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Stroke is a major cause of death and disability worldwide. Yet therapeutic strategies available to treat stroke are very limited. There is an urgent need to develop novel therapeutics that can effectively facilitate functional recovery. The injury that results from stroke is known to induce neurogenesis in penumbra of the infarct region. There is considerable interest in harnessing this response for therapeutic purposes. This review summarizes what is currently known about stroke-induced neurogenesis and the factors that have been identified to regulate it. Additionally, some key studies in this field have been highlighted and their implications on future of stroke therapy have been discussed. There is a complex interplay between neuroinflammation and neurogenesis that dictates stroke outcome and possibly recovery. This highlights the need for a better understanding of the neuroinflammatory process and how it affects neurogenesis, as well as the need to identify new mechanisms and potential modulators. Neuroinflammatory processes and their impact on post-stroke repair have therefore also been discussed.

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-1β (IL-1β). 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.

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-1αβ 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 phenotypewhen initiated 8 days after incision. Furthermore, with the generation of an IL-1β floxed(fl/fl) mouse line, we demonstrated that CRPS IgG-induced changes are in part mediated by microglia-derived IL-1β, 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.

Background Inflammation is a key contributor to central nervous system (CNS) injury such as stroke, and is a major target for therapeutic intervention. Effective treatments for CNS injuries are limited and applicable to only a minority of patients. Stem cell-based therapies are increasingly considered for the treatment of CNS disease, because they can be used as in-situ regulators of inflammation, and improve tissue repair and recovery. One promising option is the use of bone marrow-derived mesenchymal stem cells (MSCs), which can secrete anti-inflammatory and trophic factors, can migrate towards inflamed and injured sites or can be implanted locally. Here we tested the hypothesis that pre-treatment with inflammatory cytokines can prime MSCs towards an anti-inflammatory and pro-trophic phenotype in vitro. Methods Human MSCs from three different donors were cultured in vitro and treated with inflammatory mediators as follows: interleukin (IL)-1α, IL-1β, tumour necrosis factor alpha (TNF-α) or interferon-γ. After 24 h of treatment, cell supernatants were analysed by ELISA for expression of granulocyte-colony stimulating factor (G-CSF), IL-10, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), IL-1 receptor antagonist (IL-1Ra) and vascular endothelial growth factor (VEGF). To confirm the anti-inflammatory potential of MSCs, immortalised mouse microglial BV2 cells were treated with bacterial lipopolysaccharide (LPS) and exposed to conditioned media (CM) of naïve or IL-1-primed MSCs, and levels of secreted microglial-derived inflammatory mediators including TNF-α, IL-10, G-CSF and IL-6 were measured by ELISA. Results Unstimulated MSCs constitutively expressed anti-inflammatory cytokines and trophic factors (IL-10, VEGF, BDNF, G-CSF, NGF and IL-1Ra). MSCs primed with IL-1α or IL-1β showed increased secretion of G-CSF, which was blocked by IL-1Ra. Furthermore, LPS-treated BV2 cells secreted less inflammatory and apoptotic markers, and showed increased secretion of the anti-inflammatory IL-10 in response to treatment with CM of IL-1-primed MSCs compared with CM of unprimed MSCs. Conclusions Our results demonstrate that priming MSCs with IL-1 increases expression of trophic factor G-CSF through an IL-1 receptor type 1 (IL-1R1) mechanism, and induces a reduction in the secretion of inflammatory mediators in LPS-activated microglial cells. The results therefore support the potential use of preconditioning treatments of stem cells in future therapies.

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.

Background 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. Methods TLR4 mRNA expression in primary mouse glial and neuronal cultures was assessed by RT-PCR. Mouse mixed glial, neuronal or endothelial cell cultures were treated with LPS in the absence or the presence of a TLR4 specific antagonist (VIPER) or a specific JNK inhibitor (SP600125). Expression of inflammatory mediators was assayed by cytometric bead array (CBA) and ELISA. Activation of extracellular-signal regulated kinase 1/2 (ERK1/2), p38, c-Jun-N-terminal kinase (JNK) and c-Jun was assessed by Western blot. The effect of conditioned media of untreated- versus LPS-treated glial or neuronal cultures on endothelial activation was assessed by neutrophil transmigration assay, and immunocytochemistry and ELISA were used to measure expression of intercellular cell adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1). Results LPS induces strong release of the chemokines RANTES and CXCL1 (KC), tumor necrosis factor-α (TNFα) and IL-6 in primary mouse neuronal cultures. In contrast, LPS induced release of IL-1α, IL-1β and granulocyte-colony stimulating factor (G-CSF) in mixed glial, but not in neuronal cultures. LPS-induced neuronal KC expression and release were completely blocked by VIPER. In glial cultures, LPS induced activation of ERK1/2, p38 and JNK. In contrast, in neuronal cultures, LPS activated JNK but not ERK1/2 or p38, and the specific JNK inhibitor SP600125 significantly blocked LPS-induced KC expression and release. Finally, conditioned medium of LPS-treated neuronal cultures induced strong expression of ICAM-1 and VCAM-1 on endothelial cells, and induced infiltration of neutrophils across the endothelial monolayer, which was inhibited by VIPER. Conclusion These data demonstrate for the first time that neurons can play a role as key sensors of infection to initiate CNS inflammation.

Background Mesenchymal stem cells (MSCs) are one of the most promising candidates for the treatment of major neurological disorders. Desirable therapeutic properties of MSCs include reparative and regenerative potential but, despite their proven safety, the efficacy of MSCs remains controversial. Therefore, it is essential to optimise culture protocols to enhance the therapeutic potential of the MSC secretome. Here we aimed to: assess the increase in secretion of cytokines that may induce repair, regeneration, or immunomodulation when cultured in three dimensions; study the effect of interleukin (IL)-1 priming on two- (2D) and three-dimensional (3D) cultures of MSC; and evaluate the potential use of the modified secretome using microglial-MSC co-cultures. Methods We established a 3D spheroid culture of human MSCs, and compared the secretome in 2D and 3D cultures under primed (IL-1) and unprimed conditions. BV2 microglial cells were stimulated with lipopolysaccharide (LPS) and treated with spheroid conditioned media (CM) or were co-cultured with whole spheroids. Concentrations of secreted cytokines were determined by enzyme-linked immunosorbent assay (ELISA). Protein arrays were used to further evaluate the effect of IL-1 priming in 2D and 3D cultures. Results 3D culture of MSCs significantly increased secretion of the IL-1 receptor antagonist (IL-1Ra), vascular endothelial growth factor (VEGF), and granulocyte-colony stimulating factor (G-CSF) compared with 2D culture, despite priming treatments with IL-1 being more effective in 2D than in 3D. The addition of CM of 3D-MSCs reduced LPS-induced tumour necrosis factor (TNF)-α secretion from BV2 cells, while the 3D spheroid co-cultured with the BV2 cells induced an increase in IL-6, but had no effect on TNF-α release. Protein arrays indicated that priming treatments trigger a more potent immune profile which is necessary to orchestrate an effective tissue repair. This effect was lost in 3D, partly because of the overexpression of IL-6. Conclusions Increased secretion of anti-inflammatory markers occurs when MSCs are cultured in 3D, but this specific secretome did not translate into anti-inflammatory effects on LPS-treated BV2 cells in co-culture. These data highlight the importance of optimising priming treatments and culture conditions to maximise the therapeutic potential of MSC spheroids.

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-1β (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.

Breast cancer bone metastasis is currently incurable, ~75% of patients with late-stage breast cancer develop disease recurrence in bone and available treatments are only palliative. We have previously shown that production of the pro-inflammatory cytokine interleukin-1B (IL-1B) by breast cancer cells drives bone metastasis in patients and in preclinical in vivo models. In the current study, we have investigated how IL-1B from tumour cells and the microenvironment interact to affect primary tumour growth and bone metastasis through regulation of the immune system, and whether targeting IL-1 driven changes to the immune response improves standard of care therapy for breast cancer bone metastasis. Using syngeneic IL-1B/IL1R1 knock out mouse models in combination with genetic manipulation of tumour cells to overexpress IL-1B/IL1R1, we found that IL-1B signalling elicited an opposite response in primary tumours compared with bone metastases. In primary tumours, IL-1B inhibited growth, by impairing the infiltration of innate immune cell subsets with potential anti-cancer functions but promoted enhanced tumour cell migration. In bone, IL-1B stimulated the development of osteolytic metastases. In syngeneic models of breast cancer, combining standard of care treatments (Doxorubicin and Zoledronic acid) with the IL-1 receptor antagonist Anakinra inhibited both primary tumour growth and metastasis. Anakinra had opposite effects on the immune response compared to standard of care treatment, and its anti-inflammatory signature was maintained in the combination therapy. These data suggest that targeting IL-1B signalling may provide a useful therapeutic approach to inhibit bone metastasis and improve efficacy of current treatments for breast cancer patients.

Mucopolysaccharidosis IIIA is a neuronopathic lysosomal storage disease, characterised by heparan sulphate and other substrates accumulating in the brain. Patients develop behavioural disturbances and cognitive decline, a possible consequence of neuroinflammation and abnormal substrate accumulation. Interleukin (IL)‐1β and interleukin‐1 receptor antagonist (IL‐1Ra) expression were significantly increased in both murine models and human MPSIII patients. We identified pathogenic mechanisms of inflammasome activation, including that disease‐specific 2‐O‐sulphated heparan sulphate was essential for priming an IL‐1β response via the Toll‐like receptor 4 complex. However, mucopolysaccharidosis IIIA primary and secondary storage substrates, such as amyloid beta, were both required to activate the NLRP3 inflammasome and initiate IL‐1β secretion. IL‐1 blockade in mucopolysaccharidosis IIIA mice using IL‐1 receptor type 1 knockout or haematopoietic stem cell gene therapy over‐expressing IL‐1Ra reduced gliosis and completely prevented behavioural phenotypes. In conclusion, we demonstrate that IL‐1 drives neuroinflammation, behavioural abnormality and cognitive decline in mucopolysaccharidosis IIIA, highlighting haematopoietic stem cell gene therapy treatment with IL‐1Ra as a potential neuronopathic lysosomal disease treatment. Synopsis This study reports that IL‐1, stimulated by storage substrates, is a critical mediator in the mucopolysaccharidosis IIIA (MPSIIIA) inflammatory cascade. Haematopoietic stem cell gene therapy using IL‐1Ra, the natural antagonist of IL‐1, prevented cognitive and behavioural decline in mice. IL‐1 blockade in MPSIIIA mice via lentiviral dependent haematopoietic stem cell overexpression of IL‐1Ra corrected cognitive decline and reduced neuroinflammation. 2‐O‐sulphation of MPSIIIA heparan sulphate elicited production of TLR4 dependent IL‐1β. MPSIIIA primary and secondary storage substrates activated the inflammasome and induced secretion of IL‐1β. IL‐1β and IL‐1Ra are important immuno‐biomarkers in MPSIIIA patients and mice. Graphical Abstract This study reports that IL‐1, stimulated by storage substrates, is a critical mediator in the mucopolysaccharidosis IIIA (MPSIIIA) inflammatory cascade. Haematopoietic stem cell gene therapy using IL‐1Ra, the natural antagonist of IL‐1, prevented cognitive and behavioural decline in mice.