The cytokine interleukin-1 (IL-1) has two main pro-inflammatory forms, IL-1? and IL-1?, which are central to host responses to infection and to damaging sterile inflammation. Processing of IL-1 precursor proteins to active cytokines commonly occurs through activation of proteases, notably caspases and calpains. These proteases are instrumental in cell death, and inflammation and cell death are closely associated, hence we sought to determine the impact of cell death pathways on IL-1 processing and release. We discovered that apoptotic regulation of caspase-8 specifically induced the processing and release of IL-1?. Conversely, necroptosis caused the processing and release of IL-1?, and this was independent of IL-1? processing and release. These data suggest that the mechanism through which an IL-1-expressing cell dies dictates the nature of the inflammatory mechanism that follows. These insights may allow modification of inflammation through the selective targeting of cell death mechanisms during disease.
The immune system is implicated in a wide range of disorders affecting the brain and is, therefore, an attractive target for therapy. Interleukin-1 (IL-1) is a potent regulator of the innate immune system important for host defence but is also associated with injury and disease in the brain. Here, we show that IL-1 is a key mediator driving an innate immune response to inflammatory challenge in the mouse brain but is dispensable in extracerebral tissues including the lung and peritoneum. We also demonstrate that IL-1? is an important ligand contributing to the CNS dependence on IL-1 and that IL-1 derived from the CNS compartment (most likely microglia) is the major source driving this effect. These data reveal previously unknown tissue-specific requirements for IL-1 in driving innate immunity and suggest that IL-1-mediated inflammation in the brain could be selectively targeted without compromising systemic innate immune responses that are important for resistance to infection. This property could be exploited to mitigate injury- and disease-associated inflammation in the brain without increasing susceptibility to systemic infection, an important complication in several neurological disorders. This article is protected by copyright. All rights reserved.
Cerebral ischemia is one of the most common causes of disabilities in adults and leads to long-term motor and cognitive impairments with limited therapeutic possibilities. Treatment options have proven efficient in preclinical models of cerebral ischemia but have failed in the clinical setting. This limited translation may be due to the suitability of models used and outcomes measured as most studies have focused on the early period after injury with gross motor scales, which have limited correlation to the clinical situation. The aim of this study was to determine long-term functional outcomes after cerebral ischemia in rats, focusing on fine motor function, social and depressive behavior as clinically relevant measures. A secondary objective was to evaluate the effects of an anti-inflammatory treatment (interleukin-1 receptor antagonist (IL-1Ra)) on functional recovery and compensation. Infarct volume was correlated with long-term (25 days) impairments in fine motor skills, but not with emotional components of behavior. Motor impairments could not be detected using conventional neurological tests and only detailed analysis allowed differentiation between recovery and compensation. Acute systemic administration of IL-1Ra (at reperfusion) led to a faster and more complete recovery, but delayed (24h) IL-1Ra treatment had no effect. In summary functional assessment after brain injury requires detailed motor tests in order to address long-term impairments and compensation processes that are mediated by intact tissues. Functional deficits in skilled movement after brain injury represent ideal predictors of long-term outcomes and should become standard measures in the assessment of preclinical animal models.
Bacterial infection contributes to diverse noninfectious diseases and worsens outcome after stroke. Streptococcus pneumoniae, the most common infection in patients at risk of stroke, is a major cause of prolonged hospitalization and death of stroke patients, but how infection impacts clinical outcome is not known.
Acute brain injury results in peripheral inflammatory changes, although the impact of these processes on neuronal death and neuroinflammation is currently unclear. To facilitate the translation of experimental studies to clinical benefit, it is vital to characterize the mechanisms by which acute brain injury induces peripheral inflammatory changes, and how these are affected by surgical manipulation in experimental models. Here we show that in mice, even mild surgical manipulation of extracranial tissues induced marked granulocyte mobilization (300%) and systemic induction of cytokines. However, intracranial changes induced by craniotomy, or subsequent induction of focal cerebral ischemia were required to induce egress of CXCR2-positive granulocytes from the bone marrow. CXCR2 blockade resulted in reduced mobilization of granulocytes from the bone marrow, caused an unexpected increase in circulating granulocytes, but failed to affect brain injury induced by cerebral ischemia. We also demonstrate that isoflurane anaesthesia interferes with circulating leukocyte responses, which could contribute to the reported vascular and neuroprotective effects of isoflurane. In addition, no immunosuppression develops in the bone marrow after experimental stroke. Thus, experimental models of cerebral ischemia are compromised by surgery and anaesthesia in proportion to the severity of surgical intervention and overall tissue injury. Understanding the inherent confounding effects of surgical manipulation and development of new models of cerebral ischemia with minimal surgical intervention could facilitate better understanding of interactions between inflammation and brain injury.
Interleukin-1 (IL-1) is a key mediator of ischaemic brain injury induced by stroke and subarachnoid haemorrhage (SAH). IL-1 receptor antagonist (IL-1Ra) limits brain injury in experimental stroke and reduces plasma inflammatory mediators associated with poor outcome in ischaemic stroke patients. Intravenous (IV) IL-1Ra crosses the blood-brain barrier (BBB) in patients with SAH, to achieve cerebrospinal fluid (CSF) concentrations that are neuroprotective in rats.
Acute-phase proteins (APPs) are key effectors of the immune response and are routinely used as biomarkers in cerebrovascular diseases, but their role during brain inflammation remains largely unknown. Elevated circulating levels of the acute-phase protein pentraxin-3 (PTX3) are associated with worse outcome in stroke patients. Here we show that PTX3 is expressed in neurons and glia in response to cerebral ischemia, and that the proinflammatory cytokine interleukin-1 (IL-1) is a key driver of PTX3 expression in the brain after experimental stroke. Gene deletion of PTX3 had no significant effects on acute ischemic brain injury. In contrast, the absence of PTX3 strongly compromised blood-brain barrier integrity and resolution of brain edema during recovery after ischemic injury. Compromised resolution of brain edema in PTX3-deficient mice was associated with impaired glial scar formation and alterations in scar-associated extracellular matrix production. Our results suggest that PTX3 expression induced by proinflammatory signals after ischemic brain injury is a critical effector of edema resolution and glial scar formation. This highlights the potential role for inflammatory molecules in brain recovery after injury and identifies APPs, in particular PTX3, as important targets in ischemic stroke and possibly other brain inflammatory disorders.Journal of Cerebral Blood Flow & Metabolism advance online publication, 18 December 2013; doi:10.1038/jcbfm.2013.224.
Ischemic stroke is confounded by conditions such as atherosclerosis, diabetes, and infection, all of which alter peripheral inflammatory processes with concomitant impact on stroke outcome. The majority of the stroke patients are elderly, but the impact of interactions between aging and inflammation on stroke remains unknown. We thus investigated the influence of age on the outcome of stroke in animals predisposed to systemic chronic infection. Th1-polarized chronic systemic infection was induced in 18-22 month and 4-month-old C57BL/6j mice by administration of Trichuris muris (gut parasite). One month after infection, mice underwent permanent middle cerebral artery occlusion and infarct size, brain gliosis, and brain and plasma cytokine profiles were analyzed. Chronic infection increased the infarct size in aged but not in young mice at 24 h. Aged, ischemic mice showed altered plasma and brain cytokine responses, while the lesion size correlated with plasma prestroke levels of RANTES. Moreover, the old, infected mice exhibited significantly increased neutrophil recruitment and upregulation of both plasma interleukin-17? and tumor necrosis factor-? levels. Neither age nor infection status alone or in combination altered the ischemia-induced brain microgliosis. Our results show that chronic peripheral infection in aged animals renders the brain more vulnerable to ischemic insults, possibly by increasing the invasion of neutrophils and altering the inflammation status in the blood and brain. Understanding the interactions between age and infections is crucial for developing a better therapeutic regimen for ischemic stroke and when modeling it as a disease of the elderly.
Interleukin-1 (IL-1) is a key regulator of inflammation and ischaemic brain injury, but the contribution of central and peripheral sources of IL-1 to brain injury is not well understood. Here we show that haematopoietic-derived IL-1 is a key driver of ischaemic brain injury. Wild type (WT) mice transplanted with IL-1??-deficient bone marrow displayed a significant (40%) reduction in brain injury induced by focal cerebral ischaemia compared with WT mice transplanted with WT bone marrow. This was paralleled by improved neurological outcome and the almost complete absence of splenic-derived, but not liver-derived, IL-1? after stroke in WT mice lacking haematopoietic-derived IL-1. IL-1?? knockout (KO) mice transplanted with IL-1??-deficient bone marrow showed a 60% reduction in brain injury compared with WT mice receiving WT bone marrow. Transplantation of WT bone marrow in IL-1?? KO mice resulted in a similar level of blood-brain-barrier injury to that observed in WT mice receiving IL-1??-deficient bone marrow. Cerebral oedema after brain injury was reduced in IL-1?? KO recipients irrespective of donor-derived IL-1, but a lack of haematopoetic IL-1 has also been associated with smaller brain oedema independently of recipient status. Thus, both central and haematopoietic-derived IL-1 are important contributors to brain injury after cerebral ischaemia. Identification of the cellular sources of IL-1 in the periphery could allow targeted interventions at these sites.
Neuroinflammation is involved in several brain disorders and can be monitored through expression of the translocator protein 18 kDa (TSPO) on activated microglia. In recent years, several new PET radioligands for TSPO have been evaluated in disease models. [(18)F]DPA-714 is a TSPO radiotracer with great promise; however results vary between different experimental models of neuroinflammation. To further examine the potential of [(18)F]DPA-714, it was compared directly to [(11)C]PK11195 in experimental cerebral ischaemia in rats.
Macrophage can adopt several phenotypes, process call polarization, which is crucial for shaping inflammatory responses to injury. It is not known if microglia, a resident brain macrophage population, polarizes in a similar way, and whether specific microglial phenotypes modulate cell death in response to brain injury. In this study, we show that both BV2-microglia and mouse bone marrow derived macrophages (BMDMs) were able to adopt different phenotypes after LPS (M1) or IL-4 (M2) treatment in vitro, but regulated cell death differently when added to mouse organotypic hippocampal brain slices. BMDMs induced cell death when added to control slices and exacerbated damage when combined with oxygen-glucose deprivation (OGD), independently of their phenotype. In contrast, vehicle- and M2-BV2-microglia were protective against OGD-induced death. Direct treatment of brain slices with IL-4 (without cell addition) was protective against OGD and induced an M2 phenotype in the slice. In vivo, intracerebral injection of LPS or IL-4 in mice induced microglial phenotypes similar to the phenotypes observed in brain slices and in cultured cells. After injury induced by middle cerebral artery occlusion, microglial cells did not adopt classical M1/M2 phenotypes, suggesting that another subtype of regulatory phenotype was induced. This study highlights functional differences between macrophages and microglia, in response to brain injury with fundamentally different outcomes, even if both populations were able to adopt M1 or M2 phenotypes. These data suggest that macrophages infiltrating the brain from the periphery after an injury may be cytotoxic, independently of their phenotype, while microglia may be protective.
Inflammation is established as a contributor to cerebrovascular disease. Risk factors for stroke include many conditions associated with chronic or acute inflammation, and inflammatory changes in the brain after cerebrovascular events contribute to outcome in experimental studies, with growing evidence from clinical research. The brain is extremely susceptible to inflammatory challenge, but resident glia, endothelial cells and neurones can all mount a pronounced inflammatory response to infection or injury. Recent discoveries highlight the importance of peripherally-derived immune cells and inflammatory molecules in various central nervous system disorders, including stroke. The inflammatory cytokine, interleukin-1 (IL-1), plays a pivotal role in both local and systemic inflammation, and is a key driver of peripheral and central immune responses to infection or injury. Inhibition of IL-1 has beneficial effects in a variety of experimental paradigms of acute brain injury and is a promising clinical target in stroke. We propose that blockade of IL-1 could be therapeutically useful in several diseases which are risk factors for stroke, and there is already considerable pre-clinical and clinical evidence that inhibition of IL-1 by IL-1 receptor antagonist may be valuable in the management of acute stroke.
There is increasing recognition of the importance of translational pharmacokinetics in stroke research, lack of which has been cited as one of the main contributing factors to failure of Phase III trials.
The control of biochemical fluxes is distributed, and to perturb complex intracellular networks effectively it is often necessary to modulate several steps simultaneously. However, the number of possible permutations leads to a combinatorial explosion in the number of experiments that would have to be performed in a complete analysis. We used a multiobjective evolutionary algorithm to optimize reagent combinations from a dynamic chemical library of 33 compounds with established or predicted targets in the regulatory network controlling IL-1? expression. The evolutionary algorithm converged on excellent solutions within 11 generations, during which we studied just 550 combinations out of the potential search space of ~9 billion. The top five reagents with the greatest contribution to combinatorial effects throughout the evolutionary algorithm were then optimized pairwise. A p38 MAPK inhibitor together with either an inhibitor of I?B kinase or a chelator of poorly liganded iron yielded synergistic inhibition of macrophage IL-1? expression. Evolutionary searches provide a powerful and general approach to the discovery of new combinations of pharmacological agents with therapeutic indices potentially greater than those of single drugs.
Chronic systemic inflammatory conditions, such as atherosclerosis, diabetes and obesity are associated with increased risk of stroke, which suggests that systemic inflammation may contribute to the development of stroke in humans. The hypothesis that systemic inflammation may induce brain pathology can be tested in animals, and this was the key objective of the present study. First, we assessed inflammatory changes in the brain in rodent models of chronic, systemic inflammation. PET imaging revealed increased microglia activation in the brain of JCR-LA (corpulent) rats, which develop atherosclerosis and obesity, compared to the control lean strain. Immunostaining against Iba1 confirmed reactive microgliosis in these animals. An atherogenic diet in apolipoprotein E knock-out (ApoE(-/-)) mice induced microglial activation in the brain parenchyma within 8 weeks and increased expression of vascular adhesion molecules. Focal lipid deposition and neuroinflammation in periventricular and cortical areas and profound recruitment of activated myeloid phagocytes, T cells and granulocytes into the choroid plexus were also observed. In a small, preliminary study, patients at risk of stroke (multiple risk factors for stroke, with chronically elevated C-reactive protein, but negative MRI for brain pathology) exhibited increased inflammation in the brain, as indicated by PET imaging. These findings show that brain inflammation occurs in animals, and tentatively in humans, harbouring risk factors for stroke associated with elevated systemic inflammation. Thus a "primed" inflammatory environment in the brain may exist in individuals at risk of stroke and this can be adequately recapitulated in appropriate co-morbid animal models.
Inflammation is implicated in the pathogenesis and outcome of ischaemic injury. Poststroke inflammation is associated with outcome but it remains unclear whether such inflammation precedes or results from ischaemic injury. We hypothesised that inflammatory markers are associated with an increased risk of recurrent vascular events soon after transient ischaemic attack and minor stroke.
Stroke induces a systemic response that involves rapid activation of inflammatory cascades, followed later by immunodepression. Experimental stroke-induced responses in the bone marrow, which is the primary source of circulating monocytes and granulocytes, have not been investigated previously. We show that cerebral ischaemia induced early (4 ?hours) release of CXCR2-positive granulocytes from the bone marrow, which was associated with rapid systemic upregulation of CXCL1 (a ligand for CXCR2) and granulocyte-colony-stimulating factor, a key cytokine involved in the mobilisation of bone marrow leukocytes. This process involves rapid activation of nuclear factor-?B and p38 mitogen-activated protein kinase in bone marrow myeloid cells. T-cell numbers in the bone marrow increased after stroke, and bone marrow cells did not show suppressed cytokine response to bacterial endotoxin stimulation in vitro. Stroke-induced laterality observed in the brain stem and in the bone marrow indicates direct involvement of the autonomic nervous system in stroke-induced cell mobilisation. We also show that systemic inflammatory changes and leukocyte responses in the bone marrow are profoundly affected by both anaesthetic and surgical stress. We conclude that stroke influences leukocyte responses in the bone marrow through multiple mechanisms and suggest that preclinical studies should take into consideration the effect of surgical manipulation in experimental models of stroke.
Cerebrovascular inflammation is triggered by diverse central nervous system (CNS) insults and contributes to disease pathogenesis. The pro-inflammatory cytokine interleukin (IL)-1 is central to this cerebrovascular inflammatory response and understanding the underlying signalling mechanisms of IL-1 actions in brain endothelium may provide therapeutic targets for disease intervention. For the first time, we compare the contributions of p38, JNK and ERK mitogen-activated protein (MAP) kinase and NF-kB pathways to IL-1-induced brain endothelial activation. In cultures of primary mouse brain endothelium and the rat brain endothelial GPNT cell line, interleukin-1? (IL-1? induced a rapid (within 5 minutes) and transient activation of p38 and JNK (but not ERK) MAP kinases. IL-1? also induced nuclear recruitment of nuclear factor (NF)-kB p65. IL-1?-induced brain endothelial expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 was insensitive to MAP kinase inhibitors. IL-1?-induced brain endothelial expression of ICAM-1 and VCAM-1 was inhibited (80-88 %) by the proteasome inhibitor MG132 or the antioxidant caffeic acid phenethyl ester (CAPE), effects suggested to be NF-kB-dependent. IL-1?-induced brain endothelial CXCL1 expression was partially inhibited by JNK MAP kinase or MG132 (62 or 56 %, respectively). However, CXCL1 secretion from brain endothelium was reduced (65 %) only by MG132, and not MAP kinase inhibitors. Similarly, IL-1?-induced neutrophil transendothelial migration was reduced (77-89 %) by MG132, but not MAP kinase inhibitors. In summary, we show that several key components of IL-1?-induced brain endothelial activation (CAM, CXCL1 expression or release and neutrophil transmigration) are largely independent of MAP kinase activity but are reduced by proteasome inhibition, possibly reflecting a requirement for NF-kB activity. Similar mechanisms may contribute to cerebrovascular inflammation in response to CNS injury.
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.
The naturally occurring antagonist of interleukin-1, IL-1RA, is highly neuroprotective experimentally, shows few adverse effects, and inhibits the systemic acute phase response to stroke. A single regime pilot study showed slow penetration into cerebrospinal fluid (CSF) at experimentally therapeutic concentrations. Twenty-five patients with subarachnoid hemorrhage (SAH) and external ventricular drains were sequentially allocated to five administration regimes, using intravenous bolus doses of 100 to 500?mg and 4?hours intravenous infusions of IL-1RA ranging from 1 to 10?mg per kg per hour. Choice of regimes and timing of plasma and CSF sampling was informed by pharmacometric analysis of pilot study data. Data were analyzed using nonlinear mixed effects modeling. Plasma and CSF concentrations of IL-1RA in all regimes were within the predicted intervals. A 500-mg bolus followed by an intravenous infusion of IL-1RA at 10?mg per kg per hour achieved experimentally therapeutic CSF concentrations of IL-1RA within 45?minutes. Experimentally, neuroprotective CSF concentrations in patients with SAH can be safely achieved within a therapeutic time window. Pharmacokinetic analysis suggests that IL-1RA transport across the blood-CSF barrier in SAH is passive. Identification of the practicality of this delivery regime allows further studies of efficacy of IL-1RA in acute cerebrovascular disease.
Limited data on the brain penetration of potential stroke treatments have been cited as a major weakness contributing to numerous failed clinical trials. Thus, we tested whether interleukin-1 receptor antagonist (IL-1RA), established as a potent inhibitor of brain injury in animals and currently in clinical development, reaches the brain via a clinically relevant administration route, in experimental stroke.
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.
Obesity is an independent risk factor for stroke and is associated with poorer outcome after stroke. We investigated whether this poorer outcome is related to brain microvascular disruption. Focal cerebral ischaemia was induced in lean or obese (ob/ob) mice by transient middle cerebral artery occlusion. The incidence of haemorrhagic transformation and the volume of ischaemic brain damage were significantly greater in obese mice. Blood-brain barrier permeability and brain microvascular MMP-9 expression were also markedly increased in obese mice. These effects were independent of leptin or glycaemic status, suggesting that obesity potentiates brain microvascular disruption after experimental stroke.
Cerebral microvascular angiopathy (MVA) is associated with clinical vascular risk factors and is characterised by histological changes, including thickening of the walls of arterial vessels and dilatation of the Virchow-Robin spaces (VRS). We have previously described two novel biomarkers of MVA based on magnetic resonance imaging (MRI), VRS dilatation and abnormalities in the transfer of systolic arterial pulsation to the ventricular CSF, which occur as a result of decreased cerebral arterial compliance. These are associated with vascular dementia and treatment-resistant late onset depression. We studied a group of normal subjects at risk of cerebrovascular disease to determine if these biomarkers are present in patients who have no evidence of symptomatic vascular disease. We studied 31 subjects, 16 with three or more vascular risk factors and 15 with one or less significant risk factors. We measured arterial blood flow and CSF flow in the cerebral aqueduct, white matter lesion load, and the distribution and number of VRS. There were significant differences in CSF pulsatility and in VRS in the basal ganglia between the two groups, but no differences in white matter lesion load. We conclude that asymptomatic subjects at risk of stroke have MRI evidence of MVA before white matter lesions become apparent.
Increasing evidence suggests that peripheral inflammatory responses to stroke and other brain injuries have an important role in determining neurological outcome. The mediators of this response and the temporal relationships between peripheral and central inflammatory alterations are poorly understood. In this study, we show that experimental stroke in mice induces a peripheral inflammatory response that peaks 4 h after stroke, and precedes the peak in brain inflammation 24 h after stroke. This peripheral response is dominated by the induction of the chemokine CXCL-1 and the proinflammatory cytokine interleukin-6 and could serve as an accessible target for therapy and as a source of biomarkers predictive of prognosis.
Stroke is the major cause of disability in the Western world and is the third greatest cause of death, but there are no widely effective treatments to prevent the devastating effects of stroke. Extensive and growing evidence implicates inflammatory and immune processes in the occurrence of stroke and particularly in the subsequent injury. Several inflammatory mediators have been identified in the pathogenesis of stroke including specific cytokines, adhesion molecules, matrix metalloproteinases, and eicosanoids. An early clinical trial suggests that inhibiting interleukin-1 may be of benefit in the treatment of acute stroke.
Pro-inflammatory members of the interleukin-1 (IL-1) family of cytokines (IL-1alpha and beta) are important mediators of host defense responses to infection but can also exacerbate the damaging inflammation that contributes to major human diseases. IL-1alpha and beta are produced by cells of the innate immune system, such as macrophages, and act largely after their secretion by binding to the type I IL-1 receptor on responsive cells. There is evidence that IL-1alpha is also a nuclear protein that can act intracellularly. In this study, we report that both IL-1alpha and IL-1beta produced by microglia (central nervous system macrophages) in response to an inflammatory challenge are distributed between the cytosol and the nucleus. Using IL-1-beta-galactosidase and IL-1-green fluorescent protein chimeras (analyzed by fluorescence recovery after photobleaching), we demonstrate that nuclear import of IL-1alpha is exclusively active, requiring a nuclear localization sequence and Ran, while IL-1beta nuclear import is entirely passive. These data provide valuable insights into the dynamic regulation of intracellular cytokine trafficking.
Inflammatory processes induced by IL-1beta are critical for host defence responses, but are also implicated in disease. Zinc deficiency is a common consequence of, or contributor to, human inflammatory disease. However, the molecular mechanisms through which zinc contributes to inflammatory disease remain largely unknown. We report here that zinc metabolism regulates caspase-1 activation and IL-1beta secretion. One of the endogenous mediators of IL-1beta secretion is adenosine triphosphate, acting via the P2X7-receptor and caspase-1 activation in cells primed with an inflammatory stimulus such as LPS. We show that this process is selectively abolished by a brief pre-treatment with the zinc chelator N,N,N,N-tetrakis-(2-pyridylmethyl) ethylene diamine (TPEN). These effects on IL-1beta secretion were independent of rapid changes in free zinc within the cell, not a direct effect on caspase-1 activity, and upstream of caspase-1 activation. TPEN did however inhibit the activity of pannexin-1, a hemi-channel critical for adenosine triphosphate and nigericin-induced IL-1beta release. These data provide new insights into the mechanisms of caspase-1 activation and how zinc metabolism contributes to inflammatory mechanisms.
The innate immune response in the brain is initiated by pathogen-associated molecular patterns (PAMPS) or danger-associated molecular patterns (DAMPS) produced in response to central nervous system (CNS) infection or injury. These molecules activate members of the Toll-like receptor (TLR) family, of which TLR4 is the receptor for bacterial lipopolysaccharide (LPS). Although neurons have been reported to express TLR4, the function of TLR4 activation in neurons remains unknown.
The mechanism of development of delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (SAH) is poorly understood. Inflammatory processes are implicated in the development of ischemic stroke and may also predispose to the development of DCI following SAH. The objective of this study was to test whether concentrations of circulating inflammatory markers (C-reactive protein (CRP), interleukin-6 (IL-6) and interleukin 1 receptor antagonist (IL-1Ra)) were predictive for DCI following SAH. Secondary analyses considered white cell count (WCC) and erythrocyte sedimentation rate (ESR).
Whilst it is well documented that all components of the neurovascular unit contribute to the restrictive nature of the blood-brain barrier (BBB), astrocytes have been identified as the cellular component most likely to play an essential role in maintaining the barrier properties. The aim of this study was to examine the impact of the rat astrocyte cell line, CTX-TNA2, on the structural and functional characteristics of an in vitro BBB and determine the capacity of this astrocyte cell line to maintain the BBB phenotype. Co-culture of the CTX-TNA2 cells with primary porcine brain endothelial cells produced an in vitro BBB model which retains key features of the in vivo BBB. High transendothelial electrical resistances, comparable to those reported in vivo, were obtained. Ultrastructural analysis revealed distinct intercellular tight junction protein complexes and immunocytochemistry confirmed expression of the tight junction proteins ZO-1 and occludin. Western blotting and fluorescent tracer assays confirmed expression and functional activity of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) efflux transporters. Studies employing Alexa-fluor 555-conjugated human transferrin revealed temperature-sensitive internalisation indicating the BBB model retains functional receptor-mediated transferrin uptake. The findings of this study indicate that a robust BBB model has been produced and this is the first report of the inductive capacity of the CTX-TNA2 cell line. Since this in vitro BBB model possesses many key characteristics of the BBB in vivo it has the potential to be a valuable tool for the study of biochemical and physiological processes associated with the BBB.
Many neuroprotective agents have been effective in experimental stroke, yet few have translated into clinical application. One reason for this may be failure to consider clinical comorbidities/risk factors in experimental models. We have shown that a naturally occurring interleukin-1 receptor antagonist (IL-1Ra) is protective against ischemic brain damage in healthy animals. However, protective effects of IL-1Ra have not been determined in comorbid animals. Thus, we tested whether IL-1Ra protects against brain injury induced by experimental ischemia in aged JCR-LA (corpulent) rats, which have clinically relevant risk factors. Male, aged, lean, and corpulent rats exposed to transient (90 minutes) occlusion of the middle cerebral artery (tMCAO) were administered two doses of IL-1Ra (25 mg/kg, subcutaneously) during reperfusion. Brain injury and neuroinflammatory changes were assessed 24 hours after tMCAO. Our results show that IL-1Ra administered at reperfusion significantly reduced infarct volume measured by magnetic resonance imaging (50%, primary outcome) and blood-brain barrier disruption in these comorbid animals. Interleukin-1Ra also reduced microglial activation, neutrophil infiltration, and cytokines levels in the brain. These data are the first to indicate that IL-1Ra protects against ischemic brain injury when administered via a clinically relevant route and time window in animals with multiple risk factors for stroke.
Subarachnoid haemorrhage (SAH) is a major contributor to the burden of stroke on society. Treatment options are limited and animal models of SAH do not always mimic key pathophysiological hallmarks of the disease, thus hindering development of new therapeutics. Inflammation is strongly associated with brain injury after SAH in animals and patients, and inhibition of the pro-inflammatory cytokine interleukin-1 (IL-1) represents a possible therapeutic target. Here we report that a rupture of the middle cerebral artery in the rat produces heterogeneous infarct patterns similar to those observed in human SAH. Administration of the IL-1 receptor antagonist (IL-1Ra) reduced blood-brain barrier breakdown, and the extent of breakdown correlated with brain injury. After SAH, haem oxygenase-1 (HO-1) was strongly expressed around the bleed site and in the cortex and striatum, indicating the presence of free haem, a breakdown product of haemoglobin. HO-1 expression was also found in the same regions as microglial/macrophage expression of IL-1?. The direct effect of haem on IL-1? expression was confirmed in vitro using organotypic slice culture (OSC). Haem-induced cell death was dependent on IL-1 signalling, with IL-1Ra completely blocking cellular injury. Furthermore, stimulation of mouse primary mixed glial cells with haem induced the release of IL-1?, but not IL-1?. Thus, we suggest that haem, released from lysed red blood cells (RBCs) in the subarachnoid space, acts as a danger-associated molecular pattern (DAMP) driving IL-1-dependent inflammation. These data provide new insights into inflammation after SAH-induced brain injury and suggest IL-1Ra as a candidate therapeutic for the disease.
Cerebrovascular inflammation contributes to diverse CNS disorders through mechanisms that are incompletely understood. The recruitment of neutrophils to the brain can contribute to neurotoxicity, particularly during acute brain injuries, such as cerebral ischemia, trauma, and seizures. However, the regulatory and effector mechanisms that underlie neutrophil-mediated neurotoxicity are poorly understood. In this study, we show that mouse neutrophils are not inherently toxic to neurons but that transendothelial migration across IL-1-stimulated brain endothelium triggers neutrophils to acquire a neurotoxic phenotype that causes the rapid death of cultured neurons. Neurotoxicity was induced by the addition of transmigrated neutrophils or conditioned medium, taken from transmigrated neutrophils, to neurons and was partially mediated by excitotoxic mechanisms and soluble proteins. Transmigrated neutrophils also released decondensed DNA associated with proteases, which are known as neutrophil extracellular traps. The blockade of histone-DNA complexes attenuated transmigrated neutrophil-induced neuronal death, whereas the inhibition of key neutrophil proteases in the presence of transmigrated neutrophils rescued neuronal viability. We also show that neutrophil recruitment in the brain is IL-1 dependent, and release of proteases and decondensed DNA from recruited neutrophils in the brain occurs in several in vivo experimental models of neuroinflammation. These data reveal new regulatory and effector mechanisms of neutrophil-mediated neurotoxicity (i.e., the release of proteases and decondensed DNA triggered by phenotypic transformation during cerebrovascular transmigration). Such mechanisms have important implications for neuroinflammatory disorders, notably in the development of antileukocyte therapies.
Infections are common following stroke and adversely affect outcome. Cellular immune suppression associated with acute stroke may increase susceptibility to infection. Cytokines are important contributors to both stroke pathology and the response to infection. Since interleukin (IL)-1 blockade is a candidate treatment for cerebral ischemia, we examined whether administration of interleukin-1 receptor antagonist (IL-1Ra) to patients with acute stroke affected innate cellular immune responses in a phase II placebo-controlled trial.
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