RESUMEN
Natural killer (NK) cells have vital functions in human immunity and reproduction. In the innate and adaptive immune responses to infection, particularly by viruses, NK cells respond by secreting inflammatory cytokines and killing infected cells. In reproduction, NK cells are critical for genesis of the placenta, the organ that controls the supply of oxygen and nutrients to the growing fetus. Controlling NK cell functions are interactions of HLA class I with inhibitory NK cell receptors. First evolved was the conserved interaction of HLA-E with CD94:NKG2A; later established were diverse interactions of HLA-A, -B, and -C with killer cell immunoglobulin-like receptors. Characterizing the latter interactions is rapid evolution, which distinguishes human populations and all species of higher primate. Driving this evolution are the different and competing selections imposed by pathogens on NK cell-mediated immunity and by the constraints of human reproduction on NK cell-mediated placentation. Promoting rapid evolution is independent segregation of polymorphic receptors and ligands throughout human populations.
Asunto(s)
Predisposición Genética a la Enfermedad , Inmunidad , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Animales , Evolución Biológica , Sitios Genéticos , Genómica/métodos , Haplotipos , Humanos , Complejo Mayor de Histocompatibilidad/genética , Receptores KIR/genética , Receptores KIR/metabolismoRESUMEN
In all human cells, human leukocyte antigen (HLA) class I glycoproteins assemble with a peptide and take it to the cell surface for surveillance by lymphocytes. These include natural killer (NK) cells and γδ T cells of innate immunity and αß T cells of adaptive immunity. In healthy cells, the presented peptides derive from human proteins, to which lymphocytes are tolerant. In pathogen-infected cells, HLA class I expression is perturbed. Reduced HLA class I expression is detected by KIR and CD94:NKG2A receptors of NK cells. Almost any change in peptide presentation can be detected by αß CD8+ T cells. In responding to extracellular pathogens, HLA class II glycoproteins, expressed by specialized antigen-presenting cells, present peptides to αß CD4+ T cells. In comparison to the families of major histocompatibility complex (MHC) class I, MHC class II and αß T cell receptors, the antigenic specificity of the γδ T cell receptors is incompletely understood.
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Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase I/química , Inmunidad Celular , Subfamília D de Receptores Similares a Lectina de las Células NK/química , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T gamma-delta/química , Receptores KIR/química , Presentación de Antígeno , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Evolución Molecular , Regulación de la Expresión Génica , Haplotipos , Antígenos de Histocompatibilidad Clase I/clasificación , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase II/clasificación , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Inmunidad Innata , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Modelos Moleculares , Subfamília D de Receptores Similares a Lectina de las Células NK/genética , Subfamília D de Receptores Similares a Lectina de las Células NK/inmunología , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Receptores KIR/clasificación , Receptores KIR/genética , Receptores KIR/inmunología , Transducción de SeñalRESUMEN
ATP-sensitive potassium channels (KATP) couple intracellular ATP levels with membrane excitability. These channels play crucial roles in many essential physiological processes and have been implicated extensively in a spectrum of metabolic diseases and disorders. To gain insight into the mechanism of KATP, we elucidated the structure of a hetero-octameric pancreatic KATP channel in complex with a non-competitive inhibitor glibenclamide by single-particle cryoelectron microscopy to 5.6-Å resolution. The structure shows that four SUR1 regulatory subunits locate peripherally and dock onto the central Kir6.2 channel tetramer through the SUR1 TMD0-L0 fragment. Glibenclamide-bound SUR1 uses TMD0-L0 fragment to stabilize Kir6.2 channel in a closed conformation. In another structural population, a putative co-purified phosphatidylinositol 4,5-bisphosphate (PIP2) molecule uncouples Kir6.2 from glibenclamide-bound SUR1. These structural observations suggest a molecular mechanism for KATP regulation by anti-diabetic sulfonylurea drugs, intracellular adenosine nucleotide concentrations, and PIP2 lipid.
Asunto(s)
Canales KATP/química , Canales KATP/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/química , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Microscopía por Crioelectrón , Humanos , Hidrolasas/química , Hidrolasas/metabolismo , Mamíferos/metabolismo , Mesocricetus , Ratones , Modelos Moleculares , Fosfoinositido Fosfolipasa C/química , Fosfoinositido Fosfolipasa C/metabolismo , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/metabolismo , Receptores de Sulfonilureas/química , Receptores de Sulfonilureas/metabolismoRESUMEN
Ubiquitously expressed throughout the body, ATP-sensitive potassium (KATP) channels couple cellular metabolism to electrical activity in multiple tissues; their unique assembly as four Kir6 pore-forming subunits and four sulfonylurea receptor (SUR) subunits has resulted in a large armory of selective channel opener and inhibitor drugs. The spectrum of monogenic pathologies that result from gain- or loss-of-function mutations in these channels, and the potential for therapeutic correction of these pathologies, is now clear. However, while available drugs can be effective treatments for specific pathologies, cross-reactivity with the other Kir6 or SUR subfamily members can result in drug-induced versions of each pathology and may limit therapeutic usefulness. This review discusses the background to KATP channel physiology, pathology, and pharmacology and considers the potential for more specific or effective therapeutic agents.
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Canales de Potasio de Rectificación Interna , Humanos , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/metabolismo , Receptores de Sulfonilureas/genética , Receptores de Sulfonilureas/metabolismo , Mutación , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacologíaRESUMEN
Evidence is mounting that the major histocompatibility complex (MHC) molecule HLA-F (human leukocyte antigen F) regulates the immune system in pregnancy, infection, and autoimmunity by signaling through NK cell receptors (NKRs). We present structural, biochemical, and evolutionary analyses demonstrating that HLA-F presents peptides of unconventional length dictated by a newly arisen mutation (R62W) that has produced an open-ended groove accommodating particularly long peptides. Compared to empty HLA-F open conformers (OCs), HLA-F tetramers bound with human-derived peptides differentially stained leukocytes, suggesting peptide-dependent engagement. Our in vitro studies confirm that NKRs differentiate between peptide-bound and peptide-free HLA-F. The complex structure of peptide-loaded ß2m-HLA-F bound to the inhibitory LIR1 revealed similarities to high-affinity recognition of the viral MHC-I mimic UL18 and a docking strategy that relies on contacts with HLA-F as well as ß2m, thus precluding binding to HLA-F OCs. These findings provide a biochemical framework to understand how HLA-F could regulate immunity via interactions with NKRs.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/metabolismo , Células Asesinas Naturales/inmunología , Imitación Molecular , Receptores de Células Asesinas Naturales/metabolismo , Proteínas Virales/química , Presentación de Antígeno , Antígenos/inmunología , Antígenos/metabolismo , Antígenos CD/metabolismo , Evolución Biológica , Cristalografía por Rayos X , Femenino , Células HEK293 , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Receptor Leucocitario Tipo Inmunoglobulina B1 , Mutación/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Embarazo , Unión Proteica , Conformación Proteica , Receptores Inmunológicos/metabolismo , Proteínas Virales/metabolismoRESUMEN
Many receptors signal upon phosphorylation of tyrosine-based motifs in their cytosolic tail, with intrinsic disorder as a common feature. Studies on CD3ζ and CD3ε tails, which are disordered and polybasic, suggested regulation of phosphorylation through accessibility of tyrosines, governed by electrostatic interactions with membrane anionic lipids. We noticed characteristics of intrinsic disorder and previously unappreciated features in tyrosine-based motif-bearing cytosolic tails of many, especially, inhibitory receptors. They are neutral or acidic polyampholytes, with acidic and basic residues linearly segregated. To explore roles of these electrostatic features, we studied inhibitory killer-cell immunoglobulin-like receptor (KIR). Its cytosolic tail is a disordered neutrally charged polyampholyte, wherein juxtamembrane and membrane distal stretches are basic, and the intervening stretch is acidic. Despite lacking net charge, it interacted electrostatically with the plasma membrane. The juxtamembrane stretch was crucial for overall binding, which sequestered tyrosines in the lipid bilayer and restrained their constitutive phosphorylation. Human leukocyte antigen-C ligand binding to KIR released its tail from the plasma membrane to initiate signaling. Tail release occurred independently of KIR polymerization, clustering, or tyrosine phosphorylation, but required acidic residues of the acidic stretch. Tail interaction with the plasma membrane dictated signaling strength of KIR. These results revealed an electrostatic protein-lipid interaction that is unusual in being governed by segregated clusters of acidic and basic residues in polyampholytic disordered region of protein. In contrast to previously known, segregated distribution of oppositely charged residues made both binding and unbinding modules inherent to receptor tail, which could make the interaction an independent signaling switch.
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Receptores KIR , Transducción de Señal , Humanos , Electricidad Estática , Membrana Celular , Fosforilación , Lípidos de la Membrana , Tirosina/químicaRESUMEN
Neurovascular coupling (NVC), a vital physiological process that rapidly and precisely directs localized blood flow to the most active regions of the brain, is accomplished in part by the vast network of cerebral capillaries acting as a sensory web capable of detecting increases in neuronal activity and orchestrating the dilation of upstream parenchymal arterioles. Here, we report a Col4a1 mutant mouse model of cerebral small vessel disease (cSVD) with age-dependent defects in capillary-to-arteriole dilation, functional hyperemia in the brain, and memory. The fundamental defect in aged mutant animals was the depletion of the minor membrane phospholipid phosphatidylinositol 4,5 bisphosphate (PIP2) in brain capillary endothelial cells, leading to the loss of inwardly rectifying K+ (Kir2.1) channel activity. Blocking phosphatidylinositol-3-kinase (PI3K), an enzyme that diminishes the bioavailability of PIP2 by converting it to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP3), restored Kir2.1 channel activity, capillary-to-arteriole dilation, and functional hyperemia. In longitudinal studies, chronic PI3K inhibition also improved the memory function of aged Col4a1 mutant mice. Our data suggest that PI3K inhibition is a viable therapeutic strategy for treating defective NVC and cognitive impairment associated with cSVD.
Asunto(s)
Enfermedades de los Pequeños Vasos Cerebrales , Hiperemia , Acoplamiento Neurovascular , Animales , Ratones , Células Endoteliales , Fosfatidilinositol 3-Quinasas/genética , Enfermedades de los Pequeños Vasos Cerebrales/genética , Fosfatidilinositol 3-QuinasaRESUMEN
Current anesthetic theory is mostly based on neurons and/or neuronal circuits. A role for astrocytes also has been shown in promoting recovery from volatile anesthesia, while the exact modulatory mechanism and/or the molecular target in astrocytes is still unknown. In this study by animal models in male mice and electrophysiological recordings in vivo and in vitro, we found that activating astrocytes of the paraventricular thalamus (PVT) and/or knocking down PVT astrocytic Kir4.1 promoted the consciousness recovery from sevoflurane anesthesia. Single-cell RNA sequencing of the PVT reveals two distinct cellular subtypes of glutamatergic neurons: PVT GRM and PVT ChAT neurons. Patch-clamp recording results proved astrocytic Kir4.1-mediated modulation of sevoflurane on the PVT mainly worked on PVT ChAT neurons, which projected mainly to the mPFC. In summary, our findings support the novel conception that there is a specific PVTâprefrontal cortex projection involved in consciousness recovery from sevoflurane anesthesia, which is mediated by the inhibition of sevoflurane on PVT astrocytic Kir4.1 conductance.
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Astrocitos , Estado de Conciencia , Núcleos Talámicos de la Línea Media , Canales de Potasio de Rectificación Interna , Sevoflurano , Animales , Astrocitos/fisiología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Masculino , Ratones , Sevoflurano/farmacología , Estado de Conciencia/fisiología , Estado de Conciencia/efectos de los fármacos , Núcleos Talámicos de la Línea Media/fisiología , Núcleos Talámicos de la Línea Media/efectos de los fármacos , Núcleos Talámicos de la Línea Media/citología , Canales de Potasio de Rectificación Interna/metabolismo , Ratones Endogámicos C57BL , Anestésicos por Inhalación/farmacología , Vías Nerviosas/fisiología , Vías Nerviosas/efectos de los fármacos , Neuronas/fisiología , Neuronas/efectos de los fármacos , Corteza Prefrontal/fisiología , Corteza Prefrontal/efectos de los fármacos , Lóbulo Frontal/fisiología , Lóbulo Frontal/efectos de los fármacos , Periodo de Recuperación de la AnestesiaRESUMEN
Advances in DNA sequencing technologies have enabled genotyping of complex genetic regions exhibiting copy number variation and high allelic diversity, yet it is impossible to derive exact genotypes in all cases, often resulting in ambiguous genotype calls, that is, partially missing data. An example of such a gene region is the killer-cell immunoglobulin-like receptor (KIR) genes. These genes are of special interest in the context of allogeneic hematopoietic stem cell transplantation. For such complex gene regions, current haplotype reconstruction methods are not feasible as they cannot cope with the complexity of the data. We present an expectation-maximization (EM)-algorithm to estimate haplotype frequencies (HTFs) which deals with the missing data components, and takes into account linkage disequilibrium (LD) between genes. To cope with the exponential increase in the number of haplotypes as genes are added, we add three components to a standard EM-algorithm implementation. First, reconstruction is performed iteratively, adding one gene at a time. Second, after each step, haplotypes with frequencies below a threshold are collapsed in a rare haplotype group. Third, the HTF of the rare haplotype group is profiled in subsequent iterations to improve estimates. A simulation study evaluates the effect of combining information of multiple genes on the estimates of these frequencies. We show that estimated HTFs are approximately unbiased. Our simulation study shows that the EM-algorithm is able to combine information from multiple genes when LD is high, whereas increased ambiguity levels increase bias. Linear regression models based on this EM, show that a large number of haplotypes can be problematic for unbiased effect size estimation and that models need to be sparse. In a real data analysis of KIR genotypes, we compare HTFs to those obtained in an independent study. Our new EM-algorithm-based method is the first to account for the full genetic architecture of complex gene regions, such as the KIR gene region. This algorithm can handle the numerous observed ambiguities, and allows for the collapsing of haplotypes to perform implicit dimension reduction. Combining information from multiple genes improves haplotype reconstruction.
Asunto(s)
Variaciones en el Número de Copia de ADN , Modelos Genéticos , Humanos , Haplotipos , Frecuencia de los Genes , GenotipoRESUMEN
HLA-I/KIR genotypes influence HIV-1 disease progression and viral load, but their role in primary infection is uncertain. Inconsistent results from previous studies suggest that the inoculum size and transmission route-parenteral vs. sexual-may influence this association. We conducted a GWAS in a population of people living with HIV-1 and HIV-1-exposed seronegative individuals exposed to the virus through the sexual route. Our data do not support any role of the HLA/KIR system in susceptibility to sexually transmitted HIV-1 infection. The genetics basis of HIV-1 viral load and disease progression are distinct from the genetics of HIV resistance, a paradox worth exploring.
RESUMEN
We established a low background, Cre-dependent version of the inducible Tet-On system for fast, cell type-specific transgene expression in vivo Coexpression of a constitutive, Cre-dependent fluorescent marker selectively allowed single-cell analyses before and after inducible, Tet-dependent transgene expression. Here, we used this method for precise, acute manipulation of neuronal activity in the living brain. The goal was to study neuronal network homeostasis at cellular resolution. Single induction of the potassium channel Kir2.1 produced cell type-specific silencing within hours that lasted for at least 3 d. Longitudinal in vivo imaging of spontaneous calcium transients and neuronal morphology demonstrated that prolonged silencing did not alter spine densities or synaptic input strength. Furthermore, selective induction of Kir2.1 in parvalbumin interneurons increased the activity of surrounding neurons in a distance-dependent manner. This high-resolution, inducible interference and interval imaging of individual cells (high I5, HighFive) method thus allows visualizing temporally precise, genetic perturbations of defined cells.SIGNIFICANCE STATEMENT Gene function is studied by KO or overexpression of a specific gene followed by analyses of phenotypic changes. However, being able to predict and analyze exactly those cells in which genetic manipulation will occur is not possible. We combined two prominent transgene overexpression methods to fluorescently highlight the targeted cells appropriately before cell type-specific transgene induction. By inducing a potassium channel that decreases neuronal firing, we investigated how neuronal networks in the living mouse brain possibly compensate swift changes in cellular activities. Unlike in vitro, known compensatory homeostatic mechanisms, such as changes in synapses, were not observed in vivo Overall, we demonstrated with our method rapid genetic manipulation and analysis of neuronal activities as well as precision transgene expression.
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Interneuronas , Neuronas , Ratones , Animales , Neuronas/fisiología , Transgenes , Homeostasis/fisiología , Canales de Potasio/metabolismoRESUMEN
Obesity imposes deficits on adipose tissue and vascular endothelium, yet the role that distinct adipose depots play in mediating endothelial dysfunction in local arteries remains unresolved. We recently showed that obesity impairs endothelial Kir2.1 channels, mediators of nitric oxide production, in arteries of visceral adipose tissue (VAT), while Kir2.1 function in subcutaneous adipose tissue (SAT) endothelium remains intact. Therefore, we determined if VAT versus SAT from lean or diet-induced obese mice affected Kir2.1 channel function in vitro. We found that VAT from obese mice reduces Kir2.1 function without altering channel expression whereas AT from lean mice and SAT from obese mice had no effect on Kir2.1 function as compared to untreated control cells. As Kir2.1 is well known to be inhibited by fatty acid derivatives and obesity is strongly associated with elevated circulating fatty acids, we next tested the role of the fatty acid translocase CD36 in mediating VAT-induced Kir2.1 dysfunction. We found that the downregulation of CD36 restored Kir2.1 currents in endothelial cells exposed to VAT from obese mice. In addition, endothelial cells exposed to VAT from obese mice exhibited a significant increase in CD36-mediated fatty acid uptake. The importance of CD36 in obesity-induced endothelial dysfunction of VAT arteries was further supported in ex vivo pressure myography studies where CD36 ablation rescued the endothelium-dependent response to flow via restoring Kir2.1 and endothelial nitric oxide synthase function. These findings provide new insight into the role of VAT in mediating obesity-induced endothelial dysfunction and suggest a novel role for CD36 as a mediator of endothelial Kir2.1 impairment.NEW & NOTEWORTHY Our findings suggest a role for visceral adipose tissue (VAT) in the dysfunction of endothelial Kir2.1 in obesity. We further reveal a role for CD36 as a major contributor to VAT-mediated Kir2.1 and endothelial dysfunction, suggesting that CD36 offers a potential target for preventing the early development of obesity-associated cardiovascular disease.
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Antígenos CD36 , Células Endoteliales , Grasa Intraabdominal , Ratones Endogámicos C57BL , Obesidad , Canales de Potasio de Rectificación Interna , Animales , Ratones , Antígenos CD36/metabolismo , Antígenos CD36/genética , Dieta Alta en Grasa , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Grasa Intraabdominal/metabolismo , Ratones Obesos , Obesidad/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio de Rectificación Interna/genética , Grasa Subcutánea/metabolismoRESUMEN
Acute myeloid leukaemia (AML) is a prevalent haematological malignancy in which various immune and stromal cells in the bone marrow microenvironment have instrumental roles and substantially influence its progression. KIR2DL is a member of the immunoglobulin-like receptor family and a natural killer (NK) cell surface-specific receptor. However, its impact on immune infiltration regarding AML has not been addressed. We aimed to explore molecular markers associated with the immune microenvironment and prognosis of AML with a particular focus on KIR2DL family members. Analysis of data from The Cancer Genome Atlas and Genotype-Tissue Expression databases revealed that KIR2DL1, KIR2DL3 and KIR2DL4 expression were significantly upregulated in AML and associated with decreased overall survival (OS). Moreover, univariate Cox analysis implicated KIR2DL genes as independent prognostic markers of OS. Functional enrichment analysis revealed that KIR2DL genes were associated with immune cells, the immune microenvironment and NK cell-mediated cytotoxicity. Additionally, immune infiltration analyses revealed that KIR2DL upregulation was associated with stronger immune infiltration. Finally, we performed drug sensitivity profiling of KIR2DL genes using the Cellminer database. Collectively, our findings suggest that KIR2DL1, KIR2DL3 and KIR2DL4 have critical roles in AML and may represent novel biomarker genes for disease prognosis and immune infiltration.
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Leucemia Mieloide Aguda , Humanos , Pronóstico , Leucemia Mieloide Aguda/patología , Médula Ósea/patología , Microambiente Tumoral/genéticaRESUMEN
Smoking cigarettes during pregnancy is associated with adverse effects on infants including low birth weight, defective lung development, and skeletal abnormalities. Pregnant women are increasingly turning to vaping [use of electronic (e)-cigarettes] as a perceived safer alternative to cigarettes. However, nicotine disrupts fetal development, suggesting that like cigarette smoking, nicotine vaping may be detrimental to the fetus. To test the impact of maternal vaping on fetal lung and skeletal development in mice, pregnant dams were exposed to e-cigarette vapor throughout gestation. At embryonic day (E)18.5, vape exposed litter sizes were reduced, and some embryos exhibited growth restriction compared to air exposed controls. Fetal lungs were collected for histology and whole transcriptome sequencing. Maternally nicotine vaped embryos exhibited histological and transcriptional changes consistent with impaired distal lung development. Embryonic lung gene expression changes mimicked transcriptional changes observed in adult mouse lungs exposed to cigarette smoke, suggesting that the developmental defects may be due to direct nicotine exposure. Fetal skeletons were analyzed for craniofacial and long bone lengths. Nicotine directly binds and inhibits the Kcnj2 potassium channel which is important for bone development. The length of the maxilla, palatal shelves, humerus, and femur were reduced in vaped embryos, which was further exacerbated by loss of one copy of the Kcnj2 gene. Nicotine vapor exposed Kcnj2KO/+ embryos also had significantly lower birth weights than unexposed animals of either genotype. Kcnj2 mutants had severely defective lungs with and without vape exposure, suggesting that potassium channels may be broadly involved in mediating the detrimental developmental effects of nicotine vaping. These data indicate that intrauterine nicotine exposure disrupts fetal lung and skeletal development likely through inhibition of Kcnj2.
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Cigarrillo Electrónico a Vapor , Sistemas Electrónicos de Liberación de Nicotina , Vapeo , Femenino , Embarazo , Animales , Humanos , Ratones , Vapeo/efectos adversos , Nicotina/efectos adversos , Nicotina/metabolismo , Pulmón/metabolismo , Cigarrillo Electrónico a Vapor/efectos adversosRESUMEN
Macrophages and satellite glial cells are found between injured and uninjured neurons in the lumbar dorsal root ganglia (DRG). We explored the mechanism of neuro-immune and neuron-glia crosstalk leading to hyperexcitability of DRG neurons. After spared nerve injury (SNI), CX3CR1+ resident macrophages became activated, proliferated, and increased inward-rectifying potassium channel Kir 2.1 currents. Conditioned medium (CM) by macrophages, obtained from DRG of SNI mice, sensitized small DRG neurons from naïve mice. However, treatment with CM from GFAP+ glial cells did not affect neuronal excitability. When subjected to this macrophage-derived CM, DRG neurons had increased spontaneous activity, current-evoked responses and voltage-gated NaV 1.7 and NaV 1.8 currents. Silencing Kir 2.1 in macrophages after SNI prevented the induction of neuronal hyperexcitability from their CM. Blocking vesicular exocytosis or soluble tumor necrosis factor in CM or interfering with the downstream intracellular p38 pathway in neurons, also prevented neuronal hyperexcitability. Blocking protein trafficking in neurons reduced the effect of CM, suggesting that the hyperexcitable state resulted from changes in NaV channel trafficking. These results suggest that DRG macrophages, primed by peripheral nerve injury, contribute to neuron-glia crosstalk, NaV channel dysregulation and neuronal hyperexcitability implicated in the development of neuropathic pain.
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Ganglios Espinales , Canales de Potasio , Ratas , Ratones , Animales , Ganglios Espinales/metabolismo , Canales de Potasio/metabolismo , Ratas Sprague-Dawley , Neuronas/metabolismo , NeuroglíaRESUMEN
BACKGROUND: HIV-exposed uninfected infants (HEU) appear more vulnerable to infections compared to their HIV-unexposed uninfected (HUU) peers, generally attributed to poor passive immunity acquired from the mother. This may be due to some genetic factors that could alter the immune system. We thus sought to determine the distribution of Killer Cell Immunoglobulin-Like Receptors (KIRs) genes in HEU versus HUU and study their associations with the occurrence of infection-related hospitalization. METHODS: A cohort study was conducted from May 2019 to April 2020 among HEU and HUU infants, including their follow-up at weeks 6, 12, 24, and 48, in reference pediatric centers in Yaoundé-Cameroon. The infant HIV status and infections were determined. A total of 15 KIR genes were investigated using the sequence-specific primer polymerase chain reaction (PCR-SSP) method. The KIR genes that were significantly associated with HIV-1 status (HEU and HUU) were analyzed for an association with infection-related hospitalizations. This was only possible if, and to the extent that, infection-related hospitalizations varied significantly according to status. Multivariate logistic regression analyses were conducted to determine the association between KIR gene content variants and HIV status, while considering a number of potential confounding factors. Furthermore, the risk was quantified using relative risk, odds ratio, and a 95% confidence interval. The Fisher exact test was employed to compare the frequency of occurrences. A p-value of less than 0.05 was considered statistically significant. RESULTS: In this cohort, a total of 66 infants participated, but only 19 acquired infections requiring hospitalizations (14.81%, 04/27 HUU and 38.46%, 15/39 HEU, p = 0.037). At week 48 (39 HEU and 27 HUU), the relative risk (RR) for infection-related hospitalizations was 2.42 (95% CI: 1.028-5.823) for HEU versus HUU with OR 3.59 (1.037-12.448). KIR2DL1 gene was significantly underrepresented in HEU versus HUU (OR = 0.183, 95%CI: 0.053-0.629; p = 0.003), and the absence of KIR2DL1 was significantly associated with infection-related hospitalization (p < 0.001; aOR = 0.063; 95%CI: 0.017-0.229). CONCLUSION: Compared to HUU, the vulnerability of HEU is driven by KIR2DL1, indicating the protective role of this KIR against infection and hospitalizations.
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Infecciones por VIH , VIH-1 , Hospitalización , Receptores KIR2DL1 , Humanos , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/epidemiología , Camerún/epidemiología , Lactante , Hospitalización/estadística & datos numéricos , VIH-1/fisiología , Masculino , Femenino , Receptores KIR2DL1/genética , Estudios de Cohortes , Recién Nacido , Predisposición Genética a la Enfermedad , Biomarcadores , GenotipoRESUMEN
BACKGROUND: Imbalance in energy regulation is a major cause of insulin resistance and diabetes. Melanocortin-4 receptor (MC4R) signaling at specific sites in the central nervous system has synergistic but non-overlapping functions. However, the mechanism by which MC4R in the arcuate nucleus (ARC) region regulates energy balance and insulin resistance remains unclear. METHODS: The MC4Rflox/flox mice with proopiomelanocortin (POMC) -Cre mice were crossed to generate the POMC-MC4Rflox/+ mice. Then POMC-MC4Rflox/+ mice were further mated with MC4Rflox/flox mice to generate the POMC-MC4Rflox/flox mice in which MC4R is selectively deleted in POMC neurons. Bilateral injections of 200 nl of AAV-sh-Kir2.1 (AAV-sh-NC was used as control) were made into the ARC of the hypothalamus. Oxygen consumption, carbon dioxide production, respiratory exchange ratio and energy expenditure were measured by using the CLAMS; Total, visceral and subcutaneous fat was analyzed using micro-CT. Co-immunoprecipitation assays (Co-IP) were used to analyze the interaction between MC4R and Kir2.1 in GT1-7 cells. RESULTS: POMC neuron-specific ablation of MC4R in the ARC region promoted food intake, impaired energy expenditure, leading to increased weight gain and impaired systemic glucose homeostasis. Additionally, MC4R ablation reduced the activation of POMC neuron, and is not tissue-specific for peripheral regulation, suggesting the importance of its central regulation. Mechanistically, sequencing analysis and Co-IP assay demonstrated a direct interaction of MC4R with Kir2.1. Knockdown of Kir2.1 in POMC neuron-specific ablation of MC4R restored the effect of MC4R ablation on energy expenditure and systemic glucose homeostasis, indicating by reduced body weight and ameliorated insulin resistance. CONCLUSION: Hypothalamic POMC neuron-specific knockout of MC4R affects energy balance and insulin sensitivity by regulating Kir2.1. Kir2.1 represents a new target and pathway that could be targeted in obesity.
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Resistencia a la Insulina , Animales , Ratones , Glucosa , Hipotálamo , Resistencia a la Insulina/genética , Neuronas , Proopiomelanocortina/genética , Receptor de Melanocortina Tipo 4/genéticaRESUMEN
Killer immunoglobulin-like receptor (KIR) and KIR-ligand (KIRL) interactions play an important role in natural killer cell-mediated effects after haematopoietic stem cell transplantation (HCT). Previous work has shown that accounting for known KIR-KIRL interactions may identify donors with optimal NK cell-mediated alloreactivity in the adult transplant setting. Paediatric acute leukaemia patients were retrospectively analysed, and KIR-KIRL combinations and maximal inhibitory KIR ligand (IM-KIR) scores were determined. Clinical outcomes were examined using a series of graphs depicting clinical events and endpoints. The graph methodology demonstrated that prognostic variables significant in the occurrence of specific clinical endpoints remained significant for relevant downstream events. KIR-KIRL combinations were significantly predictive for reduced grade 3-4 aGVHD likelihood, in patients transplanted with increased inhibitory KIR gene content and IM-KIR = 5 scores. Improvements were also observed in associated outcomes for both ALL and AML patients, including relapse-free survival, GRFS and overall survival. This study demonstrates that NK cell KIR HLA interactions may be relevant to the paediatric acute leukaemia transplant setting. Reduction in aGVHD suggests KIR effects may extend beyond NK cells. Moving forward clinical trials utilizing donors with a higher iKIR should be considered for URD HCT in paediatric recipients with acute leukaemia to optimize clinical outcomes.
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Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Receptores KIR , Donante no Emparentado , Humanos , Receptores KIR/genética , Niño , Masculino , Femenino , Preescolar , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/mortalidad , Enfermedad Injerto contra Huésped/prevención & control , Adolescente , Estudios Retrospectivos , Lactante , Células Asesinas Naturales/inmunología , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Leucemia-Linfoma Linfoblástico de Células Precursoras/inmunología , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/inmunología , Antígenos HLA/inmunología , Antígenos HLA/genéticaRESUMEN
Macrophage polarization plays a key role in the inflammatory response. Various ion channels expressed in macrophages have been documented, but very little is known about their roles in macrophage polarization. We found that knockdown or blockade of the Kir2.1 (also known as KCNJ2) channel significantly inhibited M1 macrophage polarization, but promoted M2 macrophage polarization. Lipopolysaccharide (LPS)-induced M1 polarization was also remarkably suppressed in high extracellular K+ solutions (70â mM K+), and this inhibition was partially abolished by adding Ca2+ to the culture medium. Ca2+ imaging showed that Ca2+ influx was dependent on the hyperpolarized membrane potential generated by the Kir2.1 channel. The upregulation of phospho (p)-CaMK II, p-ERK, and p-NF-κB proteins in macrophages from the RAW264.7 cell line that were stimulated with LPS was significantly reversed by blocking the Kir2.1 channel or culturing the cells with 70â mM K+ medium. Furthermore, in vivo studies showed that mice treated with a Kir2.1 channel blocker were protected from LPS-induced peritonitis. In summary, our data reveal the essential role of the Kir2.1 channel in regulating macrophage polarization via the Ca2+/CaMK II/ERK/NF-κB signaling pathway.
Asunto(s)
Lipopolisacáridos , FN-kappa B , Animales , Calcio/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Ratones , FN-kappa B/metabolismo , Canales de Potasio de Rectificación Interna , Transducción de SeñalRESUMEN
Hypertension is associated with decreased endothelial function through reduced contributions of NO. We previously discovered that flow-induced NO production in resistance arteries of mice and humans critically depends on endothelial inwardly-rectifying K+ channels (Kir2.1). The goal of this study was to establish whether these channels contribute to the impairment of endothelial function, measured by flow-induced vasodilation (FIV) in peripheral resistance arteries of humans with hypertension. We measured FIV in vessels isolated from subcutaneous fat biopsies from normotensive (n=19; SBP: 115±27mmHg; DBP: 75.3±5.7mmHg) and hypertensive subjects (n=13; SBP: 146.1±15.2 mmHg; DBP: 94.4±6.9mmHg). We find that FIV is impaired in hypertensive adults as demonstrated by a significant reduction in FIV when compared to the normotensive adults, which is partially attributed to a reduction in Kir2.1-dependent vasodilation. Specifically, we show that pharmacologically inhibiting Kir2.1 or functionally downregulating Kir2.1 with endothelial-specific adenoviral vector dnKir2.1 result in a significant reduction in FIV in normotensive subjects but with a smaller effect in hypertensive adults. The Kir2.1-dependent vasodilation was negatively correlated to SBP and DBP, indicating that Kir2.1 contribution to FIV decreases as blood pressure increases. Furthermore, exposing vessels from normotensive adults to acute high-pressure results in loss of Kir2.1 contribution, as high-pressure impairs vasodilation. No effect is seen when these vessels were incubated with dnKir2.1. Overexpressing wtKir2.1 in the endothelium resulted in some improvement in vasodilation in arteries from all participants, with a greater recovery in hypertensive adults. Our data suggest that high pressure-induced suppression of Kir2.1 is an important mechanism underlying endothelial dysfunction in hypertension.