Jamie Moore

Commensal microbiota affects ischemic stroke outcome by regulating intestinal γδ T cells

Commensal gut bacteria impact the host immune system and can influence disease processes in several organs, including the brain. However, it remains unclear whether the microbiota has an impact on the outcome of acute brain injury. Here we show that antibiotic-induced alterations in the intestinal flora reduce ischemic brain injury in mice, an effect transmissible by fecal transplants. Intestinal dysbiosis alters immune homeostasis in the small intestine, leading to an increase in regulatory T cells and a reduction in interleukin (IL)-17–positive γδ T cells through altered dendritic cell activity. Dysbiosis suppresses trafficking of effector T cells from the gut to the leptomeninges after stroke. Additionally, IL-10 and IL-17 are required for the neuroprotection afforded by intestinal dysbiosis. The findings reveal a previously unrecognized gut-brain axis and an impact of the intestinal flora and meningeal IL-17+ γδ T cells on ischemic injury.

Inducible nitric oxide synthase in neutrophils and endothelium contributes to ischemic brain injury in mice

NO produced by inducible NO synthase (iNOS) contributes to ischemic brain injury, but the cell types expressing iNOS and mediating tissue damage have not been elucidated. To examine the relative contribution of iNOS in resident brain cells and peripheral leukocytes infiltrating the ischemic brain, we used bone marrow (BM) chimeric mice in which the middle cerebral artery was occluded and infarct volume was determined 3 d later. iNOS−/− mice engrafted with iNOS+/+ BM exhibited larger infarcts (44 ± 2 mm3; n = 13; mean ± SE) compared with autologous transplanted iNOS−/− mice (24 ± 3 mm3; n = 10; p < 0.01), implicating blood-borne leukocytes in the damage. Furthermore, iNOS+/+ mice transplanted with iNOS−/− BM had large infarcts (39 ± 6 mm3; n = 13), similar to those of autologous transplanted iNOS+/+ mice (39 ± 4 mm3; n = 14), indicating the resident brain cells also play a role. Flow cytometry and cell sorting revealed that iNOS is highly expressed in neutrophils and endothelium but not microglia. Surprisingly, postischemic iNOS expression was enhanced in the endothelium of iNOS+/+ mice transplanted with iNOS−/− BM and in leukocytes of iNOS−/− mice with iNOS+/+ BM, suggesting that endothelial iNOS suppresses iNOS expression in leukocytes and vice versa. To provide independent evidence that neutrophils mediate brain injury, neutrophils were isolated and transferred to mice 24 h after stroke. Consistent with the result in chimeric mice, transfer of iNOS+/+, but not iNOS−/−, neutrophils into iNOS−/− mice increased infarct volume. The findings establish that iNOS in both neutrophils and endothelium mediates tissue damage and identify these cell types as putative therapeutic targets for stroke injury.

The key role of transient receptor potential melastatin-2 channels in amyloid-β-induced neurovascular dysfunction

Alzheimers dementia is a devastating and incurable disease afflicting over 35 million people worldwide. Amyloid-β (Aβ), a key pathogenic factor in this disease, has potent cerebrovascular effects that contribute to brain dysfunction underlying dementia by limiting the delivery of oxygen and glucose to the working brain. However, the downstream pathways responsible for the vascular alterations remain unclear. Here we report that the cerebrovascular dysfunction induced by Aβ is mediated by DNA damage caused by vascular oxidative-nitrosative stress in cerebral endothelial cells, which, in turn, activates the DNA repair enzyme poly(ADP)-ribose polymerase. The resulting increase in ADP ribose opens transient receptor potential melastatin-2 (TRPM2) channels in endothelial cells leading to intracellular Ca(2+) overload and endothelial dysfunction. The findings provide evidence for a previously unrecognized mechanism by which Aβ impairs neurovascular regulation and suggest that TRPM2 channels are a potential therapeutic target to counteract cerebrovascular dysfunction in Alzheimers dementia and related pathologies.

Immune mechanisms in cerebral ischemic tolerance

Stressor-induced tolerance is a central mechanism in the response of bacteria, plants, and animals to potentially harmful environmental challenges. This response is characterized by immediate changes in cellular metabolism and by the delayed transcriptional activation or inhibition of genetic programs that are not generally stressor specific (cross-tolerance). These programs are aimed at countering the deleterious effects of the stressor. While induction of this response (preconditioning) can be established at the cellular level, activation of systemic networks is essential for the protection to occur throughout the organs of the body. This is best signified by the phenomenon of remote ischemic preconditioning, whereby application of ischemic stress to one tissue or organ induces ischemic tolerance (IT) in remote organs through humoral, cellular and neural signaling. The immune system is an essential component in cerebral IT acting simultaneously both as mediator and target. This dichotomy is based on the fact that activation of inflammatory pathways is necessary to establish IT and that IT can be, in part, attributed to a subdued immune activation after index ischemia. Here we describe the components of the immune system required for induction of IT and review the mechanisms by which a reprogrammed immune response contributes to the neuroprotection observed after preconditioning. Learning how local and systemic immune factors participate in endogenous neuroprotection could lead to the development of new stroke therapies.

Circulating Endothelin-1 Alters Critical Mechanisms Regulating Cerebral Microcirculation

Endothelin-1 (ET1) is a potent vasoconstrictor peptide implicated in the cerebrovascular alterations occurring in stroke, subarachnoid hemorrhage, and brain trauma. Brain or circulating levels of ET1 are elevated in these conditions and in risk factors for cerebrovascular diseases. Most studies on the cerebrovascular effects of ET1 have focused on vascular smooth muscle constriction, and little is known about the effect of the peptide on cerebrovascular regulation. We tested the hypothesis that ET1 increases cerebrovascular risk by disrupting critical mechanisms regulating cerebral blood flow. Male C57Bl6/J mice equipped with a cranial window were infused intravenously with vehicle or ET1, and somatosensory cortex blood flow was assessed by laser Doppler flowmetry. ET1 infusion increased mean arterial pressure and attenuated the blood flow increase produced by neural activity (whisker stimulation) or neocortical application of the endothelium-dependent vasodilator acetylcholine but not A23187. The cerebrovascular effects of ET1 were abrogated by the ETA receptor antagonist BQ123 and were not related to vascular oxidative stress. Rather, the dysfunction was dependent on Rho-associated protein kinase activity. Furthermore, in vitro studies demonstrated that ET1 suppresses endothelial nitric oxide (NO) production, assessed by its metabolite nitrite, an effect associated with Rho-associated protein kinase–dependent changes in the phosphorylation state of endothelial NO synthase. Collectively, these novel observations demonstrate that increased ET1 plasma levels alter key regulatory mechanisms of the cerebral circulation by modulating endothelial NO synthase phosphorylation and NO production through Rho-associated protein kinase. The ET1-induced cerebrovascular dysfunction may increase cerebrovascular risk by lowering cerebrovascular reserves and increasing the vulnerability of the brain to cerebral ischemia.

Water deprivation induces neurovascular and cognitive dysfunction through vasopressin-induced oxidative stress.

Adequate hydration is essential for normal brain function and dehydration induces cognitive deterioration. In addition, dehydration has emerged as a stroke risk factor. However, it is unknown whether alterations in cerebrovascular regulation are responsible for these effects. To address this issue, C57BI/6 mice were water deprived for 24 or 48 hours and somatosensory cortex blood flow was assessed by laser-Doppler flowmetry in a cranial window. Dehydration increased plasma osmolality and vasopressin levels, and suppressed the increase in blood flow induced by neural activity, by the endothelium-dependent vasodilator acetylcholine and the smooth muscle relaxant adenosine. The cerebrovascular dysfunction was associated with oxidative stress and cognitive deficits, assessed using the Y maze. The vasopressin la receptor antagonist SR49059 improved the dehydration-induced oxidative stress and vasomotor dysfunction. Dehydration upregulated endothelin-1 in cerebral blood vessels, an effect blocked by SR49059. Furthermore, the endothelin A receptor antagonist BQ123 ameliorated cerebrovascular function. These findings show for the first time that dehydration alters critical mechanisms regulating the cerebral circulation through vasopressin and oxidative stress. The ensuing cerebrovascular dysregulation may alter cognitive function and increase the brains susceptibility to cerebral ischemia.

COX-1-derived PGE2 and PGE2 type 1 receptors are vital for angiotensin II-induced formation of reactive oxygen species and Ca2+ influx in the subfornical organ

Regulation of blood pressure by angiotensin II (ANG II) is a process that involves the reactive oxygen species (ROS) and calcium. We have shown that ANG-II type 1 receptor (AT1R) and prostaglandin E2 (PGE2) type 1 receptors (EP1R) are required in the subfornical organ (SFO) for ROS-mediated hypertension induced by slow-pressor ANG-II infusion. However, the signaling pathway associated with this process remains unclear. We sought to determine mechanisms underlying the ANG II-induced ROS and calcium influx in mouse SFO cells. Ultrastructural studies showed that cyclooxygenase 1 (COX-1) codistributes with AT1R in the SFO, indicating spatial proximity. Functional studies using SFO cells revealed that ANG II potentiated PGE2 release, an effect dependent on AT1R, phospholipase A2 (PLA2) and COX-1. Furthermore, both ANG II and PGE2 increased ROS formation. While the increase in ROS initiated by ANG II, but not PGE2, required the activation of the AT1R/PLA2/COX-1 pathway, both ANG II and PGE2 were dependent on EP1R and Nox2 as downstream effectors. Finally, ANG II potentiated voltage-gated L-type Ca(2+) currents in SFO neurons via the same signaling pathway required for PGE2 production. Blockade of EP1R and Nox2-derived ROS inhibited ANG II and PGE2-mediated Ca(2+) currents. We propose a mechanism whereby ANG II increases COX-1-derived PGE2 through the AT1R/PLA2 pathway, which promotes ROS production by EP1R/Nox2 signaling in the SFO. ANG II-induced ROS are coupled with Ca(2+) influx in SFO neurons, which may influence SFO-mediated sympathoexcitation. Our findings provide the first evidence of a spatial and functional framework that underlies ANG-II signaling in the SFO and reveal novel targets for antihypertensive therapies.

Heritable Variation in Courtship Patterns in Drosophila melanogaster

Little is known about the genetic basis of naturally occurring variation for sexually selected behavioral traits. Drosophila melanogaster, with its rich repertoire of courtship behavior and genomic and genetic resources, is an excellent model organism for addressing this question. We assayed a genetically diverse panel of lines with full genome sequences, the Drosophila Genetic Reference Panel, to assess the heritability of variation in courtship behavior and mating progression. We subsequently used these data to quantify natural variation in transition probabilities between courtship behaviors. We found heritable variation along the expected trajectory for courtship behaviors, including the tendency to initiate courtship and rate of progression through courtship, suggesting a genetic basis to male modulation of courtship behavior based on feedback from unrelated, outbred, and genetically identical females. We assessed the genetic basis of variation of the transition with the greatest heritability—from copulation to no engagement with the female—and identified variants in Serrate and Furin 1 as well as many other polymorphisms on the chromosome 3R associated with this transition. Our findings suggest that courtship is a highly dynamic behavior with both social and genetic inputs, and that males may play an important role in courtship initiation and duration.

Endogenous Protection from Ischemic Brain Injury by Preconditioned Monocytes

Exposure to low-dose lipopolysaccharide (LPS) before cerebral ischemia is neuroprotective in stroke models, a phenomenon termed preconditioning (PC). Although it is well established that LPS-PC induces central and peripheral immune responses, the cellular mechanisms modulating ischemic injury remain unclear. Here, we investigated the role of immune cells in the brain protection afforded by PC and tested whether monocytes may be reprogrammed by ex vivo LPS exposure, thus modulating inflammatory injury after cerebral ischemia in male mice. We found that systemic injection of low-dose LPS induces a Ly6Chi monocyte response that protects the brain after transient middle cerebral artery occlusion (MCAO) in mice. Remarkably, adoptive transfer of monocytes isolated from preconditioned mice into naive mice 7 h after transient MCAO reduced brain injury. Gene expression and functional studies showed that IL-10, inducible nitric oxide synthase, and CCR2 in monocytes are essential for neuroprotection. This protective activity was elicited even if mouse or human monocytes were exposed ex vivo to LPS and then injected into male mice after stroke. Cell-tracking studies showed that protective monocytes are mobilized from the spleen and reach the brain and meninges, where they suppress postischemic inflammation and neutrophil influx into the brain parenchyma. Our findings unveil a previously unrecognized subpopulation of splenic monocytes capable of protecting the brain with an extended therapeutic window and provide the rationale for cell therapies based on the delivery of autologous or allogeneic protective monocytes in patients after ischemic stroke. SIGNIFICANCE STATEMENT Inflammation is a key component of the pathophysiology of the brain in stroke, a leading cause of death and disability with limited therapeutic options. Here, we investigate endogenous mechanisms of protection against cerebral ischemia. Using lipopolysaccharide (LPS) preconditioning (PC) as an approach to induce ischemic tolerance in mice, we found generation of neuroprotective monocytes within the spleen, from which they traffic to the brain and meninges, suppressing postischemic inflammation. Importantly, systemic LPS-PC can be mimicked by adoptive transfer of in vitro-preconditioned mouse or human monocytes at translational relevant time points after stroke. This model of neuroprotection may facilitate clinical efforts to increase the efficacy of BM mononuclear cell treatments in acute neurological diseases such as cerebral ischemia.