Analysis of brain and blood single-cell transcriptomics in acute and subacute phases after experimental stroke.

Cerebral ischemia triggers a powerful inflammatory reaction involving peripheral leukocytes and brain resident cells that contribute to both tissue injury and repair. However, their dynamics and diversity remain poorly understood. To address these limitations, we performed a single-cell transcriptomic study of brain and blood cells 2 or 14 days after ischemic stroke in mice. We observed a strong divergence of post-ischemic microglia, monocyte-derived macrophages and neutrophils over time, while endothelial cells and brain-associated macrophages showed altered transcriptomic signatures at 2 days poststroke. Trajectory inference predicted the in situ trans-differentiation of macrophages from blood monocytes into day 2 and day 14 phenotypes, while neutrophils were projected to be continuously de novo recruited from the blood. Brain single-cell transcriptomes from both female and male aged mice were similar to that of young male mice, but aged and young brains differed in their immune cell composition. Although blood leukocyte analysis also revealed altered transcriptomes after stroke, brain-infiltrating leukocytes displayed higher transcriptomic divergence than their circulating counterparts, indicating that phenotypic diversification occurs within the brain in the early and recovery phases of ischemic stroke. A portal ( https://anratherlab.shinyapps.io/strokevis/ ) is provided to allow user-friendly access to our data.

Meningeal interleukin-17-producing T cells mediate cognitive impairment in a mouse model of salt-sensitive hypertension.

Hypertension (HTN), a disease afflicting over one billion individuals worldwide, is a leading cause of cognitive impairment, the mechanisms of which remain poorly understood. In the present study, in a mouse model of HTN, we find that the neurovascular and cognitive dysfunction depends on interleukin (IL)-17, a cytokine elevated in individuals with HTN. However, neither circulating IL-17 nor brain angiotensin signaling can account for the dysfunction. Rather, IL-17 produced by T cells in the dura mater is the mediator released in the cerebrospinal fluid and activating IL-17 receptors on border-associated macrophages (BAMs). Accordingly, depleting BAMs, deleting IL-17 receptor A in brain macrophages or suppressing meningeal T cells rescues cognitive function without attenuating blood pressure elevation, circulating IL-17 or brain angiotensin signaling. Our data unveil a critical role of meningeal T cells and macrophage IL-17 signaling in the neurovascular and cognitive dysfunction in a mouse model of HTN.

Brain and blood single-cell transcriptomics in acute and subacute phases after experimental stroke.

Cerebral ischemia triggers a powerful inflammatory reaction involving both peripheral leukocytes and brain resident cells. Recent evidence indicates that their differentiation into a variety of functional phenotypes contributes to both tissue injury and repair. However, the temporal dynamics and diversity of post-stroke immune cell subsets remain poorly understood. To address these limitations, we performed a longitudinal single-cell transcriptomic study of both brain and mouse blood to obtain a composite picture of brain-infiltrating leukocytes, circulating leukocytes, microglia and endothelium diversity over the ischemic/reperfusion time. Brain cells and blood leukocytes isolated from mice 2 or 14 days after transient middle cerebral artery occlusion or sham surgery were purified by FACS sorting and processed for droplet-based single-cell transcriptomics. The analysis revealed a strong divergence of post-ischemic microglia, macrophages, and neutrophils over time, while such diversity was less evident in dendritic cells, B, T and NK cells. Conversely, brain endothelial cells and brain associated-macrophages showed altered transcriptomic signatures at 2 days post-stroke, but low divergence from sham at day 14. Pseudotime trajectory inference predicted the in-situ longitudinal progression of monocyte-derived macrophages from their blood precursors into day 2 and day 14 phenotypes, while microglia phenotypes at these two time points were not connected. In contrast to monocyte-derived macrophages, neutrophils were predicted to be continuously de-novo recruited from the blood. Brain single-cell transcriptomics from both female and male aged mice did not show major changes in respect to young mice, but aged and young brains differed in their immune cell composition. Furthermore, blood leukocyte analysis also revealed altered transcriptomes after stroke. However, brain-infiltrating leukocytes displayed higher transcriptomic divergence than their circulating counterparts, indicating that phenotypic diversification into cellular subsets occurs within the brain in the early and the recovery phase of ischemic stroke. In addition, this resource report contains a searchable database https://anratherlab.shinyapps.io/strokevis/ to allow user-friendly access to our data. The StrokeVis tool constitutes a comprehensive gene expression atlas that can be interrogated at the gene and cell type level to explore the transcriptional changes of endothelial and immune cell subsets from mouse brain and blood after stroke.

Role of microglial and endothelial CD36 in post-ischemic inflammasome activation and interleukin-1ß-induced endothelial activation.

Cerebral ischemia is associated with an acute inflammatory response that contributes to the resulting injury. The innate immunity receptor CD36, expressed in microglia and endothelium, and the pro-inflammatory cytokine interleukin-1ß (IL-1ß) are involved in the mechanisms of ischemic injury. Since CD36 has been implicated in activation of the inflammasome, the main source of IL-1ß, we investigated whether CD36 mediates brain injury through the inflammasome and IL-1ß. We found that active caspase-1, a key inflammasome component, is decreased in microglia of CD36-deficient mice subjected to transient middle cerebral artery occlusion, an effect associated with a reduction in brain IL-1ß. Conditional deletion of CD36 either in microglia or endothelium reduced ischemic injury in mice, attesting to the pathogenic involvement of CD36 in both cell types. Application of an ischemic brain extract to primary brain endothelial cell cultures from wild type (WT) mice induced IL-1ß-dependent endothelial activation, reflected by increases in the cytokine colony stimulating factor-3, a response markedly attenuated in CD36-deficient endothelia. Similarly, the increase in colony stimulating factor-3 induced by recombinant IL-1ß was attenuated in CD36-deficient compared to WT endothelia. We conclude that microglial CD36 is a key determinant of post-ischemic IL-1ß production by regulating caspase-1 activity, whereas endothelial CD36 is required for the full expression of the endothelial activation induced by IL-1ß. The data identify microglial and endothelial CD36 as critical upstream components of the acute inflammatory response to cerebral ischemia and viable putative therapeutic targets.

Endothelium-Macrophage Crosstalk Mediates Blood-Brain Barrier Dysfunction in Hypertension.

Hypertension is a leading cause of stroke and dementia, effects attributed to disrupting delivery of blood flow to the brain. Hypertension also alters the blood-brain barrier (BBB), a critical component of brain health. Although endothelial cells are ultimately responsible for the BBB, the development and maintenance of the barrier properties depend on the interaction with other vascular-associated cells. However, it remains unclear if BBB disruption in hypertension requires cooperative interaction with other cells. Perivascular macrophages (PVM), innate immune cells closely associated with cerebral microvessels, have emerged as major contributors to neurovascular dysfunction. Using 2-photon microscopy in vivo and electron microscopy in a mouse model of Ang II (angiotensin II) hypertension, we found that the vascular segments most susceptible to increased BBB permeability are arterioles and venules >10 µm and not capillaries. Brain macrophage depletion with clodronate attenuates, but does not abolish, the increased BBB permeability in these arterioles where PVM are located. Deletion of AT1R (Ang II type-1 receptors) in PVM using bone marrow chimeras partially attenuated the BBB dysfunction through the free radical-producing enzyme Nox2. In contrast, downregulation of AT1R in cerebral endothelial cells using a viral gene transfer-based approach prevented the BBB disruption completely. The results indicate that while endothelial AT1R, mainly in arterioles and venules, initiate the BBB disruption in hypertension, PVM are required for the full expression of the dysfunction. The findings unveil a previously unappreciated contribution of resident brain macrophages to increased BBB permeability of hypertension and identify PVM as a putative therapeutic target in diseases associated with BBB dysfunction.

Distinct Commensal Bacterial Signature in the Gut Is Associated With Acute and Long-Term Protection From Ischemic Stroke.

Background and Purpose- Commensal gut bacteria have a profound impact on stroke pathophysiology. Here, we investigated whether modification of the microbiota influences acute and long-term outcome in mice subjected to stroke. Methods- C57BL/6 male mice received a cocktail of antibiotics or single antibiotic. After 4 weeks, fecal bacterial density of the 16S rRNA gene was quantitated by qPCR, and phylogenetic classification was obtained by 16S rRNA gene sequencing. Infarct volume and hemispheric volume loss were measured 3 days and 5 weeks after middle cerebral artery occlusion, respectively. Neurological deficits were tested by the Tape Test and the open field test. Results- Mice treated with a cocktail of antibiotics displayed a significant reduction of the infarct volume in the acute phase of stroke. The neuroprotective effect was abolished in mice recolonized with a wild-type microbiota. Single antibiotic treatment with either ampicillin or vancomycin, but not neomycin, was sufficient to reduce the infarct volume and improved motorsensory function 3 days after stroke. This neuroprotective effect was correlated with a specific microbial population rather than the total bacterial density. In particular, random forest analysis trained for the severity of the brain damage revealed that Bacteroidetes S24.7 and the enzymatic pathway for aromatic metabolism discriminate between large versus small infarct size. Additionally, the microbiota signature in the ampicillin-treated mice was associated with a reduced gut inflammation, long-term favorable outcome shown by an amelioration of the stereotypic behavior, and a reduction of brain tissue loss in comparison to control and was predictive of a regulation of short-chain fatty acids and tryptophan pathways. Conclusions- The findings highlight the importance of the intestinal microbiota in short- and long-term outcomes of ischemic stroke and raises the possibility that targeted modification of the microbiome associated with specific microbial enzymatic pathways may provide a preventive strategy in patients at high risk for stroke. Visual Overview- An online visual overview is available for this article.

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.

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.

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.

Spatio-temporal profile, phenotypic diversity, and fate of recruited monocytes into the post-ischemic brain.

BACKGROUND: A key feature of the inflammatory response after cerebral ischemia is the brain infiltration of blood monocytes. There are two main monocyte subsets in the mouse blood: CCR2+Ly6Chi "inflammatory" monocytes involved in acute inflammation, and CX3CR1+Ly6Clo "patrolling" monocytes, which may play a role in repair processes. We hypothesized that CCR2+Ly6Chi inflammatory monocytes are recruited in the early phase after ischemia and transdifferentiate into CX3CR1+Ly6Clo "repair" macrophages in the brain. METHODS: CX3CR1GFP/+CCR2RFP/+ bone marrow (BM) chimeric mice underwent transient middle cerebral artery occlusion (MCAo). Mice were sacrificed from 1 to 28 days later to phenotype and map subsets of infiltrating monocytes/macrophages (Mo/MΦ) in the brain over time. Flow cytometry analysis 3 and 14 days after MCAo in CCR2-/- mice, which exhibit deficient monocyte recruitment after inflammation, and NR4A1-/- BM chimeric mice, which lack circulating CX3CR1+Ly6Clo monocytes, was also performed. RESULTS: Brain mapping of CX3CR1GFP/+ and CCR2RFP/+ cells 3 days after MCAo showed absence of CX3CR1GFP/+ Mo/MΦ but accumulation of CCR2RFP/+ Mo/MΦ throughout the ischemic territory. On the other hand, CX3CR1+ cells accumulated 14 days after MCAo at the border of the infarct core where CCR2RFP/+ accrued. Whereas the amoeboid morphology of CCR2RFP/+ Mo/MΦ remained unchanged over time, CX3CR1GFP/+ cells exhibited three distinct phenotypes: amoeboid cells with retracted processes, ramified cells, and perivascular elongated cells. CX3CR1GFP/+ cells were positive for the Mo/MΦ marker Iba1 and phenotypically distinct from endothelial cells, smooth muscle cells, pericytes, neurons, astrocytes, or oligodendrocytes. Because accumulation of CX3CR1+Ly6Clo Mo/MΦ was absent in the brains of CCR2 deficient mice, which exhibit deficiency in CCR2+Ly6Chi Mo/MΦ recruitment, but not in NR4A1-/- chimeric mice, which lack of circulating CX3CR1+Ly6Clo monocytes, our data suggest a local transition of CCR2+Ly6Chi Mo/MΦ into CX3CR1+Ly6Clo Mo/MΦ phenotype. CONCLUSIONS: CX3CR1+Ly6Clo arise in the brain parenchyma from CCR2+Ly6Chi Mo/MΦ rather than being de novo recruited from the blood. These findings provide new insights into the trafficking and phenotypic diversity of monocyte subtypes in the post-ischemic brain.

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.

Endothelial CD36 Contributes to Postischemic Brain Injury by Promoting Neutrophil Activation via CSF3.

The scavenger receptor CD36 is a critical factor initiating ischemic brain injury, but the cell type(s) expressing CD36 and responsible for its harmful effects remain unknown. Using bone marrow (BM) chimeras subjected to transient middle cerebral artery occlusion, we found that CD36(-/-) mice transplanted with wild-type (WT) BM (WT→CD36(-/-)) have smaller infarcts (-67%), comparable with those of mice lacking CD36 both in brain and hematogenous cells (CD36(-/-) →CD36(-/-); - 72%). Conversely, WT mice receiving CD36(-/-) BM (CD36(-/-) →WT) have infarcts similar to WT→WT mice, suggesting that CD36 in the host brain (i.e., in microglia and endothelial cells), and not in hematogenous cells is involved in the damage. As anticipated, postischemic neutrophil infiltration in CD36(-/-) →CD36(-/-) mice was attenuated. Surprisingly, however, in WT→CD36(-/-) mice, in which infarcts were small, neutrophil infiltration was large and similar to that of CD36(-/-) →WT mice, in which infarcts were not reduced. Postischemic neutrophil free radical production was attenuated in WT→CD36(-/-) mice compared with CD36(-/-) →WT mice, whereas expression of the neutrophil activator colony-stimulating factor 3 (CSF3) was suppressed in CD36(-/-) cerebral endothelial cells, but not microglia. In CD36(-/-) cerebral endothelial cultures exposed to extracts from stroke brains, the upregulation of CSF3, but not neutrophil attractant chemokines, was suppressed. Intracerebroventricular administration of CSF3, 24 h after stroke, reconstituted neutrophil radical production and increased infarct volume in WT→CD36(-/-) mice. The findings identify endothelial cells as a key player in the deleterious effects of CD36 in stroke, and unveil a novel role of endothelial CD36 in enabling neutrophil neurotoxicity through CSF3. SIGNIFICANCE STATEMENT: Ischemic stroke is a leading cause of death and disability worldwide with limited therapeutic options. The inflammatory response initiated by cerebral ischemia-reperfusion contributes to ischemic brain injury and is a potential therapeutic target. Here we report that CD36, an innate immunity receptor involved in the initiation of postischemic inflammation, is a previously unrecognized regulator of neutrophil cytotoxicity. The effect is mediated by endothelial CD36 via upregulation of the neutrophil activator CSF3 in cerebral endothelial cells. Therefore, approaches to modulate cerebral endothelial CD36 signaling or to neutralize CSF3 may provide novel therapeutic opportunities to ameliorate postischemic inflammatory 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 mm(3); n = 13; mean ± SE) compared with autologous transplanted iNOS(-/-) mice (24 ± 3 mm(3); n = 10; p < 0.01), implicating blood-borne leukocytes in the damage. Furthermore, iNOS(+/+) mice transplanted with iNOS(-/-) BM had large infarcts (39 ± 6 mm(3); n = 13), similar to those of autologous transplanted iNOS(+/+) mice (39 ± 4 mm(3); 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.

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 Alzheimer's disease (AD) that often precedes the cognitive decline and clinical diagnosis. While the exact pathogenesis of AD remains unclear, accumulation of amyloid-ß (Aß) 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 Aß. Finally, the metabolic deficits worsened as Tg2576 mice aged and amyloid burden increased in the brain. These results indicate that excess Aß 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 Aß accumulation and identify hypothalamic leptin signaling as a previously unrecognized pathogenic site of action for Aß.

Site-specific phosphorylation of the p65 protein subunit mediates selective gene expression by differential NF-κB and RNA polymerase II promoter recruitment.

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.

Monoubiquitination of nuclear RelA negatively regulates NF-κB activity independent of proteasomal degradation.

Termination and resolution of inflammation are tightly linked to the inactivation of one of its strongest inducers, NF-κB. While canonical post-stimulus inactivation is achieved by upregulation of inhibitory molecules that relocate NF-κB complexes to the cytoplasm, termination of the NF-κB response can also be accomplished directly in the nucleus by posttranslational modifications, e.g., ubiquitination of the RelA subunit. Here we reveal a functional role for RelA monoubiquitination in regulating NF-κB activity. By employing serine-to-alanine mutants, we found that hypo-phosphorylated nuclear RelA is monoubiquitinated on multiple lysine residues. Ubiquitination was reversed by IκBα expression and was reduced when nuclear translocation was inhibited. RelA monoubiquitination decreased NF-κB transcriptional activity despite prolonged nuclear presence and independently of RelA degradation, possibly through decreased CREB-binding protein (CBP) co-activator binding. Polyubiquitin-triggered proteasomal degradation has been proposed as a model for RelA inactivation. However, here we show that proteasomal inhibition, similar to RelA hypo-phosphorylation, resulted in nuclear translocation and monoubiquitination of RelA. These findings indicate a degradation-independent mechanism for regulating the activity of nuclear RelA by ubiquitination.

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

Hypo-phosphorylation leads to nuclear retention of NF-kappaB p65 due to impaired IkappaBalpha gene synthesis.

Subcellular localization guided by IkappaBalpha is crucial for regulation of nuclear factor-kappaB function. Here, we show that p65 Rel homology domain phosphorylation mutants are transported into the nucleus after IkappaBalpha degradation, but as a consequence of lower IkappaBalpha levels their relocation to the cytosol is blocked. We demonstrate that phosphorylation of residues S205, S276, and S281 of p65 is not required for interaction between p65 and IkappaBalpha, but is pivotal for regulating cellular IkappaBalpha levels by positively affecting gene synthesis. Our findings indicate that reduction of phosphorylation leads to nuclear retention of p65, which might be partly responsible for altered transcriptional behavior of p65 serine mutants.

NF-kappaB 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-kappaB, which controls the expression of a large array of genes involved in immune function and cell survival. Here we show that NF-kappaB 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 gamma treatment and was inhibited in cells constitutively expressing IkappaBalpha. Furthermore, inducible reactive oxygen species production was inhibited in IkappaBalpha 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-kappaB and we identified two potential cis-acting elements in the murine gp91(phox) promoter that control NF-kappaB-dependent regulation. The findings raise the possibility of a positive feedback loop in which NF-kappaB 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.