Developmental toxicity of bisphenol S in Caenorhabditis elegans and NODEF mice

The growing concern surrounding bisphenol A (BPA) has led to increased industrial production and application of its analog bisphenol S (BPS). The goals of this study were: (1) To examine the generational effects in the nematode C. elegans for up to three generations following developmental exposure to BPS (0.1, 1.0, 5.0 and 10.0 μM), and (2) To examine the neurotoxicity and metabolic toxicity in NODEF mouse offspring exposed to BPS (3 μg/kg BW) in utero throughout gestation once/day via oral pipette. First, worms were exposed to BPS developmentally for a single period of 48 hours and then propagated for 2 additional generations. Exposure to 0.1 and 1.0 μM BPS decreased lifespan and the number of progeny with an ability to recover in subsequent generations. In contrast, worms exposed to 5.0 or 10.0 μM BPS exhibited a continuous effect in the second generation, e.g., decreased lifespan and reduced number of progeny. Only worms exposed to 10.0 μM BPS continued to have a significant long-term effect (e.g., decreased lifespan) through the third generation. In addition, worms developmentally exposed to BPS at 5.0 μM and 10.0 μM also showed decreases in body bends. In contrast, worms exposed to 0.1 μM BPS exhibited a significant increase in head thrashes. When the multigenerational effects were examined by exposing worms to BPS for 48 hours developmentally at each generation for three generations, an accumulative effect was observed in worms treated with 0.1 or 1.0 μM BPS for two generations, but not for three generations, suggesting a threshold existed. Worms exposed to either 5.0 or 10.0 μM BPS demonstrated accumulative effects through two and three generations. When the developmental effects of BPS were studied in NODEF mice, offspring exposed gestationally exhibited behavioral deficits at 12, but not at 3, weeks of age. Specifically, female offspring had decreases in working and short-term memories while male offspring showed increases in hyperactivity and anxiety-like behaviors. In summary, this study demonstrates the sex-related effects of BPS in NODEF mouse offspring exposed in utero, along with the generational effects observed in C. elegans.     

CD4+CD25+ T Cells are Essential for Behavioral Effects of Lactobacillus rhamnosus JB-1 in Male BALB/c mice

Over the past decade there has been increasing interest in the involvement of the microbiota-gut-brain axis in mental health. However, there are major gaps in our knowledge regarding the complex signaling systems through which gut microbes modulate the CNS. The immune system is a recognized mediator in the bidirectional communication continuously occurring between gut and brain. We previously demonstrated that Lactobacillus rhamnosus JB-1 (JB-1), a bacterial strain that has anxiolytic- and antidepressant-like effects in mice, modulates the immune system through induction of immunosuppressive T regulatory cells. Here we examined a potential causal relationship between JB-1 induced regulatory T cells and the observed effects on behaviour.We found that depletion of regulatory T cells, via treatment with monoclonal antibody against CD25, inhibited the antidepressant- and anxiolytic-like effects induced by 4-week oral administration of JB-1 in mice. Ly6C^hi monocytes were found to be decreased in JB-1 fed mice with intact regulatory T cells, but not in JB-1 fed mice following depletion. Furthermore, adoptive transfer of CD4⁺CD25⁺ cells, from JB-1 treated donor mice, but not from controls, induced antidepressant- and anxiolytic-like effects in recipient mice. Ly6C^hi monocytes were also significantly decreased in mice receiving CD4⁺CD25⁺ cells from JB1 fed donors.This study identifies cells within the CD4⁺CD25⁺ population, most likely regulatory T cells, as both necessary and sufficient in JB-1-induced antidepressant- and anxiolytic-like effects in mice, providing novel mechanistic insight into microbiota-gut-brain communication in addition to highlighting the potential for immunotherapy in psychiatric disorders.     

Presence of T-cell subset abnormalities in newly diagnosed cases of multiple sclerosis and relationship with short-term clinical activity

Abnormalities of T-cell subsets in patients with multiple sclerosis are well known; in order to assess whether immunological abnormalities are relevant in the pathogenesis of the disease after its clinical onset, peripheral blood lymphocyte subsets (CD3⁺, CD4⁺, CD4⁺ CD45RA⁺, CD4⁺CD45RA^–, CD8⁺, CD8⁺CD57⁺, CD57⁺, CD25⁺) were analysed serially in 25 patients at the first clinical episode suggestive of inflammatory demyelinating disease and in an equal number of age- and sex-matched controls. During the follow-up period (12–18 months, mean 14) 6 of 25 patients presented new relapses: in this subgroup of patients, significant changes in CD4⁺ ratio (% CD4⁺CD45RA^–/%CD4⁺CD45RA^–) were detected in comparison both with healthy controls and with clinically stable patients. Patients clinically stable at follow-up did not display immunological abnormalities, regardless of the presence or absence of cerebrospinal fluid and/or magnetic resonance imaging alterations consistent with multiple sclerosis. These findings suggest a possible prognostic role of early T-cell subset imbalance in multiple sclerosis.     

CXCR4+CD45− BMMNC subpopulation is superior to unfractionated BMMNCs for protection after ischemic stroke in mice

Cell-based therapy is considered to be a promising therapeutic strategy for stroke treatment. Although unfractionated bone marrow mononuclear cells (BMMNCs) have been tried in both preclinical and clinical trials, the effective subpopulations need to be identified. In this study, we used fluorescence-activated cell sorting to harvest the CXCR4⁺CD45⁺ and CXCR4⁺CD45⁻ BMMNC subpopulations from transgenic mice that express enhanced green fluorescent protein. We then allogeneically grafted unfractionated BMMNCs or a subpopulation into mice subjected to transient middle cerebral artery occlusion (tMCAO) and compared the effects on stroke outcomes. We found that CXCR4⁺CD45⁻ BMMNCs, but not CXCR4⁺CD45⁺ BMMNCs, more effectively reduced infarction volume and neurologic deficits than did unfractionated BMMNCs. Brain tissue from the ischemic hemisphere of mice treated with CXCR4⁺CD45⁻ BMMNCs had higher levels of vascular endothelial growth factor and lower levels of TNF-α than did tissue from mice treated with unfractionated BMMNCs. In contrast, CXCR4⁺CD45⁺ BMMNCs showed an increase in TNF-α. Additionally, CXCR4⁺CD45⁺ and CXCR4⁺CD45⁻ populations exhibited more robust migration into the lesion areas and were better able to express cell-specific markers of different linages than were the unfractionated BMMNCs. Endothelial and astrocyte cell markers did not colocalize with eGFP⁺ cells in the brains of tMCAO mice that received CXCR4⁺CD45⁺ BMMNCs. In vitro, the CXCR4⁺CD45⁻ BMMNCs expressed significantly more Oct-4 and Nanog mRNA than did the unfractionated BMMNCs. However, we did not detect gene expression of these two pluripotent markers in CXCR4⁺CD45⁺ BMMNCs. Taken together, our study shows for the first time that the CXCR4⁺CD45⁻ BMMNC subpopulation is superior to unfractionated BMMNCs in ameliorating cerebral damage in a mouse model of tMCAO and could represent a new therapeutic approach for stroke treatment.     

Diet-induced obesity prolongs neuroinflammation and recruits CCR2+ monocytes to the brain following herpes simplex virus (HSV)-1 latency in mice

Herpes simplex virus (HSV)-1 is a ubiquitous human infection, with increased prevalence in obese populations. Obesity has been linked to increased inflammation, susceptibility to infection, and higher rates of anxiety disorder and cognitive impairment. To determine how obesity alters neuroinflammation and behavior following infection, we infected weanling C57BL/6 or CCR2^RFP/+/CX3CR1^GFP/+ mice with a very low dose of HSV-1. Following viral latency (14 days post infection (d p.i.)), mice were randomly assigned to remain on the low fat (LF) diet or switched to a 45% high fat (HF) diet. Eight weeks post diet shift, latently infected mice on the HF diet (HSV-HF) had greater microglial activation and infiltration of inflammatory CCR2⁺ monocytes in the hypothalamus and dentate gyrus, in comparison to both HSV-LF mice and uninfected mice on LF and HF diets. VCAM staining was present in hypothalamus and hippocampus of the HSV-HF mice in the areas of monocyte infiltration. Infiltrating monocytes also produced proinflammatory cytokines demonstrating that, along with activated microglia, monocytes contribute to sustained neuroinflammation in latently infected obese mice. Utilizing a light-dark preference test, we found that HSV-HF mice had increased anxiety-like behavior. In the marble-burying test, HF diet and HSV infection resulted in increased numbers of buried marbles. Together, these mice provide a useful, testable model to study the biobehavioral effects of obesity and latent HSV-1 infection in regards to anxiety and may provide a tool for studying diet intervention programs in the future.     

Cohabitation with a B16F10 melanoma-bearer cage mate influences behavior and dendritic cell phenotype in mice

This study evaluated the effects of cohabitation with a B16F10 melanoma-bearer cage mate on behavior and immune functions in mice. Five different experiments were conducted. In each of them, the female mice were divided into two groups: control and experimental. One mouse of each control pair was kept undisturbed and called “companion of health partner” (CHP). One mouse of each experimental pair was inoculated with B16F10 cells and the other, the subject of this study, was called “companion sick partner” (CSP). On Day 20 of cohabitation, behavior and immune parameters from CHP and CSP mice were analyzed. In comparison to the CHP, the CSP mice: (1) presented an increased general locomotion in the open field and a decreased exploration time and number of entries in the plus-maze open arms; (2) had an enhanced expression of the CD80 costimulatory molecule on Iab⁺CD11c⁺ spleen cells, but no differences were found on lymph nodes cells; (3) presented an altered differentiation of bone marrow cells in the presence of GM-CSF, IL-4, and LPS in vitro, resulting in a lower percentage of Iab⁺CD80⁺ cells; (4) had a deficit in the establishment of a Delayed Type of Hypersensitivity to ovalbumin, which was associated to an in vitro proliferation of an IL-10-producing lymphocyte subpopulation after ovalbumin stimulation. Corticosterone levels detected on Day 20 of cohabitation were similar in CHP and CSP mice. It is shown here that DCs phenotype in mice is affected by conditions associated with behavioral alterations indicative of an anxiety-like state induced by the cohabitation with a tumor-bearer conspecific. This phenomenon occurred probably through a nondependent corticosterone mechanism.     

Disruption of gallbladder smooth muscle function is an early feature in the development of cholesterol gallstone disease

Background Decreased gallbladder smooth muscle (GBSM) contractility is a hallmark of cholesterol gallstone disease, but the interrelationship between lithogenicity, biliary stasis, and inflammation are poorly understood. We studied a mouse model of gallstone disease to evaluate the development of GBSM dysfunction relative to changes in bile composition and the onset of sterile cholecystitis. Methods BALB/cJ mice were fed a lithogenic diet for up to 8 weeks, and tension generated by gallbladder muscle strips was measured. Smooth muscle Ca²⁺ transients were imaged in intact gallbladder. Key Results Lipid composition of bile was altered lithogenically as early as 1 week, with increased hydrophobicity and cholesterol saturation indexes; however, inflammation was not detectable until the fourth week. Agonist‐induced contractility was reduced from weeks 2 through 8. GBSM normally exhibits rhythmic synchronized Ca²⁺ flashes, and their frequency is increased by carbachol (3 μm ). After 1 week, lithogenic diet‐fed mice exhibited disrupted Ca²⁺ flash activity, manifesting as clustered flashes, asynchronous flashes, or prolonged quiescent periods. These changes could lead to a depletion of intracellular Ca²⁺ stores, which are required for agonist‐induced contraction, and diminished basal tone of the organ. Responsiveness of Ca²⁺ transients to carbachol was reduced in mice on the lithogenic diet, particularly after 4–8 weeks, concomitant with appearance of mucosal inflammatory changes. Conclusions & Inferences These observations demonstrate that GBSM dysfunction is an early event in the progression of cholesterol gallstone disease and that it precedes mucosal inflammation.     

Interactions of peptide amidation and copper: Novel biomarkers and mechanisms of neural dysfunction

Mammalian genomes encode only a small number of cuproenzymes. The many genes involved in coordinating copper uptake, distribution, storage and efflux make gene/nutrient interactions especially important for these cuproenzymes. Copper deficiency and copper excess both disrupt neural function. Using mice heterozygous for peptidylglycine α-amidating monooxygenase (PAM), a cuproenzyme essential for the synthesis of many neuropeptides, we identified alterations in anxiety-like behavior, thermoregulation and seizure sensitivity. Dietary copper supplementation reversed a subset of these deficits. Wildtype mice maintained on a marginally copper-deficient diet exhibited some of the same deficits observed in PAM^+/? mice and displayed alterations in PAM metabolism. Altered copper homeostasis in PAM^+/? mice suggested a role for PAM in the cell type specific regulation of copper metabolism. Physiological functions sensitive to genetic limitations of PAM that are reversed by supplemental copper and mimicked by copper deficiency may serve as indicators of marginal copper deficiency.     

Critical Role of Astrocyte NAD+ Glycohydrolase in Myelin Injury and Regeneration

Western-style diets cause disruptions in myelinating cells and astrocytes within the mouse CNS. Increased CD38 expression is present in the cuprizone and experimental autoimmune encephalomyelitis models of demyelination and CD38 is the main nicotinamide adenine dinucleotide (NAD⁺)-depleting enzyme in the CNS. Altered NAD⁺ metabolism is linked to both high fat consumption and multiple sclerosis (MS). Here, we identify increased CD38 expression in the male mouse spinal cord following chronic high fat consumption, after focal toxin [lysolecithin (LL)]-mediated demyelinating injury, and in reactive astrocytes within active MS lesions. We demonstrate that CD38 catalytically inactive mice are substantially protected from high fat-induced NAD⁺ depletion, oligodendrocyte loss, oxidative damage, and astrogliosis. A CD38 inhibitor, 78c, increased NAD⁺ and attenuated neuroinflammatory changes induced by saturated fat applied to astrocyte cultures. Conditioned media from saturated fat-exposed astrocytes applied to oligodendrocyte cultures impaired myelin protein production, suggesting astrocyte-driven indirect mechanisms of oligodendrogliopathy. In cerebellar organotypic slice cultures subject to LL-demyelination, saturated fat impaired signs of remyelination effects that were mitigated by concomitant 78c treatment. Significantly, oral 78c increased counts of oligodendrocytes and remyelinated axons after focal LL-induced spinal cord demyelination. Using a RiboTag approach, we identified a unique in vivo brain astrocyte translatome profile induced by 78c-mediated CD38 inhibition in mice, including decreased expression of proinflammatory astrocyte markers and increased growth factors. Our findings suggest that a high-fat diet impairs oligodendrocyte survival and differentiation through astrocyte-linked mechanisms mediated by the NAD⁺ase CD38 and highlights CD38 inhibitors as potential therapeutic candidates to improve myelin regeneration.SIGNIFICANCE STATEMENT Myelin disturbances and oligodendrocyte loss can leave axons vulnerable, leading to permanent neurologic deficits. The results of this study suggest that metabolic disturbances, triggered by consumption of a diet high in fat, promote oligodendrogliopathy and impair myelin regeneration through astrocyte-linked indirect nicotinamide adenine dinucleotide (NAD⁺)-dependent mechanisms. We demonstrate that restoring NAD⁺ levels via genetic inactivation of CD38 can overcome these effects. Moreover, we show that therapeutic inactivation of CD38 can enhance myelin regeneration. Together, these findings point to a new metabolic targeting strategy positioned to improve disease course in multiple sclerosis and other conditions in which the integrity of myelin is a key concern.     

Dietary phytoestrogens improve stroke outcome after transient focal cerebral ischemia in rats

As phytoestrogens are postulated as being neuroprotectants, we assessed the hypothesis that dietary isoflavone-type phytoestrogens are neuroprotective against ischemic stroke. Transient focal cerebral ischemia (90 min) was induced by middle cerebral artery occlusion (MCAO) following the intraluminal thread technique, both in rats fed with soy-based diet and in rats fed with isoflavone-free diet. Cerebro-cortical laser-Doppler flow (cortical perfusion, CP), arterial blood pressure, core temperature, PaO2, PaCO2, pH and glycemia were measured before, during and after MCAO. Neurological examination and infarct volume measurements were carried out 3 days after the ischemic insult. Dietary isoflavones (both glycosides and aglycones) were measured by high-performance liquid chromatography. Neither pre-ischemic, intra-ischemic nor post-ischemic CP values were significantly different between the soy-based diet and the isoflavone-free diet groups. Animals fed with the soy-based diet showed an infarct volume of 122 +/- 20.2 mm3 (19 +/- 3.3% of the whole ipsilateral hemisphere volume). In animals fed with the isoflavone-free diet the mean infarct volume was significantly higher, 191 +/- 26.7 mm3 (28 +/- 4.1%, P < 0.05). Neurological examination revealed significantly higher impairment in the isoflavone-free diet group compared with the soy-based diet group (3.3 +/- 0.5 vs. 1.9 +/- 0.5, P < 0.05). These results demonstrate that dietary isoflavones improve stroke outcome after transient focal cerebral ischemia in such a way that a higher dietary isoflavone content results in a lower infarct volume and a better neurological status.     

Functional recovery and facial motoneuron survival are influenced by immunodeficiency in crush-axotomized mice

Facial nerve axotomy is a well-described injury paradigm for peripheral nerve regeneration and facial motoneuron (FMN) survival. We have previously shown that CD4⁺ T helper (Th) 1 and 2 effector subsets develop in the draining cervical lymph node, and that the IL-4/STAT-6 pathway of Th2 development is critical for FMN survival after transection axotomy. In addition, delayed behavioral recovery time in immunodeficient mice may be due to the absence of T and B cells. This study utilized a crush axotomy paradigm to evaluate FMN survival and functional recovery in WT, STAT-6 KO (impaired Th2 response), T-Bet KO (impaired Th1 response), and RAG-2 KO (lacking mature T and B cells) mice to elucidate the contributions of specific CD4⁺ T cell subsets in motoneuron survival and recovery mechanisms. STAT-6 KO and RAG-2 KO mice exhibited decreased FMN survival after crush axotomy compared to WT, supporting a critical role for the Th2 effector cell in motoneuron survival before target reconnection. Long term FMN survival was sustained through 10 wpo after crush axotomy in both WT and RAG-2 KO mice, indicating that target derived neurotrophic support maintains FMN survival after target reconnection. In addition, RAG-2 KO mice exhibited delayed functional recovery compared to WT mice. Both STAT-6 and T-Bet KO mice exhibited partially delayed functional recovery compared to WT, though not to the extent of RAG-2 KO mice. Collectively, our findings indicate that both pro- and anti-inflammatory CD4⁺ T cell responses contribute to optimal functional recovery from axotomy-induced facial paralysis, while FMN survival is supported by the anti-inflammatory Th2 response alone.     

GABAergic modulation with classical benzodiazepines prevent stress-induced neuro-immune dysregulation and behavioral alterations

ObjectivePsychosocial stress is associated with altered immunity, anxiety, and depression. Repeated social defeat (RSD), a model of social stress, triggers egress of inflammatory myeloid progenitor cells (MPCs; CD11b⁺/Ly6C^hi) that traffic to the brain, promoting anxiety-like behavior. In parallel, RSD enhances neuroinflammatory signaling and long-lasting social avoidant behavior. Lorazepam and clonazepam are routinely prescribed anxiolytics that act by enhancing GABAergic activity in the brain. Besides binding to the central benzodiazepine binding site (CBBS) in the central nervous system (CNS), lorazepam binds to the translocator protein (TSPO) with high affinity causing immunomodulation. Clonazepam targets the CBBS and has low affinity for the TSPO. Here the aims were to determine if lorazepam and clonazepam would: (1) prevent stress-induced peripheral and central inflammatory responses, and (2) block anxiety and social avoidance behavior in mice subjected to RSD.MethodsC57/BL6 mice were divided into experimental groups, and treated with either lorazepam (0.10 mg/kg), clonazepam (0.25 mg/kg) or vehicle (0.9% NaCl). Behavioral data and tissues were collected the morning after the last cycle of RSD.ResultsLorazepam and clonazepam were effective in attenuating mRNA expression of CRH in the hypothalamus and corticosterone in plasma in mice subjected to RSD. Both drugs blocked stress-induced levels of IL-6 in plasma. Lorazepam and clonazepam had different effects on stress-induced enhancement of myelopoiesis and inhibited trafficking of monocytes and granulocytes in circulation. Furthermore, lorazepam, but not clonazepam, inhibited splenomegaly and the production of pro-inflammatory cytokines in the spleen following RSD. Additionally, lorazepam and clonazepam, blocked stress-induced accumulation of macrophages (CD11b⁺/CD45^high) in the CNS. In a similar manner, both lorazepam and clonazepam prevented neuroinflammatory signaling and reversed anxiety-like and depressive-like behavior in mice exposed to RSD.ConclusionThese data support the notion that lorazepam and clonazepam, aside from exerting anxiolytic and antidepressant effects, may have therapeutic potential as neuroimmunomodulators during psychosocial stress. The reversal of RSD-induced behavioral outcomes may be due to the enhancement of GABAergic neurotransmission, or some other off-target effect. The peripheral actions of lorazepam, but not clonazepam, seem to be mediated by TSPO activation.     

Serotonin reuptake transporter deficiency promotes liver steatosis and impairs intestinal barrier function in obese mice fed a Western-style diet

Background

Intestinal barrier dysfunctions have been associated with liver steatosis and metabolic diseases. Besides nutritional factors, like a Western-style diet (WSD), serotonin has been linked with leaky gut. Therefore, we aimed to evaluate the role of serotonin in the pathogenesis of intestinal barrier dysfunctions and liver steatosis in mice fed high-fat and high-sugar diets.

Methods

6–8 weeks old male serotonin reuptake transporter knockout mice (SERT^−/−) and wild-type controls (SERT^+/+) were fed either a WSD or a control diet (CD) ad libitum with or without fructose 30% (F) added to the drinking water for 12 weeks. Markers of liver steatosis and intestinal barrier function were assessed.

Key Results

SERT^−/− mice showed increased weight gain compared with SERT^+/+ mice when fed a WSD ± F for 12 weeks (p < 0.05), whereby SERT^−/− mice exhibited reduced energy (−21%) intake. Furthermore, SERT knockout resulted in a more pronounced liver steatosis (p < 0.05), enhanced levels of endotoxin in portal vein plasma (p < 0.05), and increased liver expression of Tnf and Myd88 (p < 0.05), when mice were fed a WSD ± F. Finally, SERT^−/− mice, when compared with SERT^+/+ mice, had a decreased mRNA expression of Muc2 (p < 0.01), Ocln (p < 0.05), Cldn5 (p = 0.054) and 7 (p < 0.01), Defa5 (p < 0.05) and other antimicrobial peptides in the ileum. On the protein level, ZO-1 (p < 0.01) and DEFA5 protein (p < 0.0001) were decreased.

Conclusion and Inferences

Our data demonstrate that SERT knockout causes weight gain, liver steatosis, and leaky gut, especially in mice fed a WSD. Therefore, SERT induction could be a novel therapeutic approach to improve metabolic diseases associated with intestinal barrier dysfunction.     

GPR39, a receptor of the ghrelin receptor family, plays a role in the regulation of glucose homeostasis in a mouse model of early onset diet-induced obesity

GPR39, which may function as a Zn²⁺ sensor, is a member of the G protein‐coupled receptor family that also includes the receptor for the hunger hormone ghrelin. The down‐regulation of GPR39 mRNA in adipose tissue of obese type 2 diabetic patients suggests that GPR39 may contribute to the pathogenesis of the disease. The present study aimed to investigate the role of GPR39 in the regulation of energy balance and glucose homeostasis in wild‐type (GPR39^+/+) and GPR39 knockout mice (GPR39^?/?) with obesity‐related type 2 diabetes. GPR39 mRNA levels in adipose tissue of fasted GPR39^+/+ mice fed a high‐fat diet (HFD) for 30 weeks were reduced and correlated positively with blood glucose levels. Body weight, fat percentage and energy intake were increased in the HFD group but did not differ between both genotypes. Within the HFD group, blood glucose levels were lower in GPR39^?/? than in GPR39^+/+ mice, despite significant reductions in prandial plasma insulin levels. The latter may not be a result of changes in β‐cell hyperplasia because immunohistochemical staining of pancreata of mice on a HFD showed no differences between genotypes. The lower blood glucose levels may involve alterations in insulin sensitivity as revealed by glucose tolerance tests and respiratory quotient measurements that showed a preference of obese GPR39^?/? mice for the use of carbohydrates as metabolic fuel. The increase in plasma ghrelin levels in GPR39^?/? mice fed a HFD may contribute to the alterations in glucose homeostasis, whereas changes in gastric emptying or intestinal Zn²⁺ absorption are not involved. The results obtained in the present study suggest that GPR39 plays a role in the pathogenesis of obesity‐related type 2 diabetes by affecting the regulation of glucose homeostasis.     

Loss rather than downregulation of CD4 T cells as a mechanism for remission from experimental allergic encephalomyelitis

SJL/J mice recover from clinical signs of experimental allergic encephalomyelitis (EAE) 2 to 3 days following the onset of the initial attack. The immunoregulatory events that induce clinical recovery are not well understood. In this paper we have compared the activation state of the T cells infiltrating the central nervous system (CNS) in symptomatic and remitted mice. We isolated mononuclear cells from the CNS at various time points during the course of EAE and used flow cytometry to describe the kinetics of CNS infiltration by CD45⁺, CD2⁺, CD3⁺, TCRαβ⁺, CD4⁺ cells. There was a 30-fold reduction in the number of CNS CD4⁺ T cells in remitted mice 10 days following the initial attack. More than 60% of CNS CD4⁺ cells were of a CD44^high, CD45RB^low memory/effector phenotype both in active EAE, peak EAE and in remission, contrast to lymph nodes where this phenotype never constituted more than 17%. The proportion of CD8⁺ T cells was not increased in remitted mice, and we detected no TCRγδ⁺ cells within the CNS. Our findings demonstrate an overt loss of CD4⁺ T cells from the CNS and the maintenance of an activated state by T cells within the CNS and during remission from EAE. This argues against downregulation of T cell function as a mechanism for remission.     

A case of mistaken identity: CD11c‐eYFP+ cells in the normal mouse brain parenchyma and neural retina display the phenotype of microglia,not dendritic cells

Under steady‐state conditions the central nervous system (CNS) is traditionally thought to be devoid of antigen presenting cells; however, putative dendritic cells (DCs) expressing enhanced yellow fluorescent protein (eYFP) are present in the retina and brain parenchyma of CD11c‐eYFP mice. We previously showed that these mice carry the Crb1^rd8 mutation, which causes retinal dystrophic lesions; therefore we hypothesized that the presence of CD11c‐eYFP⁺ cells within the CNS may be due to pathology associated with the Crb1^rd8 mutation. We generated CD11c‐eYFP Crb1^wt/wt mice and compared the distribution and immunophenotype of CD11c‐eYFP⁺ cells in CD11c‐eYFP mice with and without the Crb1^rd8 mutation. The number and distribution of CD11c‐eYFP⁺ cells in the CNS was similar between CD11c‐eYFP Crb1^wt/wt and CD11c‐eYFP Crb1^rd8/rd8 mice. CD11c‐eYFP⁺ cells were distributed throughout the inner retina, and clustered in brain regions that receive input from the external environment or lack a blood‐brain barrier. CD11c‐eYFP⁺ cells within the retina and cerebral cortex of CD11c‐eYFP Crb1^wt/wt mice expressed CD11b, F4/80, CD115 and Iba‐1, but not DC or antigen presentation markers, whereas CD11c‐eYFP⁺ cells within the choroid plexus and pia mater expressed CD11c, I‐A/I‐E, CD80, CD86, CD103, DEC205, CD8α and CD135. The immunophenotype of CD11c‐eYFP⁺ cells and microglia within the CNS was similar between CD11c‐eYFP Crb1^wt/wt and CD11c‐eYFP Crb1^rd8/rd8 mice; however, CD11c and I‐A/I‐E expression was significantly increased in CD11c‐eYFP Crb1^rd8/rd8 mice. This study demonstrates that the overwhelming majority of CNS CD11c‐eYFP⁺ cells do not display the phenotype of DCs or their precursors and are most likely a subpopulation of microglia. GLIA 2016. GLIA 2016;64:1331–1349     

Signs of sympathetic and endothelial cell activation in the skin of patients with restless legs syndrome

ObjectivesTo evaluate skin biopsies of patients with early- and late onset restless legs syndrome (RLS) for concomitant small fiber neuropathy (SFN) and to determine cutaneous sympathetic innervation and microvascularization in comparison to healthy individuals.MethodsDensity of intraepidermal nerve fibers (IENFD), adrenergic nerve fibers and dermal capillaries was analyzed by immunofluorescence for PGP9.5, tyrosine hydroxylase and endothelial markers CD31 and CD105 in skin biopsies of 11 individuals with RLS and 8 age- and sex-matched controls.ResultsIENFD did not differ between RLS and controls, but two RLS patients with comorbid impaired glucose metabolism fulfilled morphometric criteria of SFN according to published normative values. In contrast, dermal nerve bundles of RLS patients showed an increased density of tyrosine hydroxylase⁺ adrenergic nerve fibers (p < 0.005). Moreover, an increased ratio between immature CD105⁺ and mature CD31⁺ endothelial cells within dermal capillaries was observed in RLS (p < 0.02).ConclusionsSFN, as a potential contributing factor for RLS, should be considered in patients with predisposing comorbidities presenting with burning or shooting pain, dysesthesias and impaired sensory and temperature perception. Evidence of an increased adrenergic innervation of the skin in RLS patients is in accordance with sympathetic hyperactivity while signs of endothelial cell activation may reflect an adaptive response to tissue hypoxia.     

The prebiotics 3′Sialyllactose and 6′Sialyllactose diminish stressor-induced anxiety-like behavior and colonic microbiota alterations: Evidence for effects on the gut–brain axis

There are extensive bidirectional interactions between the gut microbiota and the central nervous system (CNS), and studies demonstrate that stressor exposure significantly alters gut microbiota community structure. We tested whether oligosaccharides naturally found in high levels in human milk, which have been reported to impact brain development and enhance the growth of beneficial commensal microbes, would prevent stressor-induced alterations in gut microbial community composition and attenuate stressor-induced anxiety-like behavior. Mice were fed standard laboratory diet, or laboratory diet containing the human milk oligosaccharides 3′Sialyllactose (3′SL) or 6′Sialyllactose (6′SL) for 2 weeks prior to being exposed to either a social disruption stressor or a non-stressed control condition. Stressor exposure significantly changed the structure of the colonic mucosa-associated microbiota in control mice, as indicated by changes in beta diversity. The stressor resulted in anxiety-like behavior in both the light/dark preference and open field tests in control mice. This effect was associated with a reduction in immature neurons in the dentate gyrus as indicated by doublecortin (DCX) immunostaining. These effects were not evident in mice fed milk oligosaccharides; stressor exposure did not significantly change microbial community structure in mice fed 3′SL or 6′SL. In addition, 3′SL and 6′SL helped maintain normal behavior on tests of anxiety-like behavior and normal numbers of DCX+ immature neurons. These studies indicate that milk oligosaccharides support normal microbial communities and behavioral responses during stressor exposure, potentially through effects on the gut microbiota–brain axis.     

The effect of a high cholesterol diet on 20,25-diazacholesterol-induced myotonia.

Myotonia induced by 20,25-diazacholesterol may be related to the accumulation of desmosterol which results when 20,25-diazacholesterol inhibits Δ²⁴ reductase; the increased (Na⁺ + K⁺)-ATPase observed in this type of myotonia may also be involved. Rats were fed a high cholesterol diet (which inhibited cholesterol and hence desmosterol biosynthesis) and were dosed daily with 20,25-diazacholesterol or water. Corresponding groups were fed a normal diet and dosed with 20,25-diazacholesterol or water. Electromyography, plasma and muscle sterols, and erythrocyte membrane ATPase were measured serially for each group. Drug-treated rats on the normal diet became myotonic within 3 weeks; their plasma and muscle desmosterol were 84 and 69% of the total sterols, respectively. Their (Na⁺ + K⁺)-ATPase was not significantly increased until 8 weeks of treatment. In contrast, drug-treated rats on the high cholesterol diet never became myotonic; the plasma and muscle desmosterol never exceeded 15%, and the (Na⁺ + K⁺)-ATPase never exceeded the control level. Changing the high cholesterol diet to normal after 100 days resulted in myotonia within 10 days in the drug-treated rats; while their plasma and muscle desmosterol increased rapidly, no increased ATPase was seen. Reinstituting the high cholesterol diet reversed these sterol changes and abolished myotonia in 2 weeks. We conclude that 20,25-diazacholesterol-induced myotonia can be prevented or reversed by a high cholesterol diet, that the time course of desmosterol accumulation is similar and may be related to the appearance and degree of myotonia, and that the increased (Na⁺ + K⁺)-ATPase is probably a secondary effect.