Graham Coutts

AIM2 and NLRC4 inflammasomes contribute with ASC to acute brain injury independently of NLRP3

Significance Cerebral ischemia (CI; stroke, brain injury, vascular dementia, neonatal hypoxia, and many other conditions) affects people at all stages of life. Of the many diseases associated with CI, stroke alone causes up to 10% of deaths worldwide and is a leading cause of disability; yet treatment options are extremely limited, so this represents an area of massive unmet clinical need. Inflammation involving the cytokine interleukin-1 is a major contributor to cell death in the ischemic brain. Inflammation can be regulated by large protein complexes called inflammasomes. Here we show that the NLRC4 (NLR family, CARD domain containing 4) and AIM2 (absent in melanoma 2) inflammasomes, but not the NLRP3 (NLR family, pyrin domain containing 3) inflammasome, contribute to inflammation and injury in an ischemic brain and are thus potential therapeutic targets for these devastating diseases. Inflammation that contributes to acute cerebrovascular disease is driven by the proinflammatory cytokine interleukin-1 and is known to exacerbate resulting injury. The activity of interleukin-1 is regulated by multimolecular protein complexes called inflammasomes. There are multiple potential inflammasomes activated in diverse diseases, yet the nature of the inflammasomes involved in brain injury is currently unknown. Here, using a rodent model of stroke, we show that the NLRC4 (NLR family, CARD domain containing 4) and AIM2 (absent in melanoma 2) inflammasomes contribute to brain injury. We also show that acute ischemic brain injury is regulated by mechanisms that require ASC (apoptosis-associated speck-like protein containing a CARD), a common adaptor protein for several inflammasomes, and that the NLRP3 (NLR family, pyrin domain containing 3) inflammasome is not involved in this process. These discoveries identify the NLRC4 and AIM2 inflammasomes as potential therapeutic targets for stroke and provide new insights into how the inflammatory response is regulated after an acute injury to the brain.

Analysis of the Drosophila Clock Promoter Reveals Heterogeneity in Expression between Subgroups of Central Oscillator Cells and Identifies a Novel Enhancer Region

The CLOCK-CYCLE (CLK-CYC) heterodimer lies at the heart of the circadian oscillator mechanism in Drosophila, yet little is known about the identity of transcription factors that regulate the expression of Clk and/or cyc. Here, the authors have used a transgenic approach to isolate regions of the Clk locus that are necessary for expression in central oscillator neurons in the adult fly brain. This analysis shows that central clock cells can be subdivided into 2 distinct groups based on Clk gene regulation. Expression in the lateral neuron (LN), dorsal neuron 1 anterior (DN1a) and 2 (DN2) clusters requires cis-elements located in a 122 base-pair (bp) region (–206 to –84) of the Clk promoter. Expression in the remaining dorsal neurons, 1 posterior (DN1p) and 3 (DN3) and the lateral posterior neurons (LPN), requires regulatory elements located in the –856 to –206 region. In addition, expression in photoreceptors of the compound eye is enhanced by cis-elements located in a 3rd region of the Clk locus (–1982 to –856). This region also enhances expression in nonoscillator cells in the brain including the Kenyon cells, but expression in these neurons is suppressed by regulatory sites located further upstream of –1982. The authors’ analysis reveals clear heterogeneity in Clk gene expression in the adult brain and provides a necessary focus to isolate novel transcription factors that bind at the Clk locus to regulate expression in different oscillator neuron subgroups. These results also suggest that the DN1a/DN2 neurons may have more molecular commonality with the LNs than they do with the DN1p/DN3/LPN neurons. Finally, this analysis has generated new transgenic lines that will enable genes to be misexpressed in subgroups of central oscillator cells that have previously been resistant to discrete genetic manipulation. Hence, these lines provide important new tools to facilitate a more complete dissection of the neural network that regulates output rhythms in physiology and behavior.

Endogenous Oils Derived From Human Adipocytes Are Potent Adjuvants That Promote IL-1α–Dependent Inflammation

Obesity is characterized by chronic inflammation associated with neutrophil and M1 macrophage infiltration into white adipose tissue. However, the mechanisms underlying this process remain largely unknown. Based on the ability of oil-based adjuvants to induce immune responses, we hypothesized that endogenous oils derived from necrotic adipocytes may function as an immunological “danger signal.” Here we show that endogenous oils of human origin are potent adjuvants, enhancing antibody responses to a level comparable to Freund’s incomplete adjuvant. The endogenous oils were capable of promoting interleukin (IL)-1α–dependent recruitment of neutrophils and M1-like macrophages, while simultaneously diminishing M2-like macrophages. We found that endogenous oils from subcutaneous and omental adipocytes, and from healthy and unhealthy obese individuals, promoted comparable inflammatory responses. Furthermore, we also confirmed that white adipocytes in visceral fat of metabolically unhealthy obese (MUO) individuals are significantly larger than those in metabolically healthy obese individuals. Since adipocyte size is positively correlated with adipocyte death, we propose that endogenous oils have a higher propensity to be released from hypertrophied visceral fat in MUO individuals and that this is the key factor in driving inflammation. In summary, this study shows that adipocytes contain a potent oil adjuvant which drives IL-1α–dependent proinflammatory responses in vivo.

IL‐1α and inflammasome‐independent IL‐1β promote neutrophil infiltration following alum vaccination

Despite its long record of successful use in human vaccines, the mechanisms underlying the immunomodulatory effects of alum are not fully understood. Alum is a potent inducer of interleukin‐1 (IL‐1) secretion in vitro in dendritic cells and macrophages via Nucleotide‐binding domain and leucine‐rich repeat‐containing (NLR) family, pyrin domain‐containing 3 (NLRP3) inflammasome activation. However, the contribution of IL‐1 to alum‐induced innate and adaptive immune responses is controversial and the role of IL‐1α following alum injection has not been addressed. This study shows that IL‐1 is dispensable for alum‐induced antibody and CD8 T cell responses to ovalbumin. However, IL‐1 is essential for neutrophil infiltration into the injection site, while recruitment of inflammatory monocytes and eosinophils is IL‐1 independent. Both IL‐1α and IL‐1β are released at the site of injection and contribute to the neutrophil response. Surprisingly, these effects are NLRP3‐inflammasome independent as is the infiltration of other cell populations. However, while NLRP3 and caspase 1 were dispensable, alum‐induced IL‐1β at the injection site was dependent on the cysteine protease cathepsin S. Overall, these data demonstrate a previously unreported role for cathepsin S in IL‐1β secretion, show that inflammasome formation is dispensable for alum‐induced innate immunity and reveal that IL‐1α and IL‐1β are both necessary for alum‐induced neutrophil influx in vivo.

Recombinant tissue plasminogen activator enhances microglial cell recruitment after stroke in mice

The effect of recombinant human tissue plasminogen activator (rtPA) on neuroinflammation after stroke remains largely unknown. Here, we tested the effect of rtPA on expression of cellular adhesion molecules, chemokines, and cytokines, and compared those with levels of inflammatory cell recruitment, brain injury, and mortality over 3 days after transient middle cerebral artery occlusion (MCAO) in mice. Mortality was dramatically increased after rtPA treatment compared with saline treatment during the first day of reperfusion. Among the animals that survived, rtPA significantly increased CCL3 expression, microglia recruitment, and cerebral infarction 6 hours after MCAO. In contrast, the extent of neutrophils and macrophages infiltration in the brain was similar in both saline- and rtPA-treated animals. Recombinant human tissue plasminogen activator induced II 1b and Tnf expression, 6 and 72 hours after MCAO, respectively, and dramatically reduced interleukin 6 (IL-6) level 24 hours after reperfusion. A dose response study confirmed the effect of rtPA on CCL3 and II1b expressions. The effect was similar at the doses of 1 and 10 mg/kg. In conclusion, we report for the first time that rtPA amplified microglia recruitment early after stroke in association with a rapid CCL3 production. This early response may take part in the higher susceptibility of rtPA-treated animals to reperfusion injury.

Requirement for interleukin‐1 to drive brain inflammation reveals tissue‐specific mechanisms of innate immunity

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 defense 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‐1α 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.

Reparative effects of interleukin-1 receptor antagonist in young and aged/co-morbid rodents after cerebral ischemia

Highlights • IL-1β is a key proinflammatory cytokine involved in ischemic brain damage.• Administration of IL-1Ra improves the stroke outcome in young and co-morbid rats.• Acute IL-1Ra administration also promotes neurogenesis after experimental stroke.

Systemic Inflammation Affects Reperfusion Following Transient Cerebral Ischaemia

Reperfusion after stroke is critical for improved patient survival and recovery and can be achieved clinically through pharmacological (recombinant tissue plasminogen activator) or physical (endovascular intervention) means. Yet these approaches remain confined to a small percentage of stroke patients, often with incomplete reperfusion, and therefore there is an urgent need to learn more about the mechanisms underlying the no-reflow phenomenon that prevents restoration of adequate microvascular perfusion. Recent evidence suggests systemic inflammation as an important contributor to no-reflow and to further investigate this here we inject interleukin 1 (IL-1) i.p. 30 min prior to an ischaemic challenge using a remote filament to occlude the middle cerebral artery (MCA) in mice. Before, during and after the injection of IL-1 and occlusion we use two-dimensional optical imaging spectroscopy to record the spatial and temporal dynamics of oxyhaemoglobin concentration in the cortical areas supplied by the MCA. Our results reveal that systemic inflammation significantly reduces oxyhaemoglobin reperfusion as early as 3 h after filament removal compared to vehicle injected animals. CD41 immunohistochemistry shows a significant increase of hyper-coagulated platelets within the microvessels in the stroked cortex of the IL-1 group compared to vehicle. We also observed an increase of pathophysiological biomarkers of ischaemic damage including elevated microglial activation co-localized with interleukin 1α (IL-1α), increased blood brain barrier breakdown as shown by IgG infiltration and increased pyknotic morphological changes of cresyl violet stained neurons. These data confirm systemic inflammation as an underlying cause of no-reflow in the post-ischaemic brain and that appropriate anti-inflammatory approaches could be beneficial in treating ischaemic stroke.

Neutrophil infiltration to the brain is platelet-dependent, and is reversed by blockade of platelet GPIbα

Neutrophils are key components of the innate immune response, providing host defence against infection and being recruited to non‐microbial injury sites. Platelets act as a trigger for neutrophil extravasation to inflammatory sites but mechanisms and tissue‐specific aspects of these interactions are currently unclear. Here, we use bacterial endotoxin in mice to trigger an innate inflammatory response in different tissues and measure neutrophil invasion with or without platelet reduction. We show that platelets are essential for neutrophil infiltration to the brain, peritoneum and skin. Neutrophil numbers do not rise above basal levels in the peritoneum and skin and are decreased (~60%) in the brain when platelet numbers are reduced. In contrast neutrophil infiltration in the lung is unaffected by platelet reduction, up‐regulation of CXCL‐1 (2·4‐fold) and CCL5 (1·4‐fold) acting as a compensatory mechanism in platelet‐reduced mice during lung inflammation. In brain inflammation targeting platelet receptor GPIbα results in a significant decrease (44%) in platelet‐mediated neutrophil invasion, while maintaining platelet numbers in the circulation. These results suggest that therapeutic blockade of platelet GPIbα could limit the harmful effects of excessive inflammation while minimizing haemorrhagic complications of platelet reduction in the brain. The data also demonstrate the ability to target damaging brain inflammation in stroke and related disorders without compromising lung immunity and hence risk of pneumonia, a major complication post stroke. In summary, our data reveal an important role for platelets in neutrophil infiltration to various tissues, including the brain, and so implicate platelets as a key, targetable component of cerebrovascular inflammatory disease or injury.

Effects of TWIN-OF-EYELESS on Clock Gene Expression and Central-Pacemaker Neuron Development in Drosophila.

Circadian oscillators are autonomous molecular rhythms that reside in cells to align whole-organism physiology and behavior to the 24-h day. In flies, as in mammals, the oscillator operates in cells that coexpress CLOCK (CLK) and CYCLE (CYC). Recent work in Drosophila has shown that CLK is unique in its ability to generate heterologous oscillators, indicating that Clk gene expression defines the circadian cell fate. Here, using standard in vitro and in vivo techniques, we show that TWIN-OF-EYELESS (TOY; dPax6) regulates Clk expression in small ventrolateral neurons (s-LNvs) that coordinate sleep-wake cycles. Crucially, toy binds multiple sites at the Clk locus, is expressed independent of CLK-CYC in LNvs, regulates CLK protein levels under optimal photoperiodic conditions, and sets clock-speed during endogenous free-run. Furthermore, TOY is necessary for the onset of Clk expression in LNvs during embryogenesis. We propose that TOY contributes to a transcription complex that functions upstream of the oscillator to promote Clk expression in s-LNvs.