RESUMEN
Gut microbiota imbalance (dysbiosis) is increasingly associated with pathological conditions, both within and outside the gastrointestinal tract. Intestinal Paneth cells are considered to be guardians of the gut microbiota, but the events linking Paneth cell dysfunction with dysbiosis remain unclear. We report a three-step mechanism for dysbiosis initiation. Initial alterations in Paneth cells, as frequently observed in obese and inflammatorybowel diseases patients, cause a mild remodeling of microbiota, with amplification of succinate-producing species. SucnR1-dependent activation of epithelial tuft cells triggers a type 2 immune response that, in turn, aggravates the Paneth cell defaults, promoting dysbiosis and chronic inflammation. We thus reveal a function of tuft cells in promoting dysbiosis following Paneth cell deficiency and an unappreciated essential role of Paneth cells in maintaining a balanced microbiota to prevent inappropriate activation of tuft cells and deleterious dysbiosis. This succinate-tuft cell inflammation circuit may also contribute to the chronic dysbiosis observed in patients.
Asunto(s)
Disbiosis , Membrana Mucosa , Humanos , Inflamación , Células de Paneth , Succinatos , Ácido SuccínicoRESUMEN
Background: Endurance athletes are prone to bradyarrhythmias, which in the long-term may underscore the increased incidence of pacemaker implantation reported in this population. Our previous work in rodent models has shown training-induced sinus bradycardia to be due to microRNA (miR)-mediated transcriptional remodeling of the HCN4 channel, leading to a reduction of the "funny" (I f) current in the sinoatrial node (SAN). Objective: To test if genetic ablation of G-protein-gated inwardly rectifying potassium channel, also known as I KACh channels prevents sinus bradycardia induced by intensive exercise training in mice. Methods: Control wild-type (WT) and mice lacking GIRK4 (Girk4 -/-), an integral subunit of I KACh were assigned to trained or sedentary groups. Mice in the trained group underwent 1-h exercise swimming twice a day for 28 days, 7 days per week. We performed electrocardiogram recordings and echocardiography in both groups at baseline, during and after the training period. At training cessation, mice were euthanized and SAN tissues were isolated for patch clamp recordings in isolated SAN cells and molecular profiling by quantitative PCR (qPCR) and western blotting. Results: At swimming cessation trained WT mice presented with a significantly lower resting HR that was reversible by acute I KACh block whereas Girk4 -/- mice failed to develop a training-induced sinus bradycardia. In line with HR reduction, action potential rate, density of I f, as well as of T- and L-type Ca2+ currents (I CaT and I CaL ) were significantly reduced only in SAN cells obtained from WT-trained mice. I f reduction in WT mice was concomitant with downregulation of HCN4 transcript and protein, attributable to increased expression of corresponding repressor microRNAs (miRs) whereas reduced I CaL in WT mice was associated with reduced Cav1.3 protein levels. Strikingly, I KACh ablation suppressed all training-induced molecular remodeling observed in WT mice. Conclusion: Genetic ablation of cardiac I KACh in mice prevents exercise-induced sinus bradycardia by suppressing training induced remodeling of inward currents I f, I CaT and I CaL due in part to the prevention of miR-mediated transcriptional remodeling of HCN4 and likely post transcriptional remodeling of Cav1.3. Strategies targeting cardiac I KACh may therefore represent an alternative to pacemaker implantation for bradyarrhythmias seen in some veteran athletes.
RESUMEN
Hyperexcitability of axotomized dorsal root ganglion neurons is thought to play a role in neuropathic pain. Numerous changes in ionic channels expression or current amplitude are reported after an axotomy, but to date no direct correlation between excitability of axotomized sensory neurons and ionic channels alteration has been provided. Following sciatic nerve injury, we examined, under whole-cell patch clamp recording, the effects of calcium homeostasis on the electrical activity of axotomized medium-sized sensory neurons isolated from lumbar dorsal root ganglia of adult mice. Axotomy induced an increase in excitability of medium sensory neurons among which 25% develop a propensity to fire repetitively. The condition necessary to get burst discharge in axotomized neurons was the presence of a high intracellular Ca2+ buffer concentration. The main effect was to amplify the increase in threshold current and apparent input resistance induced by axotomy. These data supply evidence for a role of Ca2+-dependent mechanisms in the control of excitability of axotomized sensory neurons.
Asunto(s)
Señalización del Calcio/fisiología , Ganglios Espinales/metabolismo , Neuralgia/metabolismo , Neuronas Aferentes/metabolismo , Enfermedades del Sistema Nervioso Periférico/metabolismo , Neuropatía Ciática/metabolismo , Potenciales de Acción/fisiología , Animales , Axotomía , Tampones (Química) , Calcio/metabolismo , Tamaño de la Célula , Modelos Animales de Enfermedad , Impedancia Eléctrica , Femenino , Ganglios Espinales/fisiopatología , Homeostasis/fisiología , Líquido Intracelular/metabolismo , Ratones , Neuralgia/fisiopatología , Neuronas Aferentes/patología , Técnicas de Placa-Clamp , Enfermedades del Sistema Nervioso Periférico/fisiopatología , Nervio Ciático/metabolismo , Nervio Ciático/fisiopatología , Neuropatía Ciática/fisiopatologíaRESUMEN
The doxycycline (Dox)-inducible reverse tetracycline transactivator (rtTA) is often used to control gene expression. However, the Tet-on system displays a high background activity. To overcome this unregulated expression we used the tetracycline-dependent transcriptional silencer (tTS), which binds the tetO inducible promoter in the absence of Dox. Controlled gene expression was analyzed in vivo by delivering combinations of Dox-regulated luciferase reporter construct, rtTA, and tTS expression plasmids into mouse muscle, using electrotransfer. Elevated luciferase expression levels were observed in the absence of doxycycline, and a 10-fold induction was obtained after drug administration. In contrast, when tTS was added, background expression was dramatically lowered by three to four orders of magnitude, and induction was maintained. The tTS system was then used to control expression of a therapeutic gene in experimental arthritis. DBA/1 mice were coinjected with plasmids encoding the antiinflammatory interleukin-10 cytokine under the control of the tetO promoter, the rtTA, and the tTS. Electrotransfer resulted in a dose-dependent increase in IL-10 expression, maintained over a 3-month period, and significant inhibitory effects on collagen-induced arthritis. We conclude that the use of tTS significantly improves the utility of the rtTA system for somatic gene transfer by reducing background activity.
Asunto(s)
Artritis Experimental/genética , Artritis Experimental/terapia , Regulación Viral de la Expresión Génica , Terapia Genética , Vectores Genéticos , Interleucina-10/genética , Tetraciclina/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Electroporación , Femenino , Vectores Genéticos/administración & dosificación , Inyecciones Intramusculares , Interleucina-10/administración & dosificación , Interleucina-10/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Plásmidos , TransfecciónRESUMEN
Whole cell patch-clamp recordings of calcium-activated chloride current [ICl(Ca)] were made from adult sensory neurons of naive and axotomized mouse L4-L6 lumbar dorsal root ganglia after 1 day of culture in vitro. A basal ICl(Ca) was specifically expressed in a subset of naive medium-diameter neurons (30-40 microm). Prior nerve injury, induced by sciatic nerve transection 5 days before experiments, increased both ICl(Ca) amplitude and its expression in medium-diameter neurons. Moreover, nerve injury also induced ICl(Ca) expression in a new subpopulation of neurons, the large-diameter neurons (40-50 microm). Small-diameter neurons (inferior to 30 microm) never expressed ICl(Ca). Regulated ICl(Ca) expression was strongly correlated with injury-induced regenerative growth of sensory neurons in vitro and nerve regeneration in vivo. Cell culture on a substrate not permissive for growth, D,L-polyornithine, prevented both elongation growth and ICl(Ca) expression in axotomized neurons. Regenerative growth and the induction of ICl(Ca) expression take place 2 days after injury, peak after 5 days of conditioning in vivo, slowly declining thereafter to control values. The selective expression of ICl(Ca) within medium- and large-diameter neurons conditioned for rapid, efficient growth suggests that these channels play a specific role in postinjury behavior of sensory neuron subpopulations such as neuropathic pain and/or axonal regeneration.