Adam Denes

Inflammation and brain injury: Acute cerebral ischaemia, peripheral and central inflammation

Inflammation is a classical host defence response to infection and injury that has many beneficial effects. However, inappropriate (in time, place and magnitude) inflammation is increasingly implicated in diverse disease states, now including cancer, diabetes, obesity, atherosclerosis, heart disease and, most relevant here, CNS disease. A growing literature shows strong correlations between inflammatory status and the risk of cerebral ischaemia (CI, most commonly stroke), as well as with outcome from an ischaemic event. Intervention studies to demonstrate a causal link between inflammation and CI (or its consequences) are limited but are beginning to emerge, while experimental studies of CI have provided direct evidence that key inflammatory mediators (cytokines, chemokines and inflammatory cells) contribute directly to ischaemic brain injury. However, it remains to be determined what the relative importance of systemic (largely peripheral) versus CNS inflammation is in CI. Animal models in which CI is driven by a CNS intervention may not accurately reflect the clinical condition; stroke being typically induced by atherosclerosis or cardiac dysfunction, and hence current experimental paradigms may underestimate the contribution of peripheral inflammation. Experimental studies have already identified a number of potential anti-inflammatory therapeutic interventions that may limit ischaemic brain damage, some of which have been tested in early clinical trials with potentially promising results. However, a greater understanding of the contribution of inflammation to CI is still required, and this review highlights some of the key mechanism that may offer future therapeutic targets.

Proliferating resident microglia after focal cerebral ischaemia in mice.

Cerebral ischaemia usually results in the rapid death of neurons within the immediate territory of the affected artery. Neuronal loss is accompanied by a sequence of events, including brain oedema, blood-brain barrier (BBB) breakdown, and neuroinflammation, all of which contribute to further neuronal death. Although the role of macrophages and mononuclear phagocytes in the expansion of ischaemic injury has been widely studied, the relative contribution of these cells, either of exogenous or intrinsic central nervous system (CNS) origin is still not entirely clear. The purpose of this study, therefore, was to use different durations of transient middle cerebral artery occlusion (tMCAo) in the mouse to investigate fully post-occlusion BBB permeability and cellular changes in the brain during the 72 h post-MCAo period. This was achieved using in vivo magnetic resonance imaging (MRI) and cell labelling techniques. Our results show that BBB breakdown and formation of the primary ischaemic damage after tMCAo is not associated with significant infiltration of neutrophils, although more are observed with longer periods of MCAo. In addition, we observe very few infiltrating exogenous macrophages over a 72 h period after 30 or 60 mins of occlusion, instead a profound increase in proliferating resident microglia cells was observed. Interestingly, the more severe injury associated with 60 mins of MCAo leads to a markedly reduced proliferation of resident microglial cells, suggesting that these cells may play a protective function, possibly through phagocytosis of infiltrating neutrophils. These data further support possible beneficial actions of microglial cells in the injured brain.

Role of CX3CR1 (Fractalkine Receptor) in Brain Damage and Inflammation Induced by Focal Cerebral Ischemia in Mouse

CX3CR1 (fractalkine receptor) is important for sustaining normal microglial activity in the brain. Lack of CX3CR1 reportedly results in neurotoxic microglial phenotype in disease models. The objective of this study was to test the hypothesis that the absence of CX3CR1 worsens the outcome in cerebral ischemia. We observed significantly smaller (56%) infarcts and blood—brain barrier damage in CX3CR1-deficient (CX3CR1−/−) animals compared with CX3CR1 +/− and wild-type mice after transient occlusion of the middle cerebral artery (MCAo). Functional recovery of CX3CR1−/−animals was enhanced, while less number of apoptotic cells and infiltrating leukocytes were found in the ipsilateral hemisphere. Expression of IL-1β mRNA, protein, and interleukin (IL)-1Ra and tumor necrosis factor (TNF)-α mRNAs was lower in CX3CR1−/− mice, whereas no difference was observed in the number of IL-1β-expressing microglia or plasma IL-1β concentration. We observed early IL-1β expression in astrocytes in vivo after MCAo and after oxygen—glucose deprivation in vitro, which might contribute to the ischemic damage. Our findings indicate that lack of CX3CR1 does not result in microglial neurotoxicity after MCAo, but rather significantly reduces ischemic damage and inflammation. Reduced IL-1β and TNFα expression as well as decreased leukocyte infiltration might be involved in the development of smaller infarcts in CX3CR1−/− animals.

Caspase-1: Is IL-1 just the tip of the ICEberg?

Caspase-1, formerly known as interleukin (IL)-1-converting enzyme is best established as the protease responsible for the processing of the key pro-inflammatory cytokine IL-1β from an inactive precursor to an active, secreted molecule. Thus, caspase-1 is regarded as a key mediator of inflammatory processes, and has become synonymous with inflammation. In addition to the processing of IL-1β, caspase-1 also executes a rapid programme of cell death, termed pyroptosis, in macrophages in response to intracellular bacteria. Pyroptosis is also regarded as a host response to remove the niche of the bacteria and to hasten their demise. These processes are generally accepted as the main roles of caspase-1. However, there is also a wealth of literature supporting a direct role for caspase-1 in non-infectious cell death processes. This is true in mammals, but also in non-mammalian vertebrates where caspase-1-dependent processing of IL-1β is absent because of the lack of appropriate caspase-1 cleavage sites. This literature is most prevalent in the brain where caspase-1 may directly regulate neuronal cell death in response to diverse insults. We attempt here to summarise the evidence for caspase-1 as a cell death enzyme and propose that, in addition to the processing of IL-1β, caspase-1 has an important and a conserved role as a cell death protease.

Brain inflammation is induced by co-morbidities and risk factors for stroke

Highlights ► Risk factors for stroke include atherosclerosis, obesity, diabetes and hypertension. ► Stroke risk factors are associated with peripheral inflammation. ► Corpulent rats and atherogenic mice show increased inflammation in the brain. ► Pilot data show that patients at risk of stroke may also develop brain inflammation. ► Chronic peripheral inflammation can drive inflammatory changes in the brain.

Platelet interleukin-1alpha drives cerebrovascular inflammation.

White blood cell infiltration across an activated brain endothelium contributes to neurologic disease, including cerebral ischemia and multiple sclerosis. Identifying mechanisms of cerebrovascular activation is therefore critical to our understanding of brain disease. Platelet accumulation in microvessels of ischemic mouse brain was associated with endothelial activation in vivo. Mouse platelets expressed interleukin-1alpha (IL-1alpha), but not IL-1beta, induced endothelial cell adhesion molecule expression (ICAM-1 and VCAM-1), and enhanced the release of CXC chemokine CXCL1 when incubated with primary cultures of brain endothelial cells from wild-type or IL-1alpha/beta-deficient mice. A neutralizing antibody to IL-1alpha (but not IL-1beta) or application of IL-1 receptor antagonist inhibited platelet-induced endothelial activation by more than 90%. Platelets from IL-1alpha/beta-deficient mice did not induce expression of adhesion molecules in cerebrovascular endothelial cells and did not promote CXCL1 release in vitro. Conditioned medium from activated platelets induced an IL-1alpha-dependent activation of mouse brain endothelial cells and supported the transendothelial migration of neutrophils in vitro. Thus, we have identified platelets as a key source of IL-1alpha and propose that platelet activation of brain endothelium via IL-1alpha is a critical step for the entry of white blood cells, major contributors to inflammation-mediated injury in the brain.

NLRP3-Inflammasome Activating DAMPs Stimulate an Inflammatory Response in Glia in the Absence of Priming Which Contributes to Brain Inflammation after Injury

Inflammation in the absence of infection (sterile inflammation) contributes to acute injury and chronic disease. Cerebral ischemia is a devastating condition in which the primary injury is caused by reduced blood supply and is therefore sterile. The cytokine interleukin-1β (IL-1β) is a key contributor to ischemic brain injury and central inflammatory responses. The release of IL-1β is regulated by the protease caspase-1, and its activating complex, the inflammasome. Of the known inflammasomes the best characterized, and one that is perceived to sense sterile injury is formed by a pattern recognition receptor called NOD-like receptor pyrin domain containing three (NLRP3). A key feature of NLRP3-inflammasome dependent responses in vitro in macrophages is the requirement of an initial priming stimulus by a pathogen (PAMP), or damage associated molecular pattern (DAMP) respectively. We sought to determine the inflammatory responses of NLRP3-activating DAMPs on brain derived mixed glial cells in the absence of an initial priming stimulus in vitro. In cultured mouse mixed glia the DAMPs ATP, monosodium urate, and calcium pyrophosphate dehydrate crystals had no effect on the expression of IL-1α or IL-1β and induced release only when the cells were primed with a PAMP. In the absence of priming, these DAMPs did however induce inflammation via the production of IL-6 and CXCL1, and the release of the lysosomal protease cathepsin B. Furthermore, the acute phase protein serum amyloid A (SAA) acted as a priming stimulus on glial cells resulting in levels of IL-1 expression comparable to those induced by the PAMP lipopolysaccharide. In vivo, after cerebral ischemia, IL-1 production contributed to increased IL-6 and CXCL1 since these cytokines were profoundly reduced in the ischemic hemispheres from IL-1α/β double KO mice, although injury-induced cytokine responses were not abolished. Thus, DAMPs augment brain inflammation by directly stimulating production of glial derived inflammatory mediators. This is markedly enhanced by DAMP-induced IL-1-release-dependent responses that require a sterile endogenous priming stimulus such as SAA.

Microglia protect against brain injury and their selective elimination dysregulates neuronal network activity after stroke

Microglia are the main immune cells of the brain and contribute to common brain diseases. However, it is unclear how microglia influence neuronal activity and survival in the injured brain in vivo. Here we develop a precisely controlled model of brain injury induced by cerebral ischaemia combined with fast in vivo two-photon calcium imaging and selective microglial manipulation. We show that selective elimination of microglia leads to a striking, 60% increase in infarct size, which is reversed by microglial repopulation. Microglia-mediated protection includes reduction of excitotoxic injury, since an absence of microglia leads to dysregulated neuronal calcium responses, calcium overload and increased neuronal death. Furthermore, the incidence of spreading depolarization (SD) is markedly reduced in the absence of microglia. Thus, microglia are involved in changes in neuronal network activity and SD after brain injury in vivo that could have important implications for common brain diseases.

Systemic inflammatory challenges compromise survival after experimental stroke via augmenting brain inflammation, blood- brain barrier damage and brain oedema independently of infarct size

BackgroundSystemic inflammation impairs outcome in stroke patients and experimental animals via mechanisms which are poorly understood. Circulating inflammatory mediators can activate cerebrovascular endothelium or glial cells in the brain and impact on ischaemic brain injury. One of the most serious early clinical complications of cerebral ischaemia is brain oedema, which compromises survival in the first 24-48 h. It is not understood whether systemic inflammatory challenges impair outcome after stroke by increasing brain injury only or whether they have direct effects on brain oedema, cerebrovascular inflammation and blood-brain barrier damage.MethodsWe used two different systemic inflammatory stimuli, acute endotoxin treatment and anaphylaxis to study mechanisms of brain injury after middle cerebral artery occlusion (MCAo). Ischaemic brain injury, blood-brain barrier damage and oedema were analysed by histological techniques. Systemic cytokine responses and inflammatory changes in the brain were analysed by cytometric bead array, immunofluorescence, in situ hibridization and quantitative real-time PCR.ResultsSystemic inflammatory challenges profoundly impaired survival in the first 24 h after experimental stroke in mice, independently of an increase in infarct size. Systemic lipopolysaccharide (LPS) dose-dependently increased mortality (50-100%) minutes to hours after cerebral ischaemia. Acute anaphylactic challenge in ovalbumin-sensitised mice affected stroke more seriously when induced via intraperitoneal administration compared to intravenous. Both LPS and anaphylaxis induced inflammatory changes in the blood and in the brain prior to experimental stroke. Plasma cytokine levels were significantly higher after LPS, while increased IL-10 levels were seen after anaphylaxis. After MCAo, both LPS and anaphylaxis increased microglial interleukin-1α (IL-1α) expression and blood-brain barrier breakdown. LPS caused marked granulocyte recruitment throughout the ipsilateral hemisphere. To investigate whether reduction of ischaemic damage can improve outcome in systemic inflammation, controlled hypothermia was performed. Hypothermia reduced infarct size in all treatment groups and moderately improved survival, but failed to reduce excess oedema formation after anaphylaxis and LPS-induced neuroinflammation.ConclusionsOur results suggest that systemic inflammatory conditions induce cerebrovascular inflammation via diverse mechanisms. Increased brain inflammation, blood-brain barrier injury and brain oedema formation can be major contributors to impaired outcome in mice after experimental stroke with systemic inflammatory stimuli, independently of infarct size.

Chronic Systemic Infection Exacerbates Ischemic Brain Damage via a CCL5 (Regulated on Activation, Normal T-Cell Expressed and Secreted)-Mediated Proinflammatory Response in Mice

Infection and systemic inflammation are risk factors for cerebrovascular diseases and poststroke infections impair outcome in stroke patients, although the mechanisms of their contribution are mostly unknown. No preclinical studies have identified how chronic infection affects ischemic brain damage and which key inflammatory mediators are involved. We used a well established model of gut infection (Trichuris muris) to study how chronic infection contributes to brain injury. We show that, in mice, infection that leads to a chronic Th1-polarized immune response dramatically (60%) exacerbates brain damage caused by experimental stroke. Chronic Th1-type infection resulted in systemic upregulation of proinflammatory mediators and profoundly altered stroke-induced early (40 min to 4 h) and late (48 h) inflammation in the brain and peripheral tissues. Using the same infection, we show that a Th1-, but not Th2-polarized response augments brain injury by increasing the Th1 chemokine CCL5 [regulated on activation, normal T-cell expressed and secreted (RANTES)] systemically. This infection-associated response paralleled altered regulatory T-cell response, accelerated platelet aggregation in brain capillaries, and increased microvascular injury and matrix metalloproteinase activation after stroke. Antibody neutralization of RANTES reversed the effect of chronic infection on brain damage, microvascular MMP-9 activation, and cellular inflammatory response. Our results suggest that chronic infection exacerbates ischemic brain damage via a RANTES-mediated systemic inflammatory response, which leads to delayed resolution of inflammation and augmented microvascular injury in the brain.