RESUMO
Natalizumab, a humanized monoclonal antibody (mAb) against α4-integrin, reduces the number of dendritic cells (DC) in cerebral perivascular spaces in multiple sclerosis (MS). Selective deletion of α4-integrin in CD11c+ cells should curtail their migration to the central nervous system (CNS) and ameliorate experimental autoimmune encephalomyelitis (EAE). We generated CD11c.Cre+/-ITGA4fl/fl C57BL/6 mice to selectively delete α4-integrin in CD11c+ cells. Active immunization and adoptive transfer EAE models were employed and compared with WT controls. Multiparameter flow cytometry was utilized to immunophenotype leukocyte subsets. Single-cell RNA sequencing was used to profile individual cells. α4-Integrin expression by CD11c+ cells was significantly reduced in primary and secondary lymphoid organs in CD11c.Cre+/-ITGA4fl/fl mice. In active EAE, a delayed disease onset was observed in CD11c.Cre+/-ITGA4fl/fl mice, during which CD11c+CD88+ cells were sequestered in the blood. Upon clinical EAE onset, CD11c+CD88+ cells appeared in the CNS and expressed CD317+ In adoptive transfer experiments, CD11c.Cre+/-ITGA4fl/fl mice had ameliorated clinical disease phenotype associated with significantly diminished numbers of CNS CD11c+CD88+CD317+ cells. In human cerebrospinal fluid from subjects with neuroinflammation, microglia-like cells display coincident expression of ITGAX (CD11c), C5AR1 (CD88), and BST2 (CD317). In mice, we show that only activated, but not naïve microglia expressed CD11c, CD88, and CD317. Finally, anti-CD317 treatment prior to clinical EAE substantially enhanced recovery in mice.
Assuntos
Antígenos CD/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Integrina alfa4/metabolismo , Células Mieloides/metabolismo , Animais , Apresentação de Antígeno , Células Cultivadas , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Feminino , Humanos , Masculino , Camundongos , Microglia/metabolismoAssuntos
Algoritmos , Citometria de Fluxo/normas , Imunofenotipagem/normas , Próstata/citologia , Hiperplasia Prostática/diagnóstico , Neoplasias da Próstata/diagnóstico , Adulto , Anticorpos/química , Antígenos CD/genética , Antígenos CD/imunologia , Biomarcadores/metabolismo , Estudos de Casos e Controles , Diagnóstico Diferencial , Células Epiteliais/imunologia , Células Epiteliais/patologia , Expressão Gênica , Humanos , Leucócitos/imunologia , Leucócitos/patologia , Masculino , Pessoa de Meia-Idade , Próstata/imunologia , Próstata/metabolismo , Hiperplasia Prostática/genética , Hiperplasia Prostática/imunologia , Hiperplasia Prostática/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/patologia , Células Estromais/imunologia , Células Estromais/patologiaRESUMO
Hepatocellular carcinoma (HCC) is a deadly human cancer associated with chronic inflammation. The cytosolic pathogen sensor NLRP12 has emerged as a negative regulator of inflammation, but its role in HCC is unknown. Here we investigated the role of NLRP12 in HCC using mouse models of HCC induced by carcinogen diethylnitrosamine (DEN). Nlrp12-/- mice were highly susceptible to DEN-induced HCC with increased inflammation, hepatocyte proliferation, and tumor burden. Consistently, Nlrp12-/- tumors showed higher expression of proto-oncogenes cJun and cMyc and downregulation of tumor suppressor p21. Interestingly, antibiotics treatment dramatically diminished tumorigenesis in Nlrp12-/- mouse livers. Signaling analyses demonstrated higher JNK activation in Nlrp12-/- HCC and cultured hepatocytes during stimulation with microbial pattern molecules. JNK inhibition or NLRP12 overexpression reduced proliferative and inflammatory responses of Nlrp12-/- hepatocytes. In summary, NLRP12 negatively regulates HCC pathogenesis via downregulation of JNK-dependent inflammation and proliferation of hepatocytes.
Assuntos
Carcinoma Hepatocelular/fisiopatologia , Regulação para Baixo , Hepatócitos/enzimologia , Hepatócitos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , Neoplasias Hepáticas/fisiopatologia , Animais , Carcinógenos/administração & dosagem , Carcinoma Hepatocelular/induzido quimicamente , Proliferação de Células , Dietilnitrosamina/administração & dosagem , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Neoplasias Hepáticas/induzido quimicamente , Camundongos Knockout , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
OBJECTIVE: To determine the capacity, effectiveness, efficiency, and reliability of select tissue dissociation methods to isolate mononuclear cells from the CNS of mice with experimental autoimmune encephalomyelitis (EAE). METHODS: As part of an assay qualification, we tested the isolation method Percoll PLUS vs a commercially available enzymatic Neural Tissue Dissociation Kit (Kit), and the enzymes accutase and papain in C57BL/6 mice with active EAE. In a stepwise approach, we applied the following 4 criteria to each dissociation method: (1) mononuclear cell viability post-processing was required to be ≥80% per brain or spinal cord sample, (2) absolute live mononuclear cell numbers was required to be ≥5 × 105 per brain or spinal cord sample of mice with clinical EAE, (3) test-retest reliability had to be verified, and (4) the absolute mononuclear cell numbers in brain and spinal cord had to correlate with the EAE disease course. RESULTS: Enzymatic dissociations allowed for greatly increased cell yield and specifically allowed for downstream assays from individual brains and spinal cords in C57BL/6 mice with EAE. All enzymatic dissociations provided a more efficient and effective method for isolating mononuclear cells from brains and spinal cord. Only the Kit assay provided a significant correlation between absolute mononuclear cell numbers in the spinal cord and EAE disease severity. CONCLUSIONS: Enzymatic dissociation of CNS tissue of C57BL/6 mice with active EAE with the Kit should be the standard method. The identification of optimized CNS dissociation methods in EAE has the potential to identify cellular events that are pertinent to MS pathogenesis.
RESUMO
OBJECTIVE: Natalizumab blocks α4-integrin-mediated leukocyte migration into the central nervous system (CNS). It diminishes disease activity in multiple sclerosis (MS), but carries a high risk of progressive multifocal encephalopathy (PML), an opportunistic infection with JV virus that may be prompted by diminished CNS immune surveillance. The initial host response to viral infections entails the synthesis of type I interferons (IFN) upon engagement of TLR3 receptors. We hypothesized that TLR3 agonism reestablishes CNS immune competence in the setting of α4-integrin deficiency. METHOD: We generated the conditional knock out mouse strain Mx1.Cre+ α4-integrinfl/fl, in which the α4-integrin gene is ablated upon treatment with the TLR3 agonist poly I:C. Adoptive transfer of purified lymphocytes from poly I:C-treated Mx1.Cre+ α4-integrinfl/fl donors into naive recipients recapitulates immunosuppression under natalizumab. Active experimental autoimmune encephalomyelitis (EAE) in Mx1.Cre+ α4-integrinfl/fl mice treated with poly I:C represents immune-reconstitution. RESULTS: Adoptive transfer of T cells from poly I:C treated Mx1.Cre+ α4-integrinfl/fl mice causes minimal EAE. The in vitro migratory capability of CD45+ splenocytes from these mice is reduced. In contrast, actively-induced EAE after poly I:C treatment results in full disease susceptibility of Mx1.Cre+ α4-integrinfl/fl mice, and the number and composition of CNS leukocytes is similar to controls. Extravasation of Evans Blue indicates a compromised blood-brain barrier. Poly I:C treatment results in a 2-fold increase in IFN ß transcription in the spinal cord. INTERPRETATION: Our data suggest that TLR3 agonism in the setting of relative α4-integrin deficiency can reestablish CNS immune surveillance in an experimental model. This pathway may present a feasible treatment strategy to treat and prevent PML under natalizumab therapy and should be considered for further experimental evaluation in a controlled setting.