Asunto(s)
Inmunoterapia Adoptiva , Células Asesinas Naturales/inmunología , Neumonía/terapia , COVID-19/complicaciones , COVID-19/terapia , COVID-19/virología , Humanos , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Células Asesinas Naturales/metabolismo , Neumonía/etiología , SARS-CoV-2RESUMEN
Neurofibromatosis type I (NF1) is a common dominantly inherited disorder, and one of the most common of the RASopathies. Most individuals with NF1 develop plexiform neurofibromas and cutaneous neurofibromas, nerve tumors caused by NF1 loss of function in Schwann cells. Cell culture models and mouse models of NF1 are being used to test drug efficacy in preclinical trials, which led to Food and Drug Administration approval for use of MEK inhibitors to shrink most inoperable plexiform neurofibromas. This article details methods used for testing in preclinical models, and outlines newer models that may identify additional, curative, strategies.
Asunto(s)
Neurofibroma Plexiforme , Neurofibromatosis 1 , Estados Unidos , Humanos , Animales , Ratones , Niño , Neurofibromatosis 1/complicaciones , Neurofibromatosis 1/diagnóstico , Neurofibromatosis 1/tratamiento farmacológico , Neurofibroma Plexiforme/complicaciones , Neurofibroma Plexiforme/tratamiento farmacológicoRESUMEN
The analysis of the spatial patterning of mRNA expression is critically important for assigning functional and physiological significance to a given gene product. Given the tens of thousands of mRNAs in the mammalian genome, a full assessment of individual gene functions would ideally be overlaid upon knowledge of the specific cell types expressing each mRNA. In situ hybridization approaches represent a molecular biological/histological method that can reveal cellular patterns of mRNA expression. Here, we present detailed procedures for the detection of specific mRNAs using radioactive RNA probes in tissue sections followed by autoradiographic detection. These methods allow for the specific and sensitive detection of spatial patterns of mRNA expression, thereby linking mRNA expression with cell type and function. Radioactive detection methods also facilitate semi-quantitative analyses of changes in mRNA gene expression.
Asunto(s)
Hibridación in Situ/métodos , Sondas ARN/análisis , ARN Mensajero/biosíntesis , Autorradiografía , Expresión Génica , Radioisótopos de Fósforo , Sondas ARN/síntesis química , ARN Mensajero/análisis , Radioisótopos de Azufre , TritioRESUMEN
Plexiform neurofibromas (PNF) are peripheral nerve tumors caused by bi-allelic loss of NF1 in the Schwann cell (SC) lineage. PNF are common in individuals with Neurofibromatosis type I (NF1) and can cause significant patient morbidity, spurring research into potential therapies. Immune cells are rare in peripheral nerve, whereas in PNF 30% of the cells are monocytes/macrophages. Mast cells, T cells, and dendritic cells (DCs) are also present. NF1 mutant neurofibroma SCs with elevated Ras-GTP signaling resemble injury-induced repair SCs, in producing growth factors and cytokines not normally present in SCs. This provides a cytokine-rich environment facilitating PNF immune cell recruitment and fibrosis. We propose a model based on genetic and pharmacologic evidence in which, after loss of Nf1 in the SC lineage, a lag occurs. Then, mast cells and macrophages are recruited to nerve. Later, T cell/DC recruitment through CXCL10/CXCR3 drives neurofibroma initiation and sustains PNF macrophages and tumor growth. Stat3 signaling is an additional critical mediator of neurofibroma initiation, cytokine production, and PNF growth. At each stage of PNF development therapeutic benefit should be achievable through pharmacologic modulation of leukocyte recruitment and function.
RESUMEN
Neurofibromatosis type 2 is an inherited, neoplastic disease associated with schwannomas, meningiomas, and ependymomas and that is caused by inactivation of the tumor suppressor gene NF2 The NF2 gene product, Merlin, has no intrinsic catalytic activity; its tumor suppressor function is mediated through the proteins with which it interacts. We used proximity biotinylation followed by mass spectrometry and direct binding assays to identify proteins that associated with wild-type and various mutant forms of Merlin in immortalized Schwann cells. We defined a set of 52 proteins in close proximity to wild-type Merlin. Most of the Merlin-proximal proteins were components of cell junctional signaling complexes, suggesting that additional potential interaction partners may exist in adherens junctions, tight junctions, and focal adhesions. With mutant forms of Merlin that cannot bind to phosphatidylinositol 4,5-bisphosphate (PIP2) or that constitutively adopt a closed conformation, we confirmed a critical role for PIP2 binding in Merlin function and identified a large cohort of proteins that specifically interacted with Merlin in the closed conformation. Among these proteins, we identified a previously unreported Merlin-binding protein, apoptosis-stimulated p53 protein 2 (ASPP2, also called Tp53bp2), that bound to closed-conformation Merlin predominately through the FERM domain. Our results demonstrate that Merlin is a component of cell junctional mechanosensing complexes and defines a specific set of proteins through which it acts.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Uniones Intercelulares/metabolismo , Mecanotransducción Celular , Células de Schwann/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Biotinilación , Línea Celular Transformada , Humanos , Uniones Intercelulares/genética , Ratones , Neurofibromatosis 2/genética , Neurofibromatosis 2/metabolismo , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Dominios Proteicos , Proteínas Supresoras de Tumor/genéticaRESUMEN
Plexiform neurofibroma is a major contributor to morbidity in patients with neurofibromatosis type I (NF1). Macrophages and mast cells infiltrate neurofibroma, and data from mouse models implicate these leukocytes in neurofibroma development. Antiinflammatory therapy targeting these cell populations has been suggested as a means to prevent neurofibroma development. Here, we compare gene expression in Nf1-mutant nerves, which invariably form neurofibroma, and show disruption of neuron-glial cell interactions and immune cell infiltration to mouse models, which rarely progresses to neurofibroma with or without disruption of neuron-glial cell interactions. We find that the chemokine Cxcl10 is uniquely upregulated in NF1 mice that invariably develop neurofibroma. Global deletion of the CXCL10 receptor Cxcr3 prevented neurofibroma development in these neurofibroma-prone mice, and an anti-Cxcr3 antibody somewhat reduced tumor numbers. Cxcr3 expression localized to T cells and DCs in both inflamed nerves and neurofibromas, and Cxcr3 expression was necessary to sustain elevated macrophage numbers in Nf1-mutant nerves. To our knowledge, these data support a heretofore-unappreciated role for T cells and DCs in neurofibroma initiation.
RESUMEN
Plexiform neurofibroma, a benign peripheral nerve tumor, is associated with the biallelic loss of function of the NF1 tumor suppressor in Schwann cells. Here, we show that FLLL32, a small molecule inhibitor of JAK2/STAT3 signaling, reduces neurofibroma growth in mice with conditional, biallelic deletion of Nf1 in the Schwann cell lineage. FLLL32 treatment or Stat3 deletion in tumor cells reduced inflammatory cytokine expression and tumor macrophage numbers in neurofibroma. Although STAT3 inhibition downregulated the chemokines CCL2 and CCL12, which can signal through CCR2 to recruit macrophages to peripheral nerves, deletion of Ccr2 did not improve survival or reduce macrophage numbers in neurofibroma-bearing mice. Interestingly, Iba1+; F4/80+;CD11b+ macrophages accounted for ~20-40% of proliferating cells in untreated tumors. FLLL32 suppressed macrophage proliferation, implicating STAT3-dependent, local proliferation in neurofibroma macrophage accumulation, and decreased Schwann cell proliferation and increased Schwann cell death. The functions of STAT3 signaling in neurofibroma Schwann cells and macrophages, and its relevance as a therapeutic target in neurofibroma, merit further investigation.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Curcumina/análogos & derivados , Neurofibroma Plexiforme/tratamiento farmacológico , Neurofibroma Plexiforme/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Animales , Muerte Celular/efectos de los fármacos , Quimiocina CCL2/metabolismo , Curcumina/farmacología , Citocinas/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Humanos , Janus Quinasa 2/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Proteínas Quimioatrayentes de Monocitos/metabolismo , Neurofibromatosis 1/metabolismo , Células de Schwann/efectos de los fármacos , Células de Schwann/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Neurofibromas are benign peripheral nerve tumors driven by NF1 loss in Schwann cells (SCs). Macrophages are abundant in neurofibromas, and macrophage targeted interventions may have therapeutic potential in these tumors. We generated gene expression data from fluorescence-activated cell sorted (FACS) SCs and macrophages from wild-type and mutant nerve and neurofibroma to identify candidate pathways involved in SC-macrophage cross-talk. While in 1-month-old Nf1 mutant nerve neither SCs nor macrophages significantly differed from their normal counterparts, both macrophages and SCs showed significantly altered cytokine gene expression in neurofibromas. Computationally reconstructed SC-macrophage molecular networks were enriched for inflammation-associated pathways. We verified that neurofibroma SC conditioned medium contains macrophage chemo-attractants including colony stimulation factor 1 (CSF1). Network analysis confirmed previously implicated pathways and predict novel paracrine and autocrine loops involving cytokines, chemokines, and growth factors. Network analysis also predicted a central role for decreased type-I interferon signaling. We validated type-I interferon expression in neurofibroma by protein profiling, and show that treatment of neurofibroma-bearing mice with polyethylene glycolyated (PEGylated) type-I interferon-α2b reduces the expression of many cytokines overexpressed in neurofibroma. These studies reveal numerous potential targetable interactions between Nf1 mutant SCs and macrophages for further analyses.