RESUMO
Background: The immune cell topography of solid tumors has been increasingly recognized as an important predictive factor for progression of disease and response to immunotherapy. The distribution pattern of immune cells in solid tumors is commonly classified into three categories - namely, "Immune inflamed", "Immune desert" and "Immune excluded" - which, to some degree, connect immune cell presence and positioning within the tumor microenvironment to anti-tumor activity. Materials and methods: In this review, we look at the ways immune exclusion has been defined in published literature and identify opportunities to develop consistent, quantifiable definitions, which in turn, will allow better determination of the underlying mechanisms that span cancer types and, ultimately, aid in the development of treatments to target these mechanisms. Results: The definitions of tumor immune phenotypes, especially immune exclusion, have largely been conceptual. The existing literature lacks in consistency when it comes to practically defining immune exclusion, and there is no consensus on a definition. Majority of the definitions use somewhat arbitrary cut-offs in an attempt to place each tumor into a distinct phenotypic category. Tumor heterogeneity is often not accounted for, which limits the practical application of a definition. Conclusions: We have identified two key issues in existing definitions of immune exclusion, establishing clinically relevant cut-offs within the spectrum of immune cell infiltration as well as tumor heterogeneity. We propose an approach to overcome these limitations, by reporting the degree of immune cell infiltration, tying cut-offs to clinically meaningful outcome measures, maximizing the number of regions of a tumor that are analyzed and reporting the degree of heterogeneity. This will allow for a consensus practical definition for operationalizing this categorization into clinical trial and signal-seeking endpoints.
Assuntos
Neoplasias , Evasão Tumoral , Humanos , Imunoterapia , Neoplasias/imunologia , Neoplasias/terapia , Microambiente Tumoral/imunologia , Consenso , Evasão Tumoral/imunologiaRESUMO
As an anti-inflammatory strategy, MAPK-activated protein kinase-2 (MK2) inhibition can potentially avoid the clinical failures seen for direct p38 inhibitors, especially tachyphylaxis. CC-99677, a selective targeted covalent MK2 inhibitor, employs a rare chloropyrimidine that bonds to the sulfur of cysteine 140 in the ATP binding site via a nucleophilic aromatic substitutions (SNAr) mechanism. This irreversible mechanism translates biochemical potency to cells shown by potent inhibition of heat shock protein 27 (HSP27) phosphorylation in LPS-activated monocytic THP-1 cells. The cytokine inhibitory profile of CC-99677 differentiates it from known p38 inhibitors, potentially suppressing a p38 pathway inflammatory response while avoiding tachyphylaxis. Dosed orally, CC-99677 is efficacious in a rat model of ankylosing spondylitis. Single doses, 3 to 400 mg, in healthy human volunteers show linear pharmacokinetics and apparent sustained tumor necrosis factor-α inhibition, with a favorable safety profile. These results support further development of CC-99677 for autoimmune diseases like ankylosing spondylitis.
Assuntos
Doenças Autoimunes , Espondilite Anquilosante , Trifosfato de Adenosina , Animais , Anti-Inflamatórios , Doenças Autoimunes/tratamento farmacológico , Cisteína , Proteínas de Choque Térmico HSP27/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lipopolissacarídeos , Proteínas Serina-Treonina Quinases , Ratos , Enxofre , Fator de Necrose Tumoral alfa , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Expressed strongly by myeloid cells, damage-associated molecular pattern (DAMP) proteins S100A8 and S100A9 are found in the serum of patients with infectious and autoimmune diseases. Compared to S100A9, the role of S100A8 is controversial. We investigated its biological activity in collagen-induced arthritis using the first known viable and fertile S100a8-deficient (S100a8-/-) mouse. Although comparable to the wild type (WT) in terms of lymphocyte distribution in blood and in the primary and secondary lymphoid organs, S100a8-/- mice had increased numbers of neutrophils, monocytes and dendritic cells in the blood and bone marrow, and these all expressed myeloid markers such as CD11b, Ly6G and CD86 more strongly. Granulocyte-macrophage common precursors were increased in S100a8-/- bone marrow and yielded greater numbers of macrophages and dendritic cells in culture. The animals also developed more severe arthritic disease leading to aggravated osteoclast activity and bone destruction. These findings were correlated with increased inflammatory cell infiltration and cytokine secretion in the paws. This study suggests that S100A8 is an anti-inflammatory DAMP that regulates myeloid cell differentiation, thereby mitigating the development of experimental arthritis.
Assuntos
Artrite Experimental/patologia , Calgranulina A/deficiência , Mielopoese , Animais , Artrite Experimental/diagnóstico por imagem , Medula Óssea/patologia , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/patologia , Calgranulina A/metabolismo , Cartilagem/patologia , Diferenciação Celular , Células Dendríticas/metabolismo , Feminino , Deleção de Genes , Camundongos , Células Mieloides/patologiaRESUMO
Progressive lung disease with early onset is the main cause of mortality and morbidity in cystic fibrosis patients. Here we report a reduction of sphingosine-1-phosphate (S1P) in the lung of unchallenged Cftrtm1EUR F508del CFTR mutant mice. This correlates with enhanced infiltration by inducible nitric oxide synthase (iNOS)-expressing granulocytes, B cells, and T cells. Furthermore, the ratio of macrophage-derived dendritic cells (MoDC) to conventional dendritic cells (cDC) is higher in mutant mouse lung, consistent with unprovoked inflammation. Oral application of a S1P lyase inhibitor (LX2931) increases S1P levels in mutant mouse tissues. This normalizes the lung MoDC/cDC ratio and reduces B and T cell counts. Lung granulocytes are enhanced, but iNOS expression is reduced in this population. Although lung LyC6+ monocytes are enhanced by LX2931, they apparently do not differentiate to MoDC and macrophages. After challenge with bacterial toxins (LPS-fMLP) we observe enhanced levels of proinflammatory cytokines TNF-α, KC, IFNγ, and IL-12 and the inducible mucin MUC5AC in mutant mouse lung, evidence of deficient resolution of inflammation. LX2931 does not prevent transient inflammation or goblet cell hyperplasia after challenge, but it reduces MUC5AC and proinflammatory cytokine levels toward normal values. We conclude that lung pathology in homozygous mice expressing murine F508del CFTR, which represents the most frequent mutation in CF patients, is characterized by abnormal behavior of infiltrating myeloid cells and delayed resolution of induced inflammation. This phenotype can be partially corrected by a S1P lyase inhibitor, providing a rationale for therapeutic targeting of the S1P signaling pathway in CF patients.
Assuntos
Aldeído Liases/antagonistas & inibidores , Fibrose Cística/tratamento farmacológico , Inibidores Enzimáticos/uso terapêutico , Imidazóis/uso terapêutico , Oximas/uso terapêutico , Pneumonia/tratamento farmacológico , Aldeído Liases/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Fibrose Cística/diagnóstico por imagem , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Imidazóis/farmacologia , Lipopolissacarídeos/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Lisofosfolipídeos/metabolismo , Camundongos Endogâmicos C57BL , Mucina-5AC/metabolismo , Mutação/genética , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Oximas/farmacologia , Pneumonia/diagnóstico por imagem , Pneumonia/patologia , Glândulas Salivares/efeitos dos fármacos , Glândulas Salivares/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Microtomografia por Raio-XRESUMO
The bioactive sphingolipid sphingosine-1-phosphate (S1P) and the kinase that produces it have been implicated in inflammatory bowel diseases in mice and humans; however, little is known about the role of the 2 S1P-specific phosphohydrolase isoforms, SGPP1 and SGPP2, which catalyze dephosphorylation of S1P to sphingosine. To elucidate their functions, we generated specific knockout mice. Deletion of Sgpp2, which is mainly expressed in the gastrointestinal tract, significantly reduced dextran sodium sulfate (DSS)-induced colitis severity, whereas deletion of ubiquitously expressed Sgpp1 slightly worsened colitis. Moreover, Sgpp1 deletion enhanced expression of multifunctional proinflammatory cytokines, IL-6, TNF-α, and IL-1ß, activation of the transcription factor signal transducer and activator of transcription 3, and immune cell infiltration into the colon. Conversely, Sgpp2-null mice failed to mount a DSS-induced systemic inflammatory response. Of interest, Sgpp2 deficiency suppressed DSS-induced intestinal epithelial cell apoptosis and improved mucosal barrier integrity. Furthermore, down-regulation of Sgpp2 attenuated LPS-induced paracellular permeability in cultured cells and enhanced expression of the adherens junction protein E-cadherin. Finally, in patients with ulcerative colitis, SGPP2 expression was elevated in colitis tissues relative to that in uninvolved tissues. These results indicate that induction of SGPP2 expression contributes to the pathogenesis of colitis by promoting disruption of the mucosal barrier function. SGPP2 may represent a novel therapeutic target in inflammatory bowel disease.-Huang, W.-C., Liang, J., Nagahashi, M., Avni, D., Yamada, A., Maceyka, M., Wolen, A. R., Kordula, T., Milstien, S., Takabe, K., Oravecz, T., Spiegel, S. Sphingosine-1-phosphate phosphatase 2 promotes disruption of mucosal integrity, and contributes to ulcerative colitis in mice and humans.
Assuntos
Colite Ulcerativa/metabolismo , Mucosa Intestinal/patologia , Proteínas de Membrana/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Caderinas , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/genética , Sulfato de Dextrana/toxicidade , Regulação para Baixo , Humanos , Inflamação/metabolismo , Mucosa Intestinal/enzimologia , Lipopolissacarídeos/toxicidade , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Permeabilidade , Monoéster Fosfórico Hidrolases/genéticaRESUMO
Sphingosine 1-phosphate (S1P) is a pleiotropic bioactive sphingolipid metabolite that regulates numerous processes important for immune responses. S1P is made within cells and must be transported out of cells to exert its effects through activation of 5 specific cell surface GPCRs in an autocrine or paracrine fashion. Spinster 2 (Spns2) transports S1P out of cells, and its deletion in mice reduces circulating levels of S1P, alters immune cell trafficking, and induces lymphopenia. Here we examined the effects of Spns2 deletion on adaptive immune responses and in autoimmune disease models. Airway inflammation and hypersensitivity as well as delayed-type contact hypersensitivity were attenuated in Spns2(-/-) mice. Similarly, Spns2 deletion reduced dextran sodium sulfate- and oxazolone-induced colitis. Intriguingly, Spns2(-/-) mice were protected from the development of experimental autoimmune encephalopathy, a model of the autoimmune disease multiple sclerosis. Deletion of Spns2 also strongly alleviated disease development in collagen-induced arthritis. These results point to a broad role for Spns2-mediated S1P transport in the initiation and development of adaptive immune related disorders.
Assuntos
Proteínas de Transporte de Ânions/fisiologia , Doenças Autoimunes/fisiopatologia , Inflamação/fisiopatologia , Animais , Proteínas de Transporte de Ânions/genética , Modelos Animais de Doenças , Camundongos , Camundongos KnockoutRESUMO
OBJECTIVES: To investigate the role of liver X receptors (LXRs) in experimental skin fibrosis and evaluate their potential as novel antifibrotic targets. METHODS: We studied the role of LXRs in bleomycin-induced skin fibrosis, in the model of sclerodermatous graft-versus-host disease (sclGvHD) and in tight skin-1 (Tsk-1) mice, reflecting different subtypes of fibrotic disease. We examined both LXR isoforms using LXRα-, LXRß- and LXR-α/ß-double-knockout mice. Finally, we investigated the effects of LXRs on fibroblasts and macrophages to establish the antifibrotic mode of action of LXRs. RESULTS: LXR activation by the agonist T0901317 had antifibrotic effects in bleomycin-induced skin fibrosis, in the sclGvHD model and in Tsk-1 mice. The antifibrotic activity of LXRs was particularly prominent in the inflammation-driven bleomycin and sclGvHD models. LXRα-, LXRß- and LXRα/ß-double-knockout mice showed a similar response to bleomycin as wildtype animals. Low levels of the LXR target gene ABCA-1 in the skin of bleomycin-challenged and control mice suggested a low baseline activation of the antifibrotic LXR signalling, which, however, could be specifically activated by T0901317. Fibroblasts were not the direct target cells of LXRs agonists, but LXR activation inhibited fibrosis by interfering with infiltration of macrophages and their release of the pro-fibrotic interleukin-6. CONCLUSIONS: We identified LXRs as novel targets for antifibrotic therapies, a yet unknown aspect of these nuclear receptors. Our data suggest that LXR activation might be particularly effective in patients with inflammatory disease subtypes. Activation of LXRs interfered with the release of interleukin-6 from macrophages and, thus, inhibited fibroblast activation and collagen release.
Assuntos
Fibroblastos/metabolismo , Interleucina-6/metabolismo , Macrófagos/metabolismo , Receptores Nucleares Órfãos/genética , Esclerodermia Difusa/metabolismo , Dermatopatias/metabolismo , Pele/patologia , Animais , Antibióticos Antineoplásicos/toxicidade , Bleomicina/toxicidade , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Fibrose , Humanos , Hidrocarbonetos Fluorados/farmacologia , Receptores X do Fígado , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Receptores Nucleares Órfãos/agonistas , Receptores Nucleares Órfãos/metabolismo , Pele/efeitos dos fármacos , Dermatopatias/induzido quimicamente , Sulfonamidas/farmacologiaRESUMO
UNLABELLED: Autotaxin (ENPP2/ATX) and lysophosphatidic acid (LPA) receptors represent two key players in regulating cancer progression. The present study sought to understand the mechanistic role of LPA G protein-coupled receptors (GPCR), not only in the tumor cells but also in stromal cells of the tumor microenvironment. B16F10 melanoma cells predominantly express LPA5 and LPA2 receptors but lack LPA1. LPA dose dependently inhibited invasion of cells across a Matrigel layer. RNAi-mediated knockdown of LPA5 relieved the inhibitory effect of LPA on invasion without affecting basal invasion. This suggests that LPA5 exerts an anti-invasive action in melanoma cells in response to LPA. In addition, both siRNA-mediated knockdown and pharmacologic inhibition of LPA2 reduced the basal rate invasion. Unexpectedly, when probing the role of this GPCR in host tissues, it was found that the incidence of melanoma-derived lung metastasis was greatly reduced in LPA5 knockout (KO) mice compared with wild-type (WT) mice. LPA1-KO but not LPA2-KO mice also showed diminished melanoma-derived lung metastasis, suggesting that host LPA1 and LPA5 receptors play critical roles in the seeding of metastasis. The decrease in tumor cell residence in the lungs of LPA1-KO and LPA5-KO animals was apparent 24 hours after injection. However, KO of LPA1, LPA2, or LPA5 did not affect the subcutaneous growth of melanoma tumors. IMPLICATIONS: These findings suggest that tumor and stromal LPA receptors, in particular LPA1 and LPA5, play different roles in invasion and the seeding of metastasis.
Assuntos
Neoplasias Pulmonares/genética , Melanoma Experimental/genética , Receptores de Ácidos Lisofosfatídicos/genética , Animais , Carcinogênese/genética , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Melanoma Experimental/patologia , Camundongos , Camundongos Knockout , Invasividade Neoplásica/genética , Metástase Neoplásica , Diester Fosfórico Hidrolases/genética , Transdução de Sinais/genética , Microambiente TumoralRESUMO
Lysophospholipids have emerged as biologically important chemoattractants capable of directing lymphocyte development, trafficking, and localization. Lysophosphatidic acid (LPA) is a major lysophospholipid found systemically, and its levels are elevated in certain pathological settings, such as cancer and infections. In this study, we demonstrate that BCR signal transduction by mature murine B cells is inhibited upon LPA engagement of the LPA5 (GPR92) receptor via a Gα12/13-Arhgef1 pathway. The inhibition of BCR signaling by LPA5 manifests by impaired intracellular calcium store release and most likely by interfering with inositol 1,4,5-triphosphate receptor activity. We further show that LPA5 also limits Ag-specific induction of CD69 and CD86 expression and that LPA5-deficient B cells display enhanced Ab responses. Thus, these data show that LPA5 negatively regulates BCR signaling, B cell activation, and immune response. Our findings extend the influence of lysophospholipids on immune function and suggest that alterations in LPA levels likely influence adaptive humoral immunity.
Assuntos
Formação de Anticorpos/fisiologia , Linfócitos B/imunologia , Imunidade Humoral/fisiologia , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Ácidos Lisofosfatídicos/imunologia , Transdução de Sinais/imunologia , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígeno B7-2/genética , Antígeno B7-2/imunologia , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/genética , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/imunologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos B/genética , Receptores de Ácidos Lisofosfatídicos/genética , Transdução de Sinais/genéticaRESUMO
Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4+ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1-/- B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1-/- CD4+ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4+ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.
Assuntos
Autoimunidade , Fator de Crescimento de Hepatócito/genética , Proteínas Proto-Oncogênicas/genética , Linfócitos T/imunologia , Animais , Artrite Reumatoide/imunologia , Sequência de Bases , Primers do DNA , Ativação Linfocitária , Teste de Cultura Mista de Linfócitos , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: Enterochromaffin cell-derived serotonin (5-HT) promotes intestinal inflammation. We tested hypotheses that peripheral tryptophan hydroxylase (TPH) inhibitors, administered orally, block 5-HT biosynthesis and deplete 5-HT from enterochromaffin cells sufficiently to ameliorate intestinal inflammation; moreover, peripheral TPH inhibitors fail to enter the murine enteric nervous system (ENS) or central nervous systems and thus do not affect constitutive gastrointestinal motility. DESIGN: Two peripheral TPH inhibitors, LP-920540 and telotristat etiprate (LX1032; LX1606) were given orally to mice. Effects were measured on 5-HT levels in the gut, blood and brain, 5-HT immunoreactivity in the ENS, gastrointestinal motility and severity of trinitrobenzene sulfonic acid (TNBS)-induced colitis. Quantitation of clinical scores, histological damage and intestinal expression of inflammation-associated cytokines and chemokines with focused microarrays and real-time reverse transcriptase PCR were employed to evaluate the severity of intestinal inflammation. RESULTS: LP-920540 and LX1032 reduced 5-HT significantly in the gut and blood but not in the brain. Neither LP-920540 nor LX1032 decreased 5-HT immunoreactive neurons or fibres in the myenteric plexus and neither altered total gastrointestinal transit time, colonic motility or gastric emptying in mice. In contrast, oral LP-920540 and LX1032 reduced the severity of TNBS-induced colitis; the expression of 24% of 84 genes encoding inflammation-related cytokines and chemokines was lowered at least fourfold and the reduced expression of 17% was statistically significant. CONCLUSIONS: Observations suggest that that peripheral TPH inhibitors uncouple the positive linkage of enterochromaffin cell-derived 5-HT to intestinal inflammation. Because peripheral TPH inhibitors evidently do not enter the murine ENS, they lack deleterious effects on constitutive intestinal motility in mice.
Assuntos
Colite/metabolismo , Regulação para Baixo/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Expressão Gênica/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Neurônios/metabolismo , Serotonina/biossíntese , Animais , Encéfalo/metabolismo , Colite/induzido quimicamente , Colite/patologia , Citocinas/genética , Motilidade Gastrointestinal/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Plexo Mientérico/metabolismo , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Pirimidinas/farmacologia , Serotonina/sangue , Índice de Gravidade de Doença , Ácido Trinitrobenzenossulfônico , Triptofano Hidroxilase/antagonistas & inibidoresRESUMO
The phenylalanyl-glycyl-glycyl-alanyl-prolyl (FG-GAP) domain plays an important role in protein-protein interactions, including interaction of integrins with their ligands. Integrin-α FG-GAP repeat-containing protein 2 (Itfg2) is a highly conserved protein in vertebrates that carries two FG-GAP domains, but its role in mammalian physiology is unknown. In this article, we show that Itfg2 is an intracellular protein and it plays a critical role in B cell differentiation and development of autoimmunity. Itfg2-deficient mice displayed a phenotype consistent with retention of B cells in the spleen and had a lower concentration of IgG in the blood when compared with wild-type littermates. Itfg2-deficient splenocytes also showed a defect in cell migration in vitro. After immunization with a thymus-dependent Ag, the absence of Itfg2 caused a shift in B cell maturation from the germinal centers to the extrafollicular regions of the spleen and blocked deposition of Ag-specific plasma cells in the bone marrow. In support of hematopoietic cell intrinsic activity of Itfg2, bone marrow transplantation of Itfg2-deficient cells was sufficient to impair germinal center development in wild-type mice. Furthermore, Itfg2 deficiency exacerbated development of autoimmune disease in MRL/lpr lupus-prone mice. These results identify Itfg2 as a novel contributor to B cell differentiation and a negative regulator of the autoimmune response during lupus.
Assuntos
Doenças Autoimunes/genética , Linfócitos B/citologia , Linfócitos B/metabolismo , Diferenciação Celular/genética , Cadeias alfa de Integrinas/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/metabolismo , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Sequência de Bases , Diferenciação Celular/imunologia , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Modelos Animais de Doenças , Ordem dos Genes , Marcação de Genes , Genótipo , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Imunoglobulinas/sangue , Cadeias alfa de Integrinas/química , Cadeias alfa de Integrinas/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Fenótipo , Alinhamento de Sequência , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
CD8 T lymphocytes are able to eliminate nascent tumor cells through a process referred to as immune surveillance. However, multiple inhibitory mechanisms within the tumor microenvironment have been described that impede tumor rejection by CD8 T cells, including increased signaling by inhibitory receptors. Lysophosphatidic acid (LPA) is a bioactive lysophospholipid that has been shown repeatedly to promote diverse cellular processes benefiting tumorigenesis. Accordingly, the increased expression of LPA and LPA receptors is a common feature of diverse tumor cell lineages and can result in elevated systemic LPA levels. LPA is recognized by at least 6 distinct G-protein-coupled receptors and several of which are expressed by T cells, although the precise role of LPA signaling in CD8 T cell activation and function has not been defined. Here, we demonstrate that LPA signaling via the LPA5 receptor expressed by CD8 T cells suppresses antigen receptor signaling, cell activation and proliferation in vitro and in vivo. Importantly, in a mouse melanoma model tumor-specific CD8 T cells that are LPA5-deficient are able to control tumor growth significantly better than wild-type tumor-specific CD8 T cells. Together, these data suggest that the production of LPA by tumors serves not only in an autocrine manner to promote tumorigenesis but also as a mechanism to suppress adaptive immunity and highlights a potential novel target for cancer treatment.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Ativação Linfocitária , Lisofosfolipídeos/metabolismo , Melanoma/tratamento farmacológico , Transdução de Sinais , Animais , Carcinogênese , Linhagem Celular Tumoral , Vigilância Imunológica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Processos Neoplásicos , Receptores de Ácidos Lisofosfatídicos/metabolismoRESUMO
Ribosomal protein (RP) mutations in diseases such as 5q- syndrome both disrupt hematopoiesis and increase the risk of developing hematologic malignancy. However, the mechanism by which RP mutations increase cancer risk has remained an important unanswered question. We show here that monoallelic, germline inactivation of the ribosomal protein L22 (Rpl22) predisposes T-lineage progenitors to transformation. Indeed, RPL22 was found to be inactivated in â¼ 10% of human T-acute lymphoblastic leukemias. Moreover, monoallelic loss of Rpl22 accelerates development of thymic lymphoma in both a mouse model of T-cell malignancy and in acute transformation assays in vitro. We show that Rpl22 inactivation enhances transformation potential through induction of the stemness factor, Lin28B. Our finding that Rpl22 inactivation promotes transformation by inducing expression of Lin28B provides the first insight into the mechanistic basis by which mutations in Rpl22, and perhaps some other RP genes, increases cancer risk.
Assuntos
Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/metabolismo , Neoplasias Hematológicas/genética , Proteínas de Ligação a RNA/genética , Proteínas Ribossômicas/genética , Linfócitos T/metabolismo , Animais , Ensaio de Desvio de Mobilidade Eletroforética , Citometria de Fluxo , Inativação Gênica , Neoplasias Hematológicas/metabolismo , Humanos , Immunoblotting , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
αß and γδ lineage T cells are thought to arise from a common CD4(-)CD8(-) progenitor in the thymus. However, the molecular pathways controlling fate selection and maturation of these two lineages remain poorly understood. We demonstrated recently that a ubiquitously expressed ribosomal protein, Rpl22, is selectively required for the development of αß lineage T cells. Germline ablation of Rpl22 impairs development of αß lineage, but not γδ lineage, T cells through activation of a p53-dependent checkpoint. In this study, we investigate the downstream effectors used by p53 to impair T cell development. We found that many p53 targets were induced in Rpl22(-/-) thymocytes, including miR-34a, PUMA, p21(waf), Bax, and Noxa. Notably, the proapoptotic factor Bim, while not a direct p53 target, was also strongly induced in Rpl22(-/-) T cells. Gain-of-function analysis indicated that overexpression of miR-34a caused a developmental arrest reminiscent of that induced by p53 in Rpl22-deficient T cells; however, only a few p53 targets alleviated developmental arrest when individually ablated by gene targeting or knockdown. Co-elimination of PUMA and Bim resulted in a nearly complete restoration of development of Rpl22(-/-) thymocytes, indicating that p53-mediated arrest is enforced principally through effects on cell survival. Surprisingly, co-elimination of the primary p53 regulators of cell cycle arrest (p21(waf)) and apoptosis (PUMA) actually abrogated the partial rescue caused by loss of PUMA alone, suggesting that the G1 checkpoint protein p21(waf) facilitates thymocyte development in some contexts.
Assuntos
Diferenciação Celular/imunologia , Marcação de Genes , Inibidores do Crescimento/imunologia , Proteínas Ribossômicas/deficiência , Subpopulações de Linfócitos T/imunologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Apoptose/genética , Apoptose/imunologia , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/deficiência , Proteína 11 Semelhante a Bcl-2 , Diferenciação Celular/genética , Linhagem Celular , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Marcação de Genes/métodos , Inibidores do Crescimento/genética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/genética , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia , Timo/imunologia , Timo/metabolismo , Timo/patologia , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/deficiência , Proteínas Supressoras de Tumor/biossíntese , Proteínas Supressoras de Tumor/deficiência , Proteína X Associada a bcl-2/biossínteseRESUMO
Cerebral malaria (CM) is associated with excessive inflammatory responses and endothelial activation. Sphingosine 1-phosphate (S1P) is a signaling sphingolipid implicated in regulating vascular integrity, inflammation and T-cell migration. We hypothesized that altered S1P signaling during malaria contributes to endothelial activation and inflammation, and show that plasma S1P levels were decreased in Ugandan children with CM compared with children with uncomplicated malaria. Using the Plasmodium berghei ANKA (PbA) model of experimental CM (ECM), we demonstrate that humanized S1P lyase (hS1PL)(-/-) mice with reduced S1P lyase activity (resulting in increased bio-available S1P) had improved survival compared with wild-type littermates. Prophylactic and therapeutic treatment of infected mice with compounds that modulate the S1P pathway and are in human trials for other conditions (FTY720 or LX2931) significantly improved survival in ECM. FTY720 treatment improved vascular integrity as indicated by reduced levels of soluble intercellular adhesion molecule (sICAM), increased angiopoietin 1 (Ang1) (regulator of endothelial quiescence) levels, and decreased Evans blue dye leakage into brain parenchyma. Furthermore, treatment with FTY720 decreased IFNγ levels in plasma as well as CD4(+) and CD8(+) T-cell infiltration into the brain. Finally, when administered during infection in combination with artesunate, FTY720 treatment resulted in increased survival to ECM. These findings implicate dysregulation of the S1P pathway in the pathogenesis of human and murine CM and suggest a novel therapeutic strategy to improve clinical outcome in severe malaria.
Assuntos
Aldeído Liases/metabolismo , Lisofosfolipídeos/metabolismo , Malária Cerebral/metabolismo , Plasmodium berghei/crescimento & desenvolvimento , Esfingosina/análogos & derivados , Aldeído Liases/antagonistas & inibidores , Aldeído Liases/genética , Animais , Antimaláricos/farmacologia , Artemisininas/farmacologia , Artesunato , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Moléculas de Adesão Celular/sangue , Criança , Pré-Escolar , Feminino , Cloridrato de Fingolimode , Humanos , Imidazóis/farmacologia , Imunossupressores/farmacologia , Lactente , Interferon gama/sangue , Lisofosfolipídeos/sangue , Malária Cerebral/tratamento farmacológico , Malária Cerebral/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oximas/farmacologia , Plasmodium berghei/efeitos dos fármacos , Propilenoglicóis/farmacologia , Esfingosina/sangue , Esfingosina/metabolismo , Esfingosina/farmacologia , Análise de Sobrevida , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Resultado do TratamentoRESUMO
The human proton coupled folate transporter (PCFT) is involved in low pH-dependent intestinal folate transport. In this report, we describe a new murine model of the hereditary folate malabsorption syndrome that we developed through targeted disruption of the first 3 coding exons of the murine homolog of the PCFT gene. By 4 weeks of age, PCFT-deficient (PCFT(-/-)) mice developed severe macrocytic normochromic anemia and pancytopenia. Immature erythroblasts accumulated in the bone marrow and spleen of PCFT(-/-) mice and failed to differentiate further, showing an increased rate of apoptosis in intermediate erythroblasts and reduced release of reticulocytes. In response to the inefficient hematologic development, the serum of the PCFT(-/-) animals contained elevated concentrations of erythropoietin, soluble transferrin receptor (sCD71), and thrombopoietin. In vivo folate uptake experiments demonstrated a systemic folate deficiency caused by disruption of PCFT-mediated intestinal folate uptake, thus confirming in vivo a critical and nonredundant role of the PCFT protein in intestinal folate transport and erythropoiesis. The PCFT-deficient mouse serves as a model for the hereditary folate malabsorption syndrome and is the most accurate animal model of folate deficiency anemia described to date that closely captures the spectrum of pathology typical of this disease.
Assuntos
Deficiência de Ácido Fólico/genética , Deficiência de Ácido Fólico/metabolismo , Transportador de Folato Acoplado a Próton/antagonistas & inibidores , Transportador de Folato Acoplado a Próton/genética , Animais , Sequência de Bases , Medula Óssea/patologia , Primers do DNA/genética , Modelos Animais de Doenças , Eritropoese/genética , Eritropoese/fisiologia , Feminino , Ácido Fólico/metabolismo , Deficiência de Ácido Fólico/patologia , Marcação de Genes , Humanos , Absorção Intestinal , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pancitopenia/genética , Pancitopenia/metabolismo , Pancitopenia/patologia , Transportador de Folato Acoplado a Próton/deficiência , Baço/patologiaRESUMO
An early lesion in many kidney diseases is damage to podocytes, which are critical components of the glomerular filtration barrier. A number of proteins are essential for podocyte filtration function, but the signaling events contributing to development of nephrotic syndrome are not well defined. Here we show that class II phosphoinositide 3-kinase C2α (PI3KC2α) is expressed in podocytes and plays a critical role in maintaining normal renal homeostasis. PI3KC2α-deficient mice developed chronic renal failure and exhibited a range of kidney lesions, including glomerular crescent formation and renal tubule defects in early disease, which progressed to diffuse mesangial sclerosis, with reduced podocytes, widespread effacement of foot processes, and modest proteinuria. These findings were associated with altered expression of nephrin, synaptopodin, WT-1, and desmin, indicating that PI3KC2α deficiency specifically impacts podocyte morphology and function. Deposition of glomerular IgA was observed in knockout mice; importantly, however, the development of severe glomerulonephropathy preceded IgA production, indicating that nephropathy was not directly IgA mediated. PI3KC2α deficiency did not affect immune responses, and bone marrow transplantation studies also indicated that the glomerulonephropathy was not the direct consequence of an immune-mediated disease. Thus, PI3KC2α is critical for maintenance of normal glomerular structure and function by supporting normal podocyte function.
Assuntos
Glomérulos Renais/anatomia & histologia , Glomérulos Renais/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Animais , Antígenos de Superfície/metabolismo , Transplante de Medula Óssea , Glomerulonefrite/etiologia , Glomerulonefrite/patologia , Glomerulonefrite/fisiopatologia , Glomerulonefrite por IGA/etiologia , Glomerulonefrite por IGA/patologia , Glomerulonefrite por IGA/fisiopatologia , Humanos , Imunoglobulina A/metabolismo , Imunoglobulina G/metabolismo , Glomérulos Renais/enzimologia , Glomérulos Renais/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Fosfatidilinositol 3-Quinases/deficiência , Fosfatidilinositol 3-Quinases/genética , Podócitos/enzimologia , Podócitos/patologia , Podócitos/fisiologia , Insuficiência Renal/etiologia , Insuficiência Renal/patologia , Insuficiência Renal/fisiopatologia , Quimeras de TransplanteRESUMO
Sphingosine 1-phosphate lyase (S1PL) has been characterized as a novel target for the treatment of autoimmune disorders using genetic and pharmacological methods. Medicinal chemistry efforts targeting S1PL by direct in vivo evaluation of synthetic analogues of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI, 1) led to the discovery of 2 (LX2931) and 4 (LX2932). The immunological phenotypes observed in S1PL deficient mice were recapitulated by oral administration of 2 or 4. Oral dosing of 2 or 4 yielded a dose-dependent decrease in circulating lymphocyte numbers in multiple species and showed a therapeutic effect in rodent models of rheumatoid arthritis (RA). Phase I clinical trials indicated that 2, the first clinically studied inhibitor of S1PL, produced a dose-dependent and reversible reduction of circulating lymphocytes and was well tolerated at dose levels of up to 180 mg daily. Phase II evaluation of 2 in patients with active rheumatoid arthritis is currently underway.
Assuntos
Aldeído Liases/antagonistas & inibidores , Antirreumáticos/síntese química , Imidazóis/síntese química , Isoxazóis/síntese química , Oximas/síntese química , Aldeído Liases/genética , Animais , Antirreumáticos/farmacocinética , Antirreumáticos/farmacologia , Artrite Experimental/tratamento farmacológico , Artrite Experimental/imunologia , Artrite Experimental/patologia , Pressão Sanguínea/efeitos dos fármacos , Movimento Celular , Cães , Frequência Cardíaca/efeitos dos fármacos , Imidazóis/farmacocinética , Imidazóis/farmacologia , Isoxazóis/farmacocinética , Isoxazóis/farmacologia , Linfócitos/efeitos dos fármacos , Linfócitos/fisiologia , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Oximas/farmacocinética , Oximas/farmacologia , Ratos , Ratos Sprague-Dawley , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Rpn13 is a novel mammalian proteasomal receptor that has recently been identified as an amplification target in ovarian cancer. It can interact with ubiquitin and activate the deubiquitinating enzyme Uch37 at the 26S proteasome. Since neither Rpn13 nor Uch37 is an integral proteasomal subunit, we explored whether either protein is essential for mammalian development and survival. Deletion of Uch37 resulted in prenatal lethality in mice associated with severe defect in embryonic brain development. In contrast, the majority of Rpn13-deficient mice survived to adulthood, although they were smaller at birth and fewer in number than wild-type littermates. Absence of Rpn13 produced tissue-specific effects on proteasomal function: increased proteasome activity in adrenal gland and lymphoid organs, and decreased activity in testes and brain. Adult Rpn13(-/-) mice reached normal body weight but had increased body fat content and were infertile due to defective gametogenesis. Additionally, Rpn13(-/-) mice showed increased T-cell numbers, resembling growth hormone-mediated effects. Indeed, serum growth hormone and follicular stimulating hormone levels were significantly increased in Rpn13(-/-) mice, while growth hormone receptor expression was reduced in the testes. In conclusion, this is the first report characterizing the physiological roles of Uch37 and Rpn13 in murine development and implicating a non-ATPase proteasomal protein, Rpn13, in the process of gametogenesis.