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1.
Front Immunol ; 15: 1347948, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38370417

RESUMO

Background: Anti-IgLON5 disease is a neurological disorder characterized by autoantibodies against IgLON5 and pathological evidence of neurodegeneration. IgLON5 is a cell adhesion molecule of unknown function that is highly expressed in the brain. Our aim was to investigate the impact of IgLON5 loss-of-function in evaluating brain morphology, social behavior, and the development of symptoms observed in an IgLON5 knockout (IgLON5-KO) mouse model. Methods: The IgLON5-KO mice were generated using CRISPR-Cas9 technology. Immunohistochemistry on fixed sagittal brain sections and Western blotting brain lysates were used to confirm IgLON5 silencing and to evaluate the presence of other cell surface proteins. Two- month-old IgLON5-KO and wild-type (WT) mice underwent a comprehensive battery of behavioral tests to assess 1) locomotion, 2) memory, 3) anxiety, 4) social interaction, and 5) depressive-like behavior. Brain sections were examined for the presence of anatomical abnormalities and deposits of hyperphosphorylated tau in young adult (2-month-old) and aged (22-month-old) mice. Results: Mice did not develop neurological symptoms reminiscent of those seen in patients with anti-IgLON5 disease. Behavioral testing revealed that 2-month-old IgLON5-KO mice showed subtle alterations in motor coordination and balance. IgLON5-KO females exhibited hyperactivity during night and day. Males were observed to have depressive-like behavior and excessive nest-building behavior. Neuropathological studies did not reveal brain morphological alterations or hyperphosphorylated tau deposits. Conclusion: IgLON5-KO mice showed subtle alterations in behavior and deficits in fine motor coordination but did not develop the clinical phenotype of anti-IgLON5 disease.


Assuntos
Autoanticorpos , Apneia Obstrutiva do Sono , Masculino , Feminino , Humanos , Camundongos , Animais , Lactente , Camundongos Knockout , Autoanticorpos/metabolismo , Encéfalo/metabolismo , Ansiedade , Comportamento Social , Moléculas de Adesão Celular Neuronais
2.
Hum Gene Ther ; 35(3-4): 123-131, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38299967

RESUMO

The enzyme choline acetyltransferase (ChAT) synthesizes acetylcholine from acetyl-CoA and choline at the neuromuscular junction and at the nerve terminals of cholinergic neurons. Mutations in the ChAT gene (CHAT) result in a presynaptic congenital myasthenic syndrome (CMS) that often associates with life-threatening episodes of apnea. Knockout mice for Chat (Chat-/-) die at birth. To circumvent the lethality of this model, we crossed mutant mice possessing loxP sites flanking Chat exons 4 and 5 with mice that expressed Cre-ERT2. Injection of tamoxifen (Tx) at postnatal (P) day 11 in these mice induced downregulation of Chat, autonomic failure, weakness, and death. However, a proportion of Chatflox/flox-Cre-ERT2 mice receiving at birth an intracerebroventricular injection of 2 × 1013 vg/kg adeno-associated virus type 9 (AAV9) carrying human CHAT (AAV9-CHAT) survived a subsequent Tx injection and lived to adulthood without showing signs of weakness. Likewise, injection of AA9-CHAT by intracisternal injection at P28 after the onset of weakness also resulted in survival to adulthood. The expression of Chat in spinal motor neurons of Chatflox/flox-Cre-ERT2 mice injected with Tx was markedly reduced, but AAV-injected mice showed a robust recovery of ChAT expression, which was mainly translated by the human CHAT RNA. The biodistribution of the viral genome was widespread but maximal in the spinal cord and brain of AAV-injected mice. No significant histopathological changes were observed in the brain, liver, and heart of AAV-injected mice after 1 year follow-up. Thus, AAV9-mediated gene therapy may provide an effective and safe treatment for patients severely affected with CHAT-CMS.


Assuntos
Colina O-Acetiltransferase , Dependovirus , Camundongos , Humanos , Animais , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Distribuição Tecidual , Camundongos Knockout , Terapia Genética
3.
Life Sci Alliance ; 7(4)2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38320810

RESUMO

The cellular response to a decrease in protein degradation by 26S proteasomes in chronic diseases is poorly understood. Pharmacological inhibition of proteasomes increases the expression of proteasome subunits and Proteasome Activator 200 (PA200), an alternative proteasome activator. In the S63del mouse model of the peripheral neuropathy Charcot Marie Tooth 1B (CMT1B), proteasomal protein degradation is decreased and proteasome gene expression is increased. Here, we show an increase in PA200 and PA200-bound proteasomes in the peripheral nerves of S63del mice. To test genetically whether the upregulation of PA200 was compensatory, we generated S63del//PA200-/- mice. Unexpectedly, in the sciatic nerves of these mice, there was greater proteasomal protein degradation than in S63del, less polyubiquitinated proteins and markers of the unfolded protein response, and a greater amount of assembled, active 26S proteasomes. These changes were not seen in PA200-/- controls and were therefore specific to the neuropathy. Furthermore, in S63del//PA200-/- mice, myelin thickness and nerve conduction were restored to WT levels. Thus, the upregulation of PA200 is maladaptive in S63del mice and its genetic ablation prevented neuropathy.


Assuntos
Doença de Charcot-Marie-Tooth , Complexo de Endopeptidases do Proteassoma , Camundongos , Animais , Complexo de Endopeptidases do Proteassoma/metabolismo , Camundongos Knockout , Doença de Charcot-Marie-Tooth/genética , Proteólise , Citoplasma/metabolismo
5.
Int Immunopharmacol ; 129: 111618, 2024 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-38354508

RESUMO

BACKGROUND: Acute hepatitis is a progressive inflammatory disorder that can lead to liver failure. Endothelial permeability is the vital pathophysiological change involved in infiltrating inflammatory factors. DDX24 has been implicated in immune signaling. However, the precise role of DDX24 in immune-mediated hepatitis remains unclear. Here, we investigate the phenotype of endothelium-targeted Ddx24 conditional knockout mice with Concanavalin A (ConA)-induced hepatitis. METHODS: Mice with homozygous endothelium-targeted Ddx24 conditional knockout (Ddx24flox/flox; Cdh5-Cre+) were established using the CRISPR/Cas9 mediated Cre-loxP system. We investigated the biological functions of endothelial cells derived from transgenic mice and explored the effects of Ddx24 in mice with ConA-induced hepatitis in vivo. The mass spectrometry was performed to identify the differentially expressed proteins in liver tissues of transgenic mice. RESULT: We successfully established mice with endothelium-targeted Ddx24 conditional knockout. The results showed migration and tube formation potentials of murine aortic endothelial cells with DDX24 silencing were significantly promoted. No differences were observed between Ddx24flox/flox; Cdh5-Cre+ and control regarding body weight and length, pathological tissue change and embryogenesis. We demonstrated Ddx24flox/flox; Cdh5-Cre+ exhibited exacerbation of ConA-induced hepatitis by up-regulating TNF-α and IFN-γ. Furthermore, endothelium-targeted Ddx24 conditional knockout caused vascular hyper-permeability in ConA-injected mice by down-regulating vascular integrity-associated proteins. Mechanistically, we identified Ddx24 might regulate immune-mediated hepatitis by inflammation-related permeable barrier pathways. CONCLUSION: These findings prove that endothelium-targeted Ddx24 conditional knockout exacerbates ConA-induced hepatitis in mice because of vascular hyper-permeability. The findings indicate a crucial role of DDX24 in regulating immune-mediated hepatitis, suggesting DDX24 as a potential therapeutic target in the disorder.


Assuntos
Células Endoteliais , Hepatite , Animais , Camundongos , Concanavalina A/toxicidade , Células Endoteliais/metabolismo , Endotélio/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos
6.
Transl Psychiatry ; 14(1): 101, 2024 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-38374108

RESUMO

G protein-coupled receptor 55 (GPR55) has been thought to be a putative cannabinoid receptor. However, little is known about its functional role in cannabinoid action and substance use disorders. Here we report that GPR55 is predominantly found in glutamate neurons in the brain, and its activation reduces self-administration of cocaine and nicotine in rats and mice. Using RNAscope in situ hybridization, GPR55 mRNA was identified in cortical vesicular glutamate transporter 1 (VgluT1)-positive and subcortical VgluT2-positive glutamate neurons, with no detection in midbrain dopamine (DA) neurons. Immunohistochemistry detected a GPR55-like signal in both wildtype and GPR55-knockout mice, suggesting non-specific staining. However, analysis using a fluorescent CB1/GPR55 ligand (T1117) in CB1-knockout mice confirmed GPR55 binding in glutamate neurons, not in midbrain DA neurons. Systemic administration of the GPR55 agonist O-1602 didnt impact ∆9-THC-induced analgesia, hypothermia and catalepsy, but significantly mitigated cocaine-enhanced brain-stimulation reward caused by optogenetic activation of midbrain DA neurons. O-1602 alone failed to alter extracellar DA, but elevated extracellular glutamate, in the nucleus accumbens. In addition, O-1602 also demonstrated inhibitory effects on cocaine or nicotine self-administration under low fixed-ratio and/or progressive-ratio reinforcement schedules in rats and wildtype mice, with no such effects observed in GPR55-knockout mice. Together, these findings suggest that GPR55 activation may functionally modulate drug-taking and drug-seeking behavior possibly via a glutamate-dependent mechanism, and therefore, GPR55 deserves further study as a new therapeutic target for treating substance use disorders.


Assuntos
Canabidiol/análogos & derivados , Cocaína , Transtornos Relacionados ao Uso de Substâncias , Ratos , Camundongos , Animais , Ácido Glutâmico/metabolismo , Preparações Farmacêuticas/metabolismo , Nicotina/farmacologia , Cocaína/farmacologia , Neurônios Dopaminérgicos/metabolismo , Camundongos Knockout , Receptores de Canabinoides/metabolismo , Receptores Acoplados a Proteínas G/genética
7.
J Endocrinol ; 261(1)2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38305305

RESUMO

Metabolic syndrome (MetS) is an increasing global health threat and strong risk factor for type 2 diabetes (T2D). MetS causes both hyperinsulinemia and islet size overexpansion, and pancreatic ß-cell failure impacts insulin and proinsulin secretion, mitochondrial density, and cellular identity loss. The low-density lipoprotein receptor knockout (LDLr-/-) model combined with high-fat diet (HFD) has been used to study alterations in multiple organs, but little is known about the changes to ß-cell identity resulting from MetS. Osteocalcin (OC), an insulin-sensitizing protein secreted by bone, shows promising impact on ß-cell identity and function. LDLr-/- mice at 12 months were fed chow or HFD for 3 months ± 4.5 ng/h OC. Islets were examined by immunofluorescence for alterations in nuclear Nkx6.1 and PDX1 presence, insulin-glucagon colocalization, islet size and %ß-cell and islet area by insulin and synaptophysin, and mitochondria fluorescence intensity by Tomm20. Bone mineral density (BMD) and %fat changes were examined by Piximus Dexa scanning. HFD-fed mice showed fasting hyperglycemia by 15 months, increased weight gain, %fat, and fasting serum insulin and proinsulin; concurrent OC treatment mitigated weight increase and showed lower proinsulin-to-insulin ratio, and higher BMD. HFD increased %ß and %islet area, while simultaneous OC-treatment with HFD was comparable to chow-fed mice. Significant reductions in nuclear PDX1 and Nkx6.1 expression, increased insulin-glucagon colocalization, and reduction in ß-cell mitochondria fluorescence intensity were noted with HFD, but largely prevented with OC administration. OC supplementation here suggests a benefit to ß-cell identity in LDLr-/- mice and offers intriguing clinical implications for countering metabolic syndrome.


Assuntos
Diabetes Mellitus Tipo 2 , Hiperinsulinismo , Células Secretoras de Insulina , Ilhotas Pancreáticas , Síndrome Metabólica , Animais , Camundongos , Diabetes Mellitus Tipo 2/metabolismo , Dieta Hiperlipídica/efeitos adversos , Glucagon/metabolismo , Hiperinsulinismo/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Lipoproteínas LDL , Síndrome Metabólica/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteocalcina/metabolismo , Proinsulina/metabolismo , Aumento de Peso
8.
Curr Biol ; 34(4): R133-R134, 2024 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-38412819

RESUMO

Serotonergic circuits in the central nervous system play important roles in regulating mood and behavior, yet the functions of peripheral serotonergic neurons are less understood. Here, we engineered mice lacking the serotonin-producing enzyme Tph2 in peripheral neurons but with intact Tph2 in central neurons. In contrast to mice lacking Tph2 in all neurons, mice lacking Tph2 in peripheral serotonergic neurons did not exhibit increased territorial aggression. However, similar to the total body Tph2 knockout (KO) mice, the conditional KO animals exhibited reduced gut motility and decreased anxiety-like behavior. These observations reveal that peripheral serotonergic neurons contribute to control of intestinal motility and anxiety-like behavior and suggest that therapeutics targeting this subset of peripheral neurons could be beneficial.


Assuntos
Neurônios Serotoninérgicos , Serotonina , Camundongos , Animais , Serotonina/fisiologia , Ansiedade/genética , Camundongos Knockout , Sistema Nervoso Central
9.
Autoimmunity ; 57(1): 2319202, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38389178

RESUMO

BACKGROUNDS: The role of O-GlcNAc transferase (OGT)-induced O-linked N-acetylglucosaminylation (O-GlcNAcylation) has been reported in multiple human diseases. However, its specific functions in osteoarthritis (OA) progression remain undetermined. OBJECTIVE: This study focused on the target proteins of OGT-induced O-GlcNAcylation in OA and the specific functional mechanism. METHODS: The levels of total O-GlcNAc and OGT were measured in both in vitro and in vivo OA models using western blot. The effects of OGT knockout on OA progression were detected through Safranin O staining, immunohistochemical staining and OARSI score evaluation. The effects of OGT silencing on LPS-induced chondrocyte injury were assessed by performing loss-of function assays. Co-immunoprecipitation (co-IP) was conducted to verify the effect of OGT-induced O-GlcNAcylation on the interaction between NEK7 and NLRP3. The role of OGT in modulating the O-GlcNAcylation and phosphorylation levels of NEK7 was analysed using western blot. RESULTS: The OGT-indued O-GlcNAcylation level was increased in both in vitro and in vivo OA models. Knockout of OGT mitigated OA progression in model mice. Additionally, silencing of OGT suppressed LPS-induced chondrocyte pyroptosis. Moreover, silencing of OGT inhibited the O-GlcNAcylation and enhanced the phosphorylation of NEK7 at S260 site, thereby blocking the binding of NEK7 with NLRP3. CONCLUSION: OGT-induced NEK7 O-GlcNAcylation promotes OA progression by promoting chondrocyte pyroptosis via the suppressing interaction between NEK7 and NLRP3.


Assuntos
N-Acetilglucosaminiltransferases , Proteína 3 que Contém Domínio de Pirina da Família NLR , Osteoartrite , Humanos , Camundongos , Animais , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Lipopolissacarídeos , Camundongos Knockout , Quinases Relacionadas a NIMA/genética
10.
Sci Rep ; 14(1): 3472, 2024 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-38342939

RESUMO

MicroRNAs play a crucial role in regulating the epithelial barrier and immune response, which are implicated in the pathogenesis of ulcerative colitis (UC). This study aimed to investigate the role and molecular mechanism of miR-30c in the pathogenesis of UC using a dextran sulfate sodium salt (DSS)-induced colitis model, which is similar to ulcerative colitis. Wild-type (WT) and miR-30c knockout (KO) mice were assigned to either control or DSS-treated groups to evaluate the influence of aberrant miR-30c expression on UC pathogenesis. The disease activity index, inflammatory factors, and the extent of pathological and histological damage in colon tissues were analyzed. The effect of miR-30c on vasoactive intestinal peptide (VIP) gene expression was validated through luciferase reporter assay, qRT-PCR, Western blotting, and immunohistochemistry. The results showed that miR-30c KO mice with DSS-induced colitis model showed more severe phenotypes: significantly higher disease activity indices, significant body weight loss, reduced length of the colon of mice, increased number of aberrant crypt structures, reduced mucus secretion, and significant differences in inflammatory factors. These findings suggested that the absence of miR-30c might promote DSS-induced colitis, and the targe-regulatory effect of miR-30c on VIP might play an important role in the development of colitis.


Assuntos
Colite Ulcerativa , Colite , MicroRNAs , Camundongos , Animais , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/genética , Colite Ulcerativa/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Colite/induzido quimicamente , Camundongos Knockout , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Colo/patologia
11.
Nat Commun ; 15(1): 1281, 2024 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-38346959

RESUMO

Patients with Type 2 Diabetes Mellitus are increasingly susceptible to atherosclerotic plaque vulnerability, leading to severe cardiovascular events. In this study, we demonstrate that elevated serum levels of palmitic acid, a type of saturated fatty acid, are significantly linked to this enhanced vulnerability in patients with Type 2 Diabetes Mellitus. Through a combination of human cohort studies and animal models, our research identifies a key mechanistic pathway: palmitic acid induces macrophage Delta-like ligand 4 signaling, which in turn triggers senescence in vascular smooth muscle cells. This process is critical for plaque instability due to reduced collagen synthesis and deposition. Importantly, our findings reveal that macrophage-specific knockout of Delta-like ligand 4 in atherosclerotic mice leads to reduced plaque burden and improved stability, highlighting the potential of targeting this pathway. These insights offer a promising direction for developing therapeutic strategies to mitigate cardiovascular risks in patients with Type 2 Diabetes Mellitus.


Assuntos
Diabetes Mellitus Tipo 2 , Placa Aterosclerótica , Animais , Humanos , Camundongos , Apolipoproteínas E/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Macrófagos/metabolismo , Camundongos Knockout , Miócitos de Músculo Liso/metabolismo , Ácido Palmítico/metabolismo , Placa Aterosclerótica/metabolismo
12.
J Ovarian Res ; 17(1): 37, 2024 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-38336796

RESUMO

BACKGROUND: Premature ovarian insufficiency (POI) is a condition defined as women developing menopause before 40 years old. These patients display low ovarian reserve at young age and difficulties to conceive even with assisted reproductive technology. The pathogenesis of ovarian insufficiency is not fully understood. Genetic factors may underlie most of the cases. Actin cytoskeleton plays a pivotal role in ovarian folliculogenesis. Calponin 2 encoded by the Cnn2 gene is an actin associated protein that regulates motility and mechanical signaling related cellular functions. RESULTS: The present study compared breeding of age-matched calponin 2 knockout (Cnn2-KO) and wild type (WT) mice and found that Cnn2-KO mothers had significantly smaller litter sizes. Ovaries from 4 weeks old Cnn2-KO mice showed significantly lower numbers of total ovarian follicles than WT control with the presence of multi-oocyte follicles. Cnn2-KO mice also showed age-progressive earlier depletion of ovarian follicles. Cnn2 expression is detected in the cumulus cells of the ovarian follicles of WT mice and colocalizes with actin stress fiber, tropomyosin and myosin II in primary cultures of cumulus cells. CONCLUSIONS: The findings demonstrate that the loss of calponin 2 impairs ovarian folliculogenesis with premature depletion of ovarian follicles. The role of calponin 2 in ovarian granulosa cells suggests a molecular target for further investigations on the pathogenesis of POI and for therapeutic development.


Assuntos
Insuficiência Ovariana Primária , Adulto , Animais , Feminino , Humanos , Camundongos , Actinas/genética , Actinas/metabolismo , /metabolismo , Menopausa Precoce , Camundongos Knockout , Insuficiência Ovariana Primária/genética , Insuficiência Ovariana Primária/metabolismo
13.
Neurobiol Dis ; 192: 106431, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38331351

RESUMO

Mutations of the human TRAFFICKING PROTEIN PARTICLE COMPLEX SUBUNIT 9 (TRAPPC9) cause a neurodevelopmental disorder characterised by microcephaly and intellectual disability. Trappc9 constitutes a subunit specific to the intracellular membrane-associated TrappII complex. The TrappII complex interacts with Rab11 and Rab18, the latter being specifically associated with lipid droplets (LDs). Here we used non-invasive imaging to characterise Trappc9 knock-out (KO) mice as a model of the human hereditary disorder. KOs developed postnatal microcephaly with many grey and white matter regions being affected. In vivo magnetic resonance imaging (MRI) identified a disproportionately stronger volume reduction in the hippocampus, which was associated with a significant loss of Sox2-positive neural stem and progenitor cells. Diffusion tensor imaging indicated a reduced organisation or integrity of white matter areas. Trappc9 KOs displayed behavioural abnormalities in several tests related to exploration, learning and memory. Trappc9-deficient primary hippocampal neurons accumulated a larger LD volume per cell following Oleic Acid stimulation, and the coating of LDs by Perilipin-2 was much reduced. Additionally, Trappc9 KOs developed obesity, which was significantly more severe in females than in males. Our findings indicate that, beyond previously reported Rab11-related vesicle transport defects, dysfunctions in LD homeostasis might contribute to the neurobiological symptoms of Trappc9 deficiency.


Assuntos
Microcefalia , Animais , Feminino , Humanos , Masculino , Camundongos , Imagem de Tensor de Difusão , Gotículas Lipídicas , Camundongos Knockout , Microcefalia/genética , Microcefalia/metabolismo , Neurônios/metabolismo
14.
J Immunother Cancer ; 12(2)2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38413223

RESUMO

BACKGROUND: Targeting of solid cancers with chimeric antigen receptor (CAR)-T cells is limited by the lack of suitable tumor-specific antigens and the immunosuppressive, desmoplastic tumor microenvironment that impedes CAR-T cell infiltration, activity and persistence. We hypothesized that targeting the endosialin (CD248) receptor, strongly expressed by tumor-associated pericytes and perivascular cancer-associated fibroblasts, would circumvent these challenges and offer an exciting antigen for CAR-T cell therapy due to the close proximity of target cells to the tumor vasculature, the limited endosialin expression in normal tissues and the lack of phenotype observed in endosialin knockout mice. METHODS: We generated endosialin-directed E3K CAR-T cells from three immunocompetent mouse strains, BALB/c, FVB/N and C57BL/6. E3K CAR-T cell composition (CD4+/CD8+ ratio), activity in vitro against endosialin+ and endosialin- cells, and expansion and activity in vivo in syngeneic tumor models as well as in tumor-naive healthy and wounded mice and tumor-bearing endosialin knockout mice was assessed. RESULTS: E3K CAR-T cells were active in vitro against both mouse and human endosialin+, but not endosialin-, cells. Adoptively transferred E3K CAR-T cells exhibited no activity in endosialin knockout mice, tumor-naive endosialin wildtype mice or in wound healing models, demonstrating an absence of off-target and on-target/off-tumor activity. By contrast, adoptive transfer of E3K CAR-T cells into BALB/c, FVB/N or C57BL/6 mice bearing syngeneic breast or lung cancer lines depleted target cells in the tumor stroma resulting in increased tumor necrosis, reduced tumor growth and a substantial impairment in metastatic outgrowth. CONCLUSIONS: Together these data highlight endosialin as a viable antigen for CAR-T cell therapy and that targeting stromal cells closely associated with the tumor vasculature avoids CAR-T cells having to navigate the harsh immunosuppressive tumor microenvironment. Further, the ability of E3K CAR-T cells to recognize and target both mouse and human endosialin+ cells makes a humanized and optimized E3K CAR a promising candidate for clinical development applicable to a broad range of solid tumor types.


Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Camundongos , Animais , Pericitos/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Camundongos Endogâmicos C57BL , Neoplasias/metabolismo , Linfócitos T/metabolismo , Camundongos Knockout , Microambiente Tumoral , Antígenos de Neoplasias/metabolismo , Antígenos CD/metabolismo
15.
Mol Med ; 30(1): 21, 2024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38317079

RESUMO

BACKGROUND: Pericytes are a vital component of the blood-brain barrier, and their involvement in acute inflammation was recently suggested. However, it remains unclear whether pericytes contribute to hypothalamic chronic inflammation and energy metabolism in obesity. The present study investigated the impact of pericytes on the pathophysiology of obesity by focusing on platelet-derived growth factor (PDGF) signaling, which regulates pericyte functions. METHODS: Tamoxifen-inducible systemic conditional PDGF receptor ß knockout mice (Pdgfrb∆SYS-KO) and Calcium/calmodulin-dependent protein kinase type IIa (CaMKIIa)-positive neuron-specific PDGF receptor ß knockout mice (Pdgfrb∆CaMKII-KO) were fed a high-fat diet, and metabolic phenotypes before and 3 to 4 weeks after dietary loading were examined. Intracellular energy metabolism and relevant signal transduction in lipopolysaccharide- and/or platelet-derived growth factor-BB (PDGF-BB)-stimulated human brain pericytes (HBPCs) were assessed by the Seahorse XFe24 Analyzer and Western blotting. The pericyte secretome in conditioned medium from HBPCs was studied using cytokine array kit, and its impact on polarization was examined in bone marrow-derived macrophages (BMDMs), which are microglia-like cells. RESULTS: Energy consumption increased and body weight gain decreased after high-fat diet loading in Pdgfrb∆SYS-KO mice. Cellular oncogene fos (cFos) expression increased in proopiomelanocortin (POMC) neurons, whereas microglial numbers and inflammatory gene expression decreased in the hypothalamus of Pdgfrb∆SYS-KO mice. No significant changes were observed in Pdgfrb∆CaMKII-KO mice. In HBPCs, a co-stimulation with lipopolysaccharide and PDGF-BB shifted intracellular metabolism towards glycolysis, activated mitogen-activated protein kinase (MAPK), and modulated the secretome to the inflammatory phenotype. Consequently, the secretome showed an increase in various proinflammatory chemokines and growth factors including Epithelial-derived neutrophil-activating peptide 78 (C-X-C motif chemokine ligand (CXCL)5), Thymus and activation-regulated chemokine (C-C motif chemokine (CCL)17), Monocyte chemoattractant protein 1 (CCL2), and Growth-regulated oncogene α (CXCL1). Furthermore, conditioned medium from HBPCs stimulated the inflammatory priming of BMDMs, and this change was abolished by the C-X-C motif chemokine receptor (CXCR) inhibitor. Consistently, mRNA expression of CXCL5 was elevated by lipopolysaccharide and PDGF-BB treatment in HBPCs, and the expression was significantly lower in the hypothalamus of Pdgfrb∆SYS-KO mice than in control Pdgfrbflox/flox mice (FL) following 4 weeks of HFD feeding. CONCLUSIONS: PDGF receptor ß signaling in hypothalamic pericytes promotes polarization of macrophages by changing their secretome and contributes to the progression of obesity.


Assuntos
Pericitos , Fator de Crescimento Derivado de Plaquetas , Camundongos , Humanos , Animais , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Pericitos/metabolismo , Becaplermina/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Meios de Cultivo Condicionados/metabolismo , Lipopolissacarídeos , Transdução de Sinais , Inflamação/metabolismo , Camundongos Knockout , Obesidade/metabolismo , Hipotálamo , Proteínas Proto-Oncogênicas c-sis/genética , Proteínas Proto-Oncogênicas c-sis/metabolismo
16.
FASEB J ; 38(3): e23457, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38318648

RESUMO

Aging is associated with chronic, low-level inflammation which may contribute to cardiovascular pathologies such as hypertension and atherosclerosis. This chronic inflammation may be opposed by endogenous mechanisms to limit inflammation, for example, by the actions of annexin A1 (ANXA1), an endogenous glucocorticoid-regulated protein that has anti-inflammatory and pro-resolving activity. We hypothesized the pro-resolving mediator ANXA1 protects against age-induced changes in blood pressure (BP), cardiovascular structure and function, and cardiac senescence. BP was measured monthly in conscious mature (4-month) and middle-aged (12-month) ANXA1-deficient (ANXA1-/- ) and wild-type C57BL/6 mice. Body composition was measured using EchoMRI, and both cardiac and vascular function using ultrasound imaging. Cardiac hypertrophy, fibrosis and senescence, vascular fibrosis, elastin, and calcification were assessed histologically. Gene expression relevant to structural remodeling, inflammation, and cardiomyocyte senescence were also quantified. In C57BL/6 mice, progression from 4 to 12 months of age did not affect the majority of cardiovascular parameters measured, with the exception of mild cardiac hypertrophy, vascular calcium, and collagen deposition. Interestingly, ANXA1-/- mice exhibited higher BP, regardless of age. Additionally, age progression had a marked impact in ANXA1-/- mice, with markedly augmented vascular remodeling, impaired vascular distensibility, and body composition. Consistent with vascular dysfunction, cardiac dysfunction, and hypertrophy were also evident, together with markers of senescence and inflammation. These findings suggest that endogenous ANXA1 plays a critical role in regulating BP, cardiovascular function, and remodeling and delays cardiac senescence. Our findings support the development of novel ANXA1-based therapies to prevent age-related cardiovascular pathologies.


Assuntos
Anexina A1 , Camundongos , Animais , Anexina A1/genética , Anexina A1/metabolismo , Pressão Sanguínea , Camundongos Knockout , Camundongos Endogâmicos C57BL , Inflamação/patologia , Fibrose , Cardiomegalia
17.
Cell Commun Signal ; 22(1): 96, 2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-38308301

RESUMO

BACKGROUND: Neutrophil extracellular trap (NET) has been implicated in the pathology of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). However, the specific contributions of NLRP3, a NET-associated molecule, to EAE pathogenesis and its regulatory role in NET formation remain unknown. METHODS: To investigate the detrimental effect of NETs supported by NLRP3 in MS pathogenesis, we induced EAE in WT and NLRP3 KO mice and monitored the disease severity. At the peak of the disease, NET formation was assessed by flow cytometry, immunoblotting, and immunofluorescence staining. To further identify the propensity of infiltrated neutrophils, NET-related chemokine receptors, degranulation, ROS production, and PAD4 expression levels were evaluated by flow cytometry. In some experiments, mice were injected with DNase-1 to eliminate the formed NETs. RESULTS: Our data revealed that neutrophils significantly infiltrate the brain and spinal cord and form NETs during EAE pathogenesis. NLRP3 significantly elevates NET formation, primarily in the brain. NLRP3 also modulated the phenotypes of brain-infiltrated and circulating neutrophils, augmenting CXCR2 and CXCR4 expression, thereby potentially enhancing NET formation. NLRP3 facilitates NET formation in a ROS-dependent and PAD4-independent manner in brain-infiltrated neutrophils. Finally, NLRP3-supported NET formation exacerbates disease severity, triggering Th1 and Th17 cells recruitment. CONCLUSIONS: Collectively, our findings suggest that NLRP3-supported NETs may be an etiological factor in EAE pathogenesis, primarily in the brain. This study provides evidence that targeting NLRP3 could be a potential therapeutic strategy for MS, specifically by attenuating NET formation.


Assuntos
Encefalomielite Autoimune Experimental , Armadilhas Extracelulares , Camundongos , Animais , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Espécies Reativas de Oxigênio/metabolismo , Armadilhas Extracelulares/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Camundongos Knockout , Encéfalo/metabolismo , Neutrófilos/metabolismo , Camundongos Endogâmicos C57BL
18.
Sci Adv ; 10(5): eadj7813, 2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-38306420

RESUMO

Metabolic syndrome (MetS) is closely associated with an increased risk of dementia and cognitive impairment, and a complex interaction of genetic and environmental dietary factors may be implicated. Free fatty acid receptor 4 (Ffar4) may bridge the genetic and dietary aspects of MetS development. However, the role of Ffar4 in MetS-related cognitive dysfunction is unclear. In this study, we found that Ffar4 expression is down-regulated in MetS mice and MetS patients with cognitive impairment. Conventional and microglial conditional knockout of Ffar4 exacerbated high-fat diet (HFD)-induced cognitive dysfunction and anxiety, whereas microglial Ffar4 overexpression improved HFD-induced cognitive dysfunction and anxiety. Mechanistically, we found that microglial Ffar4 regulated microglial activation through type I interferon signaling. Microglial depletion and NF-κB inhibition partially reversed cognitive dysfunction and anxiety in microglia-specific Ffar4 knockout MetS mice. Together, these findings uncover a previously unappreciated role of Ffar4 in negatively regulating the NF-κB-IFN-ß signaling and provide an attractive therapeutic target for delaying MetS-associated cognitive decline.


Assuntos
Disfunção Cognitiva , Síndrome Metabólica , Receptores Acoplados a Proteínas G , Animais , Humanos , Camundongos , Disfunção Cognitiva/genética , Disfunção Cognitiva/complicações , Síndrome Metabólica/complicações , Síndrome Metabólica/genética , Camundongos Knockout , Microglia/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Receptores Acoplados a Proteínas G/metabolismo
19.
Sci Adv ; 10(5): eadj0146, 2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-38306436

RESUMO

Acute pancreatitis (AP) is one of the most common gastrointestinal diseases. Bile acids (BAs) were proposed to be a cause of AP nearly 170 years ago, though the underlying mechanisms remain unclear. Here, we report that two G protein-coupled receptors, GPR39 and GHSR, mediated cellular responses to BAs. Our results revealed GPR39 as an evolutionarily conserved receptor for BAs, particularly 3-O-sulfated lithocholic acids. In cultured cell lines, GPR39 is sufficient for BA-induced Ca2+ elevation. In pancreatic acinar cells, GPR39 mediated BA-induced Ca2+ elevation and necrosis. Furthermore, AP induced by BAs was significantly reduced in GPR39 knockout mice. Our findings provide in vitro and in vivo evidence demonstrating that GPR39 is necessary and sufficient to mediate BA signaling, highlighting its involvement in biliary AP pathogenesis, and suggesting it as a promising therapeutic target for biliary AP.


Assuntos
Pancreatite , Receptores Acoplados a Proteínas G , Animais , Camundongos , Doença Aguda , Ácidos e Sais Biliares , Proteínas de Transporte/metabolismo , Camundongos Knockout , Pancreatite/genética , Pancreatite/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais
20.
Nat Commun ; 15(1): 1337, 2024 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-38351043

RESUMO

Tyrosine kinase 2 (TYK2), a member of the JAK family, has attracted attention as a potential therapeutic target for autoimmune diseases. However, the role of TYK2 in CD8+ T cells and autoimmune type 1 diabetes (T1D) is poorly understood. In this study, we generate Tyk2 gene knockout non-obese diabetes (NOD) mice and demonstrate that the loss of Tyk2 inhibits the development of autoreactive CD8+ T-BET+ cytotoxic T lymphocytes (CTLs) by impairing IL-12 signaling in CD8+ T cells and the CD8+ resident dendritic cell-driven cross-priming of CTLs in the pancreatic lymph node (PLN). Tyk2-deficient CTLs display reduced cytotoxicity. Increased inflammatory responses in ß-cells with aging are dampened by Tyk2 deficiency. Furthermore, treatment with BMS-986165, a selective TYK2 inhibitor, inhibits the expansion of T-BET+ CTLs, inflammation in ß-cells and the onset of autoimmune T1D in NOD mice. Thus, our study reveals the diverse roles of TYK2 in driving the pathogenesis of T1D.


Assuntos
Antineoplásicos , Diabetes Mellitus Tipo 1 , Camundongos , Animais , Linfócitos T CD8-Positivos , Linfócitos T Citotóxicos , Diabetes Mellitus Tipo 1/genética , TYK2 Quinase/genética , Camundongos Knockout , Camundongos Endogâmicos NOD
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