Genetic Cell Ablation Reveals Clusters of Local Self-Renewing Microglia in the Mammalian Central Nervous System.

During early embryogenesis, microglia arise from yolk sac progenitors that populate the developing central nervous system (CNS), but how the tissue-resident macrophages are maintained throughout the organism's lifespan still remains unclear. Here, we describe a system that allows specific, conditional ablation of microglia in adult mice. We found that the microglial compartment was reconstituted within 1 week of depletion. Microglia repopulation relied on CNS-resident cells, independent from bone-marrow-derived precursors. During repopulation, microglia formed clusters of highly proliferative cells that migrated apart once steady state was achieved. Proliferating microglia expressed high amounts of the interleukin-1 receptor (IL-1R), and treatment with an IL-1R antagonist during the repopulation phase impaired microglia proliferation. Hence, microglia have the potential for efficient self-renewal without the contribution of peripheral myeloid cells, and IL-1R signaling participates in this restorative proliferation process.

Transfer of complex regional pain syndrome to mice via human autoantibodies is mediated by interleukin-1-induced mechanisms.

Neuroimmune interactions may contribute to severe pain and regional inflammatory and autonomic signs in complex regional pain syndrome (CRPS), a posttraumatic pain disorder. Here, we investigated peripheral and central immune mechanisms in a translational passive transfer trauma mouse model of CRPS. Small plantar skin-muscle incision was performed in female C57BL/6 mice treated daily with purified serum immunoglobulin G (IgG) from patients with longstanding CRPS or healthy volunteers followed by assessment of paw edema, hyperalgesia, inflammation, and central glial activation. CRPS IgG significantly increased and prolonged swelling and induced stable hyperalgesia of the incised paw compared with IgG from healthy controls. After a short-lasting paw inflammatory response in all groups, CRPS IgG-injected mice displayed sustained, profound microglia and astrocyte activation in the dorsal horn of the spinal cord and pain-related brain regions, indicating central sensitization. Genetic deletion of interleukin-1 (IL-1) using IL-1αß knockout (KO) mice and perioperative IL-1 receptor type 1 (IL-1R1) blockade with the drug anakinra, but not treatment with the glucocorticoid prednisolone, prevented these changes. Anakinra treatment also reversed the established sensitization phenotype when initiated 8 days after incision. Furthermore, with the generation of an IL-1ß floxed(fl/fl) mouse line, we demonstrated that CRPS IgG-induced changes are in part mediated by microglia-derived IL-1ß, suggesting that both peripheral and central inflammatory mechanisms contribute to the transferred disease phenotype. These results indicate that persistent CRPS is often contributed to by autoantibodies and highlight a potential therapeutic use for clinically licensed antagonists, such as anakinra, to prevent or treat CRPS via blocking IL-1 actions.

IL-1 signaling is critical for expansion but not generation of autoreactive GM-CSF+ Th17 cells.

Interleukin-1 (IL-1) is implicated in numerous pathologies, including multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). However, the exact mechanism by which IL-1 is involved in the generation of pathogenic T cells and in disease development remains largely unknown. We found that following EAE induction, pertussis toxin administration leads to IL-1 receptor type 1 (IL-1R1)-dependent IL-1ß expression by myeloid cells in the draining lymph nodes. This myeloid-derived IL-1ß did not vitally contribute to the generation and plasticity of Th17 cells, but rather promoted the expansion of a GM-CSF+ Th17 cell subset, thereby enhancing its encephalitogenic potential. Lack of expansion of GM-CSF-producing Th17 cells led to ameliorated disease in mice deficient for IL-1R1 specifically in T cells. Importantly, pathogenicity of IL-1R1-deficient T cells was fully restored by IL-23 polarization and expansion in vitro Therefore, our data demonstrate that IL-1 functions as a mitogenic mediator of encephalitogenic Th17 cells rather than qualitative inducer of their generation.

ß1 integrin is a sensor of blood flow direction.

Endothelial cell (EC) sensing of fluid shear stress direction is a critical determinant of vascular health and disease. Unidirectional flow induces EC alignment and vascular homeostasis, whereas bidirectional flow has pathophysiological effects. ECs express several mechanoreceptors that respond to flow, but the mechanism for sensing shear stress direction is poorly understood. We determined, by using in vitro flow systems and magnetic tweezers, that ß1 integrin is a key sensor of force direction because it is activated by unidirectional, but not bidirectional, shearing forces. ß1 integrin activation by unidirectional force was amplified in ECs that were pre-sheared in the same direction, indicating that alignment and ß1 integrin activity has a feedforward interaction, which is a hallmark of system stability. En face staining and EC-specific genetic deletion studies in the murine aorta revealed that ß1 integrin is activated and is essential for EC alignment at sites of unidirectional flow but is not activated at sites of bidirectional flow. In summary, ß1 integrin sensing of unidirectional force is a key mechanism for decoding blood flow mechanics to promote vascular homeostasis.This article has an associated First Person interview with the first author of the paper.

Interleukin 1 alpha administration is neuroprotective and neuro-restorative following experimental ischemic stroke.

BACKGROUND: Stroke remains a leading cause of death and disability worldwide despite recent treatment breakthroughs. A primary event in stroke pathogenesis is the development of a potent and deleterious local and peripheral inflammatory response regulated by the pro-inflammatory cytokine interleukin-1 (IL-1). While the role of IL-1ß (main released isoform) has been well studied in stroke, the role of the IL-1α isoform remains largely unknown. With increasing utilization of intravenous tissue plasminogen activator (t-PA) or thrombectomy to pharmacologically or mechanically remove ischemic stroke causing blood clots, respectively, there is interest in pairing successful cerebrovascular recanalization with neurotherapeutic pharmacological interventions (Fraser et al., J Cereb Blood Flow Metab 37:3531-3543, 2017; Hill et al., Lancet Neurol 11:942-950, 2012; Amaro et al., Stroke 47:2874-2876, 2016). METHODS: Transient stroke was induced in mice via one of two methods. One group of mice were subjected to tandem ipsilateral common carotid artery and middle cerebral artery occlusion, while another group underwent the filament-based middle cerebral artery occlusion. We have recently developed an animal model of intra-arterial (IA) drug administration after recanalization (Maniskas et al., J Neurosci Met 240:22-27, 2015). Sub groups of the mice were treated with either saline or Il-1α, wherein the drug was administered either acutely (immediately after surgery) or subacutely (on the third day after stroke). This was followed by behavioral and histological analyses. RESULTS: We now show in the above-mentioned mouse stroke models (transient tandem ipsilateral common carotid artery (CCA) and middle cerebral artery occlusion (MCA) occlusion, MCA suture occlusion) that IL-1α is neuroprotective when acutely given either intravenously (IV) or IA at low sub-pathologic doses. Furthermore, while IV administration induces transient hemodynamic side effects without affecting systemic markers of inflammation, IA delivery further improves overall outcomes while eliminating these side effects. Additionally, we show that delayed/subacute IV IL-1α administration ameliorates functional deficit and promotes neurorepair. CONCLUSIONS: Taken together, our present study suggests for the first time that IL-1α could, unexpectedly, be an effective ischemic stroke therapy with a broad therapeutic window.

Interleukin-1 mediates ischaemic brain injury via distinct actions on endothelial cells and cholinergic neurons.

The cytokine interleukin-1 (IL-1) is a key contributor to neuroinflammation and brain injury, yet mechanisms by which IL-1 triggers neuronal injury remain unknown. Here we induced conditional deletion of IL-1R1 in brain endothelial cells, neurons and blood cells to assess site-specific IL-1 actions in a model of cerebral ischaemia in mice. Tamoxifen treatment of IL-1R1 floxed (fl/fl) mice crossed with mice expressing tamoxifen-inducible Cre-recombinase under the Slco1c1 promoter resulted in brain endothelium-specific deletion of IL-1R1 and a significant decrease in infarct size (29%), blood-brain barrier (BBB) breakdown (53%) and neurological deficit (40%) compared to vehicle-treated or control (IL-1R1fl/fl) mice. Absence of brain endothelial IL-1 signalling improved cerebral blood flow, followed by reduced neutrophil infiltration and vascular activation 24 h after brain injury. Conditional IL-1R1 deletion in neurons using tamoxifen inducible nestin-Cre mice resulted in reduced neuronal injury (25%) and altered microglia-neuron interactions, without affecting cerebral perfusion or vascular activation. Deletion of IL-1R1 specifically in cholinergic neurons reduced infarct size, brain oedema and improved functional outcome. Ubiquitous deletion of IL-1R1 had no effect on brain injury, suggesting beneficial compensatory mechanisms on other cells against the detrimental effects of IL-1 on endothelial cells and neurons. We also show that IL-1R1 signalling deletion in platelets or myeloid cells does not contribute to brain injury after experimental stroke. Thus, brain endothelial and neuronal (cholinergic) IL-1R1 mediate detrimental actions of IL-1 in the brain in ischaemic stroke. Cell-specific targeting of IL-1R1 in the brain could therefore have therapeutic benefits in stroke and other cerebrovascular diseases.

Characterization of a conditional interleukin-1 receptor 1 mouse mutant using the Cre/LoxP system.

IL-1 is a key cytokine known to drive chronic inflammation and to regulate many physiological, immunological, and neuroimmunological responses via actions on diverse cell types of the body. To determine the mechanisms of IL-1 actions as part of the inflammatory response in vivo, we generated a conditional IL-1 receptor 1 (IL-1R1) mouse mutant using the Cre/LoxP system (IL-1R1(fl/fl) ). In the mutant generated, exon 5, which encodes part of the extracellular-binding region of the receptor, is flanked by LoxP sites, thereby inactivating the two previously described functional IL-1R1 gene transcripts after Cre-mediated recombination. Using keratin 14-Cre driver mice, new IL-1R1 deficient (-/-) mice were subsequently generated, in which all signaling IL-1 receptor isoforms are deleted ubiquitously. Furthermore, using vav-iCre driver mice, we deleted IL-1 receptor isoforms in the hematopoietic system. In these mice, we show that both the IL-17 and IL-22 cytokine response is reduced, when mice are challenged by the helminth Trichuris muris. We are currently crossing IL-1R1(fl/fl) mice with different Cre-expressing mice in order to study mechanisms of acute and chronic inflammatory diseases.

Involvement of interleukin-1 type 1 receptors in lipopolysaccharide-induced sickness responses.

Sickness responses to lipopolysaccharide (LPS) were examined in mice with deletion of the interleukin (IL)-1 type 1 receptor (IL-1R1). IL-1R1 knockout (KO) mice displayed intact anorexia and HPA-axis activation to intraperitoneally injected LPS (anorexia: 10 or 120µg/kg; HPA-axis: 120µg/kg), but showed attenuated but not extinguished fever (120µg/kg). Brain PGE2 synthesis was attenuated, but Cox-2 induction remained intact. Neither the tumor necrosis factor-α (TNFα) inhibitor etanercept nor the IL-6 receptor antibody tocilizumab abolished the LPS induced fever in IL-1R1 KO mice. Deletion of IL-1R1 specifically in brain endothelial cells attenuated the LPS induced fever, but only during the late, 3rd phase of fever, whereas deletion of IL-1R1 on neural cells or on peripheral nerves had little or no effect on the febrile response. We conclude that while IL-1 signaling is not critical for LPS induced anorexia or stress hormone release, IL-1R1, expressed on brain endothelial cells, contributes to the febrile response to LPS. However, also in the absence of IL-1R1, LPS evokes a febrile response, although this is attenuated. This remaining fever seems not to be mediated by IL-6 receptors or TNFα, but by some yet unidentified pyrogenic factor.

IL-1alpha induces angiogenesis in brain endothelial cells in vitro: implications for brain angiogenesis after acute injury.

Inflammation is a major contributor to neuronal injury and is associated with poor outcome after acute brain injury such as stroke. The pro-inflammatory cytokine interleukin (IL)-1 is a critical regulator of cerebrovascular inflammation after ischemic injury, mainly through action of both of its isoforms, IL-1α and IL-1ß, at the brain endothelium. In contrast, the differential action of these ligands on endothelial activation and post-stroke angiogenesis is largely unknown. Here, we demonstrate that IL-1α is chronically elevated in the brain after experimental stroke suggesting that it is present during post-stroke angiogenic periods. Furthermore, we demonstrate that IL-1α is a potent mediator of endothelial activation and inducer of angiogenic markers in endothelial cells in vitro. Using brain endothelial cell lines, we found that IL-1α was significantly more potent than IL-1ß at inducing endothelial cell activation, as measured by expression of the pro-angiogenic chemokine CXCL-1. IL-1α also induced strong expression of the angiogenic mediator IL-6 in a concentration-dependent manner. Furthermore, IL-1α induced significant proliferation and migration of endothelial cells, and promoted formation of tube-like structures that are established key hallmarks of angiogenesis in vitro. Finally, all of those responses were blocked by the IL-1 receptor antagonist (IL-1RA). In conclusion, our data highlights a potential new role for IL-1 in brain repair mechanisms and identifies IL-1α as a potential new therapy to promote post-stroke angiogenesis. Inflammation is a major contributor to neuronal injury and is associated with poor outcome after neurotrauma. We demonstrate that cytokine IL-1α is chronically elevated in the brain after experimental stroke suggesting that it is present chronically post-stroke. We demonstrate that IL-1α is a potent mediator of endothelial activation and inducer of angiogenic markers in endothelial cells. Our data highlights a new role for IL-1 in brain repair mechanisms and identifies IL-1α as a potential therapy to promote post-stroke angiogenesis.

The extracellular matrix protein laminin-10 promotes blood-brain barrier repair after hypoxia and inflammation in vitro.

BACKGROUND: The blood-brain barrier (BBB) of the central nervous system (CNS) is essential for normal brain function. However, the loss of BBB integrity that occurs after ischaemic injury is associated with extracellular matrix (ECM) remodelling and inflammation, and contributes to poor outcome. ECM remodelling also contributes to BBB repair after injury, but the precise mechanisms and contribution of specific ECM molecules involved are unknown. Here, we investigated the mechanisms by which hypoxia and inflammation trigger loss of BBB integrity and tested the hypothesis ECM changes could contribute to BBB repair in vitro. METHODS: We used an in vitro model of the BBB, composed of primary rat brain endothelial cells grown on collagen (Col) I-, Col IV-, fibronectin (FN)-, laminin (LM) 8-, or LM10-coated tissue culture plates, either as a single monolayer culture or on Transwell® inserts above mixed glial cell cultures. Cultures were exposed to oxygen-glucose deprivation (OGD) and/or reoxygenation, in the absence or the presence of recombinant interleukin-1ß (IL-1ß). Cell adhesion to ECM molecules was assessed by cell attachment and cell spreading assays. BBB dysfunction was assessed by immunocytochemistry for tight junction proteins occludin and zona occludens-1 (ZO-1) and measurement of trans-endothelial electrical resistance (TEER). Change in endothelial expression of ECM molecules was assessed by semi-quantitative RT-PCR. RESULTS: OGD and/or IL-1 induce dramatic changes associated with loss of BBB integrity, including cytoplasmic relocalisation of membrane-associated tight junction proteins occludin and ZO-1, cell swelling, and decreased TEER. OGD and IL-1 also induced gene expression of key ECM molecules associated with the BBB, including FN, Col IV, LM 8, and LM10. Importantly, we found that LM10, but not FN, Col IV, nor LM8, plays a key role in maintenance of BBB integrity and reversed most of the key hallmarks of BBB dysfunction induced by IL-1. CONCLUSIONS: Our data unravel new mechanisms of BBB dysfunction induced by hypoxia and inflammation and identify LM10 as a key ECM molecule involved in BBB repair after hypoxic injury and inflammation.

Pentraxin 3 mediates neurogenesis and angiogenesis after cerebral ischaemia.

BACKGROUND: The acute phase protein pentraxin 3 (PTX3) is a new biomarker of stroke severity and is a key regulator of oedema resolution and glial responses after cerebral ischaemia, emerging as a possible target for brain repair after stroke. Neurogenesis and angiogenesis are essential events in post-stroke recovery. Here, we investigated for the first time the role of PTX3 in neurogenesis and angiogenesis after stroke. METHODS: PTX3 knockout (KO) or wild-type (WT) mice were subjected to experimental cerebral ischaemia (induced by middle cerebral artery occlusion (MCAo)). Poststroke neurogenesis was assessed by nestin, doublecortin (DCX) and bromodeoxyuridine (BrdU) immunostaining, whereas angiogenesis was assessed by BrdU, vascular endothelial growth factor receptor 2 (VEGFR2) and PECAM-1 immunostaining. In vitro neurogenesis and angiogenesis assays were carried out on neurospheres derived from WT or interleukin-1ß (IL-1ß) KO mice, and mouse endothelial cell line bEnd.5 respectively. Behavioural function was assessed in WT and PTX3 KO mice using open-field, motor and Y-maze tests. RESULTS: Neurogenesis was significantly reduced in the dentate gyrus (DG) of the hippocampus of PTX3 KO mice, compared to WT mice, 6 days after MCAo. In addition, recombinant PTX3 was neurogenic in vitro when added to neurospheres, which was mediated by IL-1ß. In vivo poststroke angiogenesis was significantly reduced in PTX3 KO mice compared to WT mice 14 days after MCAo, as revealed by reduced vascular density, less newly formed blood vessels and decreased expression of VEGFR2. In vitro, recombinant PTX3 induced marked endothelial cellular proliferation and promoted formation of tube-like structures of endothelial cell line bEnd.5. Finally, a lack of PTX3 potentiated motor deficits 14 days after MCAo. CONCLUSIONS: These results indicate that PTX3 mediates neurogenesis and angiogenesis and contributes to functional recovery after stroke, highlighting a key role of PTX3 as a mediator of brain repair and suggesting that PTX3 could be used as a new target for stroke therapy.

Neutrophil cerebrovascular transmigration triggers rapid neurotoxicity through release of proteases associated with decondensed DNA.

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.

Activation of brain endothelial cells by interleukin-1 is regulated by the extracellular matrix after acute brain injury.

The extracellular matrix (ECM) of the central nervous system (CNS) is essential for normal brain function, whilst ECM remodelling is associated with cerebrovascular inflammation driven by the cytokine interleukin-1 (IL-1) after acute brain injury. The effect of ECM remodelling on endothelial activation during neuroinflammation remains unknown. Here we report that ECM remodelling in the cerebrovasculature critically regulates IL-1-induced endothelial cell activation after cerebral ischaemia; Expression levels of ECM molecules associated with the cerebrovasculature, namely fibronectin (FN) and collagen IV (Col IV), strongly increased in brain blood vessels after middle cerebral artery occlusion (MCAo) in a time-dependent manner, reaching a peak of vascular expression 48 h after MCAo. In cultures, FN and Col IV (but also laminin-1 and fibrillin-1) promoted strong attachment of the GPNT endothelial cell line and primary rat brain endothelial cells, which was markedly inhibited by RGD (Arg-Gly-Asp) peptide, or specific integrin ß1, α4, α5 and αv blockade. IL-1ß-induced activation of extracellular-regulated kinase 1/2 (ERK1/2) and nuclear factor κB (NFκB), and synthesis of cytokine-induced neutrophil chemoattractant (CINC-1) were enhanced in cells plated onto ECM molecules, and these responses were inhibited by selective integrin blockade. Finally, increased ECM expression in vessels after MCAo was found associated with vinculin clustering, increased integrin ß1 expression, and increased IL-1 receptor associated kinase-1 (IRAK-1) activity in endothelial cells and perivascular astrocytes. Therefore, our data indicate a novel function for the ECM in the regulation of cerebrovascular inflammation triggered by IL-1 during acute brain injury.

Hypoxia and interleukin-1-primed mesenchymal stem/stromal cells as novel therapy for stroke.

Promising preclinical stroke research has not yielded meaningful and significant success in clinical trials. This lack of success has prompted the need for refinement of preclinical studies with the intent to optimize the chances of clinical success. Regenerative medicine, especially using mesenchymal stem/stromal cells (MSCs), has gained popularity in the last decade for treating many disorders, including central nervous system (CNS), such as stroke. In addition to less stringent ethical constraints, the ample availability of MSCs also makes them an attractive alternative to totipotent and other pluripotent stem cells. The ability of MSCs to differentiate into neurons and other brain parenchymal and immune cells makes them a promising therapy for stroke. However, these cells also have some drawbacks that, if not addressed, will render MSCs unfit for treating ischaemic stroke. In this review, we highlighted the molecular and cellular changes that occur following an ischaemic stroke (IS) incidence and discussed the physiological properties of MSCs suitable for tackling these changes. We also went further to discuss the major drawbacks of utilizing MSCs in IS and how adequate priming using both hypoxia and interleukin-1 can optimize the beneficial properties of MSCs while eliminating these drawbacks.

Endothelial to mesenchymal transition in the interleukin-1 pathway during aortic aneurysm formation.

OBJECTIVE: Endothelial to mesenchymal transition may represent a key link between inflammatory stress and endothelial dysfunction seen in aortic aneurysm disease. Endothelial to mesenchymal transition is regulated by interleukin-1ß, and previous work has demonstrated an essential role of interleukin-1 signaling in experimental aortic aneurysm models. We hypothesize that endothelial to mesenchymal transition is present in murine aortic aneurysms, and loss of interleukin-1 signaling attenuates this process. METHODS: Murine aortic aneurysms were created in novel CDH5-Cre lineage tracking mice by treating the intact aorta with peri-adventitial elastase. Endothelial to mesenchymal transition transcription factors as well as endothelial and mesenchymal cell markers were analyzed via immunohistochemistry and immunofluorescence (n = 10/group). To determine the role of interleukin-1 signaling, endothelial-specific interleukin-1 receptor 1 knockout and wild-type mice (n = 10/group) were treated with elastase. Additionally, C57/BL6 mice were treated with the interleukin-1 receptor 1 antagonist Anakinra (n = 7) or vehicle (n = 8). RESULTS: Elastase treatment yielded greater aortic dilation compared with controls (elastase 97.0% ± 34.0%; control 5.3% ± 4.8%; P < .001). Genetic deletion of interleukin-1 receptor 1 attenuated aortic dilation (control 126.7% ± 38.7%; interleukin-1 receptor 1 knockout 35.2% ± 14.7%; P < .001), as did pharmacologic inhibition of interleukin-1 receptor 1 with Anakinra (vehicle 146.3% ± 30.1%; Anakinra 63.5% ± 23.3%; P < .001). Elastase treatment resulted in upregulation of endothelial to mesenchymal transition transcription factors (Snail, Slug, Twist, ZNF) and mesenchymal cell markers (S100, alpha smooth muscle actin) and loss of endothelial cell markers (vascular endothelial cadherin, endothelial nitric oxide synthase, von Willebrand factor). These changes were attenuated by interleukin-1 receptor 1 knockout and Anakinra treatment. CONCLUSIONS: Endothelial to mesenchymal transition occurs in aortic aneurysm disease and is attenuated by loss of interleukin-1 signaling. Endothelial dysfunction through endothelial to mesenchymal transition represents a new and novel pathway in understanding aortic aneurysm disease and may be a potential target for future treatment.

Selective deletion of interleukin-1 alpha in microglia does not modify acute outcome but regulates neurorepair processes after experimental ischemic stroke.

Inflammation is a key contributor to stroke pathogenesis and exacerbates brain damage leading to poor outcome. Interleukin-1 (IL-1) is an important regulator of post-stroke inflammation, and blocking its actions is beneficial in pre-clinical stroke models and safe in the clinical setting. However, the distinct roles of the two major IL-1 receptor type 1 agonists, IL-1α and IL-1ß, and the specific role of IL-1α in ischemic stroke remain largely unknown. Here we show that IL-1α and IL-1ß have different spatio-temporal expression profiles in the brain after experimental stroke, with early microglial IL-1α expression (4 h) and delayed IL-1ß expression in infiltrated neutrophils and a small microglial subset (24-72 h). We examined for the first time the specific role of microglial-derived IL-1α in experimental permanent and transient ischemic stroke through microglial-specific tamoxifen-inducible Cre-loxP-mediated recombination. Microglial IL-1α deletion did not influence acute brain damage, cerebral blood flow, IL-1ß expression, neutrophil infiltration, microglial nor endothelial activation after ischemic stroke. However, microglial IL-1α knock out (KO) mice showed reduced peri-infarct vessel density and reactive astrogliosis at 14 days post-stroke, alongside long-term impaired functional recovery. Our study identifies for the first time a critical role for microglial IL-1α on neurorepair and functional recovery after stroke, highlighting the importance of targeting specific IL-1 mechanisms in brain injury to develop more effective therapies.

Neuroimmmune interactions of cannabinoids in neurogenesis: focus on interleukin-1ß (IL-1ß) signalling.

Neuroimmune networks and the brain endocannabinoid system contribute to the maintenance of neurogenesis. Activation of cannabinoid receptors suppresses chronic inflammatory responses through the attenuation of pro-inflammatory mediators. Moreover, the endocannabinoid system directs cell fate specification of NSCs (neural stem cells) in the CNS (central nervous system). The aim of our work is to understand better the relationship between the endocannabinoid and the IL-1ß (interleukin-1ß) associated signalling pathways and NSC biology, in order to develop therapeutical strategies on CNS diseases that may facilitate brain repair. NSCs express functional CB1 and CB2 cannabinoid receptors, DAGLα (diacylglycerol lipase α) and the NSC markers SOX-2 and nestin. We have investigated the role of CB1 and CB2 cannabinoid receptors in the control of NSC proliferation and in the release of immunomodulators [IL-1ß and IL-1Ra (IL-1 receptor antagonist)] that control NSC fate decisions. Pharmacological blockade of CB1 and/or CB2 cannabinoid receptors abolish or decrease NSC proliferation, indicating a critical role for both CB1 and CB2 receptors in the proliferation of NSC via IL-1 signalling pathways. Thus the endocannabinoid system, which has neuroprotective and immunomodulatory actions mediated by IL-1 signalling cascades in the brain, could assist the process of proliferation and differentiation of embryonic or adult NSCs, and this may be of therapeutic interest in the emerging field of brain repair.

Loss of substance P and inflammation precede delayed neurodegeneration in the substantia nigra after cerebral ischemia.

Focal cerebral ischemia leads to delayed neurodegeneration in remote brain regions. The substantia nigra (SN) does not normally show primary neuronal death after ischemic events affecting the striatum, but can exhibit delayed neuronal loss after the ischemic injury through mechanisms that are unknown. No data are available in mice showing acute post-stroke inflammation and remote injury in the SN. Substance P (SP), a mediator of neurogenic inflammation, is a key element of the striato-nigral circuitry, but alterations of SP in the SN have not been studied after acute striatal injury. Inflammation, a key contributor to neuronal death, is found in the SN after striatal ischemia, but it is unknown whether it precedes or occurs concomitantly with neuronal death. We hypothesised that focal striatal ischemia induces changes in SP levels in the SN and that inflammation precedes neuronal death in the SN. Using the middle cerebral artery occlusion model, we found a significant loss of SP in the ipsilateral SN 24h after striatal ischemia in mice. In the same area where SP loss occurs, significant glial and vascular activation, but no neuronal death, were observed. In contrast, a marked neuronal loss was observed within six days in the area of SP loss and inflammation. Our data suggest that focal loss of SP and early inflammatory changes in the SN precede remote neuronal injury after striatal ischemic damage. These observations may have important implications for motor impairment in stroke patients and indicate that striatal ischemia might facilitate Parkinson's disease development.

Preconditioning with interleukin-1 alpha is required for the neuroprotective properties of mesenchymal stem cells after ischemic stroke in mice.

Mesenchymal stem cell (MSC) pre-conditioning with interleukin-1 alpha (IL-1ɑ) drives MSCs toward a potent anti-inflammatory phenotype. The aim of this study was to assess the therapeutic potential of intra-arterially administered IL-1ɑ preconditioned MSCs, after experimental cerebral ischaemia in mice. After 3 h from the start of middle cerebral artery occlusion, animals were treated with vehicle, 9.1 × 104 non-conditioned or IL-1ɑ preconditioned MSCs by intra-arterial administration. Animals were allowed to recover for 1.5 h after treatment to measure cerebral blood flow (CBF), and 3 days or 14 days post-stroke to evaluate lesion volume and functional outcomes. At 3-days post-stroke preconditioned MSCs reduced (by 67%) lesion volume and increased CBF (by 32%) compared to vehicle, while non-conditioned MSCs had no effect. A separate cohort of animals recovered to 14 days post-stroke also showed reduced infarct volume (by 51%) at 48 h (assessed by MRI) and better functional recovery at 14 days when treated with preconditioned MSCs when compared to vehicle. Preconditioning MSCs with IL-1α increases their neuroprotective capability and improves functional recovery after delayed intra-arterial administration. With increasing use of thrombectomy, the adjunct use of preconditioned MSCs therefore represents a highly relevant therapy to improve outcomes in ischemic stroke.

Intracerebral Administration of a Novel Self-Assembling Peptide Hydrogel Is Safe and Supports Cell Proliferation in Experimental Intracerebral Haemorrhage.

Intracerebral haemorrhage (ICH) is the deadliest form of stroke, but current treatment options are limited, meaning ICH survivors are often left with life-changing disabilities. The significant unmet clinical need and socioeconomic burden of ICH mean novel regenerative medicine approaches are gaining interest. To facilitate the regeneration of the ICH lesion, injectable biomimetic hydrogels are proposed as both scaffolds for endogenous repair and delivery platforms for pro-regenerative therapies. In this paper, the objective was to explore whether injection of a novel self-assembling peptide hydrogel (SAPH) Alpha2 was feasible, safe and could stimulate brain tissue regeneration, in a collagenase-induced ICH model in rats. Alpha2 was administered intracerebrally at 7 days post ICH and functional outcome measures, histological markers of damage and repair and RNA-sequencing were investigated for up to 8 weeks. The hydrogel Alpha2 was safe, well-tolerated and was retained in the lesion for several weeks, where it allowed infiltration of host cells. The hydrogel had a largely neutral effect on functional outcomes and expression of angiogenic and neurogenic markers but led to increased numbers of proliferating cells. RNAseq and pathway analysis showed that ICH altered genes related to inflammatory and phagocytic pathways, and these changes were also observed after administration of hydrogel. Overall, the results show that the novel hydrogel was safe when injected intracerebrally and had no negative effects on functional outcomes but increased cell proliferation. To elicit a regenerative effect, future studies could use a functionalised hydrogel or combine it with an adjunct therapy.