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 reduces 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 IL-17+ γδ T cells, through altered dendritic cell activity. Dysbiosis suppresses trafficking of effector T cells from the gut to the leptomeninges after stroke. Interleukin-10 (IL-10) and IL-17 are required for the neuroprotection afforded by intestinal dysbiosis. The findings reveal a previously unrecognized gut-brain axis and the impact of the intestinal flora and meningeal IL-17+ γδ T cells on ischemic injury.

In this study we investigated the contribution of PVMs to the neurovascular and cognitive dysfunction induced by hypertension. We found that depletion of PVMs in models of chronic hypertension suppresses vascular oxidative stress and ameliorates the attendant impairment in neurovascular coupling and endothelium-dependent responses. Studies in bone marrow (BM) chimeras provided evidence that the dysfunction is mediated by ANGII acting on PVM AT1Rs resulting in NOX2-dependent ROS production. Importantly, concomitant to the neurovascular improvement, PVM depletion also rescued cognitive dysfunction. The findings unveil a previously unrecognized role of PVMs in the neurovascular and cognitive dysfunction induced by hypertension, and identify PVMs as a novel pathogenic component of the NVU of critical importance for brain health.

A diet rich in salt is linked to an increased risk of cerebrovascular diseases and dementia, but it remains unclear how dietary salt harms the brain. We report that, in mice, excess dietary salt suppresses resting cerebral blood flow and endothelial function, leading to cognitive impairment. The effect depends on expansion of TH17 cells in the small intestine, resulting in a marked increase in plasma interleukin-17 (IL-17). Circulating IL-17, in turn, promotes endothelial dysfunction and cognitive impairment by the Rho kinase-dependent inhibitory phosphorylation of endothelial nitric oxide synthase and reduced nitric oxide production in cerebral endothelial cells. The findings reveal a new gut-brain axis linking dietary habits to cognitive impairment through a gut-initiated adaptive immune response compromising brain function via circulating IL-17. Thus, the TH17 cell-IL-17 pathway is a putative target to counter the deleterious brain effects induced by dietary salt and other diseases associated with TH17 polarization.

The superoxide-generating phagocytic NADPH oxidase is an important component of the innate immune response against microbial agents, and is involved in shaping the cellular response to a variety of physiological and pathological signals. One of the downstream targets of NADPH oxidase-derived radicals is the ubiquitous transcription factor NF-B, which controls the expression of a large array of genes involved in immune function and cell survival. Here we show that NF-B itself is a key factor in controlling NADPH oxidase expression and function. In monocytic and microglial cell lines, the expression of the NADPH oxidase subunit gp91 phox was induced by lipopolysaccharide/interferon ␥ treatment and was inhibited in cells constitutively expressing IB␣. Furthermore, inducible reactive oxygen species production was inhibited in IB␣ overexpressing cells. gp91 phox expression was very low in RelA ؊/؊ fibroblasts and could be induced by reconstituting these cells with p65/RelA. Thus, gp91 phox expression is dependent on the presence of p65/RelA. We also found that gp91 phox transcription is dependent on NF-B and we identified two potential cis-acting elements in the murine gp91 phox promoter that control NF-B-dependent regulation. The findings raise the possibility of a positive feedback loop in which NF-B activation by oxidative stress leads to further radical production via NADPH oxidase.The superoxide-generating phagocytic NADPH oxidase is an essential component of the antimicrobial host defense system and is therefore highly expressed in granulocytes and macrophages. It catalyzes the reduction of molecular oxygen to superoxide (O 2 . ) by using NADPH as an electron donor. Superoxide is a ROS 2 intermediate, which is the starting material for generation of a variety of reactive oxidants including hydrogen peroxide, hydroxyl radical, singlet oxygen, reactive halogens, and peroxynitrite. Because of the destructive action of most of these radicals, NADPH oxidase activity must be tightly regulated. NADPH oxidase is composed of five subunits, p40phox (phox for phagocytic oxidase), p47 phox , p67 phox , p22 phox , and gp91 phox . The latter two are membrane associated and together constitute the flavocytochrome b 558 , whereas the other components are located in the cytoplasm of resting cells. Upon activation, the cytoplasmic components translocate to the cell membrane where they bind to flavocytochrome b 558 , thus forming the active NADPH oxidase (reviewed in Ref. 1). In addition to the five classical subunits, the small GTPases Rac1 (2) and Rac2 (3) have emerged as important functional units that are integrated in the membrane bound enzymatic complex and are indispensable for NADPH oxidase activity. More recent evidence indicates that NADPH oxidase is also present in non-myeloid cells, such as myocytes (4), fibroblasts (5, 6), as well as endothelial (7,8) and neuronal cells (9). However, in these cells gp91 phox (Nox2) might not constitute the dominant catalytic subunit, as Nox1 is the major form expressed in vascular ...

The class B scavenger receptor CD36 is involved in the cytotoxicity associated with inflammation, but its role in the inflammatory reaction that accompanies cerebral ischemia has not been examined. In this study, we investigated whether CD36 contributes to the brain damage produced by cerebral ischemia. The middle cerebral artery was transiently occluded in wild-type mice and in mice deficient in CD36. In wild-type mice, CD36 protein expression was increased in the ischemic brain, such that it was located predominantly in cells expressing the microglia/macrophage marker CD11b. The infarct produced by middle cerebral artery occlusion was 49% smaller in CD36-null mice than in wild-type controls, an effect associated with improved neurological function. The attenuation in brain injury in CD36 nulls could not be attributed to differences in cerebral blood flow during ischemia-reperfusion. However, the increase in reactive oxygen species (ROS) produced by cerebral ischemia was markedly attenuated in CD36-null mice in the early stage after reperfusion. The data unveil a previously unrecognized role of CD36 in ischemia-induced ROS production and brain injury. Modulation of CD36 signaling may provide a new strategy for the treatment of ischemic stroke.

Phosphorylation of nuclear factor-B (NF-B) subunits emerges as a mechanism by which transcriptional activity of nuclear NF-B complexes is regulated in an inhibitor B-independent fashion. As the main transactivator, the p65 subunit of NF-B has an outstanding position in the hierarchy of NF-B proteins. p65 is a multiply phosphorylated protein with phosphorylation sites in the C-terminal transactivation domain and the N-terminal Rel homology domain (RHD). In this study, we describe two previously non-reported phospho-acceptor sites within the p65 RHD. We show that differential phosphorylation of serine residues within the RHD modulates transcriptional activity in a cis-acting element and promoter-specific context, thus leading to a phosphorylation state-dependent gene expression profile. RelA ؊/؊ mouse embryonic fibroblasts reconstituted with wild-type p65 or p65 phosphorylation-deficient mutants showed a distinctive expression profile of synthetic B-dependent reporters as well as endogenous genes. Hypophosphorylated p65 did not display cis-acting element-specific changes in DNA binding or dimerization behavior. This study shows for the first time that site-specific phosphorylation can target a transcription factor to a particular subset of genes.Protein phosphorylation is used in many different ways to control the activity of transcription factors. It directs subcellular localization (e.g. nuclear factor of activated cells (1)), selectively controls binding of dimerization partners (e.g. signal transducers and activators of transcription (2)), or alters transcriptional activity by facilitating the interaction with compo-

Weight loss is a prominent early feature of Alzheimer's disease (AD) that often precedes the cognitive decline and clinical diagnosis. While the exact pathogenesis of AD remains unclear, accumulation of amyloid-␤ (A␤) derived from the amyloid precursor protein (APP) in the brain is thought to lead to the neuronal dysfunction and death underlying the dementia. In this study, we examined whether transgenic mice overexpressing the Swedish mutation of APP (Tg2576), recapitulating selected features of AD, have hypothalamic leptin signaling dysfunction leading to early body weight deficits. We found that 3-month-old Tg2576 mice, before amyloid plaque formation, exhibit decreased weight with markedly decreased adiposity, low plasma leptin levels, and increased energy expenditure without alterations in feeding behavior. The expression of the orexigenic neuropeptide Y (NPY) in the hypothalamus to the low leptin state was abnormal at basal and fasting conditions. In addition, arcuate NPY neurons exhibited abnormal electrophysiological responses to leptin in Tg2576 hypothalamic slices or wild-type slices treated with A␤. Finally, the metabolic deficits worsened as Tg2576 mice aged and amyloid burden increased in the brain. These results indicate that excess A␤ can potentially disrupt hypothalamic arcuate NPY neurons leading to weight loss and a pathologically low leptin state early in the disease process that progressively worsens as the amyloid burden increases. Collectively, these findings suggest that weight loss is an intrinsic pathological feature of A␤ accumulation and identify hypothalamic leptin signaling as a previously unrecognized pathogenic site of action for A␤.

CD36, a class-B scavenger receptor involved in multiple functions, including inflammatory signaling, may also contribute to ischemic brain injury through yet unidentified mechanisms. We investigated whether CD36 participates in the molecular events underlying the inflammatory reaction that accompanies cerebral ischemia and may contribute to the tissue damage. We found that activation of nuclear factor-B, a transcription factor that coordinates postischemic gene expression, is attenuated in CD36-null mice subjected to middle cerebral artery occlusion. The infiltration of neutrophils and the glial reaction induced by cerebral ischemia were suppressed. Treatment with an inhibitor of inducible nitric oxide synthase, an enzyme that contributes to the tissue damage, reduced ischemic brain injury in wild-type mice, but not in CD36 nulls. In contrast to cerebral ischemia, the molecular and cellular inflammatory changes induced by intracerebroventricular injection of interleukin-1␤ were not attenuated in CD36-null mice. The findings unveil a novel role of CD36 in early molecular events leading to nuclear factor-B activation and postischemic inflammation. Inhibition of CD36 signaling may be a valuable therapeutic approach to counteract the deleterious effects of postischemic inflammation.