Paternal microbiome perturbations impact offspring fitness.

The gut microbiota operates at the interface of host-environment interactions to influence human homoeostasis and metabolic networks1-4. Environmental factors that unbalance gut microbial ecosystems can therefore shape physiological and disease-associated responses across somatic tissues5-9. However, the systemic impact of the gut microbiome on the germline-and consequently on the F1 offspring it gives rise to-is unexplored10. Here we show that the gut microbiota act as a key interface between paternal preconception environment and intergenerational health in mice. Perturbations to the gut microbiota of prospective fathers increase the probability of their offspring presenting with low birth weight, severe growth restriction and premature mortality. Transmission of disease risk occurs via the germline and is provoked by pervasive gut microbiome perturbations, including non-absorbable antibiotics or osmotic laxatives, but is rescued by restoring the paternal microbiota before conception. This effect is linked with a dynamic response to induced dysbiosis in the male reproductive system, including impaired leptin signalling, altered testicular metabolite profiles and remapped small RNA payloads in sperm. As a result, dysbiotic fathers trigger an elevated risk of in utero placental insufficiency, revealing a placental origin of mammalian intergenerational effects. Our study defines a regulatory 'gut-germline axis' in males, which is sensitive to environmental exposures and programmes offspring fitness through impacting placenta function.

A kinase-dead Csf1r mutation associated with adult-onset leukoencephalopathy has a dominant inhibitory impact on CSF1R signalling.

Amino acid substitutions in the kinase domain of the human CSF1R gene are associated with autosomal dominant adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). To model the human disease, we created a disease-associated mutation (pGlu631Lys; E631K) in the mouse Csf1r locus. Homozygous mutation (Csf1rE631K/E631K) phenocopied the Csf1r knockout, with prenatal mortality or severe postnatal growth retardation and hydrocephalus. Heterozygous mutation delayed the postnatal expansion of tissue macrophage populations in most organs. Bone marrow cells from Csf1rE631K/+mice were resistant to CSF1 stimulation in vitro, and Csf1rE631K/+ mice were unresponsive to administration of a CSF1-Fc fusion protein, which expanded tissue macrophage populations in controls. In the brain, microglial cell numbers and dendritic arborisation were reduced in Csf1rE631K/+ mice, as in patients with ALSP. The microglial phenotype is the opposite of microgliosis observed in Csf1r+/- mice. However, we found no evidence of brain pathology or impacts on motor function in aged Csf1rE631K/+ mice. We conclude that heterozygous disease-associated CSF1R mutations compromise CSF1R signalling. We speculate that leukoencephalopathy associated with dominant human CSF1R mutations requires an environmental trigger and/or epistatic interaction with common neurodegenerative disease-associated alleles.

Independent endothelial functions of PIEZO1 and TRPV4 in hepatic portal vein and predominance of PIEZO1 in mechanical and osmotic stress.

BACKGROUND & AIMS: PIEZO1 and TRPV4 are mechanically and osmotically regulated calcium-permeable channels. The aim of this study was to determine the relevance and relationship of these channels in the contractile tone of the hepatic portal vein, which experiences mechanical and osmotic variations as it delivers blood to the liver from the intestines, gallbladder, pancreas and spleen. METHODS: Wall tension was measured in freshly dissected portal veins from adult male mice, which were genetically unmodified or modified for either a non-disruptive tag in native PIEZO1 or endothelial-specific PIEZO1 deletion. Pharmacological agents were used to activate or inhibit PIEZO1, TRPV4 and associated pathways, including Yoda1 and Yoda2 for PIEZO1 and GSK1016790A for TRPV4 agonism, respectively. RESULTS: PIEZO1 activation leads to nitric oxide synthase- and endothelium-dependent relaxation of the portal vein. TRPV4 activation causes contraction, which is also endothelium-dependent but independent of nitric oxide synthase. The TRPV4-mediated contraction is suppressed by inhibitors of phospholipase A2 and cyclooxygenases and mimicked by prostaglandin E2 , suggesting mediation by arachidonic acid metabolism. TRPV4 antagonism inhibits the effect of agonising TRPV4 but not PIEZO1. Increased wall stretch and hypo-osmolality inhibit TRPV4 responses while lacking effects on or amplifying PIEZO1 responses. CONCLUSIONS: The portal vein contains independently functioning PIEZO1 channels and TRPV4 channels in the endothelium, the pharmacological activation of which leads to opposing effects of vessel relaxation (PIEZO1) and contraction (TRPV4). In mechanical and osmotic strain, the PIEZO1 mechanism dominates. Modulators of these channels could present important new opportunities for manipulating liver perfusion and regeneration in disease and surgical procedures.

Nuclear receptor REVERBα is a state-dependent regulator of liver energy metabolism.

The nuclear receptor REVERBα is a core component of the circadian clock and proposed to be a dominant regulator of hepatic lipid metabolism. Using antibody-independent ChIP-sequencing of REVERBα in mouse liver, we reveal a high-confidence cistrome and define direct target genes. REVERBα-binding sites are highly enriched for consensus RORE or RevDR2 motifs and overlap with corepressor complex binding. We find no evidence for transcription factor tethering and DNA-binding domain-independent action. Moreover, hepatocyte-specific deletion of Reverbα drives only modest physiological and transcriptional dysregulation, with derepressed target gene enrichment limited to circadian processes. Thus, contrary to previous reports, hepatic REVERBα does not repress lipogenesis under basal conditions. REVERBα control of a more extensive transcriptional program is only revealed under conditions of metabolic perturbation (including mistimed feeding, which is a feature of the global Reverbα-/- mouse). Repressive action of REVERBα in the liver therefore serves to buffer against metabolic challenge, rather than drive basal rhythmicity in metabolic activity.

Quantitative live imaging of Venus::BMAL1 in a mouse model reveals complex dynamics of the master circadian clock regulator.

Evolutionarily conserved circadian clocks generate 24-hour rhythms in physiology and behaviour that adapt organisms to their daily and seasonal environments. In mammals, the suprachiasmatic nucleus (SCN) of the hypothalamus is the principal co-ordinator of the cell-autonomous clocks distributed across all major tissues. The importance of robust daily rhythms is highlighted by experimental and epidemiological associations between circadian disruption and human diseases. BMAL1 (a bHLH-PAS domain-containing transcription factor) is the master positive regulator within the transcriptional-translational feedback loops (TTFLs) that cell-autonomously define circadian time. It drives transcription of the negative regulators Period and Cryptochrome alongside numerous clock output genes, and thereby powers circadian time-keeping. Because deletion of Bmal1 alone is sufficient to eliminate circadian rhythms in cells and the whole animal it has been widely used as a model for molecular disruption of circadian rhythms, revealing essential, tissue-specific roles of BMAL1 in, for example, the brain, liver and the musculoskeletal system. Moreover, BMAL1 has clock-independent functions that influence ageing and protein translation. Despite the essential role of BMAL1 in circadian time-keeping, direct measures of its intra-cellular behaviour are still lacking. To fill this knowledge-gap, we used CRISPR Cas9 to generate a mouse expressing a knock-in fluorescent fusion of endogenous BMAL1 protein (Venus::BMAL1) for quantitative live imaging in physiological settings. The Bmal1Venus mouse model enabled us to visualise and quantify the daily behaviour of this core clock factor in central (SCN) and peripheral clocks, with single-cell resolution that revealed its circadian expression, anti-phasic to negative regulators, nuclear-cytoplasmic mobility and molecular abundance.

A Transgenic Line That Reports CSF1R Protein Expression Provides a Definitive Marker for the Mouse Mononuclear Phagocyte System.

The proliferation, differentiation, and survival of cells of the mononuclear phagocyte system (MPS; progenitors, monocytes, macrophages, and classical dendritic cells) are controlled by signals from the M-CSF receptor (CSF1R). Cells of the MPS lineage have been identified using numerous surface markers and transgenic reporters, but none is both universal and lineage restricted. In this article, we report the development and characterization of a CSF1R reporter mouse. A FusionRed (FRed) cassette was inserted in-frame with the C terminus of CSF1R, separated by a T2A-cleavable linker. The insertion had no effect of CSF1R expression or function. CSF1R-FRed was expressed in monocytes and macrophages and absent from granulocytes and lymphocytes. In bone marrow, CSF1R-FRed was absent in lineage-negative hematopoietic stem cells, arguing against a direct role for CSF1R in myeloid lineage commitment. It was highly expressed in marrow monocytes and common myeloid progenitors but significantly lower in granulocyte-macrophage progenitors. In sections of bone marrow, CSF1R-FRed was also detected in osteoclasts, CD169+ resident macrophages, and, consistent with previous mRNA analysis, in megakaryocytes. In lymphoid tissues, CSF1R-FRed highlighted diverse MPS populations, including classical dendritic cells. Whole mount imaging of nonlymphoid tissues in mice with combined CSF1R-FRed/Csf1r-EGFP confirmed the restriction of CSF1R expression to MPS cells. The two markers highlight the remarkable abundance and regular distribution of tissue MPS cells, including novel macrophage populations within tendon and skeletal muscle and underlying the mesothelial/serosal/capsular surfaces of every major organ. The CSF1R-FRed mouse provides a novel reporter with exquisite specificity for cells of the MPS.

Pak2 as a Novel Therapeutic Target for Cardioprotective Endoplasmic Reticulum Stress Response.

RATIONALE: Secreted and membrane-bound proteins, which account for 1/3 of all proteins, play critical roles in heart health and disease. The endoplasmic reticulum (ER) is the site for synthesis, folding, and quality control of these proteins. Loss of ER homeostasis and function underlies the pathogenesis of many forms of heart disease. OBJECTIVE: To investigate mechanisms responsible for regulating cardiac ER function, and to explore therapeutic potentials of strengthening ER function to treat heart disease. METHODS AND RESULTS: Screening a range of signaling molecules led to the discovery that Pak (p21-activated kinase)2 is a stress-responsive kinase localized in close proximity to the ER membrane in cardiomyocytes. We found that Pak2 cardiac deleted mice (Pak2-CKO) under tunicamycin stress or pressure overload manifested a defective ER response, cardiac dysfunction, and profound cell death. Small chemical chaperone tauroursodeoxycholic acid treatment of Pak2-CKO mice substantiated that Pak2 loss-induced cardiac damage is an ER-dependent pathology. Gene array analysis prompted a detailed mechanistic study, which revealed that Pak2 regulation of protective ER function was via the IRE (inositol-requiring enzyme)-1/XBP (X-box-binding protein)-1-dependent pathway. We further discovered that this regulation was conferred by Pak2 inhibition of PP2A (protein phosphatase 2A) activity. Moreover, IRE-1 activator, Quercetin, and adeno-associated virus serotype-9-delivered XBP-1s were able to relieve ER dysfunction in Pak2-CKO hearts. This provides functional evidence, which supports the mechanism underlying Pak2 regulation of IRE-1/XBP-1s signaling. Therapeutically, inducing Pak2 activation by genetic overexpression or adeno-associated virus serotype-9-based gene delivery was capable of strengthening ER function, improving cardiac performance, and diminishing apoptosis, thus protecting the heart from failure. CONCLUSIONS: Our findings uncover a new cardioprotective mechanism, which promotes a protective ER stress response via the modulation of Pak2. This novel therapeutic strategy may present as a promising option for treating cardiac disease and heart failure.

ADAMTS10-mediated tissue disruption in Weill-Marchesani syndrome.

Fibrillin microfibrils are extracellular matrix assemblies that form the template for elastic fibres, endow blood vessels, skin and other elastic tissues with extensible properties. They also regulate the bioavailability of potent growth factors of the TGF-ß superfamily. A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)10 is an essential factor in fibrillin microfibril function. Mutations in fibrillin-1 or ADAMTS10 cause Weill-Marchesani syndrome (WMS) characterized by short stature, eye defects, hypermuscularity and thickened skin. Despite its importance, there is poor understanding of the role of ADAMTS10 and its function in fibrillin microfibril assembly. We have generated an ADAMTS10 WMS mouse model using Clustered Regularly Spaced Interspaced Short Palindromic Repeats and CRISPR associated protein 9 (CRISPR-Cas9) to introduce a truncation mutation seen in WMS patients. Homozygous WMS mice are smaller and have shorter long bones with perturbation to the zones of the developing growth plate and changes in cell proliferation. Furthermore, there are abnormalities in the ciliary apparatus of the eye with decreased ciliary processes and abundant fibrillin-2 microfibrils suggesting perturbation of a developmental expression switch. WMS mice have increased skeletal muscle mass and more myofibres, which is likely a consequence of an altered skeletal myogenesis. These results correlated with expression data showing down regulation of Growth differentiation factor (GDF8) and Bone Morphogenetic Protein (BMP) growth factor genes. In addition, the mitochondria in skeletal muscle are larger with irregular shape coupled with increased phospho-p38 mitogen-activated protein kinase (MAPK) suggesting muscle remodelling. Our data indicate that decreased SMAD1/5/8 and increased p38/MAPK signalling are associated with ADAMTS10-induced WMS. This model will allow further studies of the disease mechanism to facilitate the development of therapeutic interventions.

Distinct Cellular Mechanisms Underlie Smooth Muscle Turnover in Vascular Development and Repair.

RATIONALE: Vascular smooth muscle turnover has important implications for blood vessel repair and for the development of cardiovascular diseases, yet lack of specific transgenic animal models has prevented it's in vivo analysis. OBJECTIVE: The objective of this study was to characterize the dynamics and mechanisms of vascular smooth muscle turnover from the earliest stages of embryonic development to arterial repair in the adult. METHODS AND RESULTS: We show that CD146 is transiently expressed in vascular smooth muscle development. By using CRISPR-Cas9 genome editing and in vitro smooth muscle differentiation assay, we demonstrate that CD146 regulates the balance between proliferation and differentiation. We developed a triple-transgenic mouse model to map the fate of NG2+CD146+ immature smooth muscle cells. A series of pulse-chase experiments revealed that the origin of aortic vascular smooth muscle cells can be traced back to progenitor cells that reside in the wall of the dorsal aorta of the embryo at E10.5. A distinct population of CD146+ smooth muscle progenitor cells emerges during embryonic development and is maintained postnatally at arterial branch sites. To characterize the contribution of different cell types to arterial repair, we used 2 injury models. In limited wire-induced injury response, existing smooth muscle cells are the primary contributors to neointima formation. In contrast, microanastomosis leads to early smooth muscle death and subsequent colonization of the vascular wall by proliferative adventitial cells that contribute to the repair. CONCLUSIONS: Extensive proliferation of immature smooth muscle cells in the primitive embryonic dorsal aorta establishes the long-lived lineages of smooth muscle cells that make up the wall of the adult aorta. A discrete population of smooth muscle cells forms in the embryo and is postnatally sustained at arterial branch sites. In response to arterial injuries, existing smooth muscle cells give rise to neointima, but on extensive damage, they are replaced by adventitial cells.

Immunity to gastrointestinal nematodes: mechanisms and myths.

Immune responses to gastrointestinal nematodes have been studied extensively for over 80 years and intensively investigated over the last 30-40 years. The use of laboratory models has led to the discovery of new mechanisms of protective immunity and made major contributions to our fundamental understanding of both innate and adaptive responses. In addition to host protection, it is clear that immunoregulatory processes are common in infected individuals and resistance often operates alongside modulation of immunity. This review aims to discuss the recent discoveries in both host protection and immunoregulation against gastrointestinal nematodes, placing the data in context of the specific life cycles imposed by the different parasites studied and the future challenges of considering the mucosal/immune axis to encompass host, parasite, and microbiome in its widest sense.

Overexpression of IκB⍺ modulates NF-κB activation of inflammatory target gene expression.

Cells respond to inflammatory stimuli such as cytokines by activation of the nuclear factor-κB (NF-κB) signalling pathway, resulting in oscillatory translocation of the transcription factor p65 between nucleus and cytoplasm in some cell types. We investigate the relationship between p65 and inhibitor-κB⍺ (IκBα) protein levels and dynamic properties of the system, and how this interaction impacts on the expression of key inflammatory genes. Using bacterial artificial chromosomes, we developed new cell models of IκB⍺-eGFP protein overexpression in a pseudo-native genomic context. We find that cells with high levels of the negative regulator IκBα remain responsive to inflammatory stimuli and maintain dynamics for both p65 and IκBα. In contrast, canonical target gene expression is dramatically reduced by overexpression of IκBα, but can be partially rescued by overexpression of p65. Treatment with leptomycin B to promote nuclear accumulation of IκB⍺ also suppresses canonical target gene expression, suggesting a mechanism in which nuclear IκB⍺ accumulation prevents productive p65 interaction with promoter binding sites. This causes reduced target promoter binding and gene transcription, which we validate by chromatin immunoprecipitation and in primary cells. Overall, we show how inflammatory gene transcription is modulated by the expression levels of both IκB⍺ and p65. This results in an anti-inflammatory effect on transcription, demonstrating a broad mechanism to modulate the strength of inflammatory response.

RAC1B function is essential for breast cancer stem cell maintenance and chemoresistance of breast tumor cells.

Breast cancer stem cells (BCSC) are presumed to be responsible for treatment resistance, tumor recurrence and metastasis of breast tumors. However, development of BCSC-targeting therapies has been held back by their heterogeneity and the lack of BCSC-selective molecular targets. Here, we demonstrate that RAC1B, the only known alternatively spliced variant of the small GTPase RAC1, is expressed in a subset of BCSCs in vivo and its function is required for the maintenance of BCSCs and their chemoresistance to doxorubicin. In human breast cancer cell line MCF7, RAC1B is required for BCSC plasticity and chemoresistance to doxorubicin in vitro and for tumor-initiating abilities in vivo. Unlike Rac1, Rac1b function is dispensable for normal mammary gland development and mammary epithelial stem cell (MaSC) activity. In contrast, loss of Rac1b function in a mouse model of breast cancer hampers the BCSC activity and increases their chemosensitivity to doxorubicin treatment. Collectively, our data suggest that RAC1B is a clinically relevant molecular target for the development of BCSC-targeting therapies that may improve the effectiveness of doxorubicin-mediated chemotherapy.

A rare human variant that disrupts GPR10 signalling causes weight gain in mice.

Disruption of brain-expressed G protein-coupled receptor-10 (GPR10) causes obesity in animals. Here, we identify multiple rare variants in GPR10 in people with severe obesity and in normal weight controls. These variants impair ligand binding and G protein-dependent signalling in cells. Transgenic mice harbouring a loss of function GPR10 variant found in an individual with obesity, gain excessive weight due to decreased energy expenditure rather than increased food intake. This evidence supports a role for GPR10 in human energy homeostasis. Therapeutic targeting of GPR10 may represent an effective weight-loss strategy.

PIEZO1 and PECAM1 interact at cell-cell junctions and partner in endothelial force sensing.

Two prominent concepts for the sensing of shear stress by endothelium are the PIEZO1 channel as a mediator of mechanically activated calcium ion entry and the PECAM1 cell adhesion molecule as the apex of a triad with CDH5 and VGFR2. Here, we investigated if there is a relationship. By inserting a non-disruptive tag in native PIEZO1 of mice, we reveal in situ overlap of PIEZO1 with PECAM1. Through reconstitution and high resolution microscopy studies we show that PECAM1 interacts with PIEZO1 and directs it to cell-cell junctions. PECAM1 extracellular N-terminus is critical in this, but a C-terminal intracellular domain linked to shear stress also contributes. CDH5 similarly drives PIEZO1 to junctions but unlike PECAM1 its interaction with PIEZO1 is dynamic, increasing with shear stress. PIEZO1 does not interact with VGFR2. PIEZO1 is required in Ca2+-dependent formation of adherens junctions and associated cytoskeleton, consistent with it conferring force-dependent Ca2+ entry for junctional remodelling. The data suggest a pool of PIEZO1 at cell junctions, the coming together of PIEZO1 and PECAM1 mechanisms and intimate cooperation of PIEZO1 and adhesion molecules in tailoring junctional structure to mechanical requirement.

Helminth products bypass the need for TSLP in Th2 immune responses by directly modulating dendritic cell function.

Thymic stromal lymphopoietin (TSLP) is an interleukin (IL)-7-like cytokine, mainly expressed by epithelial cells, and key to the development of allergic responses. The well-documented involvement of TSLP in allergy has led to the conviction that TSLP promotes the development of inflammatory Th2 cell responses. However, we now report that the interaction of TSLP with its receptor (TSLPR) has no functional impact on the development of protective Th2 immune responses after infection with 2 helminth pathogens, Heligmosomoides polygyrus and Nippostrongylus brasiliensis. Mice deficient in the TSLP binding chain of the TSLPR (TSLPR(-/-)) exhibited normal Th2 cell differentiation, protective immunity and memory responses against these two distinct rodent helminths. In contrast TSLP was found to be necessary for the development of protective Th2 responses upon infection with the helminth Trichuris muris (T. muris). TSLP inhibited IL-12p40 production in response to T. muris infection, and treatment of TSLPR(-/-) animals with neutralizing anti-IL-12p40 monoclonal antibody (mAb) was able to reverse susceptibility and attenuate IFN-gamma production. We additionally demonstrated that excretory-secretory (ES) products from H. polygyrus and N. brasiliensis, but not T. muris, were capable of directly suppressing dendritic cell (DC) production of IL-12p40, thus bypassing the need for TSLP. Taken together, our data show that the primary function of TSLP is to directly suppress IL-12 secretion, thus supporting Th2 immune responses.

Chronic systemic infection exacerbates ischemic brain damage via a CCL5 (regulated on activation, normal T-cell expressed and secreted)-mediated proinflammatory response in mice.

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

IL-1-dependent, IL-1R1-independent resistance to gastrointestinal nematodes.

IL-1 null mice are unable to expel the intestinal nematode Trichuris muris; whereas WT littermates exhibit sterile immunity. Intriguingly the essential signalling components IL-1R1 and IL-1R accessory protein (AcP) are dispensable for expulsion of this parasite. IL-1 is thus critical for CD4(+) Th2-mediated immunity to T. muris; however, this action is independent of the established IL-1 signalling receptor. We also present data demonstrating that both IL-1alpha and IL-1beta induce measurable effects on T. muris primed cells isolated from IL-1R1 or IL-1R AcP null mice. MLN cells from these mice restimulated with parasite antigen proliferated at a greater rate and produced more cytokines in response to exogenous IL-1. This ability to respond to IL-1 was restricted to these parasite-primed cells and importantly was not evident in cells from naïve gene null mice. These in vitro data are consistent with the observed ability of mice with compromised IL-1 signalling to expel the parasite, bolstering the premise that an alternative IL-1 signalling mechanism is accessible in the context of an intestinal helminth-driven Th2 immune response.

Cell-specific conditional deletion of interleukin-1 (IL-1) ligands and its receptors: a new toolbox to study the role of IL-1 in health and disease.

The pro-inflammatory cytokine interleukin-1 (IL-1) plays a key role in many physiological processes and during the inflammatory and immune response to most common diseases. IL-1 exists as two agonists, IL-1α and IL-1ß that bind to the only signaling IL-1 type 1 receptor (IL-1R1), while a second decoy IL-1 type 2 receptor (IL-1R2) binds both forms of IL-1 without inducing cell signaling. The field of immunology and inflammation research has, over the past 35 years, unraveled many mechanisms of IL-1 actions, through in vitro manipulation of the IL-1 system or by using genetically engineered mouse models that lack either member of the IL-1 family in ubiquitous constitutive manner. However, the limitation of global mouse knockout technology has significantly hampered our understanding of the precise mechanisms of IL-1 actions in animal models of disease. Here we report and review the recent generation of new conditional mouse mutants in which exons of Il1a, Il1b, Il1r1, and Il1r2 genes flanked by loxP sites (fl/fl) can be deleted in cell-/tissue-specific constitutive or inducible manner by Cre recombinase expression. Hence, IL-1αfl/fl, IL-1ßfl/fl, IL-1R1fl/fl, and IL-1R2fl/fl mice constitute a new toolbox that will provide a step change in our understanding of the cell-specific role of IL-1 and its receptor in health and disease and the potential development of targeted IL-1 therapies.

LRRC8A is essential for hypotonicity-, but not for DAMP-induced NLRP3 inflammasome activation.

The NLRP3 inflammasome is a multi-molecular protein complex that converts inactive cytokine precursors into active forms of IL-1ß and IL-18. The NLRP3 inflammasome is frequently associated with the damaging inflammation of non-communicable disease states and is considered an attractive therapeutic target. However, there is much regarding the mechanism of NLRP3 activation that remains unknown. Chloride efflux is suggested as an important step in NLRP3 activation, but which chloride channels are involved is still unknown. We used chemical, biochemical, and genetic approaches to establish the importance of chloride channels in the regulation of NLRP3 in murine macrophages. Specifically, we identify LRRC8A, an essential component of volume-regulated anion channels (VRAC), as a vital regulator of hypotonicity-induced, but not DAMP-induced, NLRP3 inflammasome activation. Although LRRC8A was dispensable for canonical DAMP-dependent NLRP3 activation, this was still sensitive to chloride channel inhibitors, suggesting there are additional and specific chloride sensing and regulating mechanisms controlling NLRP3.

Inflammation is a critical part of a healthy immune system, which protects us against harmful pathogens (such as bacteria or viruses) and works to restore damaged tissues. In the immune cells of our body, the inflammatory process can be activated through a group of inflammatory proteins that together are known as the NLRP3 inflammasome complex. While inflammation is a powerful mechanism that protects the human body, persistent or uncontrolled inflammation can cause serious, long-term damage. The inappropriate activation of the NLRP3 inflammasome has been implicated in several diseases, including Alzheimer's disease, heart disease, and diabetes. The NLRP3 inflammasome can be activated by different stimuli, including changes in cell volume and exposure to either molecules produced by damaged cells or toxins from bacteria. However, the precise mechanism through which the NLRP3 becomes activated in response to these stimuli was not clear. The exit of chloride ions from immune cells is known to activate the NLRP3 inflammasome. Chloride ions exit the cell through proteins called anion channels, including volume-regulated anion channels (VRACs), which respond to changes in cell volume. Green et al. have found that, in immune cells from mice grown in the lab called macrophages, VRACs are the only chloride channels involved in activating the NLRP3 inflammasome when the cell's volume changes. However, when the macrophages are exposed to molecules produced by damaged cells or toxins from bacteria, Green et al. discovered that other previously unidentified chloride channels are involved in activating the NLRP3 inflammasome. These results suggest that it might be possible to develop drugs to prevent the activation of the NLRP3 inflammasome that selectively target specific sets of chloride channels depending on which stimuli are causing the inflammation. Such a selective approach would minimise the side effects associated with drugs that generically suppress all NLRP3 activity by directly binding to NLRP3 itself. Ultimately, this may help guide the development of new, targeted anti-inflammatory drugs that can help treat the symptoms of a variety of diseases in humans.