Common PIEZO1 Allele in African Populations Causes RBC Dehydration and Attenuates Plasmodium Infection.

Hereditary xerocytosis is thought to be a rare genetic condition characterized by red blood cell (RBC) dehydration with mild hemolysis. RBC dehydration is linked to reduced Plasmodium infection in vitro; however, the role of RBC dehydration in protection against malaria in vivo is unknown. Most cases of hereditary xerocytosis are associated with gain-of-function mutations in PIEZO1, a mechanically activated ion channel. We engineered a mouse model of hereditary xerocytosis and show that Plasmodium infection fails to cause experimental cerebral malaria in these mice due to the action of Piezo1 in RBCs and in T cells. Remarkably, we identified a novel human gain-of-function PIEZO1 allele, E756del, present in a third of the African population. RBCs from individuals carrying this allele are dehydrated and display reduced Plasmodium infection in vitro. The existence of a gain-of-function PIEZO1 at such high frequencies is surprising and suggests an association with malaria resistance.

GPR68 Senses Flow and Is Essential for Vascular Physiology.

Mechanotransduction plays a crucial role in vascular biology. One example of this is the local regulation of vascular resistance via flow-mediated dilation (FMD). Impairment of this process is a hallmark of endothelial dysfunction and a precursor to a wide array of vascular diseases, such as hypertension and atherosclerosis. Yet the molecules responsible for sensing flow (shear stress) within endothelial cells remain largely unknown. We designed a 384-well screening system that applies shear stress on cultured cells. We identified a mechanosensitive cell line that exhibits shear stress-activated calcium transients, screened a focused RNAi library, and identified GPR68 as necessary and sufficient for shear stress responses. GPR68 is expressed in endothelial cells of small-diameter (resistance) arteries. Importantly, Gpr68-deficient mice display markedly impaired acute FMD and chronic flow-mediated outward remodeling in mesenteric arterioles. Therefore, GPR68 is an essential flow sensor in arteriolar endothelium and is a critical signaling component in cardiovascular pathophysiology.

LRRC8 Proteins Form Volume-Regulated Anion Channels that Sense Ionic Strength.

The volume-regulated anion channel (VRAC) is activated when a cell swells, and it plays a central role in maintaining cell volume in response to osmotic challenges. SWELL1 (LRRC8A) was recently identified as an essential component of VRAC. However, the identity of the pore-forming subunits of VRAC and how the channel is gated by cell swelling are unknown. Here, we show that SWELL1 and up to four other LRRC8 subunits assemble into heterogeneous complexes of ∼800 kDa. When reconstituted into bilayers, LRRC8 complexes are sufficient to form anion channels activated by osmolality gradients. In bilayers, as well as in cells, the single-channel conductance of the complexes depends on the LRRC8 composition. Finally, low ionic strength (Γ) in the absence of an osmotic gradient activates the complexes in bilayers. These data demonstrate that LRRC8 proteins together constitute the VRAC pore and that hypotonic stress can activate VRAC through a decrease in cytoplasmic Γ.

Endothelial cells are central orchestrators of cytokine amplification during influenza virus infection.

Cytokine storm during viral infection is a prospective predictor of morbidity and mortality, yet the cellular sources remain undefined. Here, using genetic and chemical tools to probe functions of the S1P(1) receptor, we elucidate cellular and signaling mechanisms that are important in initiating cytokine storm. Whereas S1P(1) receptor is expressed on endothelial cells and lymphocytes within lung tissue, S1P(1) agonism suppresses cytokines and innate immune cell recruitment in wild-type and lymphocyte-deficient mice, identifying endothelial cells as central regulators of cytokine storm. Furthermore, our data reveal immune cell infiltration and cytokine production as distinct events that are both orchestrated by endothelial cells. Moreover, we demonstrate that suppression of early innate immune responses through S1P(1) signaling results in reduced mortality during infection with a human pathogenic strain of influenza virus. Modulation of endothelium with a specific agonist suggests that diseases in which amplification of cytokine storm is a significant pathological component could be chemically tractable.

Piezo1, a mechanically activated ion channel, is required for vascular development in mice.

Mechanosensation is perhaps the last sensory modality not understood at the molecular level. Ion channels that sense mechanical force are postulated to play critical roles in a variety of biological processes including sensing touch/pain (somatosensation), sound (hearing), and shear stress (cardiovascular physiology); however, the identity of these ion channels has remained elusive. We previously identified Piezo1 and Piezo2 as mechanically activated cation channels that are expressed in many mechanosensitive cell types. Here, we show that Piezo1 is expressed in endothelial cells of developing blood vessels in mice. Piezo1-deficient embryos die at midgestation with defects in vascular remodeling, a process critically influenced by blood flow. We demonstrate that Piezo1 is activated by shear stress, the major type of mechanical force experienced by endothelial cells in response to blood flow. Furthermore, loss of Piezo1 in endothelial cells leads to deficits in stress fiber and cellular orientation in response to shear stress, linking Piezo1 mechanotransduction to regulation of cell morphology. These findings highlight an essential role of mammalian Piezo1 in vascular development during embryonic development.

Bitopic Sphingosine 1-Phosphate Receptor 3 (S1P3) Antagonist Rescue from Complete Heart Block: Pharmacological and Genetic Evidence for Direct S1P3 Regulation of Mouse Cardiac Conduction.

The molecular pharmacology of the G protein-coupled receptors for sphingosine 1-phosphate (S1P) provides important insight into established and new therapeutic targets. A new, potent bitopic S1P3 antagonist, SPM-354, with in vivo activity, has been used, together with S1P3-knockin and S1P3-knockout mice to define the spatial and functional properties of S1P3 in regulating cardiac conduction. We show that S1P3 is a key direct regulator of cardiac rhythm both in vivo and in isolated perfused hearts. 2-Amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol in vivo and S1P in isolated hearts induced a spectrum of cardiac effects, ranging from sinus bradycardia to complete heart block, as measured by a surface electrocardiogram in anesthetized mice and in volume-conducted Langendorff preparations. The agonist effects on complete heart block are absent in S1P3-knockout mice and are reversed in wild-type mice with SPM-354, as characterized and described here. Homologous knockin of S1P3-mCherry is fully functional pharmacologically and is strongly expressed by immunohistochemistry confocal microscopy in Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4 (HCN4)-positive atrioventricular node and His-Purkinje fibers, with relative less expression in the HCN4-positive sinoatrial node. In Langendorff studies, at constant pressure, SPM-354 restored sinus rhythm in S1P-induced complete heart block and fully reversed S1P-mediated bradycardia. S1P3 distribution and function in the mouse ventricular cardiac conduction system suggest a direct mechanism for heart block risk that should be further studied in humans. A richer understanding of receptor and ligand usage in the pacemaker cells of the cardiac system is likely to be useful in understanding ventricular conduction in health, disease, and pharmacology.

Chemical and genetic tools to explore S1P biology.

The zwitterionic lysophospholipid Sphingosine 1-Phosphate (S1P) is a pleiotropic mediator of physiology and pathology. The synthesis, transport, and degradation of S1P are tightly regulated to ensure that S1P is present in the proper concentrations in the proper location. The binding of S1P to five G protein-coupled S1P receptors regulates many physiological systems, particularly the immune and vascular systems. Our understanding of the functions of S1P has been aided by the tractability of the system to both chemical and genetic manipulation. Chemical modulators have been generated to affect most of the known components of S1P biology, including agonists of S1P receptors and inhibitors of enzymes regulating S1P production and degradation. Genetic knockouts and manipulations have been similarly engineered to disrupt the functions of individual S1P receptors or enzymes involved in S1P metabolism. This chapter will focus on the development and utilization of these chemical and genetic tools to explore the complex biology surrounding S1P and its receptors, with particular attention paid to the in vivo findings that these tools have allowed for.

Sphingosine 1-phosphate receptor 1 (S1P(1)) upregulation and amelioration of experimental autoimmune encephalomyelitis by an S1P(1) antagonist.

Sphingosine 1-phosphate receptor 1 (S1P(1)) is a G protein-coupled receptor that is critical for proper lymphocyte development and recirculation. Agonists to S1P(1) are currently in use clinically for the treatment of multiple sclerosis, and these drugs may act on both S1P(1) expressed on lymphocytes and S1P(1) expressed within the central nervous system. Agonists to S1P(1) and deficiency in S1P(1) both cause lymphocyte sequestration in the lymph nodes. In the present study, we show that S1P(1) antagonism induces lymphocyte sequestration in the lymph nodes similar to that observed with S1P(1) agonists while upregulating S1P(1) on lymphocytes and endothelial cells. Additionally, we show that S1P(1) antagonism reverses experimental autoimmune encephalomyelitis in mice without acting on S1P(1) expressed within the central nervous system, demonstrating that lymphocyte sequestration via S1P(1) antagonism is sufficient to alleviate autoimmune pathology.

Actions of a picomolar short-acting S1P1 agonist in S1P1-eGFP knock-in mice.

Sphingosine 1-phosphate receptor 1 (S1P(1)) is critical for lymphocyte recirculation and is a clinical target for treatment of multiple sclerosis. By generating a short-duration S1P(1) agonist and mice in which fluorescently tagged S1P(1) replaces wild-type receptor, we elucidate physiological and agonist-perturbed changes in expression of S1P(1) at a subcellular level in vivo. We demonstrate differential downregulation of S1P(1) on lymphocytes and endothelia after agonist treatment.

Real-time differential labeling of blood, interstitium, and lymphatic and single-field analysis of vasculature dynamics in vivo.

Lymph nodes are highly organized structures specialized for efficient regulation of adaptive immunity. The blood and lymphatic systems within a lymph node play essential roles by providing functionally distinct environments for lymphocyte entry and egress, respectively. Direct imaging and measurement of vascular microenvironments by intravital multiphoton microscopy provide anatomical and mechanistic insights into the essential events of lymphocyte trafficking. Lymphocytes, blood endothelial cells, and lymphatic endothelial cells express sphingosine 1-phosphate receptor 1, a key G protein-coupled receptor regulating cellular egress and a modulator of endothelial permeability. Here we report the development of a differential vascular labeling (DVL) technique in which a single intravenous injection of a fluorescent dextran, in combination with fluorescent semiconductor quantum dot particles, differentially labels multiple blood and lymphatic compartments in a manner dependent on the size of the fluorescent particle used. Thus DVL allows measurement of endothelial integrity in multiple vascular compartments and the affects or pharmacological manipulation in vascular integrity. In addition, this technique allows for real-time observation of lymphocyte trafficking across physiological barriers differentiated by DVL. Last, single-field fluid movement dynamics can be derived, allowing for the simultaneous determination of fluid flow rates in diverse blood and lymphatic compartments.

S1P(1) receptor modulation with cyclical recovery from lymphopenia ameliorates mouse model of multiple sclerosis.

Multiple sclerosis (MS) therapies modulate T-cell autoimmunity in the central nervous system (CNS) but may exacerbate latent infections. Fingolimod, a nonselective sphingosine-1-phosphate (S1P) receptor agonist that induces sustained lymphopenia and accumulates in the CNS, represents a new treatment modality for MS. We hypothesized that sustained lymphopenia would not be required for efficacy and that a selective, CNS-penetrant, peripherally short-acting, S1P(1) agonist would show full efficacy in a mouse MS model. Using daily treatment with 10 mg/kg 2-(4-(5-(3,4-diethoxyphenyl)-1,2,4-oxadiazol-3-yl)-2,3-dihydro-1H-inden-1-yl amino)ethanol (CYM-5442) at the onset of clinical signs in myelin oligodendrocyte glycoprotein MOG(35-55)- induced experimental allergic encephalomyelitis (EAE), we assessed clinical scores, CNS cellular infiltration, demyelination, and gliosis for 12 days with CYM-5442, vehicle, or fingolimod. CYM-5442 levels in CNS and plasma were determined at experiment termination, and blood lymphopenia was measured 3 and 24 h after the last injection. Plasma levels of cytokines were assayed at the end of the protocol. Changes in S1P(1)-enhanced green fluorescent protein expression on neurons and astrocytes during active EAE and upon CYM-5442 treatment were quantified with flow cytometry and Western blotting by using native-locus enhanced green fluorescent protein-tagged S1P(1) mice. S1P(1) agonism alone reduced pathological features as did fingolimod (maximally lymphopenic throughout), despite full reversal of lymphopenia within each dosing interval. CYM-5442 levels in CNS but not in plasma were sustained. Neuronal and astrocytic S1P(1) expression in EAE was suppressed by CYM-5442 treatment, relative to vehicle, and levels of key cytokines, such as interleukin 17A, were also significantly reduced in drug-treated mice. S1P(1)-selective agonists that induce reversible lymphopenia while persisting in the CNS may be effective MS treatments.

Modulating tone: the overture of S1P receptor immunotherapeutics.

SUMMARY: Modulation of complex functions within the immune system has proven to be surprisingly sensitive to alterations in the lysophospholipid sphingosine 1-phosphate (S1P) receptor-ligand rheostat. This has become increasingly evident from both chemical and genetic manipulation of the S1P system, with pharmacological effects upon lymphoid cells, dendritic cell function, as well as vascular interfaces. The integrated immune system, perhaps as a result of its relatively recent evolutionary ontogeny, has selected for a number of critical control points regulated by five distinct high affinity G-protein-coupled receptor subtypes with a shared ligand, with receptors distributed on lymphocytes, dendritic cells, and endothelium. All of these cellular components of the axis are capable of modulating immune responses in vivo, with the impact on the immune response being very different from classical immunosuppressants, by virtue of selective spatial and temporal sparing of humoral and myeloid elements of host defense. Pharmacological subversion of the S1P rheostat is proving to be clinically efficacious in multiple sclerosis, and both the scope and limitations of therapeutic modulation of the S1P axis in immunotherapy are becoming clearer as understanding of the integrated chemical physiology of the S1P system emerges.

Piezo1 channels restrain regulatory T cells but are dispensable for effector CD4+ T cell responses.

T lymphocytes encounter complex mechanical cues during an immune response. The mechanosensitive ion channel, Piezo1, drives inflammatory responses to bacterial infections, wound healing, and cancer; however, its role in helper T cell function remains unclear. In an animal model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we found that mice with genetic deletion of Piezo1 in T cells showed diminished disease severity. Unexpectedly, Piezo1 was not essential for lymph node homing, interstitial motility, Ca2+ signaling, T cell proliferation, or differentiation into proinflammatory T helper 1 (TH1) and TH17 subsets. However, Piezo1 deletion in T cells resulted in enhanced transforming growth factor-ß (TGFß) signaling and an expanded pool of regulatory T (Treg) cells. Moreover, mice with deletion of Piezo1 specifically in Treg cells showed significant attenuation of EAE. Our results indicate that Piezo1 selectively restrains Treg cells, without influencing activation events or effector T cell functions.

OSCA/TMEM63 are an Evolutionarily Conserved Family of Mechanically Activated Ion Channels.

Mechanically activated (MA) ion channels convert physical forces into electrical signals, and are essential for eukaryotic physiology. Despite their importance, few bona-fide MA channels have been described in plants and animals. Here, we show that various members of the OSCA and TMEM63 family of proteins from plants, flies, and mammals confer mechanosensitivity to naïve cells. We conclusively demonstrate that OSCA1.2, one of the Arabidopsis thaliana OSCA proteins, is an inherently mechanosensitive, pore-forming ion channel. Our results suggest that OSCA/TMEM63 proteins are the largest family of MA ion channels identified, and are conserved across eukaryotes. Our findings will enable studies to gain deep insight into molecular mechanisms of MA channel gating, and will facilitate a better understanding of mechanosensory processes in vivo across plants and animals.

Structure of the human volume regulated anion channel.

SWELL1 (LRRC8A) is the only essential subunit of the Volume Regulated Anion Channel (VRAC), which regulates cellular volume homeostasis and is activated by hypotonic solutions. SWELL1, together with four other LRRC8 family members, potentially forms a vastly heterogeneous cohort of VRAC channels with different properties; however, SWELL1 alone is also functional. Here, we report a high-resolution cryo-electron microscopy structure of full-length human homo-hexameric SWELL1. The structure reveals a trimer of dimers assembly with symmetry mismatch between the pore-forming domain and the cytosolic leucine-rich repeat (LRR) domains. Importantly, mutational analysis demonstrates that a charged residue at the narrowest constriction of the homomeric channel is an important pore determinant of heteromeric VRAC. Additionally, a mutation in the flexible N-terminal portion of SWELL1 affects pore properties, suggesting a putative link between intracellular structures and channel regulation. This structure provides a scaffold for further dissecting the heterogeneity and mechanism of activation of VRAC.

Endogenous Piezo1 Can Confound Mechanically Activated Channel Identification and Characterization.

A gold standard for characterizing mechanically activated (MA) currents is via heterologous expression of candidate channels in naive cells. Two recent studies described MA channels using this paradigm. TMEM150c was proposed to be a component of an MA channel partly based on a heterologous expression approach (Hong et al., 2016). In another study, Piezo1's N-terminal "propeller" domain was proposed to constitute an intrinsic mechanosensitive module based on expression of a chimera between a pore-forming domain of the mechanically insensitive ASIC1 channel and Piezo1 (Zhao et al., 2016). When we attempted to replicate these results, we found each construct conferred modest MA currents in a small fraction of naive HEK cells similar to the published work. Strikingly, these MA currents were not detected in cells in which endogenous Piezo1 was CRISPR/Cas9 inactivated. These results highlight the importance of choosing cells lacking endogenous MA channels to assay the mechanotransduction properties of various proteins. This Matters Arising paper is in response to Hong et al. (2016) and Zhao et al. (2016) in Neuron. See also the response papers by Hong et al. (2017) and Zhao et al. (2017) published concurrently with this Matters Arising.

Piezo1 links mechanical forces to red blood cell volume.

Red blood cells (RBCs) experience significant mechanical forces while recirculating, but the consequences of these forces are not fully understood. Recent work has shown that gain-of-function mutations in mechanically activated Piezo1 cation channels are associated with the dehydrating RBC disease xerocytosis, implicating a role of mechanotransduction in RBC volume regulation. However, the mechanisms by which these mutations result in RBC dehydration are unknown. In this study, we show that RBCs exhibit robust calcium entry in response to mechanical stretch and that this entry is dependent on Piezo1 expression. Furthermore, RBCs from blood-cell-specific Piezo1 conditional knockout mice are overhydrated and exhibit increased fragility both in vitro and in vivo. Finally, we show that Yoda1, a chemical activator of Piezo1, causes calcium influx and subsequent dehydration of RBCs via downstream activation of the KCa3.1 Gardos channel, directly implicating Piezo1 signaling in RBC volume control. Therefore, mechanically activated Piezo1 plays an essential role in RBC volume homeostasis.

Impaired PIEZO1 function in patients with a novel autosomal recessive congenital lymphatic dysplasia.

Piezo1 ion channels are mediators of mechanotransduction in several cell types including the vascular endothelium, renal tubular cells and erythrocytes. Gain-of-function mutations in PIEZO1 cause an autosomal dominant haemolytic anaemia in humans called dehydrated hereditary stomatocytosis. However, the phenotypic consequence of PIEZO1 loss of function in humans has not previously been documented. Here we discover a novel role of this channel in the lymphatic system. Through whole-exome sequencing, we identify biallelic mutations in PIEZO1 (a splicing variant leading to early truncation and a non-synonymous missense variant) in a pair of siblings affected with persistent lymphoedema caused by congenital lymphatic dysplasia. Analysis of patients' erythrocytes as well as studies in a heterologous system reveal greatly attenuated PIEZO1 function in affected alleles. Our results delineate a novel clinical category of PIEZO1-associated hereditary lymphoedema.

Dehydrated hereditary stomatocytosis linked to gain-of-function mutations in mechanically activated PIEZO1 ion channels.

Dehydrated hereditary stomatocytosis is a genetic condition with defective red blood cell membrane properties that causes an imbalance in intracellular cation concentrations. Recently, two missense mutations in the mechanically activated PIEZO1 (FAM38A) ion channel were associated with dehydrated hereditary stomatocytosis. However, it is not known how these mutations affect PIEZO1 function. Here, by combining linkage analysis and whole-exome sequencing in a large pedigree and Sanger sequencing in two additional kindreds and 11 unrelated dehydrated hereditary stomatocytosis cases, we identify three novel missense mutations and one recurrent duplication in PIEZO1, demonstrating that it is the major gene for dehydrated hereditary stomatocytosis. All the dehydrated hereditary stomatocytosis-associated mutations locate at C-terminal half of PIEZO1. Remarkably, we find that all PIEZO1 mutations give rise to mechanically activated currents that inactivate more slowly than wild-type currents. This gain-of-function PIEZO1 phenotype provides insight that helps to explain the increased permeability of cations in red blood cells of dehydrated hereditary stomatocytosis patients. Our findings also suggest a new role for mechanotransduction in red blood cell biology and pathophysiology.