RESUMO
ATRX, a chromatin remodeler protein, is recurrently mutated in H3F3A-mutant pediatric glioblastoma (GBM) and isocitrate dehydrogenase (IDH)-mutant grade 2/3 adult glioma. Previous work has shown that ATRX-deficient GBM cells show enhanced sensitivity to irradiation, but the etiology remains unclear. We find that ATRX binds the regulatory elements of cell-cycle phase transition genes in GBM cells, and there is a marked reduction in Checkpoint Kinase 1 (CHEK1) expression with ATRX loss, leading to the early release of G2/M entry after irradiation. ATRX-deficient cells exhibit enhanced activation of master cell-cycle regulator ATM with irradiation. Addition of the ATM inhibitor AZD0156 doubles median survival in mice intracranially implanted with ATRX-deficient GBM cells, which is not seen in ATRX-wild-type controls. This study demonstrates that ATRX-deficient high-grade gliomas (HGGs) display Chk1-mediated dysregulation of cell-cycle phase transitions, which opens a window for therapies targeting this phenotype.
Assuntos
Quinase 1 do Ponto de Checagem/metabolismo , Glioma/metabolismo , Proteína Nuclear Ligada ao X/metabolismo , Animais , Neoplasias Encefálicas/metabolismo , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/fisiologia , Feminino , Histonas/metabolismo , Humanos , Isocitrato Desidrogenase/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Recidiva Local de Neoplasia/metabolismo , Cultura Primária de Células , Proteína Nuclear Ligada ao X/genéticaRESUMO
Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) proteins work in concert to regulate the levels of reactive oxygen species (ROS). The Keap1-Nrf2 antioxidant system also participates in T cell differentiation and inflammation, but its role in innate T cell development and functions remains unclear. We report that T cell-specific deletion of Keap1 results in defective development and reduced numbers of invariant natural killer T (NKT) cells in the thymus and the peripheral organs in a cell-intrinsic manner. The frequency of NKT2 and NKT17 cells increases while NKT1 decreases in these mice. Keap1-deficient NKT cells show increased rates of proliferation and apoptosis, as well as increased glucose uptake and mitochondrial function, but reduced ROS, CD122, and Bcl2 expression. In NKT cells deficient in Nrf2 and Keap1, all these phenotypic and metabolic defects are corrected. Thus, the Keap1-Nrf2 system contributes to NKT cell development and homeostasis by regulating cell metabolism.
Assuntos
Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Células T Matadoras Naturais/metabolismo , Animais , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Glucose/metabolismo , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Subunidade beta de Receptor de Interleucina-2/genética , Subunidade beta de Receptor de Interleucina-2/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/deficiência , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/deficiência , Fator 2 Relacionado a NF-E2/genética , Células T Matadoras Naturais/citologia , Células T Matadoras Naturais/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Timo/metabolismoRESUMO
Cellular metabolism and signaling pathways are key regulators to determine conventional T cell fate and function, but little is understood about the role of cell metabolism for natural killer T (NKT) cell survival, proliferation, and function. We found that NKT cells operate distinct metabolic programming from CD4 T cells. NKT cells are less efficient in glucose uptake than CD4 T cells with or without activation. Gene-expression data revealed that, in NKT cells, glucose is preferentially metabolized by the pentose phosphate pathway and mitochondria, as opposed to being converted into lactate. In fact, glucose is essential for the effector functions of NKT cells and a high lactate environment is detrimental for NKT cell survival and proliferation. Increased glucose uptake and IFN-γ expression in NKT cells is inversely correlated with bacterial loads in response to bacterial infection, further supporting the significance of glucose metabolism for NKT cell function. We also found that promyelocytic leukemia zinc finger seemed to play a role in regulating NKT cells' glucose metabolism. Overall, our study reveals that NKT cells use distinct arms of glucose metabolism for their survival and function.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Mitocôndrias/metabolismo , Células T Matadoras Naturais/imunologia , Fosforilação Oxidativa , Via de Pentose Fosfato/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Glucose/genética , Glucose/imunologia , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Células T Matadoras Naturais/citologia , Via de Pentose Fosfato/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/imunologiaRESUMO
Type I or invariant natural killer T cells belong to a unique lineage of innate T cells, which express markers of both T lymphocytes and NK cells, namely T cell receptor (TCR) and NK1.1 (CD161C), respectively. Thus, apart from direct killing of target cells like NK cells, and they also produce a myriad of cytokines which modulate the adaptive immune responses. Unlike traditional T cells which carry a conventional αß TCR, NKT cells express semi-invariant TCR - Vα14-Jα18, coupled with Vß8, Vß7 and Vß2 in mice. In humans, the invariant TCR is composed of Vα24-Jα18, coupled with Vß11.
RESUMO
We show the presence of lymphoid tissue-resident PLZF+ CD45RA+ RO+ CD4 T cells in humans. They express HLA-DR, granzyme B, and perforin and are low on CCR7 like terminally differentiated effector memory (Temra) cells and are likely generated from effector T cells (Te) or from central (Tcm) or effector (Tem) memory T (Tcm) cells during immune responses. Tn, Naïve T cells.
Assuntos
Linfócitos T CD4-Positivos/fisiologia , Genótipo , Tecido Linfoide/imunologia , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Subpopulações de Linfócitos T/fisiologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Células Cultivadas , Granzimas/metabolismo , Antígenos HLA-DR/metabolismo , Humanos , Imunidade Celular , Memória Imunológica , Perforina/metabolismoRESUMO
Reactive oxygen species (ROS) are byproducts of aerobic metabolism and contribute to both physiological and pathological conditions as second messengers. ROS are essential for activation of T cells, but how ROS influence NKT cells is unknown. In the present study, we investigated the role of ROS in NKT cell function. We found that NKT cells, but not CD4 or CD8 T cells, have dramatically high ROS in the spleen and liver of mice but not in the thymus or adipose tissues. Accordingly, ROS-high NKT cells exhibited increased susceptibility and apoptotic cell death with oxidative stress. High ROS in the peripheral NKT cells were primarily produced by NADPH oxidases and not mitochondria. We observed that sorted ROS-high NKT cells were enriched in NKT1 and NKT17 cells, whereas NKT2 cells were dominant in ROS-low cells. Furthermore, treatment of NKT cells with antioxidants led to reduced frequencies of IFN-γ- and IL-17-expressing cells, indicating that ROS play a role in regulating the inflammatory function of NKT cells. The transcription factor promyelocytic leukemia zinc finger (PLZF) seemed to control the ROS levels. NKT cells from adipose tissues that do not express PLZF and those from PLZF haplodeficient mice have low ROS. Conversely, ROS were highly elevated in CD4 T cells from mice ectopically expressing PLZF. Thus, our findings demonstrate that PLZF controls ROS levels, which in turn governs the inflammatory function of NKT cells.
Assuntos
Fígado/imunologia , Células T Matadoras Naturais/imunologia , Proteína com Dedos de Zinco da Leucemia Promielocítica/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Baço/imunologia , Animais , Apoptose , Células Cultivadas , Interferon gama/metabolismo , Interleucina-17/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , NADPH Oxidases/metabolismo , Estresse Oxidativo , Proteína com Dedos de Zinco da Leucemia Promielocítica/genéticaRESUMO
BACKGROUND & AIMS: The liver is an immunologically-privileged organ. Breakdown of liver immune privilege has been reported in chronic liver disease; however, the role of adaptive immunity in liver injury is poorly defined. Nuclear factor-κB-inducing kinase (NIK) is known to regulate immune tissue development, but its role in maintaining liver homeostasis remains unknown. This study aimed to assess the role of NIK, particularly thymic NIK, in regulating liver adaptive immunity. METHODS: NIK was deleted systemically or conditionally using the Cre/loxp system. Cluster of differentiation [CD]4+ or CD8+ T cells were depleted using anti-CD4 or anti-CD8 antibody. Donor bone marrows or thymi were transferred into recipient mice. Immune cells were assessed by immunohistochemistry and flow cytometry. RESULTS: Global, but not liver-specific or hematopoietic lineage cell-specific, deletion of NIK induced fatal liver injury, inflammation, and fibrosis. Likewise, adoptive transfer of NIK-null, but not wild-type, thymi into immune-deficient mice induced liver inflammation, injury, and fibrosis in recipients. Liver inflammation was characterized by a massive expansion of T cells, particularly the CD4+ T cell subpopulation. Depletion of CD4+, but not CD8+, T cells fully protected against liver injury, inflammation, and fibrosis in NIK-null mice. NIK deficiency also resulted in inflammation in the lung, kidney, and pancreas, but to a lesser degree relative to the liver. CONCLUSIONS: Thymic NIK suppresses development of autoreactive T cells against liver antigens, and NIK deficiency in the thymus results in CD4+ T cell-orchestrated autoimmune hepatitis and liver fibrosis. Thus, thymic NIK is essential for the maintenance of liver immune privilege and liver homeostasis. LAY SUMMARY: We found that global or thymus-specific ablation of the NIK gene results in fatal autoimmune liver disease in mice. NIK-deficient mice develop liver inflammation, injury, and fibrosis. Our findings indicate that thymic NIK is essential for the maintenance of liver integrity and homeostasis.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Hepatite Autoimune/etiologia , Cirrose Hepática Experimental/etiologia , Fígado/imunologia , Proteínas Serina-Treonina Quinases/fisiologia , Timo/fisiologia , Imunidade Adaptativa , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Quinase Induzida por NF-kappaBRESUMO
The mammalian target of rapamycin (mTOR) senses and incorporates different environmental cues via the two signaling complexes mTOR complex 1 (mTORC1) and mTORC2. As a result, mTOR controls cell growth and survival, and also shapes different effector functions of the cells including immune cells such as T cells. We demonstrate in this article that invariant NKT (iNKT) cell development is controlled by mTORC2 in a cell-intrinsic manner. In mice deficient in mTORC2 signaling because of the conditional deletion of the Rictor gene, iNKT cell numbers were reduced in the thymus and periphery. This is caused by decreased proliferation of stage 1 iNKT cells and poor development through subsequent stages. Functionally, iNKT cells devoid of mTORC2 signaling showed reduced number of IL-4-expressing cells, which correlated with a decrease in the transcription factor GATA-3-expressing cells. However, promyelocytic leukemia zinc-finger (PLZF), a critical transcription factor for iNKT cell development, is expressed at a similar level in mTORC2-deficient iNKT cells compared with that in the wild type iNKT cells. Furthermore, cellular localization of PLZF was not altered in the absence of mTOR2 signaling. Thus, our study reveals the PLZF-independent mechanisms of the development and function of iNKT cells regulated by mTORC2.
Assuntos
Proteínas de Transporte/imunologia , Fator de Transcrição GATA3/biossíntese , Fatores de Transcrição Kruppel-Like/biossíntese , Complexos Multiproteicos/imunologia , Células T Matadoras Naturais/citologia , Serina-Treonina Quinases TOR/imunologia , Animais , Proteínas de Transporte/genética , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Interferon gama/biossíntese , Interleucina-17/biossíntese , Interleucina-4/biossíntese , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Complexos Multiproteicos/genética , Células T Matadoras Naturais/imunologia , Proteína com Dedos de Zinco da Leucemia Promielocítica , Proteína Companheira de mTOR Insensível à Rapamicina , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR/genéticaRESUMO
Developmental endothelial locus-1 (Del-1) is an endothelial cell-secreted protein that limits the recruitment of neutrophils by antagonizing the interaction between the LFA-1 integrin on neutrophils and the intercellular adhesion molecule (ICAM)-1 on endothelial cells. Mice with genetic or age-associated Del-1 deficiency exhibit increased neutrophil infiltration in the periodontium resulting in inflammatory bone loss. Here we investigated additional novel mechanisms whereby Del-1 could interfere with neutrophil recruitment and inflammation. Treatment of human endothelial cells with Del-1 did not affect the expression of endothelial molecules involved in the leukocyte adhesion cascade (ICAM-1, VCAM-1, and E-selectin). Moreover, genetic or age-associated Del-1 deficiency did not significantly alter the expression of these adhesion molecules in the murine periodontium, further ruling out altered adhesion molecule expression as a mechanism whereby Del-1 regulates leukocyte recruitment. Strikingly, Del-1 inhibited ICAM-1-dependent chemokine release (CXCL2, CCL3) by neutrophils. Therefore, Del-1 could potentially suppress the amplification of inflammatory cell recruitment mediated through chemokine release by infiltrating neutrophils. Interestingly, Del-1 was itself regulated by inflammatory stimuli, which generally exerted opposite effects on adhesion molecule expression. The reciprocal regulation between Del-1 and inflammation may contribute to optimally balance the protective and the potentially harmful effects of inflammatory cell recruitment.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Endoteliais/metabolismo , Periodonto/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Quimiocinas/biossíntese , Expressão Gênica , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Camundongos , Camundongos Knockout , Neutrófilos/metabolismo , Osteoporose/genética , Osteoporose/metabolismo , Periodontite/genética , Periodontite/imunologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
Kaposica, the complement regulator of Kaposi's sarcoma-associated herpesvirus, inhibits complement by supporting factor I-mediated inactivation of the proteolytically activated form of C3 (C3b) and C4 (C4b) (cofactor activity [CFA]) and by accelerating the decay of classical and alternative pathway C3-convertases (decay-accelerating activity [DAA]). Previous data suggested that electrostatic interactions play a critical role in the binding of viral complement regulators to their targets, C3b and C4b. We therefore investigated how electrostatic potential on Kaposica influences its activities. We built a homology structure of Kaposica and calculated the electrostatic potential of the molecule, using the Poisson-Boltzmann equation. Mutants were then designed to alter the overall positive potential of the molecule or of each of its domains and linkers by mutating Lys/Arg to Glu/Gln, and the functional activities of the expressed mutants were analyzed. Our data indicate that 1) positive potential at specific sites and not the overall positive potential on the molecule guides the CFAs and classical pathway DAA; 2) positive potential around the linkers between complement control protein domains (CCPs) 1-2 and 2-3 is more important for DAAs than for CFAs; 3) positive potential in CCP1 is crucial for binding to C3b and C4b, and thereby its functional activities; 4) conversion to negative or enhancement of negative potential for CCPs 2-4 has a marked effect on C3b-linked activities as opposed to C4b-linked activities; and 5) reversal of the electrostatic potential of CCP4 to negative has a differential effect on classical and alternative pathway DAAs. Together, our data provide functional relevance to conservation of positive potential in CCPs 1 and 4 and the linkers of viral complement regulators.
Assuntos
Proteínas Inativadoras do Complemento/fisiologia , Herpesvirus Humano 8/imunologia , Eletricidade Estática , Proteínas Virais/fisiologia , Proteínas Inativadoras do Complemento/genética , Via Alternativa do Complemento/genética , Via Alternativa do Complemento/imunologia , Herpesvirus Humano 8/genética , Humanos , Mutagênese Sítio-Dirigida , Proteínas Virais/genéticaRESUMO
The complement system is a principal bastion of innate immunity designed to combat a myriad of existing as well as newly emerging pathogens. Since viruses are obligatory intracellular parasites, they are continuously exposed to host complement assault and, therefore, have imbibed various strategies to subvert it. One of them is molecular mimicry of the host complement regulators. Large DNA viruses such as pox and herpesviruses encode proteins that are structurally and functionally similar to human regulators of complement activation (RCA), a family of proteins that regulate complement. In this review, we have presented the structural and functional aspects of virally encoded RCA homologs (vRCA), in particular two highly studied vRCAs, vaccinia virus complement control protein (VCP) and Kaposi's sarcoma-associated herpesvirus complement regulator (kaposica). Importance of these evasion molecules in viral pathogenesis and their role beyond complement regulation are also discussed.