High-salt diet suppresses autoimmune demyelination by regulating the blood-brain barrier permeability

Sodium chloride, "salt," is an essential component of daily food and vitally contributes to the body's homeostasis. However, excessive salt intake has often been held responsible for numerous health risks associated with the cardiovascular system and kidney. Recent reports linked a high-salt diet (HSD) to the exacerbation of artificially induced central nervous system (CNS) autoimmune pathology through changes in microbiota and enhanced T(H)17 cell differentiation [M. Kleinewietfeld et al., Nature 496, 518-522 (2013); C. Wu et al., Nature 496, 513-517 (2013); N. Wilck et al., Nature 551, 585-589 (2017)]. However, there is no evidence that dietary salt promotes or worsens a spontaneous autoimmune disease. Here we show that HSD suppresses autoimmune disease development in a mouse model of spontaneous CNS autoimmunity. We found that HSD consumption increased the circulating serum levels of the glucocorticoid hormone corticosterone. Corticosterone enhanced the expression of tight junction molecules on the brain endothelial cells and promoted the tightening of the blood-brain barrier (BBB) thereby controlling the entry of inflammatory T cells into the CNS. Our results demonstrate the multifaceted and potentially beneficial effects of moderately increased salt consumption in CNS autoimmunity.We thank the Mass Spectrometry and NGS Core Facilities at the Max Planck Institute of Biochemistry for performing sample analysis for proteomics and mRNA-seq experiments

I RAPPORTI ECONOMICO-FINANZIARI TRA ITALIA E REPUBBLICA DI SAN MARINO

Circadian clocks coordinate 24-hr rhythms of behavior and physiology. In mammals, a master clock residing in the suprachiasmatic nucleus (SCN) is reset by the light-dark cycle, while timed food intake is a potent synchronizer of peripheral clocks such as the liver. Alterations in food intake rhythms can uncouple peripheral clocks from the SCN, resulting in internal desynchrony, which promotes obesity and metabolic disorders. Pancreas-derived hormones such as insulin and glucagon have been implicated in signaling mealtime to peripheral clocks. In this study, we identify a novel, more direct pathway of food-driven liver clock resetting involving oxyntomodulin (OXM). In mice, food intake stimulates OXM secretion from the gut, which resets liver transcription rhythms via induction of the core clock genes Per1 and 2. Inhibition of OXM signaling blocks food-mediated resetting of hepatocyte clocks. These data reveal a direct link between gastric filling with food and circadian rhythm phasing in metabolic tissues

Circadian Clocks in Mouse and Human CD4+ T Cells

Though it has been shown that immunological functions of CD4+ T cells are time of day-dependent, the underlying molecular mechanisms remain largely obscure. To address the question whether T cells themselves harbor a functional clock driving circadian rhythms of immune function, we analyzed clock gene expression by qPCR in unstimulated CD4+ T cells and immune responses of PMA/ionomycin stimulated CD4+ T cells by FACS analysis purified from blood of healthy subjects at different time points throughout the day. Molecular clock as well as immune function was further analyzed in unstimulated T cells which were cultured in serum-free medium with circadian clock reporter systems. We found robust rhythms of clock gene expression as well as, after stimulation, IL-2, IL-4, IFN-γ production and CD40L expression in freshly isolated CD4+ T cells. Further analysis of IFN-γ and CD40L in cultivated T cells revealed that these parameters remain rhythmic in vitro. Moreover, circadian luciferase reporter activity in CD4+ T cells and in thymic sections from PER2::LUCIFERASE reporter mice suggest that endogenous T cell clock rhythms are self-sustained under constant culture conditions. Microarray analysis of stimulated CD4+ T cell cultures revealed regulation of the NF-κB pathway as a candidate mechanism mediating circadian immune responses. Collectively, these data demonstrate for the first time that CD4+ T cell responses are regulated by an intrinsic cellular circadian oscillator capable of driving rhythmic CD4+ T cell immune responses

Circadian clocks, stress, and immunity

In mammals, molecular circadian clocks are present in most cells of the body, and this circadian network plays an important role in synchronizing physiological processes and behaviors to the appropriate time of day. The hypothalamic–pituitary–adrenal endocrine axis regulates the response to acute and chronic stress, acting through its final effectors – glucocorticoids – released from the adrenal cortex. Glucocorticoid secretion, characterized by its circadian rhythm, has an important role in synchronizing peripheral clocks and rhythms downstream of the master circadian pacemaker in the suprachiasmatic nucleus. Finally, glucocorticoids are powerfully anti-inflammatory, and recent work has implicated the circadian clock in various aspects and cells of the immune system, suggesting a tight interplay of stress and circadian systems in the regulation of immunity. This mini-review summarizes our current understanding of the role of the circadian clock network in both the HPA axis and the immune system, and discusses their interactions

Physiological functions of the adrenocortical circadian clock.

In the course of evolution most organisms have evolved endogenous circadian clocks that help them to anticipate daily environmental changes in light, temperature and food availability and therefore adjust physiology and behavior in a more efficient manner. The mammalian genome encodes a number of dedicated clock proteins, which coordinate the rhythmic transcription and translation of hundreds of genes in almost every cell of the body. The transcriptional activator BMAL1 (also known as ARNTL or MOP3) is at the core of this molecular clockwork and indispensable for circadian clock function. Information about external time, so called Zeitgeber input, is integrated by a circadian master pacemaker in the hypothalamic suprachiasmatic nuclei (SCN) that synchronizes subordinate clocks in other brain regions and peripheral tissues via different routes including hormonal signals, autonomic innervation, and regulation of the sleep-wake cycle. Glucocorticoid (GC) hormones (mainly cortisol in humans and corticosterone (CORT) in rodents) produced by the adrenal cortex in a rhythmic fashion are essential for the coordination of responses to stress, but also act as systemic synchronizers of circadian rhythms in various tissues. It has previously been shown that adrenocortical clocks modulate the sensitivity of the steroidogenic machinery to external stimuli. Their role in the regulation of stress responses, however, remains unclear. To close this gap and to study adrenocortical clock function in vivo, two genetic mouse models with full and conditional deletion of the Bmal1 gene were used in this study. In the first part of the thesis I studied GC production and stress responses in Bmal1-deficient mice. Under unstressed conditions Bmal1−/− mutants suffer from hypocortisolism, associated with impaired adrenal responsiveness to adrenocorticotropin (ACTH) and down-regulated transcription of genes involved in cholesterol transport and steroidogenesis in the adrenal gland, such as Star, Stard4, Ldlr and Por. Bmal1-deficient mice show reduced GC responses to acute stress, but preserved ACTH responses. Furthermore, they develop behavioral resistance to acute and sub-chronic stressors, as shown using forced swim, tail suspension and sucrose preference tests. These data suggest that the clock gene Bmal1 regulates circadian and acute secretion of GCs by the adrenal gland and contributes to behavioral resistance to stress, probably via its effects on adrenocortical function. The second part of the thesis focuses on the generation of conditional knockout mice that lack a functional circadian clock specifically in adrenocortical cells. For this purpose Bmal1fl/fl mice were cross-bred with Cyp11a1-Cre mice that express the CRE recombinase in steroid-producing cells of the adrenal gland and the gonads. Immunohistochemical stainings reveal high efficiency of BMAL1 deletion in the adrenal cortex of Cyp11a1Cre/+;Bmal1fl/fl (ACD) mice compared to Cyp11a1Cre/+ controls. Moreover, abolished rhythms in the expression of clock and clock-controlled genes in the adrenal cortex, but not in the kidney, of ACD mice confirm a disrupted functionality of the adrenocortical clock. However, circadian rhythms of CORT and aldosterone are not significantly altered in ACD mice kept in constant darkness. This suggests that the adrenocortical clock itself is dispensable for maintenance of circadian GC rhythms under unstressed conditions. Considering that GCs play a crucial role in the entrainment of circadian clocks, I analyzed behavioral responses of ACD and control mice to a rapid shift of the light-dark cycle. After a 6-hour phase advance the photic re-entrainment of locomotor activity of ACD mice occurs significantly faster compared to control mice, indicating that the adrenocortical clock contributes to the robustness of the circadian system under conditions of persistent external noise. In conclusion, this study shows that the adrenocortical clock plays a role in regulation of hormonal and behavioral responses to stress and contributes to the phase stability of the circadian system. Since many people live under conditions of regular Zeitgeber contamination, e.g. during shift work, manipulation of the adrenocortical clock may help buffering our endogenous clocks against exogenous perturbation. On the other hand, suppressing the stabilizing effect of the adrenocortical clock could speed up adaption of the circadian system when this is wanted, such as during jetlag

Impaired glucocorticoid production and response to stress in Arntl-deficient male mice.

The basic helix-loop-helix transcription factor Aryl Hydrocarbon Receptor Nuclear Translocator-Like (ARNTL, also known as BMAL1 or MOP3) is a core component of the circadian timing system in mammals, which orchestrates 24-hour rhythms of physiology and behavior. Genetic ablation of Arntl in mice leads to behavioral and physiological arrhythmicity, including loss of circadian baseline regulation of glucocorticoids (GCs). GCs are important downstream regulators of circadian tissue clocks and have essential functions in the physiological adaptation to stress. The role of the clock machinery in the regulation of stress-induced GC release, however, is not well understood. Here we show that already under unstressed conditions Arntl-deficient mice suffer from hypocortisolism with impaired adrenal responsiveness to ACTH and down-regulated transcription of genes involved in cholesterol transport in adrenocortical cells. Under stress they show diminished GC and behavioral responses and develop behavioral resistance to acute and subchronic stressors, as shown using forced swim, tail suspension, and sucrose preference tests. These data suggest that the clock gene Arntl regulates circadian and acute secretion of GCs by the adrenal gland. Arntl disruption, probably via its effect on adrenal clock function, modulates stress axis activity and, thus, may promote resistance to both acute and repeated stress

Zirkadiane Uhren in Gehirn und Peripherie: biologische Funktion und Relevanz für die Klinik.

In den meisten Organismen - von Cyanobakterien bis zum Menschen - haben sich genetisch kodierte zirkadiane Uhren entwickelt, die Verhalten und Physiologie an im Tagesverlauf veränderliche Umweltbedingungen adaptieren. Störungen der Uhr, zum Beispiel durch Schichtarbeit, beeinträchtigen diese Anpassung und fördern so die Entwicklung von metabolischen, immunologischen und neuropsychiatrischen Erkrankungen. Das zirkadiane System der Säugetiere besteht aus einem zentralen Schrittmacher im Nucleus suprachiasmaticus des Hypothalamus und untergeordneten, semi-autonomen Uhren in, unter anderem, der Leber, der Niere, der Nebenniere, aber auch in vielen weiteren Hirnregionen. Während periphere Oszillatoren endokrine, metabolische und immunologische Prozesse regulieren, modulieren zentrale Uhren grundlegende wie höhere Hirnfunktionen. In Klinik und Praxis hilft die Kenntnis dieser physiologischen Rhythmen bei der Interpretation von Labordaten und anderen Krankheitssymptomen. Die Chronomedizin kann durch Anpassung der Behandlungszeiten die Wirksamkeit und Nebenwirkungen von Therapien optimieren oder über eine Stabilisierung des internen zirkadianen Rhythmus den Krankheitsstatus direkt beeinflussen