RESUMO
BACKGROUND: Breast cancer is the most common cancer in women diagnosed in the U.S. and worldwide. Obesity increases breast cancer risk without clear underlying molecular mechanisms. Our studies demonstrate that circulating adipose fatty acid binding protein (A-FABP, or FABP4) links obesity-induced dysregulated lipid metabolism and breast cancer risk, thus potentially offering a new target for breast cancer treatment. METHODS: We immunized FABP4 knockout mice with recombinant human FABP4 and screened hybridoma clones with specific binding to FABP4. The potential effects of antibodies on breast cancer cells in vitro were evaluated using migration, invasion, and limiting dilution assays. Tumor progression in vivo was evaluated in various types of tumorigenesis models including C57BL/6 mice, Balb/c mice, and SCID mice. The phenotype and function of immune cells in tumor microenvironment were characterized with multi-color flow cytometry. Tumor stemness was detected by ALDH assays. To characterize antigen-antibody binding capacity, we determined the dissociation constant of selected anti-FABP4 antibodies via surface plasmon resonance. Further analyses in tumor tissue were performed using 10X Genomics Visium spatial single cell technology. RESULTS: Herein, we report the generation of humanized monoclonal antibodies blocking FABP4 activity for breast cancer treatment in mouse models. One clone, named 12G2, which significantly reduced circulating levels of FABP4 and inhibited mammary tumor growth, was selected for further characterization. After confirming the therapeutic efficacy of the chimeric 12G2 monoclonal antibody consisting of mouse variable regions and human IgG1 constant regions, 16 humanized 12G2 monoclonal antibody variants were generated by grafting its complementary determining regions to selected human germline sequences. Humanized V9 monoclonal antibody showed consistent results in inhibiting mammary tumor growth and metastasis by affecting tumor cell mitochondrial metabolism. CONCLUSIONS: Our current evidence suggests that targeting FABP4 with humanized monoclonal antibodies may represent a novel strategy for the treatment of breast cancer and possibly other obesity- associated diseases.
Assuntos
Neoplasias da Mama , Proteínas de Ligação a Ácido Graxo , Animais , Proteínas de Ligação a Ácido Graxo/antagonistas & inibidores , Proteínas de Ligação a Ácido Graxo/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/imunologia , Humanos , Feminino , Camundongos , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Camundongos Knockout , Ensaios Antitumorais Modelo de Xenoenxerto , Microambiente Tumoral/imunologia , Modelos Animais de Doenças , Camundongos SCIDRESUMO
Induction of muscle pain triggers a local immune response to produce pain and this mechanism may be sex and activity level dependent. The purpose of this study was to measure the immune system response in the muscle following induction of pain in sedentary and physically active mice. Muscle pain was produced via an activity-induced pain model using acidic saline combined with fatiguing muscle contractions. Prior to induction of muscle pain, mice (C57/BL6) were sedentary or physically active (24hr access to running wheel) for 8 weeks. The ipsilateral gastrocnemius was harvested 24hr after induction of muscle pain for RNA sequencing or flow cytometry. RNA sequencing revealed activation of several immune pathways in both sexes after induction of muscle pain, and these pathways were attenuated in physically active females. Uniquely in females, the antigen processing and presentation pathway with MHC II signaling was activated after induction of muscle pain; activation of this pathway was blocked by physical activity. Blockade of MHC II attenuated development of muscle hyperalgesia exclusively in females. Induction of muscle pain increased the number of macrophages and T-cells in the muscle in both sexes, measured by flow cytometry. In both sexes, the phenotype of macrophages shifted toward a pro-inflammatory state after induction of muscle pain in sedentary mice (M1 + M1/2) but toward an anti-inflammatory state in physically active mice (M2 + M0). Thus, induction of muscle pain activates the immune system with sex-specific differences in the transcriptome while physical activity attenuates immune response in females and alters macrophage phenotype in both sexes.
Assuntos
Hiperalgesia , Mialgia , Masculino , Feminino , Animais , Camundongos , Hiperalgesia/metabolismo , Macrófagos/metabolismo , Medição da Dor , ImunidadeRESUMO
Anti-CD20 monoclonal antibody (mAb) therapy is a mainstay of therapy for B cell malignancies, however many patients fail to respond or eventually develop resistance. The current understanding of mechanisms responsible for this resistance is limited. When peripheral blood mononuclear cells of healthy donors were cultured with Raji cells for 7 days, rituximab (RTX) induced NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC), enhanced NK cell viability and increased or maintained NK expression of CD56, CD16, CD57 and KIR. T cells, mainly CD4+, mediated these changes in a contact-dependent manner, with local T cell production of IL2 playing a central role. Similar findings were found when autologous B cells were used as target cells demonstrating the need for T cell help was not due to allogenic reaction. Results with other anti-CD20 and anti-EGFR antibodies were consistent. Small numbers of T cells activated by anti-CD3/CD28 beads or bispecific antibody enhanced RTX-mediated NK cell ADCC, viability and phenotypical changes. Pathway analysis of bulk NK cell mRNA sequencing after activation by RTX with and without T cells was consistent with T cells maintaining the viability of the activated NK cells. These findings suggest T cell help, mediated in large part by local production of IL2, contributes to NK cell ADCC and viability, and that activating T cells in the tumor microenvironment, such as through the use of anti-CD3 based bispecific antibodies, could enhance the efficacy of anti-CD20 and other mAb therapies where NK-mediated ADCC is a primary mechanism of action.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/imunologia , Antígenos CD20/química , Linfócitos T CD4-Positivos/metabolismo , Células Matadoras Naturais/imunologia , Leucócitos Mononucleares/imunologia , Neoplasias/imunologia , Rituximab/farmacologia , Antineoplásicos Imunológicos/farmacologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Ativação Linfocitária , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Células Tumorais CultivadasRESUMO
MOTIVATION: Single-cell RNA-sequencing (scRNA-seq) provides more granular biological information than bulk RNA-sequencing; bulk RNA sequencing remains popular due to lower costs which allows processing more biological replicates and design more powerful studies. As scRNA-seq costs have decreased, collecting data from more than one biological replicate has become more feasible, but careful modeling of different layers of biological variation remains challenging for many users. Here, we propose a statistical model for scRNA-seq gene counts, describe a simple method for estimating model parameters and show that failing to account for additional biological variation in scRNA-seq studies can inflate false discovery rates (FDRs) of statistical tests. RESULTS: First, in a simulation study, we show that when the gene expression distribution of a population of cells varies between subjects, a naïve approach to differential expression analysis will inflate the FDR. We then compare multiple differential expression testing methods on scRNA-seq datasets from human samples and from animal models. These analyses suggest that a naïve approach to differential expression testing could lead to many false discoveries; in contrast, an approach based on pseudobulk counts has better FDR control. AVAILABILITY AND IMPLEMENTATION: A software package, aggregateBioVar, is freely available on Bioconductor (https://www.bioconductor.org/packages/release/bioc/html/aggregateBioVar.html) to accommodate compatibility with upstream and downstream methods in scRNA-seq data analysis pipelines. SUPPLEMENTARY INFORMATION: Raw gene-by-cell count matrices for pig scRNA-seq data are available as GEO accession GSE150211. Supplementary data are available at Bioinformatics online.
RESUMO
BACKGROUND: Traumatic brain injury (TBI) is a leading cause of death and disability that lacks neuroprotective therapies. Following a TBI, secondary injury response pathways are activated and contribute to ongoing neurodegeneration. Microglia and astrocytes are critical neuroimmune modulators with early and persistent reactivity following a TBI. Although histologic glial reactivity is well established, a precise understanding of microglia and astrocyte function following trauma remains unknown. METHODS: Adult male C57BL/6J mice underwent either fluid percussion or sham injury. RNA sequencing of concurrently isolated microglia and astrocytes was conducted 7 days post-injury to evaluate cell-type-specific transcriptional responses to TBI. Dual in situ hybridization and immunofluorescence were used to validate the TBI-induced gene expression changes in microglia and astrocytes and to identify spatial orientation of cells expressing these genes. Comparative analysis was performed between our glial transcriptomes and those from prior reports in mild TBI and other neurologic diseases to determine if severe TBI induces unique states of microglial and astrocyte activation. RESULTS: Our findings revealed sustained, lineage-specific transcriptional changes in both microglia and astrocytes, with microglia showing a greater transcriptional response than astrocytes at this subacute time point. Microglia and astrocytes showed overlapping enrichment for genes related to type I interferon signaling and MHC class I antigen presentation. The microglia and astrocyte transcriptional response to severe TBI was distinct from prior reports in mild TBI and other neurodegenerative and neuroinflammatory diseases. CONCLUSION: Concurrent lineage-specific analysis revealed novel TBI-specific transcriptional changes; these findings highlight the importance of cell-type-specific analysis of glial reactivity following TBI and may assist with the identification of novel, targeted therapies.
Assuntos
Astrócitos/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Interferon Tipo I/biossíntese , Microglia/metabolismo , Transcriptoma/fisiologia , Animais , Astrócitos/patologia , Lesões Encefálicas Traumáticas/genética , Lesões Encefálicas Traumáticas/patologia , Interferon Tipo I/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/patologiaRESUMO
Life scientists are increasingly turning to high-throughput sequencing technologies in their research programs, owing to the enormous potential of these methods. In a parallel manner, the number of core facilities that provide bioinformatics support are also increasing. Notably, the generation of complex large datasets has necessitated the development of bioinformatics support core facilities that aid laboratory scientists with cost-effective and efficient data management, analysis, and interpretation. In this article, we address the challenges-related to communication, good laboratory practice, and data handling-that may be encountered in core support facilities when providing bioinformatics support, drawing on our own experiences working as support bioinformaticians on multidisciplinary research projects. Most importantly, the article proposes a list of guidelines that outline how these challenges can be preemptively avoided and effectively managed to increase the value of outputs to the end user, covering the entire research project lifecycle, including experimental design, data analysis, and management (i.e., sharing and storage). In addition, we highlight the importance of clear and transparent communication, comprehensive preparation, appropriate handling of samples and data using monitoring systems, and the employment of appropriate tools and standard operating procedures to provide effective bioinformatics support.
Assuntos
Biologia Computacional/economia , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Pesquisa Biomédica/economia , Pesquisa Biomédica/métodos , Comunicação , Biologia Computacional/normas , Sequenciamento de Nucleotídeos em Larga Escala/economia , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Projetos de Pesquisa/normasRESUMO
Type I interferons (IFNs) are required for spontaneous lacrimal gland inflammation in the nonobese diabetic (NOD) mouse model of Sjögren's disease, but the consequences of type I IFN signaling are not well-defined. Here, we use RNA sequencing to define cytokine and chemokine genes upregulated in lacrimal glands of NOD mice in a type I IFN-dependent manner. Interleukin (IL)-21 was the highest differentially expressed cytokine gene, and Il21 knockout NOD mice were relatively protected from lacrimal gland inflammation. We defined a set of chemokines upregulated early in disease including Cxcl9 and Cxcl10, which share a receptor, CXCR3. CXCR3+ T cells were enriched in lacrimal glands with a dominant proportion of CXCR3+ regulatory T cells. Together these data define the early cytokine and chemokine signals associated with type I IFN-signaling in the development of lacrimal gland inflammation in NOD mice providing insight into the role of type I IFN in autoimmunity development.
Assuntos
Quimiocina CXCL10/imunologia , Quimiocina CXCL9/imunologia , Interleucinas/imunologia , Aparelho Lacrimal/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Quimiocina CXCL10/genética , Quimiocina CXCL9/genética , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucinas/genética , Aparelho Lacrimal/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Receptores CXCR3/genética , Receptores CXCR3/imunologia , Transdução de Sinais/genética , Linfócitos T Reguladores/patologiaRESUMO
Sjögren syndrome (SS) is an immunologically complex, chronic autoimmune disease targeting lacrimal and salivary glands. Nonobese diabetic (NOD) mice spontaneously develop inflammation of lacrimal and salivary glands with histopathological features similar to SS in humans including focal lymphocytic infiltrates in the affected glands. The innate immune signals driving lymphocytic infiltration of these glands are not well-defined. Here we evaluate the role of Toll-like receptor (TLR) 7 in the development of SS-like manifestations in NOD mice. We created a Tlr7 knockout NOD mouse strain and performed histological and gene expression studies to characterize the effects of TLR7 on autoimmunity development. TLR7 was required for male-specific lacrimal gland inflammation but not for female-specific salivary gland inflammation. Moreover, TLR7 was required for type 1 diabetes development in male but not female NOD mice. RNA sequencing demonstrated that TLR7 was associated with a type I interferon (IFN) response and a type I IFN-independent B cell response in the lacrimal glands. Together these studies identify a previously unappreciated pathogenic role for TLR7 in lacrimal gland autoimmunity and T1D development in male NOD mice adding to the growing body of evidence supporting sex differences in mechanisms of autoimmune disease in NOD mice.
Assuntos
Autoimunidade/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Aparelho Lacrimal/imunologia , Glicoproteínas de Membrana/imunologia , Síndrome de Sjogren/imunologia , Receptor 7 Toll-Like/imunologia , Animais , Linfócitos B/imunologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interferon Tipo I/metabolismo , Aparelho Lacrimal/citologia , Aparelho Lacrimal/patologia , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , RNA-Seq , Glândulas Salivares/citologia , Glândulas Salivares/imunologia , Glândulas Salivares/metabolismo , Sexo , Receptor 7 Toll-Like/genéticaRESUMO
Emerging evidence indicates that aneurysmal subarachnoid hemorrhage (aSAH) elicits a response from both innate and adaptive immune systems. An upregulation of CD8 + CD161 + cells has been observed in the cerebrospinal fluid (CSF) after aSAH, yet the precise role of these cells in the context of aSAH is unkown. CSF samples from patients with aSAH and non-aneurysmal SAH (naSAH) were analyzed. Single-cell RNA sequencing (scRNAseq) was performed on CD8 + CD161 + sorted samples from aSAH patients. Cell populations were identified using "clustering." Gene expression levels of ten previously described genes involved in inflammation were quantified from aSAH and naSAH samples using RT-qPCR. The study focused on the following genes: CCL5, CCL7, APOE, SPP1, CXCL8, CXCL10, HMOX1, LTB, MAL, and HLA-DRB1. Gene clustering analysis revealed that monocytes, NK cells, and T cells expressed CD8 + CD161 + in the CSF of patients with aSAH. In comparison to naSAH samples, aSAH samples exhibited higher mRNA levels of CXCL10 (median, IQR = 90, 16-149 vs. 0.5, 0-6.75, p = 0.02). A trend towards higher HMOX1 levels was also observed in aSAH (median, IQR = 12.6, 9-17.6 vs. 2.55, 1.68-5.7, p = 0.076). Specifically, CXCL10 and HMOX1 were expressed by the monocyte subpopulation. Monocytes, NK cells, and T cells can potentially express CD8 + CD161 + in patients with aSAH. Notably, monocytes show high levels of CXCL10. The elevated expression of CXCL10 in aSAH compared to naSAH indicates its potential significance as a target for future studies.
RESUMO
Elevated airborne PCB levels in older schools are concerning due to their health impacts, including cancer, metabolic dysfunction-associated steatotic liver disease (MASLD), cardiovascular issues, neurodevelopmental diseases, and diabetes. During a four-week inhalation exposure to PCB52, an air pollutant commonly found in school environments, adolescent rats exhibited notable presence of PCB52 and its hydroxylated forms in their livers, alongside changes in gene expression. Female rats exhibited more pronounced changes in gene expression compared to males, particularly in fatty acid synthesis genes regulated by the transcription factor SREBP1. In vitro studies with human liver cells showed that the hydroxylated metabolite of PCB52, 4-OH-PCB52, but not the parent compound, upregulated genes involved in fatty acid biosynthesis similar to in vivo exposure. These findings highlight the sex-specific effects of PCB52 exposure on livers, particularly in females, suggesting a potential pathway for increased MASLD susceptibility.
RESUMO
A group of diverse proteins reversibly binds to growing microtubule plus ends through interactions with end-binding proteins (EBs). These +TIPs control microtubule dynamics and microtubule interactions with other intracellular structures. Here, we use cytoplasmic linker-associated protein 2 (CLASP2) binding to EB1 to determine how multisite phosphorylation regulates interactions with EB1. The central, intrinsically disordered region of vertebrate CLASP proteins contains two SXIP EB1 binding motifs that are required for EB1-mediated plus-end-tracking in vitro. In cells, both EB1 binding motifs can be functional, but most of the binding free energy results from nearby electrostatic interactions. By employing molecular dynamics simulations of the EB1 interaction with a minimal CLASP2 plus-end-tracking module, we find that conserved arginine residues in CLASP2 form extensive hydrogen-bond networks with glutamate residues predominantly in the unstructured, acidic C-terminal tail of EB1. Multisite phosphorylation of glycogen synthase kinase 3 (GSK3) sites near the EB1 binding motifs disrupts this electrostatic "molecular Velcro." Molecular dynamics simulations and (31)P NMR spectroscopy indicate that phosphorylated serines participate in intramolecular interactions with and sequester arginine residues required for EB1 binding. Multisite phosphorylation of these GSK3 motifs requires priming phosphorylation by interphase or mitotic cyclin-dependent kinases (CDKs), and we find that CDK- and GSK3-dependent phosphorylation completely disrupts CLASP2 microtubule plus-end-tracking in mitosis.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Motivos de Aminoácidos , Quinase 3 da Glicogênio Sintase/química , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/química , Microtúbulos/genética , Microtúbulos/metabolismo , Mitose/fisiologia , Simulação de Dinâmica Molecular , Fosforilação/fisiologia , Estrutura Terciária de Proteína , Eletricidade EstáticaRESUMO
Polychlorinated biphenyls (PCBs) accumulate in adipose tissue and are linked to obesity and diabetes. The congener, PCB52 (2,2',5,5'-tetrachorobiphenyl), is found at high levels in school air. Hydroxylation of PCB52 to 4-OH-PCB52 (4-hydroxy-2,2',5,5'-tetrachorobiphenyl) may increase its toxicity. To understand PCB52's role in causing adipose dysfunction, we exposed human preadipocytes to PCB52 or 4-OH-PCB52 across a time course and assessed transcript changes using RNAseq. 4-OH-PCB52 caused considerably more changes in the number of differentially expressed genes as compared to PCB52. Both PCB52 and 4-OH-PCB52 upregulated transcript levels of the sulfotransferase SULT1E1 at early time points, but cytochrome P450 genes were generally not affected. A set of genes known to be transcriptionally regulated by PPARα were consistently downregulated by PCB52 at all time points. In contrast, 4-OH-PCB52 affected a variety of pathways, including those involving cytokine responses, hormone responses, focal adhesion, Hippo, and Wnt signaling. Sets of genes known to be transcriptionally regulated by IL17A or parathyroid hormone (PTH) were found to be consistently downregulated by 4-OH-PCB52. Most of the genes affected by PCB52 and 4-OH-PCB52 were different and, of those that were the same, many were changed in an opposite direction. These studies provide insight into how PCB52 or its metabolites may cause adipose dysfunction to cause disease.
Assuntos
Bifenilos Policlorados , Humanos , Bifenilos Policlorados/toxicidade , Hidroxilação , Sistema Enzimático do Citocromo P-450/metabolismo , Expressão GênicaRESUMO
Polychlorinated biphenyls (PCBs) were used extensively in building materials, including those used in schools. PCBs accumulate in fat, and exposure to PCBs is associated with the development of cancer, neurodevelopmental disorders, cardiovascular disease, obesity, and diabetes. The non-dioxin-like PCB congener, PCB52 (2,2',5,5'-tetrachlorobiphenyl), is found at one of the highest levels of any congener in school air. PCB52 is oxidized in the liver to hydroxylated forms, mainly 4-OH-PCB52 (2,2',5,5'-tetrachlorobiphenyl-4-ol). In a previous study, we reported on RNAseq data generated from exposure of human preadipocytes to the dioxin-like PCB congener, PCB126. In this new dataset, we used identical techniques to examine alterations in gene transcript levels in human preadipocytes exposed to PCB52 or 4-OH-PCB52 over a time course. This updated set of data provides a comprehensive transcriptional profile of changes that occur in preadipocytes exposed to PCB52 or 4-OH-PCB52 over time and allows for comparison of these changes between the parent compound and its hydroxy metabolite. The datasets will allow others to explore how PCB52 and 4-OH-PCB52 impact biological pathways in preadipocytes. Further studies can be performed to determine how these changes might lead to disease.
RESUMO
One of the hallmarks of intractable psoriasis is neutrophil infiltration in skin lesions. However, detailed molecular mechanisms of neutrophil chemotaxis and activation remain unclear. Here, we demonstrate a significant upregulation of epidermal fatty acid binding protein (E-FABP, FABP5) in the skin of human psoriasis and psoriatic mouse models. Genetic deletion of FABP5 in mice by global knockout and keratinocyte conditional (Krt6a-Cre) knockout, but not myeloid cell conditional (LysM-Cre) knockout, attenuates psoriatic symptoms. Immunophenotypic analysis shows that FABP5 deficiency specifically reduces skin recruitment of Ly6G+ neutrophils. Mechanistically, activated keratinocytes produce chemokines and cytokines that trigger neutrophil chemotaxis and activation in an FABP5-dependent manner. Proteomic analysis further identifies that FABP5 interacts with valosin-containing protein (VCP), a key player in NF-κB signaling activation. Silencing of FABP5, VCP, or both inhibits NF-κB/neutrophil chemotaxis signaling. Collectively, these data demonstrate dysregulated FABP5 as a molecular mechanism promoting NF-κB signaling and neutrophil infiltration in psoriasis pathogenesis.
Assuntos
Neutrófilos , Psoríase , Animais , Humanos , Camundongos , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Inflamação/metabolismo , Queratinócitos/metabolismo , Neutrófilos/metabolismo , NF-kappa B/metabolismo , Proteômica , Psoríase/patologia , Proteína com Valosina/metabolismoRESUMO
Schizophrenia is marked by poor social functioning that can have a severe impact on quality of life and independence, but the underlying neural circuity is not well understood. Here we used a translational model of subanesthetic ketamine in mice to delineate neural pathways in the brain linked to social deficits in schizophrenia. Mice treated with chronic ketamine (30 mg/kg/day for 10 days) exhibit profound social and sensorimotor deficits as previously reported. Using three- dimensional c-Fos immunolabeling and volume imaging (iDISCO), we show that ketamine treatment resulted in hypoactivation of the lateral septum (LS) in response to social stimuli. Chemogenetic activation of the LS rescued social deficits after ketamine treatment, while chemogenetic inhibition of previously active populations in the LS (i.e. social engram neurons) recapitulated social deficits in ketamine-naïve mice. We then examined the translatome of LS social engram neurons and found that ketamine treatment dysregulated genes implicated in neuronal excitability and apoptosis, which may contribute to LS hypoactivation. We also identified 38 differentially expressed genes (DEGs) in common with human schizophrenia, including those involved in mitochondrial function, apoptosis, and neuroinflammatory pathways. Chemogenetic activation of LS social engram neurons induced downstream activity in the ventral part of the basolateral amygdala, subparafascicular nucleus of the thalamus, intercalated amygdalar nucleus, olfactory areas, and dentate gyrus, and it also reduces connectivity of the LS with the piriform cortex and caudate-putamen. In sum, schizophrenia-like social deficits may emerge via changes in the intrinsic excitability of a discrete subpopulation of LS neurons that serve as a central hub to coordinate social behavior via downstream projections to reward, fear extinction, motor and sensory processing regions of the brain.
RESUMO
Accumulating evidence suggests that type I interferon (IFN-I) signaling is a key contributor to immune cell-mediated neuropathology in neurodegenerative diseases. Recently, we demonstrated a robust upregulation of type I interferon-stimulated genes in microglia and astrocytes following experimental traumatic brain injury (TBI). The specific molecular and cellular mechanisms by which IFN-I signaling impacts the neuroimmune response and neuropathology following TBI remains unknown. Using the lateral fluid percussion injury model (FPI) in adult male mice, we demonstrated that IFN α/ß receptor (IFNAR) deficiency resulted in selective and sustained blockade of type I interferon-stimulated genes following TBI as well as decreased microgliosis and monocyte infiltration. Phenotypic alteration of reactive microglia also occurred with diminished expression of molecules needed for MHC class I antigen processing and presentation following TBI. This was associated with decreased accumulation of cytotoxic T cells in the brain. The IFNAR-dependent modulation of the neuroimmune response was accompanied by protection from secondary neuronal death, white matter disruption, and neurobehavioral dysfunction. These data support further efforts to leverage the IFN-I pathway for novel, targeted therapy of TBI.
RESUMO
Accumulating evidence suggests that type I interferon (IFN-I) signaling is a key contributor to immune cell-mediated neuropathology in neurodegenerative diseases. Recently, we demonstrated a robust upregulation of type I interferon-stimulated genes in microglia and astrocytes following experimental traumatic brain injury (TBI). The specific molecular and cellular mechanisms by which IFN-I signaling impacts the neuroimmune response and neuropathology following TBI remains unknown. Using the lateral fluid percussion injury model (FPI) in adult male mice, we demonstrated that IFN α/ß receptor (IFNAR) deficiency resulted in selective and sustained blockade of type I interferon-stimulated genes following TBI as well as decreased microgliosis and monocyte infiltration. Molecular alteration of reactive microglia also occurred with diminished expression of genes needed for MHC class I antigen processing and presentation following TBI. This was associated with decreased accumulation of cytotoxic T cells in the brain. The IFNAR-dependent modulation of the neuroimmune response was accompanied by protection from secondary neuronal death, white matter disruption, and neurobehavioral dysfunction. These data support further efforts to leverage the IFN-I pathway for novel, targeted therapy of TBI.
Assuntos
Lesões Encefálicas Traumáticas , Interferon Tipo I , Masculino , Animais , Camundongos , Neuropatologia , Lesões Encefálicas Traumáticas/complicações , Encéfalo , AnticorposRESUMO
PURPOSE: Malignant peripheral nerve sheath tumors (MPNST) are lethal, Ras-driven sarcomas that lack effective therapies. We investigated effects of targeting cyclin-dependent kinases 4 and 6 (CDK4/6), MEK, and/or programmed death-ligand 1 (PD-L1) in preclinical MPNST models. EXPERIMENTAL DESIGN: Patient-matched MPNSTs and precursor lesions were examined by FISH, RNA sequencing, IHC, and Connectivity-Map analyses. Antitumor activity of CDK4/6 and MEK inhibitors was measured in MPNST cell lines, patient-derived xenografts (PDX), and de novo mouse MPNSTs, with the latter used to determine anti-PD-L1 response. RESULTS: Patient tumor analyses identified CDK4/6 and MEK as actionable targets for MPNST therapy. Low-dose combinations of CDK4/6 and MEK inhibitors synergistically reactivated the retinoblastoma (RB1) tumor suppressor, induced cell death, and decreased clonogenic survival of MPNST cells. In immune-deficient mice, dual CDK4/6-MEK inhibition slowed tumor growth in 4 of 5 MPNST PDXs. In immunocompetent mice, combination therapy of de novo MPNSTs caused tumor regression, delayed resistant tumor outgrowth, and improved survival relative to monotherapies. Drug-sensitive tumors that regressed contained plasma cells and increased cytotoxic T cells, whereas drug-resistant tumors adopted an immunosuppressive microenvironment with elevated MHC II-low macrophages and increased tumor cell PD-L1 expression. Excitingly, CDK4/6-MEK inhibition sensitized MPNSTs to anti-PD-L1 immune checkpoint blockade (ICB) with some mice showing complete tumor regression. CONCLUSIONS: CDK4/6-MEK inhibition induces a novel plasma cell-associated immune response and extended antitumor activity in MPNSTs, which dramatically enhances anti-PD-L1 therapy. These preclinical findings provide strong rationale for clinical translation of CDK4/6-MEK-ICB targeted therapies in MPNST as they may yield sustained antitumor responses and improved patient outcomes.
Assuntos
Neurofibrossarcoma , Camundongos , Humanos , Animais , Neurofibrossarcoma/tratamento farmacológico , Plasmócitos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Quinases de Proteína Quinase Ativadas por Mitógeno , Linhagem Celular Tumoral , Microambiente Tumoral , Quinase 4 Dependente de CiclinaRESUMO
Background: Breast milk has abundant extracellular vesicles (EVs) containing various biological molecules (cargo), including miRNAs. EVs are not degraded in the gastrointestinal system and circulation; thus, breast milk EVs (bEVs) are expected to interact with other organs in breastfed infants and modify the gene expression of recipient cells using miRNAs. Maternal pre-pregnancy BMI is a critical factor influencing the composition of breast milk. Thus, in mothers with obesity, miRNAs in bEVs can be altered, which might be associated with adverse health outcomes in infants. In this study, we examined 798 miRNAs to determine which miRNAs are altered in the bEVs of mothers with obesity and their potential impact on breastfed infants. Methods: We recruited healthy nursing mothers who were either of normal weight (BMI < 25) or with obesity (BMI ≥ 30) based on their pre-pregnancy BMI, and delivered a singleton baby in the prior 6 months. EVs were isolated from breast milk with ultracentrifugation. bEV characteristics were examined by flow cytometry and fluorescence imaging of EV markers. A total of 798 miRNAs were screened using a NanoString human miRNA panel to find differentially expressed miRNAs in bEVs of mothers with obesity compared to mothers of normal weight. Results: We included 65 nursing mothers: 47 of normal weight and 18 with obesity based on pre-pregnancy BMI. After bEV isolation, we confirmed the expression of various EV markers. Out of 37 EV markers, CD326 (EpCaM) was the most highly expressed in bEVs. The most abundant miRNAs in bEVs include miR-30b-5p, miR-4454, miR-494-3p, and let-7 miRNAs. Target genes of the top 10 miRNAs were associated with cancer, prolactin pathway, EGFR, ErbB, and FoxO signaling pathway. In bEVs of mothers with obesity, 19 miRNAs were differentially expressed (adjusted p < 0.05 cut-off), which include miR-575, miR-630, miR-642a-3p, and miR-652-5p. These miRNAs and their target genes were associated with neurological diseases and psychological disorders. Conclusion: In this study, we characterized bEVs and demonstrated altered miRNAs in bEVs of mothers with obesity and identified the pathways of their potential target genes. Our findings will provide insight for future studies investigating the role of bEVs in breastfed infants.
RESUMO
Exposure to polychlorinated biphenyls (PCBs) has been associated with the development of metabolic syndrome, a cluster of diseases that includes obesity, diabetes, liver steatosis, and cardiovascular problems. PCBs accumulate and fat and are known to act on adipocytes and their precursors, termed preadipocytes. The PCB congener, PCB126, has been shown to activate the aryl hydrocarbon receptor (AhR) as well as proinflammatory genes. Here, we used RNAseq to assess gene transcript changes that occur in PCB126-exposed human preadipocytes over a time course. RNA was collected from 4 replicates of PCB126-exposed and control-treated preadipocytes at 9 h, 24 h, and 72 h post-exposure. RNA was processed for RNAseq analysis using a NovaSeq 6000 with an obtained minimum of 25 million paired-end 50 bp reads per sample. Reads were aligned using the salmon aligner and transcript expression values were summarized to the gene level using tximport. Gene transcript level counts comparing treated- versus control-treated cells were used for differential expression analysis using DESeq2. Differential expression Excel tables (one for each time point) were generated displaying average differential expression (log2 fold change) of the 4 replicates of treated versus control samples with cutoffs of 0.3 log2 fold change (increase or decrease) and p-values of less than 0.05. FastQ, raw, and differential expression tables were uploaded to GEO. A heat map of genes that were changed in common across all time points was generated using GraphPrism. The data generated from this analysis provides a full transcriptional profile of changes that occur over time in preadipocytes that have been exposed to PCB126. The rich datasets can be mined by other researchers to understand how PCB126 and other dioxin-like compounds, including other PCB congeners such as PCB77 and PCB118, affect biological pathways in preadipocytes and other cell types to cause disease.