Gianfranco Racchumi

NF-κB Regulates Phagocytic NADPH Oxidase by Inducing the Expression of gp91phox

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-κB, which controls the expression of a large array of genes involved in immune function and cell survival. Here we show that NF-κB 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 gp91phox was induced by lipopolysaccharide/interferon γ treatment and was inhibited in cells constitutively expressing IκBα. Furthermore, inducible reactive oxygen species production was inhibited in IκBα overexpressing cells. gp91phox expression was very low in RelA-/- fibroblasts and could be induced by reconstituting these cells with p65/RelA. Thus, gp91phox expression is dependent on the presence of p65/RelA. We also found that gp91phox transcription is dependent on NF-κB and we identified two potential cis-acting elements in the murine gp91phox promoter that control NF-κB-dependent regulation. The findings raise the possibility of a positive feedback loop in which NF-κB activation by oxidative stress leads to further radical production via NADPH oxidase.

The Class B Scavenger Receptor CD36 Mediates Free Radical Production and Tissue Injury in Cerebral Ischemia

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.

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.

Nuclear Factor-κB Activation and Postischemic Inflammation Are Suppressed in CD36-Null Mice after Middle Cerebral Artery Occlusion

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-κB, 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-1β were not attenuated in CD36-null mice. The findings unveil a novel role of CD36 in early molecular events leading to nuclear factor-κB activation and postischemic inflammation. Inhibition of CD36 signaling may be a valuable therapeutic approach to counteract the deleterious effects of postischemic inflammation.

Interaction Between Inducible Nitric Oxide Synthase and Poly(ADP-ribose) Polymerase in Focal Ischemic Brain Injury

Background and Purpose— Overactivation of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP) contributes to ischemic brain injury. Because PARP upregulates proinflammatory genes, we investigated whether inducible nitric oxide synthase (iNOS), a gene involved in the deleterious effects of postischemic inflammation, participates in the mechanisms by which PARP activation contributes to cerebral ischemic injury. Methods— The middle cerebral artery (MCA) was occluded in mice for 20 minutes using an intravascular filament, and injury volume was measured 72 hours later in Nissl-stained brain sections. mRNA expression was assessed in the postischemic brain by the quantitative “real-time” polymerase chain reaction. Results— The PARP inhibitor PJ34 reduced infarct volume and attenuated postischemic iNOS mRNA upregulation by 72%. To determine whether iNOS contributes to the toxicity of PARP, the iNOS inhibitor aminoguanidine was co-administered with PARP inhibitors. Unexpectedly, co-administration of PARP and iNOS inhibitors, or treatment of iNOS-null mice with PARP inhibitors, abrogated the protective effect afforded by iNOS or PARP inhibition alone. The loss of neuroprotection was associated with upregulation of the inflammatory genes iNOS, intercellular adhesion molecule-1, and gp91phox. Conclusions— The results suggest that iNOS expression contributes to the deleterious effects exerted by PARP activation in cerebral ischemia. However, iNOS activity is required for the protective effect of PARP inhibition and, conversely, PARP activity must be present for iNOS inhibition to be effective. The findings unveil a previously unrecognized deleterious interaction between iNOS and PARP that is relevant to the development of combination therapies for ischemic stroke.

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.

Perivascular macrophages mediate the neurovascular and cognitive dysfunction associated with hypertension

Hypertension is a leading risk factor for dementia, but the mechanisms underlying its damaging effects on the brain are poorly understood. Due to a lack of energy reserves, the brain relies on continuous delivery of blood flow to its active regions in accordance with their dynamic metabolic needs. Hypertension disrupts these vital regulatory mechanisms, leading to the neuronal dysfunction and damage underlying cognitive impairment. Elucidating the cellular bases of these impairments is essential for developing new therapies. Perivascular macrophages (PVMs) represent a distinct population of resident brain macrophages that serves key homeostatic roles but also has the potential to generate large amounts of reactive oxygen species (ROS). Here, we report that PVMs are critical in driving the alterations in neurovascular regulation and attendant cognitive impairment in mouse models of hypertension. This effect was mediated by an increase in blood-brain barrier permeability that allowed angiotensin II to enter the perivascular space and activate angiotensin type 1 receptors in PVMs, leading to production of ROS through the superoxide-producing enzyme NOX2. These findings unveil a pathogenic role of PVMs in the neurovascular and cognitive dysfunction associated with hypertension and identify these cells as a putative therapeutic target for diseases associated with cerebrovascular oxidative stress.

Site-specific Phosphorylation of the p65 Protein Subunit Mediates Selective Gene Expression by Differential NF-κB and RNA Polymerase II Promoter Recruitment

Background: Phosphorylation of nuclear factor-κB (NF-κB) subunits is critical for NF-κB activity. Results: Mutation of phospho-acceptor sites within the p65 Rel homology domain influences NF-κB activity in a gene-dependent manner by altering p65 and RNA polymerase II promoter recruitment. Conclusion: Differential p65 phosphorylation serves as a code to target NF-κB transcriptional activity to distinct gene subsets. Significance: Our data provide insight into how NF-κB transcriptional specificity is achieved. Phosphorylation of NF-κB plays an important role in modulating transcriptional activity of NF-κB independently of inhibitor of κB (IκB) proteins. For the p65 subunit, multiple phosphorylation sites have been mapped in and adjacent to both the N-terminal Rel homology domain and the C-terminal transactivation domain. Their impact on NF-κB-dependent transcription, however, has never been assessed at a broader level. In this study, we evaluate the importance of differential p65 phosphorylation on four serine acceptor sites in the Rel homology domain for the expression of an array of NF-κB-dependent genes in endothelial cells. We find that inhibition of p65 phosphorylation on these serine residues targets NF-κB activity to distinctive gene subsets in a κB enhancer element-specific context. We show that the phosphorylation-dependent alterations in gene and protein expression are reflective of the amount of p65 and phosphorylated RNA polymerase II (p-RNAP II) bound to respective gene promoter regions. Depending on the gene subset, impaired gene expression was either a result of decreased p65 promoter recruitment or of a failure of bound p65 to recruit p-RNAP II. In conclusion, our findings demonstrate that site-specific p65 phosphorylation targets NF-κB activity to particular gene subsets on a global level by influencing p65 and p-RNAP II promoter recruitment.

Transgenic Mice Overexpressing Amyloid Precursor Protein Exhibit Early Metabolic Deficits and a Pathologically Low Leptin State Associated with Hypothalamic Dysfunction in Arcuate Neuropeptide Y Neurons

Weight loss is a prominent early feature of Alzheimers disease (AD) that often precedes the cognitive decline and clinical diagnosis. While the exact pathogenesis of AD remains unclear, accumulation of amyloid-β (Aβ) 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 Aβ. Finally, the metabolic deficits worsened as Tg2576 mice aged and amyloid burden increased in the brain. These results indicate that excess Aβ 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 Aβ accumulation and identify hypothalamic leptin signaling as a previously unrecognized pathogenic site of action for Aβ.

Dietary salt promotes neurovascular and cognitive dysfunction through a gut-initiated TH17 response

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.A salt-rich diet promotes cerebrovascular diseases and dementia. This study shows that high dietary salt in mice induces a TH17 response in the gut leading to cerebral endothelial dysfunction and cognitive impairment via circulating IL-17.