Derme/patologia , Feminino , Citometria de Fluxo , Humanos , Immunoblotting , Inflamação/genética , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , Neutrófilos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo , Quinase Syk , Quinases da Família src/imunologia , Quinases da Família src/metabolismoRESUMO
BACKGROUND: Spinal cord injury elicits widespread inflammation that can exacerbate long-term neurologic deficits. Neutrophils are the most abundant immune cell type to invade the spinal cord in the early acute phase after injury, however, their role in secondary pathogenesis and functional recovery remains unclear. We have previously shown that neutrophil functional responses during inflammation are augmented by spleen tyrosine kinase, Syk, a prominent intracellular signaling enzyme. In this study, we evaluated the contribution of Syk towards neutrophil function and long-term neurologic deficits after spinal cord injury. METHODS: Contusive spinal cord injury was performed at thoracic vertebra level 9 in mice with conditional deletion of Syk in neutrophils (Sykf/fMRP8-Cre). Hindlimb locomotor recovery was evaluated using an open-field test for 35 days following spinal cord injury. Long-term white matter sparing was assessed using eriochrome cyanide staining. Blood-spinal cord barrier disruption was evaluated by immunoblotting. Neutrophil infiltration, activation, effector functions, and cell death were determined by flow cytometry. Cytokine and chemokine expression in neutrophils was assessed using a gene array. RESULTS: Syk deficiency in neutrophils improved long-term functional recovery after spinal cord injury, but did not promote long-term white matter sparing. Neutrophil activation, cytokine expression, and cell death in the acutely injured spinal cord were attenuated by the genetic loss of Syk while neutrophil infiltration and effector functions were not affected. Acute blood-spinal cord barrier disruption was also unaffected by Syk deficiency in neutrophils. CONCLUSIONS: Syk facilitates specific neutrophil functional responses to spinal cord injury including activation, cytokine expression, and cell death. Long-term neurologic deficits are exacerbated by Syk signaling in neutrophils independent of acute blood-spinal cord barrier disruption and long-term white matter sparing. These findings implicate Syk in pathogenic neutrophil activities that worsen long-term functional recovery after spinal cord injury.
Assuntos
Doenças do Sistema Nervoso/etiologia , Doenças do Sistema Nervoso/patologia , Ativação de Neutrófilo , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/patologia , Baço/enzimologia , Quinase Syk/genética , Animais , Apoptose , Morte Celular , Quimiocinas/biossíntese , Citocinas/biossíntese , Feminino , Membro Posterior/inervação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos , Recuperação de Função Fisiológica , Substância Branca/patologiaRESUMO
C-type lectin receptors (CLRs) play key roles in antifungal defense. CLR-induced NF-κB is central to CLR functions in immunity, and thus, molecules that control the amplitude of CLR-induced NF-κB could profoundly influence host defense against fungal pathogens. However, little is known about the mechanisms that negatively regulate CLR-induced NF-κB, and molecules which act on the CLR family broadly and which directly regulate acute CLR-signaling cascades remain unidentified. Here, we identify the ubiquitin-editing enzyme A20 as a negative regulator of acute NF-κB activation downstream of multiple CLR pathways. Absence of A20 suppression results in exaggerated CLR responses in cells which are A20 deficient and also cells which are A20 haplosufficient, including multiple primary immune cells. Loss of a single allele of A20 results in enhanced defense against systemic Candida albicans infection and prolonged host survival. Thus, A20 restricts CLR-induced innate immune responses in vivo and is a suppressor of host defense against systemic fungal infection.
Assuntos
Candida albicans/imunologia , Candidíase/imunologia , Interações entre Hospedeiro e Microrganismos/imunologia , Lectinas Tipo C/imunologia , Processamento de Proteína Pós-Traducional , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/microbiologia , Candida albicans/patogenicidade , Candidíase/genética , Candidíase/microbiologia , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Feminino , Feto , Interações entre Hospedeiro e Microrganismos/genética , Imunidade Inata , Lectinas Tipo C/genética , Fígado/imunologia , Fígado/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Cultura Primária de Células , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/imunologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/deficiência , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Ubiquitina/genética , Ubiquitina/imunologia , UbiquitinaçãoRESUMO
Neutrophil recruitment to the site of inflammation plays a pivotal role in host defense. However, overwhelming activation and accumulation of neutrophils in the tissue may cause tissue damage and autoimmunity due to the release of cytokines, oxidants, and proteases. Neutrophil adhesion in acute inflammation is initiated by activation of αLß2 (LFA-1), which can be induced by rolling on E-selectin (slowly) or by exposure to the chemokine CXCL1 (rapidly). Despite the clinical importance, cell-intrinsic molecular mechanisms of negative regulation of integrin adhesiveness and neutrophil recruitment are poorly understood. Mice deficient in the tyrosine phosphatase Src homology 2 domain-containing protein tyrosine phosphatase 1 (Shp1) show increased leukocyte adhesion, but the interpretation of these data is limited by the severe global phenotype of these mice. In this study, we used mice with global and myeloid-restricted deletion of Shp1 to study neutrophil arrest, adhesion, crawling, and transendothelial migration in vitro and in vivo. Shp1 deficiency results in increased neutrophil adhesion in vivo; however, neutrophil crawling, transmigration, and chemotaxis were reduced in these mice. Mechanistically, Shp1 binds and controls PIPKIγ activity and, thereby, modulates phosphatidylinositol (4,5)-bisphosphate levels and adhesion. Thus, Shp1 is involved in the deactivation of integrins and regulation of neutrophil recruitment into inflamed tissue.
Assuntos
Adesão Celular/imunologia , Infiltração de Neutrófilos/imunologia , Fosfatidilinositóis/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Animais , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/imunologia , Quimiocina CXCL1/imunologia , Selectina E/imunologia , Ativação Enzimática/imunologia , Células HL-60 , Humanos , Inflamação/imunologia , Migração e Rolagem de Leucócitos/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infiltração de Neutrófilos/genética , Neutrófilos/imunologia , Fosfatidilinositóis/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismoRESUMO
Autoinflammatory diseases include a number of monogenic systemic inflammatory diseases, as well as acquired autoinflammatory diseases such as gout. Here, we show that the myeloid Src-family kinases Hck, Fgr, and Lyn are critical for experimental models of gout, as well as for genetically determined systemic inflammation in the Ptpn6me-v/me-v (motheaten viable) mouse model. The Hck-/-Fgr-/-Lyn-/- mutation abrogated various monosodium urate (MSU) crystal-induced pro-inflammatory responses of neutrophils, and protected mice from the development of gouty arthritis. The Src-family inhibitor dasatinib abrogated MSU crystal-induced responses of human neutrophils and reduced experimental gouty arthritis in mice. The Hck-/-Fgr-/-Lyn-/- mutation also abrogated spontaneous inflammation and prolonged the survival of the Ptpn6me-v/me-v mice. Spontaneous adhesion and superoxide release of Ptpn6me-v/me-v neutrophils were also abolished by the Hck-/-Fgr-/-Lyn-/- mutation. Excessive activation of tyrosine phosphorylation pathways in myeloid cells may characterize a subset of autoinflammatory diseases.
Assuntos
Artrite Gotosa , Gota , Doenças Hereditárias Autoinflamatórias , Camundongos , Humanos , Animais , Quinases da Família src/metabolismo , Neutrófilos/metabolismo , Proteínas Proto-Oncogênicas c-hck/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Artrite Gotosa/metabolismo , Gota/metabolismo , Inflamação/metabolismo , Doenças Hereditárias Autoinflamatórias/metabolismoRESUMO
Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq), to profile mouse autoantibodies. This system and library were validated using seven genetic mouse models across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2-/- and µMT) were used to model non-specific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor (BCR)-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate three distinct autoimmune disease models. First, serum from Lyn-/- IgD+/- mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities, lending support to the hypothesis that apoptosis is a shared origin of these antigens. Second, serum from non-obese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, enriched peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire-/- mouse sera were used to identify numerous auto-antigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 (APS1) carrying recessive mutations in AIRE. Among these were peptides derived from Perilipin-1, a validated autoimmune biomarker of generalized acquired lipodystrophy in humans. Autoreactivity to Perilipin-1 correlated with lymphocyte infiltration in adipose tissue and underscores the approach in revealing previously unknown specificities. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity.
RESUMO
Autoimmunity is characterized by loss of tolerance to tissue-specific as well as systemic antigens, resulting in complex autoantibody landscapes. Here, we introduce and extensively validate the performance characteristics of a murine proteome-wide library for phage display immunoprecipitation and sequencing (PhIP-seq) in profiling mouse autoantibodies. This library was validated using 7 genetically distinct mouse lines across a spectrum of autoreactivity. Mice deficient in antibody production (Rag2-/- and µMT) were used to model nonspecific peptide enrichments, while cross-reactivity was evaluated using anti-ovalbumin B cell receptor-restricted OB1 mice as a proof of principle. The PhIP-seq approach was then utilized to interrogate 3 distinct autoimmune disease models. First, serum from Lyn-/- IgD+/- mice with lupus-like disease was used to identify nuclear and apoptotic bleb reactivities. Second, serum from nonobese diabetic (NOD) mice, a polygenic model of pancreas-specific autoimmunity, was enriched in peptides derived from both insulin and predicted pancreatic proteins. Lastly, Aire-/- mouse sera were used to identify numerous autoantigens, many of which were also observed in previous studies of humans with autoimmune polyendocrinopathy syndrome type 1 carrying recessive mutations in AIRE. These experiments support the use of murine proteome-wide PhIP-seq for antigenic profiling and autoantibody discovery, which may be employed to study a range of immune perturbations in mouse models of autoimmunity profiling.
Assuntos
Autoanticorpos , Bacteriófagos , Humanos , Animais , Camundongos , Proteoma , Autoimunidade , Peptídeos , Camundongos Endogâmicos NODRESUMO
DAP12 is an adapter protein that associates with several receptors in macrophages. Little is known about the biological role of DAP12 in alveolar macrophages. In genome-wide profiling, we previously found that two DAP12-associated receptors, myeloid DAP12-associated lectin-1 and triggering receptor expressed on myeloid cells 2 (TREM2), were highly induced in alveolar macrophages from habitual smokers. Here, we found that transcript levels for these receptors in alveolar macrophages increased with packs per day of cigarettes smoked and expression of TREM2 protein was increased in lung macrophages of former smokers with emphysema compared with that in controls. In vitro, cigarette smoke directly induced expression of myeloid DAP12-associated lectin-1 and TREM2 and activation of DAP12 signaling in mouse macrophages. To determine whether DAP12 plays a role in cigarette smoke-induced pulmonary inflammation, we exposed wild-type and DAP12-deficient mice to chronic cigarette smoke and found significant reduction in recruitment of alveolar macrophages in DAP12-deficient mice. Because cigarette smoking induces the macrophage chemoattractant CCL2, we tested the chemotactic ability of DAP12-deficient macrophages and found abrogation of chemotaxis toward CCL2 in vitro. Airway administration of CCL2 also resulted in a significant reduction of macrophage recruitment to the lungs of DAP12-deficient mice compared with that in controls. DAP12 was also required for normal macrophage migration in a "scratch" assay. Reconstitution studies revealed that phosphorylation of the DAP12 ITAM was required for normal migration in vitro and association with TREM2 was sufficient for normal migration. These findings indicate that DAP12, possibly through association with TREM2, contributes to alveolar macrophage chemotaxis and recruitment to the lung and may mediate macrophage accumulation in lung diseases such as emphysema.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Quimiocina CCL2/metabolismo , Quimiotaxia de Leucócito/imunologia , Pulmão/imunologia , Macrófagos Alveolares/metabolismo , Proteínas de Membrana/metabolismo , Fumaça/efeitos adversos , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Western Blotting , Separação Celular , Quimiocina CCL2/imunologia , Citometria de Fluxo , Imunofluorescência , Humanos , Imunoprecipitação , Macrófagos Alveolares/imunologia , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doença Pulmonar Obstrutiva Crônica/imunologia , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Background: Psoriasis is a chronic skin disease associated with deregulated interplays between immune cells and keratinocytes. Neutrophil accumulation in the skin is a histological feature that characterizes psoriasis. However, the role of neutrophils in psoriasis onset and development remains poorly understood. Methods: In this study, we utilized the model of psoriasiform dermatitis, caused by the repeated topical application of an imiquimod containing cream, in neutrophil-depleted mice or in mice carrying impairment in neutrophil functions, including p47phox -/- mice (lacking a cytosolic subunit of the phagocyte nicotinamide adenine dinucleotide phosphate - NADPH - oxidase) and Sykfl/fl MRP8-cre+ mice (carrying the specific deletion of the Syk kinase in neutrophils only), to elucidate the specific contribution of neutrophils to psoriasis development. Results: By analyzing disease development/progression in neutrophil-depleted mice, we now report that neutrophils act as negative modulators of disease propagation and exacerbation by inhibiting gammadelta T cell effector functions via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated reactive oxygen species (ROS) production. We also report that Syk functions as a crucial molecule in determining the outcome of neutrophil and γδ T cell interactions. Accordingly, we uncover that a selective impairment of Syk-dependent signaling in neutrophils is sufficient to reproduce the enhancement of skin inflammation and γδ T cell infiltration observed in neutrophil-depleted mice. Conclusions: Overall, our findings add new insights into the specific contribution of neutrophils to disease progression in the IMQ-induced mouse model of psoriasis, namely as negative regulatory cells.
Assuntos
Eczema , Psoríase , Camundongos , Animais , Imiquimode , Neutrófilos , NADP , Psoríase/induzido quimicamente , Modelos Animais de Doenças , NADPH Oxidases/genética , Progressão da DoençaRESUMO
Shp1, encoded by the gene Ptpn6, is a protein tyrosine phosphatase that transduces inhibitory signals downstream of immunoreceptors in many immune cell types. Blocking Shp1 activity represents an exciting potential immunotherapeutic strategy for the treatment of cancer, as Shp1 inhibition would be predicted to unleash both innate and adaptive immunity against tumor cells. Antibodies blocking the interaction between CD47 on tumor cells and SIRPα on macrophages enhance macrophage phagocytosis, show efficacy in preclinical tumor models, and are being evaluated in the clinic. Here we found that Shp1 bound to phosphorylated peptide sequences derived from SIRPα and transduced the anti-phagocytic signal, as Shp1 loss in mouse bone marrow-derived macrophages increased phagocytosis of tumor cells in vitro. We also generated a novel mouse model to evaluate the impact of global, inducible Ptpn6 deletion on anti-tumor immunity. We found that inducible Shp1 loss drove an inflammatory disease in mice that was phenotypically similar to that seen when Ptpn6 is knocked out from birth. This indicates that acute perturbation of Shp1 in vivo could drive hyperactivation of immune cells, which could be therapeutically beneficial, though at the risk of potential toxicity. In this model, we found that Shp1 loss led to robust anti-tumor immunity against two immune-rich syngeneic tumor models that are moderately inflamed though not responsive to checkpoint inhibitors, MC38 and E0771. Shp1 loss did not promote anti-tumor activity in the non-inflamed B16F10 model. The observed activity in MC38 and E0771 tumors was likely due to effects of both innate and adaptive immune cells. Following Shp1 deletion, we observed increases in intratumoral myeloid cells in both models, which was more striking in E0771 tumors. E0771 tumors also contained an increased ratio of effector to regulatory T cells following Shp1 loss. This was not observed for MC38 tumors, though we did find increased levels of IFNγ, a cytokine produced by effector T cells, in these tumors. Overall, our preclinical data suggested that targeting Shp1 may be an attractive therapeutic strategy for boosting the immune response to cancer via a mechanism involving both innate and adaptive leukocytes.
Assuntos
Adenocarcinoma/enzimologia , Neoplasias da Mama/enzimologia , Neoplasias do Colo/enzimologia , Melanoma Experimental/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/deficiência , Neoplasias Cutâneas/enzimologia , Macrófagos Associados a Tumor/enzimologia , Imunidade Adaptativa , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Animais , Antígenos de Diferenciação/metabolismo , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Neoplasias do Colo/terapia , Feminino , Humanos , Imunidade Inata , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Receptores Imunológicos/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , Células THP-1 , Carga Tumoral , Microambiente Tumoral , Macrófagos Associados a Tumor/imunologiaRESUMO
CARD9 is an immune adaptor protein in myeloid cells that is involved in C-type lectin signaling and antifungal immunity. CARD9 is implicated in autoimmune and inflammatory-related diseases, such as rheumatoid arthritis, IgA nephropathy, ankylosing spondylitis, and inflammatory bowel disease (IBD). Given that Lyn-deficient (Lyn-/-) mice are susceptible to both autoimmunity and IBD, we investigated the immunological role of CARD9 in the development of these diseases using the Lyn-/- mouse model. We found that genetic deletion of CARD9 was sufficient to reduce the development of both spontaneous autoimmune disease as well as DSS- or IL-10 deficiency-associated colitis in Lyn-/- mice. Mechanistically, CARD9 was a vital component of the Lyn-mediated regulation of Toll-like receptor (TLR2 and TLR4) signaling in dendritic cells, but not in macrophages. In the absence of Lyn, signaling through a CD11b-Syk-PKCδ-CARD9 pathway was amplified, leading to increased TLR-induced production of inflammatory cytokines. Dendritic cell-specific deletion of CARD9 reversed the development of autoimmune and experimental colitis observed in dendritic cell-specific, Lyn-deficient mice. These findings suggest that targeting CARD9 may suppress the development of colitis and autoimmunity by reducing dendritic cell-driven inflammation.
Assuntos
Autoimunidade , Proteínas Adaptadoras de Sinalização CARD/deficiência , Células Dendríticas/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Quinases da Família src/deficiência , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Modelos Animais de Doenças , Doenças Inflamatórias Intestinais/genética , Interleucina-10/deficiência , Interleucina-10/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína Quinase C-delta/genética , Proteína Quinase C-delta/metabolismo , Interferência de RNA , Transdução de Sinais , Quinase Syk/genética , Quinase Syk/metabolismo , Receptores Toll-Like/metabolismo , Quinases da Família src/genéticaRESUMO
Macrophages are key components of the innate immune response. These cells possess a diverse repertoire of receptors that allow them to respond to a host of external stimuli including cytokines, chemokines, and pathogen-associated molecules. Signals resulting from these stimuli activate a number of macrophage functional responses such as adhesion, migration, phagocytosis, proliferation, survival, cytokine release and production of reactive oxygen and nitrogen species. The cytoplasmic tyrosine kinase Src and its family members (SFKs) have been implicated in many intracellular signaling pathways in macrophages, initiated by a diverse set of receptors ranging from integrins to Toll-like receptors. However, it has been difficult to implicate any given member of the family in any specific pathway. SFKs appear to have overlapping and complementary functions in many pathways. Perhaps the function of these enzymes is to modulate the overall intracellular signaling network in macrophages, rather than operating as exclusive signaling switches for defined pathways. In general, SFKs may function more like rheostats, influencing the amplitude of many pathways.
Assuntos
Macrófagos/enzimologia , Quinases da Família src/metabolismo , Animais , Humanos , Células de Kupffer/enzimologia , Células de Kupffer/imunologia , Células de Langerhans/enzimologia , Células de Langerhans/imunologia , Macrófagos/imunologia , Macrófagos Alveolares/enzimologia , Macrófagos Alveolares/imunologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Citocinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Toll-Like/metabolismoRESUMO
The immunoreceptor tyrosine-based activation motif (ITAM) is the primary signaling domain used by classical immunoreceptors, such as the antigen receptors on B and T lymphocytes and the Fc receptors (FcRs) on myeloid cells. The ITAM is contained in the intracellular region of subunits associated with these receptors, often in pairs, or is part of the cytoplasmic domain of the receptors themselves. Data from many investigators have demonstrated that ITAMs are both necessary and sufficient for initiation of signaling downstream of all immunoreceptors. More recent reports indicate that ITAM signaling is used by additional receptors beyond the classical immunoreceptors: Cell adhesion molecules (integrins and PSGL-1), chemokine receptors (CXCR4), plexins, and lectin receptors all mediate immune cell function through ITAM-like signaling pathways. This convergence of intracellular signaling pathways in leukocytes illuminates the importance of tyrosine-based activation motifs in the immune system and suggests that inhibitors of ITAM signaling may have broader effects than originally envisioned.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Leucócitos/metabolismo , Transdução de Sinais , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Genes MHC da Classe II/imunologia , Humanos , Integrinas/metabolismo , Leucócitos/imunologia , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Quimiocinas/metabolismo , Receptores Mitogênicos/metabolismo , Proteínas Virais/metabolismoRESUMO
The motheaten mouse was first described in 1975 as a model of systemic inflammation and autoimmunity, as a result of immune system dysregulation. The phenotype was later ascribed to mutations in the cytoplasmic tyrosine phosphatase Shp1. This phosphatase is expressed widely throughout the hematopoietic system and has been shown to impact a multitude of cell signaling pathways. The determination of which cell types contribute to the different aspects of the phenotype caused by global Shp1 loss or mutation and which pathways within these cell types are regulated by Shp1 is important to further our understanding of immune system regulation. In this review, we focus on the role of Shp1 in myeloid cells and how its dysregulation affects immune function, which can impact human disease.
Assuntos
Doenças Autoimunes/imunologia , Mutação , Células Mieloides/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Transdução de Sinais/imunologia , Animais , Doenças Autoimunes/enzimologia , Doenças Autoimunes/genética , Doenças Autoimunes/patologia , Camundongos , Células Mieloides/enzimologia , Células Mieloides/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Transdução de Sinais/genéticaAssuntos
Plaquetas/metabolismo , Cadeias beta de Integrinas/metabolismo , Síndrome da Aderência Leucocítica Deficitária/imunologia , Leucócitos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Actinas/imunologia , Actinas/metabolismo , Regulação Alostérica , Plaquetas/imunologia , Plaquetas/patologia , Adesão Celular/genética , Adesão Celular/imunologia , Predisposição Genética para Doença , Humanos , Cadeias beta de Integrinas/genética , Cadeias beta de Integrinas/imunologia , Síndrome da Aderência Leucocítica Deficitária/etiologia , Síndrome da Aderência Leucocítica Deficitária/genética , Leucócitos/imunologia , Leucócitos/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Polimorfismo Genético , Ligação Proteica , Transdução de Sinais/imunologiaRESUMO
Since the first example of conditional gene targeting in mice in 1994, the use of Cre recombinase and loxP flanked sequences has become an invaluable technique to generate tissue and temporal specific gene knockouts. The number of mouse strains expressing floxed-gene sequences, and tissue-specific or temporal-specific Cre-recombinase that have been reported in the literature has grown exponentially. However, increased use of this technology has highlighted several problems that can impact the interpretation of any phenotype observed in these mouse models. In particular, accurate knowledge of the specificity of Cre expression in each strain is critical in order to make conclusions about the role of specific cell types in the phenotypes observed. Cre-mediated deletion specificity and efficiency have been described in many different ways in the literature, making direct comparisons between these Cre strains impossible. Here we report crossing thirteen different myeloid-Cre mouse strains to ROSA-EYFP reporter mice and assaying YFP expression in a variety of naïve unstimulated hematopoietic cells, in parallel. By focusing on myeloid subsets, we directly compare the relative efficiency and specificity of myeloid deletion in these strains under steady-state conditions.
Assuntos
Proteínas de Bactérias/biossíntese , Deleção de Genes , Marcação de Genes , Genes Reporter , Integrases/genética , Proteínas Luminescentes/biossíntese , Células Mieloides/metabolismo , RNA não Traduzido/genética , Animais , Proteínas de Bactérias/genética , Citometria de Fluxo , Genótipo , Proteínas Luminescentes/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , FenótipoRESUMO
The TEL-Syk fusion protein was isolated from a patient with myelodysplasia with megakaryocyte blasts. Expression of TEL-Syk transforms interleukin-3 (IL-3)-dependent Ba/F3 cells in vitro by deregulating STAT5-mediated signal transduction pathways. In vivo, TEL-Syk expression in pre-B cells blocks B cell differentiation, leading to lymphoid leukemia. Here, we demonstrate that TEL-Syk introduced into fetal liver hematopoietic cells, which are then adoptively transferred into lethally irradiated recipients, leads to an aggressive myelodysplasia with myelofibrosis that is lethal in mice by 60-75 days. Expression of TEL-Syk induces a short-lived myeloexpansion that is rapidly followed by bone marrow failure and extreme splenic/hepatic fibrosis accompanied by extensive apoptosis. The disease is dependent on Syk kinase activity. Analysis of serum from TEL-Syk mice reveals an inflammatory cytokine signature reminiscent of that found in the sera from patients and mouse models of myeloproliferative neoplasms. TEL-Syk expressing cells showed constitutive STAT5 phosphorylation, which was resistant to JAK inhibition, consistent with deregulated cytokine signaling. These data indicate that expression of TEL-Syk in fetal liver hematopoietic cells results in JAK-independent STAT5 phosphorylation ultimately leading to a uniquely aggressive and lethal form of myelofibrosis.
Assuntos
Expressão Gênica , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Proteínas de Fusão Oncogênica/genética , Mielofibrose Primária/genética , Anemia/genética , Animais , Medula Óssea/patologia , Proliferação de Células , Quimera , Células HEK293 , Hematopoese Extramedular , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , Mielofibrose Primária/patologia , Fator de Transcrição STAT5/metabolismo , Esplenomegalia/genética , Trombocitopenia/genéticaRESUMO
Integrins are the principal cell adhesion receptors that mediate leukocyte migration and activation in the immune system. These receptors signal bidirectionally through the plasma membrane in pathways referred to as inside-out and outside-in signaling. Each of these pathways is mediated by conformational changes in the integrin structure. Such changes allow high-affinity binding of the receptor with counter-adhesion molecules on the vascular endothelium or extracellular matrix and lead to association of the cytoplasmic tails of the integrins with intracellular signaling molecules. Leukocyte functional responses resulting from outside-in signaling include migration, proliferation, cytokine secretion, and degranulation. Here, we review the key signaling events that occur in the inside-out versus outside-in pathways, highlighting recent advances in our understanding of how integrins are activated by a variety of stimuli and how they mediate a diverse array of cellular responses.