Pharmacological inhibition of PI5P4Kα/β disrupts cell energy metabolism and selectively kills p53-null tumor cells

Most human cancer cells harbor loss-of-function mutations in the p53 tumor suppressor gene. Genetic experiments have shown that phosphatidylinositol 5-phosphate 4-kinase α and β (PI5P4Kα and PI5P4Kβ) are essential for the development of late-onset tumors in mice with germline p53 deletion, but the mechanism underlying this acquired dependence remains unclear. PI5P4K has been previously implicated in metabolic regulation. Here, we show that inhibition of PI5P4Kα/β kinase activity by a potent and selective small-molecule probe disrupts cell energy homeostasis, causing AMPK activation and mTORC1 inhibition in a variety of cell types. Feedback through the S6K/insulin receptor substrate (IRS) loop contributes to insulin hypersensitivity and enhanced PI3K signaling in terminally differentiated myotubes. Most significantly, the energy stress induced by PI5P4Kαβ inhibition is selectively toxic toward p53-null tumor cells. The chemical probe, and the structural basis for its exquisite specificity, provide a promising platform for further development, which may lead to a novel class of diabetes and cancer drugs.

There are two synthetic routes for phosphatidylinositol 4,5-bisphosphate, or PI(4,5)P₂, a versatile phospholipid with both structural and signaling functions in most eukaryotic cells (1 –3). The bulk of PI(4,5)P₂ is found at the inner leaflet of the plasma membrane and is synthesized from phosphatidylinositol 4-phosphate, or PI(4)P, by type 1 phosphatidylinositol phosphate kinase PI4P5K (4, 5). A smaller fraction of PI(4,5)P₂ is generated from the much rarer phosphatidylinositol 5-phosphate, or PI(5)P, through the activity of type 2 phosphatidylinositol phosphate kinase PI5P4K (6, 7). Although PI5P4K is as abundantly expressed as PI4P5K (8), its function is less well understood (9). It has been proposed that PI5P4K may play a role in suppressing PI(5)P, which is often elevated by stress (10, 11), or produce local pools of PI(4,5)P₂ at subcellular compartments such as Golgi and nucleus (12).Higher animals have three PI5P4K isoforms, α, β, and γ, which are encoded by three different genes, PIP4K2A, PIP4K2B, and PIP4K2C. The three isoforms differ, at least in vitro, significantly in enzymatic activity: PI5P4Kα is two orders of magnitude more active than PI5P4Kβ, while PI5P4K-γ has very little activity (13). PI5P4Ks are dimeric proteins (14), and the possibility that they can form heterodimers may have important functional implications, especially for the lesser active isoforms (15, 16). PI5P4Kβ is the only isoform that preferentially localizes to the nucleus (17).Genetic studies have implicated PI5P4Kβ in metabolic regulation (18, 19). Mice with both PIP4K2B genes inactivated manifest hypersensitivity to insulin stimulation (adult males are also leaner). Although this is consistent with the observation that PI(5)P levels, which can be manipulated by overexpressing PI5P4K or a bacterial phosphatase that robustly produces PI(5)P from PI(4,5)P₂, correlate positively with PI3K/Akt signaling, the underlying molecular mechanisms remain undefined (20). Both male and female PIP4K2B ^−/− mice are mildly growth retarded. Inactivation of the only PI5P4K isoform in Drosophila also produced small and developmentally delayed animals (21). These phenotypes may be related to suppressed TOR signaling (22, 23), but again, the underlying mechanism is unclear since TORC1 is downstream of, and positively regulated by, PI3K/Akt. Knocking out the enzymatically more active PI5P4Kα, in contrast, did not produce any overt metabolic or developmental phenotypes (19).Malignant transformation is associated with profound changes in cell metabolism (24, 25). Although metabolic reprograming generally benefits tumor cells by increasing energy and material supplies, it can also, counterintuitively, generate unique dependencies (26, 27). Loss of p53, a tumor suppressor that is mutated in most human cancers, has been shown to render cells more susceptible to nutrient stress (28, 29) and to the antidiabetic drug metformin (30, 31). Although TP53 ^−/− and PIP4K2B ^−/− mice are themselves viable, combining the two is embryonically lethal (19). Knocking out three copies of PI5P4K (PIP4K2A ^−/− PIP4K2B ^+/− ) greatly reduces tumor formation and cancer-related death in TP53 ^−/− animals (19). The synthetic lethal interaction between p53 and PI5P4Kα/β was thought to result from suppressed glycolysis and increased reactive oxygen species (19), although how the lipid kinases impact glucose metabolism remains uncertain.Given the interest in the physiological function of this alternative synthetic route for PI(4,5)P₂, and the potential of PI5P4K inactivation in treating type 2 diabetes and cancer, several attempts have been made to identify chemical probes that target various PI5P4K isoforms, which yielded compounds with micromolar affinity and unknown selectivity (32 –35). Here, we report the development of a class of PI5P4Kα/β inhibitors that have much improved potency and better-defined selectivity. Using the chemical probe, we show that transient inhibition of the lipid kinases alters cell energy metabolism and induces different responses in muscle and cancer cells.     

The β-encapsulation cage of rearrangement hotspot (Rhs) effectors is required for type VI secretion

Bacteria deploy rearrangement hotspot (Rhs) proteins as toxic effectors against both prokaryotic and eukaryotic target cells. Rhs proteins are characterized by YD-peptide repeats, which fold into a large β-cage structure that encapsulates the C-terminal toxin domain. Here, we show that Rhs effectors are essential for type VI secretion system (T6SS) activity in Enterobacter cloacae (ECL). ECL rhs⁻ mutants do not kill Escherichia coli target bacteria and are defective for T6SS-dependent export of hemolysin-coregulated protein (Hcp). The RhsA and RhsB effectors of ECL both contain Pro−Ala−Ala−Arg (PAAR) repeat domains, which bind the β-spike of trimeric valine−glycine repeat protein G (VgrG) and are important for T6SS activity in other bacteria. Truncated RhsA that retains the PAAR domain is capable of forming higher-order, thermostable complexes with VgrG, yet these assemblies fail to restore secretion activity to ∆rhsA ∆rhsB mutants. Full T6SS-1 activity requires Rhs that contains N-terminal transmembrane helices, the PAAR domain, and an intact β-cage. Although ∆rhsA ∆rhsB mutants do not kill target bacteria, time-lapse microscopy reveals that they assemble and fire T6SS contractile sheaths at ∼6% of the frequency of rhs⁺ cells. Therefore, Rhs proteins are not strictly required for T6SS assembly, although they greatly increase secretion efficiency. We propose that PAAR and the β-cage provide distinct structures that promote secretion. PAAR is clearly sufficient to stabilize trimeric VgrG, but efficient assembly of T6SS-1 also depends on an intact β-cage. Together, these domains enforce a quality control checkpoint to ensure that VgrG is loaded with toxic cargo before assembling the secretion apparatus.

Bacteria use many strategies to compete against other microorganisms in the environment. Research over the past 15 y has uncovered several distinct mechanisms by which bacteria deliver inhibitory toxins directly into neighboring competitors (1–8). Cell contact-dependent competition systems have been characterized most extensively in Gram-negative bacteria, and the most widespread mechanism is mediated by the type VI secretion system (T6SS) (9). T6SSs are multiprotein complexes related in structure and function to the contractile tails of Myoviridae bacteriophages. T6SS loci vary considerably between bacterial species, but all encode 13 core type VI secretion (Tss) proteins that are required to build a functional apparatus. TssJ, TssL, and TssM form a multimeric complex that spans the cell envelope and serves as the secretion conduit. The phage-like baseplate is composed of TssE, TssF, TssG, and TssK proteins, which form a sixfold symmetrical array surrounding a central “hub” of trimeric valine−glycine repeat protein G (VgrG/TssI). VgrG is structurally homologous to the gp27−gp5 tail spike of phage T4 (10, 11). The T4 tail spike is further acuminated with gp5.4, a small protein that forms a sharpened apex at the tip of the gp5 spike (12). Proline−alanine−alanine−arginine (PAAR) repeat proteins form an orthologous structure on VgrG; and PAAR is thought to facilitate penetration of the target cell outer membrane (13). The T6SS duty cycle begins when the baseplate docks onto TssJLM at the cytoplasmic face of the inner membrane (14). The baseplate then serves as the assembly origin for the contractile sheath and inner tube. The sheath is built from TssB−TssC subunits, and the tube is formed by stacked hexameric rings of hemolysin-coregulated protein (Hcp/TssD). TssA coordinates this assembly process to ensure that the sheath and tube are polymerized at equivalent rates (15). After elongating across the width of the cell, the sheath undergoes rapid contraction to expel the PAAR•VgrG-capped Hcp tube through the transenvelope complex. The ejected tube impales neighboring cells and delivers a variety of toxic effector proteins into the target. After firing, the contracted sheath is disassembled by the ClpV (TssH) ATPase (16), and the recycled TssBC subunits are used to support additional rounds of sheath assembly and contraction.T6SSs were originally identified through their ability to intoxicate eukaryotic host cells (17), and VgrG proteins were the first effectors to be recognized. VgrG-1 from Vibrio cholerae V52 carries a C-terminal domain that cross-links actin and blocks macrophage phagocytosis (10). Similarly, the VgrG1 protein from Aeromonas hydrophila American Type Culture Collection (ATCC) 7966 carries a C-terminal actin adenosine 5′-diphosphate (ADP) ribosyltransferase domain that disrupts the host cytoskeleton (18). Although the T6SS clearly plays a role in pathogenesis, most of the systems characterized to date deliver toxic effectors into competing bacteria. Because antibacterial effectors are potentially autoinhibitory, these latter toxins are invariably encoded with specific immunity proteins. Antibacterial effectors commonly disrupt the integrity of the bacterial cell envelope. VgrG-3 from V. cholerae carries a lysozyme-like domain that degrades the peptidoglycan cell wall (19, 20). Other peptidoglycan-cleaving amidase toxins are packaged within the lumen of Hcp hexamers for T6SS-mediated delivery (21–24). Phospholipase toxins collaborate with peptidoglycan degrading enzymes to lyse target bacteria (25–27). Other T6SS effectors act in the cytosol to degrade nucleic acids and nicotinamide adenine dinucleotide cofactors (3, 28, 29). Most recently, Whitney and coworkers described a novel T6SS effector that produces the inhibitory nucleotide ppApp (30). These latter toxins are commonly delivered through noncovalent interactions with VgrG. Many effectors contain PAAR domains, which enable direct binding to the C-terminal β-spike of VgrG (13), whereas others are indirectly tethered to VgrG through adaptor proteins (31–33). This combinatorial strategy allows multiple different toxins to be delivered with each firing event.Rearrangement hotspot (Rhs) proteins are potent effectors deployed by many T6SS⁺ bacteria (3, 34–37). T6SS-associated Rhs effectors range from ∼150 kDa to 180 kDa in mass and carry highly variable C-terminal toxin domains. The N-terminal region of Rhs proteins often contains two predicted transmembrane (TM) helix regions followed by a PAAR domain. The central region is composed of many Rhs/YD-peptide repeats, which form a β-cage structure that fully encapsulates the toxin domain (38). Genes coding for Rhs were first identified in Escherichia coli K-12 as elements that promote chromosomal duplication (39, 40). This genomic rearrangement was the result of unequal recombination between the rhsA and rhsB loci, which share 99.4% sequence identity over some 3,700 nucleotides. Subsequently, Hill and coworkers recognized that rhs genes are genetic composites (41), and that the variable C-terminal extension domains inhibit cell growth (42). Although E. coli K-12 encodes four full-length Rhs proteins, it lacks a T6SS, and there is no evidence that it deploys Rhs in competition. However, other Rhs/YD-peptide repeat proteins are known to deliver toxins in a T6SS-independent manner. Gram-positive bacteria export antibacterial YD-repeat proteins through the Sec pathway (3), and the tripartite insecticidal toxin complexes released by Photorhabdus and Yersinia species contain subunits with Rhs/YD repeats (38, 43). Thus, the Rhs encapsulation structure has been incorporated into at least three different toxin delivery platforms.Here, we report that Rhs effectors are critical for the activity of the T6SS-1 locus of Enterobacter cloacae ATCC 13047 (ECL). ECL encodes two Rhs effectors—RhsA and RhsB—which are each exported in a constitutive manner by T6SS-1 (35). Deletion of either rhs gene has little effect on T6SS-1 activity, but mutants lacking both rhsA and rhsB are defective for Hcp1 secretion and no longer inhibit target bacteria. Although ∆rhsA ∆rhsB mutants lose T6SS-1−mediated inhibition activity, they still assemble and fire contractile sheaths at a significantly reduced frequency. We further show that truncated RhsA that retains the PAAR domain still interacts with cognate VgrG2, but the resulting complex does not support Hcp1 secretion or target-cell killing. Full T6SS-1 function requires wild-type Rhs effectors that retain the N-terminal TM helices and PAAR domain together with an intact β-cage. These findings suggest that the Rhs β-cage mediates a quality control checkpoint on T6SS-1 assembly to ensure that VgrG is loaded with a toxic effector prior to export.     

IDO1 scavenges reactive oxygen species in myeloid-derived suppressor cells to prevent graft-versus-host disease

Tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) also has an immunological function to suppress T cell activation in inflammatory circumstances, including graft-versus-host disease (GVHD), a fatal complication after allogeneic bone marrow transplantation (allo-BMT). Although the mononuclear cell expression of IDO1 has been associated with improved outcomes in GVHD, the underlying mechanisms remain unclear. Herein, we used IDO-deficient (Ido1^−/−) BMT to understand why myeloid IDO limits the severity of GVHD. Hosts with Ido1^−/− BM exhibited increased lethality, with enhanced proinflammatory and reduced regulatory T cell responses compared with wild type (WT) allo-BMT controls. Despite the comparable expression of the myeloid-derived suppressor cell (MDSC) mediators, arginase-1, inducible nitric oxide synthase, and interleukin 10, Ido1^−/− Gr-1⁺CD11b⁺ cells from allo-BMT or in vitro BM culture showed compromised immune-suppressive functions and were skewed toward the Ly6C^lowLy6G^hi subset, compared with the WT counterparts. Importantly, Ido1^−/−Gr-1⁺CD11b⁺ cells exhibited elevated levels of reactive oxygen species (ROS) and neutrophil numbers. These characteristics were rescued by human IDO1 with intact heme-binding and catalytic activities and were recapitulated by the treatment of WT cells with the IDO1 inhibitor L1-methyl tryptophan. ROS scavenging by N-acetylcysteine reverted the Ido1^−/−Gr-1⁺CD11b⁺ composition and function to an MDSC state, as well as improved the survival of GVHD hosts with Ido1^−/− BM. In summary, myeloid-derived IDO1 enhances GVHD survival by regulating ROS levels and limiting the ability of Gr-1⁺CD11b⁺ MDSCs to differentiate into proinflammatory neutrophils. Our findings provide a mechanistic insight into the immune-regulatory roles of the metabolic enzyme IDO1.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-binding metabolic enzyme that catalyzes the conversion of tryptophan (Trp) into kynurenine (Kyn). In addition to Trp catabolism, IDO1 has long been recognized to have immune-regulatory roles, preventing excessive inflammation (1). IDO1 is up-regulated in response to inflammatory stimuli, including Toll-like receptor (TLR) and type I/II interferon (IFN) signaling (1, 2). The induction of IDO1 after TLR9 stimulation has been demonstrated to mitigate experimental colitis (3). Catalytic function blockade in mice by pharmacological inhibition or genetic ablation of IDO1 (Ido1^−/−) enhanced inflammation and aggravated autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE) (4). The enhanced immune responses induced by IDO1 deficiency were associated with increased T helper (Th)1/Th17 responses; in contrast, regulatory T cell (Treg) responses were repressed (4–6). Consistently, IDO1 inhibition enhanced antitumor immune responses (7–9). The immune-regulatory effects of IDO1 have been ascribed to the depletion of Trp (10, 11) and the production of toxic catabolites along the Kyn pathway (4, 12–14). However, it remains unclear whether additional mechanisms are involved in IDO1-mediated immune suppression.Graft-versus-host disease (GVHD) is a severe inflammatory disease for which IDO1 has been shown to play a protective role (2, 14, 15). GVHD often develops as an adverse systemic complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is induced by activation of donor T cells reactive to the recipient’s major histocompatibility complexes (MHCs) and/or minor histocompatibility antigens (MiHAs) (16). Allo-reactivity of the activated donor T cells promotes tissue inflammation in the host, leading to morbidity and mortality. IDO1 deficiency in the bone marrow (BM) of the donor or the recipient has been linked to increased lethality (2, 14, 15), indicating a crucial role of IDO1 expression in the parenchymal and hematopoietic compartments in preventing GVHD. Kyn produced in IDO1-expressing lung epithelial cells and tissue macrophages suppressed T cell activation by binding to and activating immunomodulatory aryl hydrocarbon receptors (AhRs), which could explain the GVHD aggravation in Ido1^−/− recipients (14). Nevertheless, the mechanisms behind GVHD exacerbation by Ido1^−/− BM transfer remain obscure. Wild-type (WT) donor antigen-presenting cells prolonged survival in GVHD regardless of epithelial cell expression of IDO1, and IDO1 up-regulation after treatment of donor BM with TLR ligands reduced GVHD severity (2). These findings suggest an important role of IDO1 expressed by donor-derived myeloid cells in preventing severe GVHD. However, the immune-regulatory roles of IDO1 expressed in myeloid cells (termed myeloid IDO1 hereafter) remain elusive.Myeloid-derived suppressor cells (MDSCs) are innate cells that have immune-suppressive functions (17). Conventionally, MDSCs are identified as Gr-1⁺CD11b⁺ cells and can be further classified into Ly6C^hiLy6G^low monocytic (M) or Ly6C^lowLy6G^hi polymorphonuclear (PMN) subsets. MDSCs produce various immune-suppressive mediators, including arginase-1 (Arg-1), inducible nitric oxide synthase (iNOS), and interleukin 10 (IL-10) (17, 18). Their ability to enhance Treg responses has also been reported (19, 20). As immature cells, MDSCs maintain the ability to differentiate into dendritic cells (DCs), macrophages, or neutrophils (21, 22). In GVHD, MDSCs derived from donor BM are the major population of myeloid cells expanding in the host (23), and along with Tregs they suppress GVHD (24–26). We previously reported that transplantation of MyD88-deficient (Myd88^−/−) BM suppressed Gr-1⁺CD11b⁺ cell expansion and polarized the differentiation of Gr-1⁺CD11b⁺ cells into DCs, aggravating GVHD (27, 28). These findings indicate that increasing the number of undifferentiated Gr-1⁺CD11b⁺ cells is essential for MDSC-mediated immune suppression in GVHD. Additionally, the finding that IDO1 expression in mononuclear cells, rather than in parenchymal cells, correlated positively with the survival of GVHD patients (29) suggested that IDOl expression in myeloid cells might be involved in the MDSC-mediated suppression of GVHD. Understanding the role of IDO1 in the function of MDSCs derived from the donor BM could lead to novel therapeutic strategies for the treatment of GVHD.In this study, we investigated the mechanisms underlying GVHD aggravation in hosts transplanted with IDO1-deficient BM. We found that IDO1 deficiency in donor BM did not affect the expansion of Gr-1⁺CD11b⁺ cells in GVHD hosts but polarized them toward a Ly6C^lowLy6G^hi phenotype, reducing their immune-regulatory potential. This phenomenon was ascribed to increased reactive oxygen species (ROS) generation in the Ido1^−/− Gr-1⁺CD11b⁺ cells and their skewing to neutrophil differentiation. Treatment of ROS-scavenging chemical reversed this phenomenon. Our findings suggest that the immune-regulatory roles of IDO1 are mediated by ROS scavenging and suppression of the differentiation of Gr-1⁺CD11b⁺ cells.     

Characterization of the strain-rate–dependent mechanical response of single cell–cell junctions

Cell–cell adhesions are often subjected to mechanical strains of different rates and magnitudes in normal tissue function. However, the rate-dependent mechanical behavior of individual cell–cell adhesions has not been fully characterized due to the lack of proper experimental techniques and therefore remains elusive. This is particularly true under large strain conditions, which may potentially lead to cell–cell adhesion dissociation and ultimately tissue fracture. In this study, we designed and fabricated a single-cell adhesion micro tensile tester (SCAµTT) using two-photon polymerization and performed displacement-controlled tensile tests of individual pairs of adherent epithelial cells with a mature cell–cell adhesion. Straining the cytoskeleton–cell adhesion complex system reveals a passive shear-thinning viscoelastic behavior and a rate-dependent active stress-relaxation mechanism mediated by cytoskeleton growth. Under low strain rates, stress relaxation mediated by the cytoskeleton can effectively relax junctional stress buildup and prevent adhesion bond rupture. Cadherin bond dissociation also exhibits rate-dependent strengthening, in which increased strain rate results in elevated stress levels at which cadherin bonds fail. This bond dissociation becomes a synchronized catastrophic event that leads to junction fracture at high strain rates. Even at high strain rates, a single cell–cell junction displays a remarkable tensile strength to sustain a strain as much as 200% before complete junction rupture. Collectively, the platform and the biophysical understandings in this study are expected to build a foundation for the mechanistic investigation of the adaptive viscoelasticity of the cell–cell junction.

Adhesive organelles between neighboring epithelial cells form an integrated network as the foundation of complex tissues (1). As part of normal physiology, this integrated network is constantly exposed to mechanical stress and strain, which is essential to normal cellular activities, such as proliferation (2–4), migration (5, 6), differentiation (7), and gene regulation (7, 8) associated with a diverse set of functions in tissue morphogenesis (9–11) and wound healing (9). A host of developmental defects or clinical pathologies in the form of compromised cell–cell associations will arise when cells fail to withstand external mechanical stress due to genetic mutations or pathological perturbations (12, 13). Indeed, since the mechanical stresses are mainly sustained by the intercellular junctions, which may represent the weakest link and limit the stress tolerance within the cytoskeleton network of a cell sheet, mutations or disease-induced changes in junction molecules and components in adherens junctions and desmosomes lead to cell layer fracture and tissue fragility, which exacerbate the pathological conditions (14–17). This clinical relevance gives rise to the importance of understanding biophysical transformations of the cell–cell adhesion interface when cells are subjected to mechanical loads.As part of their normal functions, cells often experience strains of tens to a few hundred percent at strain rates of 10⁻⁴ to 1 s⁻¹ (18–21). For instance, embryonic epithelia are subjected to strain rates in the range of 10⁻⁴ to 10⁻³ s⁻¹ during normal embryogenesis (22). Strain rates higher than 0.1 s⁻¹ are often experienced by adult epithelia during various normal physiological functions (21, 23, 24), such as breathing motions in the lung (1 to 10 s⁻¹) (25), cardiac pulses in the heart (1 to 6.5 s⁻¹) (20), peristaltic movements in the gut (0.4 to 1.5 s⁻¹), and normal stretching of the skin (0.1 to 5 s⁻¹). Cells have different mechanisms to dissipate the internal stress produced by external strain to avoid fracture, often via cytoskeleton remodeling and cell–cell adhesion enhancement (26, 27). These coping mechanisms may have different characteristic timescales. Cytoskeleton remodeling can dissipate mechanical stress promptly due to its viscoelastic nature and the actomyosin-mediated cell contractility (17, 28–32). Adhesion enhancement at the cell–cell contact is more complex in terms of timescale. Load-induced cell–cell adhesion strengthening has been shown via the increase in the number of adhesion complexes (33–35) or by the clustering of adhesion complexes (36–39), which occurs on a timescale ranging from a few minutes up to a few hours after cells experience an initial load (28). External load on the cell–cell contact also results in a prolonged cell–cell adhesion dissociation time (40, 41), suggesting cadherin bonds may transition to catch bonds under certain loading conditions (42, 43), which can occur within seconds (44). With the increase in cellular tension, failure to dissipate the stress within the cell layer at a rate faster than the accumulation rate will inevitably lead to the fracture of the cell layer (45). Indeed, epithelial fracture often aggravates the pathological outcomes in several diseases, such as acute lung injuries (46), skin disorders (47), and development defects (48). It is generally accepted that stress accumulation in the cytoskeleton network (49, 50) and potentially in the cytoplasm is strain-rate–dependent (51). However, to date, there is a lack of understanding about the rate-dependent behavior of cell–cell adhesions, particularly about which of the stress-relaxation mechanisms are at play across the spectrum of strain rates. In addition, it remains unclear how the stress relaxation interplays with adhesion enhancement under large strains, especially at high strain rates which may lead to fracture, that is, a complete separation of mature cell–cell adhesions under a tensile load (45, 52, 53). Yet, currently, there is a lack of quantitative technology that enables the investigation of these mechanobiological processes in a precisely controlled manner. This is especially true at high strain rates.To delineate this mechanical behavior, the cleanest characterization method is to directly measure stress dynamics at a single mature cell–cell adhesion interface. Specifically, just as a monolayer cell sheet is a reduction from three-dimensional (3D) tissue, a single cell–cell adhesion interface, as a reduction from a monolayer system, represents the smallest unit to study the rheological behavior of cellular junctions. The mechanistic understanding uncovered with this single unit will inform cellular adaptations to a more complex stress microenvironment in vivo and in vitro, in healthy and diseased conditions. To this end, we developed a single-cell adhesion micro tensile tester (SCAµTT) platform based on nanofabricated polymeric structures using two-photon polymerization (TPP). This platform allows in situ investigation of stress–strain characteristics of a mature cell–cell junction through defined strains and strain rates. With SCAµTT, we reveal some interesting biophysical phenomena at the single cell–cell junction that were previously not possible to observe using existing techniques. We show that cytoskeleton growth can effectively relax intercellular stress between an adherent cell pair in a strain-rate–dependent manner. Along with cadherin-clustering–induced bond strengthening, it prevents failure to occur at low strain rates. At high strain rates, insufficient relaxation leads to stress accumulation, which results in cell–cell junction rupture. We show that a remarkably large strain can be sustained before junction rupture (>200%), even at a strain rate as high as 0.5 s⁻¹. Collectively, the rate-dependent mechanical characterization of the cell–cell junction builds the foundation for an improved mechanistic understanding of junction adaptation to an external load and potentially the spatiotemporal coordination of participating molecules at the cell–cell junction.     

High-dimensional mass cytometry analysis of NK cell alterations in AML identifies a subgroup with adverse clinical outcome

Natural killer (NK) cells are major antileukemic immune effectors. Leukemic blasts have a negative impact on NK cell function and promote the emergence of phenotypically and functionally impaired NK cells. In the current work, we highlight an accumulation of CD56⁻CD16⁺ unconventional NK cells in acute myeloid leukemia (AML), an aberrant subset initially described as being elevated in patients chronically infected with HIV-1. Deep phenotyping of NK cells was performed using peripheral blood from patients with newly diagnosed AML (n = 48, HEMATOBIO cohort, {"type":"clinical-trial","attrs":{"text":"NCT02320656","term_id":"NCT02320656"}}NCT02320656) and healthy subjects (n = 18) by mass cytometry. We showed evidence of a moderate to drastic accumulation of CD56⁻CD16⁺ unconventional NK cells in 27% of patients. These NK cells displayed decreased expression of NKG2A as well as the triggering receptors NKp30 and NKp46, in line with previous observations in HIV-infected patients. High-dimensional characterization of these NK cells highlighted a decreased expression of three additional major triggering receptors required for NK cell activation, NKG2D, DNAM-1, and CD96. A high proportion of CD56⁻CD16⁺ NK cells at diagnosis was associated with an adverse clinical outcome and decreased overall survival (HR = 0.13; P = 0.0002) and event-free survival (HR = 0.33; P = 0.018) and retained statistical significance in multivariate analysis. Pseudotime analysis of the NK cell compartment highlighted a disruption of the maturation process, with a bifurcation from conventional NK cells toward CD56⁻CD16⁺ NK cells. Overall, our data suggest that the accumulation of CD56⁻CD16⁺ NK cells may be the consequence of immune escape from innate immunity during AML progression.

Natural killer (NK) cells are critical cytotoxic effectors involved in leukemic blast recognition, tumor cell clearance, and maintenance of long-term remission (1). NK cells directly kill target cells without prior sensitization, enabling lysis of cells stressed by viral infections or tumor transformation. NK cells are divided into different functional subsets according to CD56 and CD16 expression (2–4). CD56^bright NK cells are the most immature NK cells found in peripheral blood. This subset is less cytotoxic than mature NK cells and secretes high amounts of chemokines and cytokines such as IFNγ and TNFα. These cytokines have a major effect on the infected or tumor target cells and play a critical role in orchestration of the adaptive immune response through dendritic cell activation. CD56^dimCD16⁺ NK cells, which account for the majority of circulating human NK cells, are the most cytotoxic NK cells. NK cell activation is finely tuned by integration of signals from inhibitory and triggering receptors, in particular, those of NKp30, NKp46 and NKp44, DNAM-1, and NKG2D (5). Upon target recognition, CD56^dimCD16⁺ NK cells release perforin and granzyme granules and mediate antibody-dependent cellular cytotoxicity through CD16 (FcɣRIII) to clear transformed cells.NK cells are a major component of the antileukemic immune response, and NK cell alterations have been associated with adverse clinical outcomes in acute myeloid leukemia (AML) (6–9). Therefore, it is crucial to better characterize AML-induced NK cell alterations in order to optimize NK cell–targeted therapies. During AML progression, NK cell functions are deeply altered, with decreased expression of NK cell–triggering receptors and reduced cytotoxic functions as well as impaired NK cell maturation (6, 9–13). Cancer-induced NK cell impairment occurs through various mechanisms of immune escape, including shedding and release of ligands for NK cell–triggering receptors; release of immunosuppressive soluble factors such as TGFβ, adenosine, PGE2, or L-kynurenine; and interference with NK cell development, among others (14).Interestingly, these mechanisms of immune evasion are also seen to some extent in chronic viral infections, notably HIV (2). In patients with HIV, NK cell functional anergy is mediated by the release of inflammatory cytokines and TGFβ, the presence of MHC^low target cells, and the shedding of ligands for NK cell–triggering receptors (2). As a consequence, some phenotypical alterations described in cancer patients are also induced by chronic HIV infections, with decreased expression of major triggering receptors such as NKp30, NKp46, and NKp44 (15, 16); decreased expression of CD16 (17); and increased expression of inhibitory receptors such as T cell immunoreceptor with Ig and ITIM domains (TIGIT) (18) all observed. In addition, patients with HIV display an accumulation of CD56⁻CD16⁺ unconventional NK cells, a highly dysfunctional NK cell subset (19, 20). Mechanisms leading to the loss of CD56 are still poorly described, and the origin of this subset of CD56⁻ NK cells is still unknown. To date, two hypotheses have been considered: CD56⁻ NK cells could be terminally differentiated cells arising from a mixed population of mature NK cells with altered characteristics or could expand from a pool of immature precursor NK cells (21). Expansion of CD56⁻CD16⁺ NK cells is mainly observed in viral noncontrollers (19, 20). Indeed, CD56 is an important adhesion molecule involved in NK cell development, motility, and pathogen recognition (22–27). CD56 is also required for the formation of the immunological synapse between NK cells and target cells, lytic functions, and cytokine production (26, 28). As a consequence, CD56⁻CD16⁺ NK cells display lower degranulation capacities and decreased expression of triggering receptors, perforin, and granzyme B, dramatically reducing their cytotoxic potential, notably against tumor target cells (2, 19, 20, 29, 30). In line with this loss of the cytotoxic functions against tumor cells, patients with concomitant Burkitt lymphoma and Epstein-Barr virus infection display a dramatic increase of CD56⁻CD16⁺ NK cells (30), which could represent an important hallmark of escape to NK cell immunosurveillance in virus-driven hematological malignancies.To our knowledge, this population has not been characterized in the context of nonvirally induced hematological malignancies. In the present work, we investigated the presence of this population of unconventional NK cells in patients with AML, its phenotypical characteristics, and the consequences of its accumulation on disease control. Finally, we explored NK cell developmental trajectories leading to the emergence of this phenotype.     

Inverse correlation between fatty acid transport protein 4 and vision in Leber congenital amaurosis associated with RPE65 mutation

Fatty acid transport protein 4 (FATP4), a transmembrane protein in the endoplasmic reticulum (ER), is a recently identified negative regulator of the ER-associated retinal pigment epithelium (RPE)65 isomerase necessary for recycling 11-cis-retinal, the light-sensitive chromophore of both rod and cone opsin visual pigments. The role of FATP4 in the disease progression of retinal dystrophies associated with RPE65 mutations is completely unknown. Here we show that FATP4-deficiency in the RPE results in 2.8-fold and 1.7-fold increase of 11-cis- and 9-cis-retinals, respectively, improving dark-adaptation rates as well as survival and function of rods in the Rpe65 R91W knockin (KI) mouse model of Leber congenital amaurosis (LCA). Degradation of S-opsin in the proteasomes, but not in the lysosomes, was remarkably reduced in the KI mouse retinas lacking FATP4. FATP4-deficiency also significantly rescued S-opsin trafficking and M-opsin solubility in the KI retinas. The number of S-cones in the inferior retinas of 4- or 6-mo-old KI;Fatp4^−/− mice was 7.6- or 13.5-fold greater than those in age-matched KI mice. Degeneration rates of S- and M-cones are negatively correlated with expression levels of FATP4 in the RPE of the KI, KI;Fatp4^+/−, and KI;Fatp4^−/− mice. Moreover, the visual function of S- and M-cones is markedly preserved in the KI;Fatp4^−/− mice, displaying an inverse correlation with the FATP4 expression levels in the RPE of the three mutant lines. These findings establish FATP4 as a promising therapeutic target to improve the visual cycle, as well as survival and function of cones and rods in patients with RPE65 mutations.

Retinal pigment epithelium 65 (RPE65) is a key retinoid isomerase (1–4) in the visual cycle responsible for recycling 11-cis-retinal (11cRAL), which functions not only as a molecular switch for initiating the phototransduction in response to light stimuli, but also as a chaperone for normal trafficking of cone opsins to the outer segments (OS) of cones (5, 6). RPE65 is also the isomerase responsible for the production of meso-zeaxanthin (7), one of the three macular pigments in the human retina that function as potent antioxidants and light-screening pigments to protect the macula (8). Expression levels and activities of RPE65 are positively correlated with an increase in both retinal susceptibility to light-induced degeneration (9, 10) and the accumulation rates of the visual cycle-derived cytotoxic bisretinoids, the major autofluorescent components of lipofuscin implicated in Stargardt disease and geographic atrophy of age-related macular degeneration (11–13).Mutations in the RPE65 gene cause vision impairment and retinal degeneration in affected patients, canines, and mice. In humans, more than 100 DNA variants in the RPE65 gene are reported as pathogenic mutations causing retinal degenerative diseases (Global Variome shared LOVD: https://databases.lovd.nl/shared/genes/RPE65). Although night blindness is the first significant symptom in most patients with RPE65 mutations, in vivo microscopy of the fovea demonstrated that many patients exhibited severe cone degeneration at very early ages (14, 15). The potentially important role of RPE65 in maintaining human cone photoreceptor health and vision is also supported by its abundant expression and higher activity in the macaque central RPE layer localized to the cone-rich area (16).With the exception of adeno-associated virus (AAV)-based gene therapy, there is no approved effective treatment available for diseases caused by RPE65 mutations. In clinical trials, subretinaly injected AAV-RPE65 has improved vision in some patients (17–20). However, subsequent studies showed that the gene therapy could not stop progressive retinal degeneration in patients (21–23). In addition, more than half of the subjects injected with a higher dose of AAV-RPE65 developed various degrees of intraocular inflammation (23). AAV cis-regulatory sequences are associated with toxic effects on the RPE and microglial cells (24). Lower dose of AAV-RPE65 may reduce the side effects but it will limit the beneficial outcome of this very high-cost therapy because only a small population of RPE cells will express the exogenous RPE65 (17). These studies suggest the need for alternative interventions and improved gene therapy to prevent progressive retinal degeneration in patients.A recent study showed that systemic administration of 4-phenylbutyrate (PBA) could partially rescue the function of mutated RPE65, thereby improving the preservation of photoreceptors and vision in a mouse model of Leber congenital amaurosis (LCA) (25). This study suggests that rescuing the intrinsic function of mutated RPE65 has the potential to mitigate retinal degeneration in patients with RPE65 mutations. Studies in cultured cells have shown that many RPE65 mutants could be rescued by chemical and physical treatments (26, 27). These studies have provided the basis to explore new therapeutic strategies for diseases associated with RPE65 mutations. One of the possible approaches that can rescue RPE65 mutants, and thereby enhance the efficacy of the gene therapy and PBA-treatment, is to modulate endogenous regulators of RPE65.Through screening of RPE cDNA libraries, we have previously identified fatty acid transport protein 4 (FATP4) as a negative regulator of RPE65 (28). FATP4 is a transmembrane protein with an endoplasmic reticulum (ER)-localization domain (29). Among the six members of the FATP family, FATP4 is the most abundant FATP in the RPE. It has fatty acyl-CoA synthetase activity with specificity toward saturated and monounsaturated very long-chain fatty acids. Activation of C24:0, but not C16:0, fatty acid was reduced in the FATP4-null mouse cells (30, 31). In an in vitro assay for RPE65 isomerase, lignoceroyl (C24:0)-CoA inhibited the synthesis of 11-cis-retinol (11cROL), whereas palmitoyl (C16:0)-CoA promoted the synthesis of 11cROL (28). In addition, FATP4 has been shown to interact with RPE65 and inhibits 11cROL synthesis catalyzed by RPE65 (32). Consistent with these studies, the retinoid isomerase activity and the visual cycle rates are increased in a mouse line lacking FATP4 in the RPE (28).In the present study, we investigated the role and mechanisms of FATP4 function in regulating the visual cycle, as well as survival and function of rod and cone photoreceptors in pathological conditions caused by hypomorphic R91W RPE65, the most common RPE65 mutant linked to LCA. The R91W mutation has been shown to cause early degeneration of cones in affected patients and animal models (33, 34). We found that FATP4 is a promising therapeutic target to preserve cones and vision in patients with RPE65 mutation.     

Wild-type α-synuclein inherits the structure and exacerbated neuropathology of E46K mutant fibril strain by cross-seeding

Heterozygous point mutations of α-synuclein (α-syn) have been linked to the early onset and rapid progression of familial Parkinson’s diseases (fPD). However, the interplay between hereditary mutant and wild-type (WT) α-syn and its role in the exacerbated pathology of α-syn in fPD progression are poorly understood. Here, we find that WT mice inoculated with the human E46K mutant α-syn fibril (hE46K) strain develop early-onset motor deficit and morphologically different α-syn aggregation compared with those inoculated with the human WT fibril (hWT) strain. By using cryo-electron microscopy, we reveal at the near-atomic level that the hE46K strain induces both human and mouse WT α-syn monomers to form the fibril structure of the hE46K strain. Moreover, the induced hWT strain inherits most of the pathological traits of the hE46K strain as well. Our work suggests that the structural and pathological features of mutant strains could be propagated by the WT α-syn in such a way that the mutant pathology would be amplified in fPD.

α-Synuclein (α-Syn) is the main component of Lewy bodies, which serve as the common histological hallmark of Parkinson’s disease (PD) and other synucleinopathies (1, 2). α-Syn fibrillation and cell-to-cell transmission in the brain play essential roles in disease progression (3–5). Interestingly, WT α-syn could form fibrils with distinct polymorphs, which exhibit disparate seeding capability in vitro and induce distinct neuropathologies in mouse models (6–10). Therefore, it is proposed that α-syn fibril polymorphism may underlie clinicopathological variability of synucleinopathies (6, 9). In fPD, several single-point mutations of SNCA have been identified, which are linked to early-onset, severe, and highly heterogeneous clinical symptoms (11–13). These mutations have been reported to influence either the physiological or pathological function of α-syn (14). For instance, A30P weakens while E46K strengthens α-syn membrane binding affinity that may affect its function in synaptic vesicle trafficking (14, 15). E46K, A53T, G51D, and H50Q have been found to alter the aggregation kinetics of α-syn in different manners (15–17). Recently, several cryogenic electron microscopy (cryo-EM) studies revealed that α-syn with these mutations forms diverse fibril structures that are distinct from the WT α-syn fibrils (18–26). Whether and how hereditary mutations induced fibril polymorphism contributes to the early-onset and exacerbated pathology in fPD remains to be elucidated. More importantly, most fPD patients are heterozygous for SNCA mutations (12, 13, 27, 28), which leads to another critical question: could mutant fibrils cross-seed WT α-syn to orchestrate neuropathology in fPD patients?E46K mutation is one of the eight disease-causing mutations on SNCA originally identified from a Spanish family with autosomal-dominant PD (11). E46K-associated fPD features early-onset motor symptoms and rapid progression of dementia with Lewy bodies (11). Studies have shown that E46K mutant has higher neurotoxicity than WT α-syn in neurons and mouse models overexpressing α-syn (29–32). The underlying mechanism is debatable. Some reported that E46K promotes the formation of soluble species of α-syn without affecting the insoluble fraction (29, 30), while others suggested that E46K mutation may destabilize α-syn tetramer and induce aggregation (31, 32). Our previous study showed that E46K mutation disrupts the salt bridge between E46 and K80 in the WT fibril strain and rearranges α-syn into a different polymorph (33). Compared with the WT strain, the E46K fibril strain is prone to be fragmented due to its smaller and less stable fibril core (33). Intriguingly, the E46K strain exhibits higher seeding ability in vitro, suggesting that it might induce neuropathology different from the WT strain in vivo (33).In this study, we found that human E46K and WT fibril strains (referred to as hE46K and hWT strains) induced α-syn aggregates with distinct morphologies in mice. Mice injected with the hE46K strain developed more α-syn aggregation and early-onset motor deficits compared with the mice injected with the hWT strain. Notably, the hE46K strain was capable of cross-seeding both human and mouse WT (mWT) α-syn to form fibrils (named as hWT_cs and mWT_cs). The cross-seeded fibrils replicated the structure and seeding capability of the hE46K template both in vitro and in vivo. Our results suggest that the hE46K strain could propagate its structure as well as the seeding properties to the WT monomer so as to amplify the α-syn pathology in fPD.     

Stable metal anodes enabled by a labile organic molecule bonded to a reduced graphene oxide aerogel

Metallic anodes (lithium, sodium, and zinc) are attractive for rechargeable battery technologies but are plagued by an unfavorable metal–electrolyte interface that leads to nonuniform metal deposition and an unstable solid–electrolyte interphase (SEI). Here we report the use of electrochemically labile molecules to regulate the electrochemical interface and guide even lithium deposition and a stable SEI. The molecule, benzenesulfonyl fluoride, was bonded to the surface of a reduced graphene oxide aerogel. During metal deposition, this labile molecule not only generates a metal-coordinating benzenesulfonate anion that guides homogeneous metal deposition but also contributes lithium fluoride to the SEI to improve Li surface passivation. Consequently, high-efficiency lithium deposition with a low nucleation overpotential was achieved at a high current density of 6.0 mA cm⁻². A Li|LiCoO₂ cell had a capacity retention of 85.3% after 400 cycles, and the cell also tolerated low-temperature (−10 °C) operation without additional capacity fading. This strategy was applied to sodium and zinc anodes as well.

Rechargeable batteries based on metal anodes including lithium (Li), sodium (Na), and zinc (Zn) show great promise in achieving high energy density (1–3). Unfortunately, the electrochemical interface of the metal anodes is not favorable for metal deposition. Metal nucleation is inhomogeneous at the surface, leading to the growth of metal dendrites (4–7) and the formation of an unstable solid–electrolyte interphase (SEI) that is incapable of protecting metals from the side reactions with the electrolyte (8–12).Substantial efforts have been devoted to stabilizing the interface of metal anodes, especially for Li metal. These include the design of artificial protective layers (13–17), alternative electrolytes (18–24), and sacrificial additives (25–30) to stabilize the metal–electrolyte interface, the development of mechanically robust coatings (31–34) to block Li dendrite growth, and the use of structured scaffolds to host dendrite-free Li deposition by reducing local current densities (35–43). However, the performance of metal anodes remains poor under high-current or low-temperature conditions. This is because the inhomogeneous Li nucleation and unstable SEI problems have not been well addressed, and these problems at the interface are even exacerbated under critical operating conditions, especially high-current densities and low temperatures (5, 6, 44).Toward this end, we report a simple molecular approach for regulating the electrochemical interface of metal anodes, which enables even Li deposition and stable SEI formation in a conventional electrolyte. This was realized by bonding a labile organic molecule, benzenesulfonyl fluoride (BSF), to a reduced graphene oxide (rGO) aerogel surface as the Li anode host (Fig. 1A). During Li deposition, BSF molecules electrochemically decompose at the interface and generate benzenesulfonate anions bonded to the rGO aerogel (Fig. 1B). The conjugated anions have a strong binding affinity for Li, serving as lithiophilic sites on the rGO surface to synergistically induce homogeneous Li nucleation of Li on the rGO surface. At the same time, BSF molecules contribute LiF to the SEI layer, which facilitates Li surface passivation (Fig. 1C). As a result, high-efficiency (99.2%) Li deposition was achieved at a Li deposition amount of 6.0 mAh cm⁻² and a current density of 6.0 mA cm⁻²; the barrier to Li nucleation was markedly reduced, as evidenced by the low nucleation overpotentials at high-current density (6.0 mA cm⁻²) or at a low temperature (−10 °C). A 400-cycle life with a capacity retention of 83.6% was achieved for a Li|LiCoO₂ (LCO) cell in a conventional carbonate electrolyte. Moreover, with the organic molecule-tuned interface, the Li|LCO cell can be stably cycled at a low operating temperature (−10 °C). This approach was applied to Na and Zn metal anodes as well.Open in a separate windowFig. 1.Illustration of a stable interface for Li deposition using a labile organic molecule, benzenesulfonyl fluoride (BSF). (A) Covalently bonded BSF on the rGO aerogel surface. (B) In situ generation of a lithiophilic conjugated anion (benzenesulfonate) and LiF on the surface during Li deposition. (C) Li nucleation preferentially occurs at the conjugated anion sites owing to the strong Li binding affinity, which leads to uniform Li deposition. In addition, the LiF that is formed is in the SEI layer and passivates the Li surface.     

Mitochondrial metabolism is essential for invariant natural killer T cell development and function

Conventional T cell fate and function are determined by coordination between cellular signaling and mitochondrial metabolism. Invariant natural killer T (iNKT) cells are an important subset of “innate-like” T cells that exist in a preactivated effector state, and their dependence on mitochondrial metabolism has not been previously defined genetically or in vivo. Here, we show that mature iNKT cells have reduced mitochondrial respiratory reserve and iNKT cell development was highly sensitive to perturbation of mitochondrial function. Mice with T cell-specific ablation of Rieske iron-sulfur protein (RISP; T-Uqcrfs1^−/−), an essential subunit of mitochondrial complex III, had a dramatic reduction of iNKT cells in the thymus and periphery, but no significant perturbation on the development of conventional T cells. The impaired development observed in T-Uqcrfs1^−/− mice stems from a cell-autonomous defect in iNKT cells, resulting in a differentiation block at the early stages of iNKT cell development. Residual iNKT cells in T-Uqcrfs1^−/− mice displayed increased apoptosis but retained the ability to proliferate in vivo, suggesting that their bioenergetic and biosynthetic demands were not compromised. However, they exhibited reduced expression of activation markers, decreased T cell receptor (TCR) signaling and impaired responses to TCR and interleukin-15 stimulation. Furthermore, knocking down RISP in mature iNKT cells diminished their cytokine production, correlating with reduced NFATc2 activity. Collectively, our data provide evidence for a critical role of mitochondrial metabolism in iNKT cell development and activation outside of its traditional role in supporting cellular bioenergetic demands.

Cellular metabolic pathways are interwoven with traditional signaling pathways to regulate the function and differentiation of T cells (1–3). Upon activation, effector T cells display a marked increase in glycolytic metabolism even in the presence of ample oxygen, termed aerobic glycolysis (4). We have previously shown that despite increased aerobic glycolysis, T cell activation depends on mitochondrial metabolism for generation of reactive oxygen species (ROS) for signaling (5). As activated T cells progress to a memory or regulatory phenotype, they preferentially oxidize fatty acids to support mitochondrial metabolism, and enhanced fatty acid oxidation (FAO) and spare respiratory capacity (SRC) are essential to maintenance of their phenotype (6, 7).CD1d-restricted invariant natural killer T (iNKT) cells are a unique subset of lymphocytes that exhibit a preactivated phenotype with rapid effector responses (8, 9). iNKT cells are capable of producing large amount of proinflammatory and antiinflammatory cytokines thus have broad immunomodulatory roles (8–10). Given that these cells are poised for rapid proliferation and cytokine production, we hypothesized that coordination of cellular signaling with cellular metabolism will be especially critical for optimal iNKT function. In support of this hypothesis, several studies suggest that modulation of cellular metabolism affects iNKT cell development and function. iNKT cell development is diminished upon deletion of the miR-181 a1b1 cluster, which regulates phosphoinositide 3-kinase signaling and decreases aerobic glycolysis (11, 12). In addition, T cell-specific deletion of Raptor (a component of mTORC1), a metabolic regulator, leads to defects in iNKT cell development and function (13, 14). Loss of folliculin-interacting protein 1 (Fnip1), an adaptor protein that physically interacts with AMP-activated protein kinase, also results in defective NKT cell development, and interestingly conventional T cells develop normally (15). Furthermore, a number of studies targeting bioenergetics processes or related molecules, like alteration of glucose metabolism, mitochondrial-targeted antioxidant treatment, and receptor-interacting protein kinase 3-dependent activation of mitochondrial phosphatase, showed significant effects on iNKT cell ratio and function (16–19). A recent study showed that iNKT cells are less efficient in glucose uptake than CD4⁺ T cells. Furthermore, activated iNKT cells preferentially metabolize glucose by the pentose phosphate pathway and mitochondria, instead of converting into lactate, since high lactate environment is detrimental to their homeostasis and effector function (20).In conventional lymphocytes, mitochondria clearly play a role in coordination of cell signaling and cell fate decisions outside of production of energy (5, 21–23). During T cell activation mitochondria localize at immune synapses that T cells form with antigen-presenting cells (22). T cell receptor (TCR) stimulation triggers mitochondrial ROS (mROS) production as well as mitochondrial ATP production that are released at the immune synapses and are critical for Ca²⁺ homeostasis and modulation of TCR-induced downstream signaling pathways (22). We previously showed that mice with T-cell–specific deletion of Rieske iron sulfur protein (RISP), a component of mitochondrial complex III of the mitochondrial electron transport chain (ETC), are defective in antigen-specific T cell activation due to deficiency of mROS required for cellular signaling (5). Several recent studies showed that ROS or factors that affect ROS production are also important in iNKT cell development and effector functions (24–27). In addition, inhibition of mitochondrial oxidative phosphorylation (OXPHOS) by oligomycin has been shown to decreased survival and cytokine production by splenic iNKT cells (20). However, the requirement of mitochondrial metabolism for iNKT cell development and function has not been previously defined genetically or in vivo.Here we showed that iNKT cells have comparable basal mitochondrial oxygen consumption to conventional T cells but displayed lower SRC and FAO, which are thought to impart cells with mitochondrial reserve under stress. Using Uqcrfs1^fl/fl;CD4-Cre⁺ (hereafter referred as T-Uqcrfs1^−/−) mice, we showed that abrogation of mitochondrial metabolism resulted in a cell-autonomous defect in iNKT cell development in thymus and periphery. The iNKT cells were able to proliferate but exhibited impaired activation, suggesting that they were not lacking bioenergetically but rather had aberrant TCR signaling in vivo, leading to altered expression of downstream factors required for their terminal maturation. Accordingly, T-Uqcrfs1^−/− iNKT cells displayed lower T-bet and CD122 levels and did not respond to interleukin (IL)-15 stimulation. Knockdown of RISP in mature iNKT cells also limited NFATc2 translocation to the nucleus. Collectively, our data highlighted an important role of mitochondrial metabolism in modulating TCR signaling in vivo and regulating iNKT cell development and function.     

An isolated water droplet in the aqueous solution of a supramolecular tetrahedral cage

Water under nanoconfinement at ambient conditions has exhibited low-dimensional ice formation and liquid–solid phase transitions, but with structural and dynamical signatures that map onto known regions of water’s phase diagram. Using terahertz (THz) absorption spectroscopy and ab initio molecular dynamics, we have investigated the ambient water confined in a supramolecular tetrahedral assembly, and determined that a dynamically distinct network of 9 ± 1 water molecules is present within the nanocavity of the host. The low-frequency absorption spectrum and theoretical analysis of the water in the Ga₄L₆¹²⁻ host demonstrate that the structure and dynamics of the encapsulated droplet is distinct from any known phase of water. A further inference is that the release of the highly unusual encapsulated water droplet creates a strong thermodynamic driver for the high-affinity binding of guests in aqueous solution for the Ga₄L₆¹²⁻ supramolecular construct.

Supramolecular capsules create internal cavities that are thought to act like enzyme active sites (1). As aqueous enzymes provide inspiration for the design of supramolecular catalysts, one of the goals of supramolecular chemistry is the creation of synthetic “receptors” that have both a high affinity and a high selectivity for the binding of guests in water (2, 3). The Ga₄L₆¹²⁻ tetrahedral assembly formulated by Raymond and coworkers represents an excellent example of a water-soluble supramolecular cage that has provided host interactions that promotes guest encapsulation. Using steric interactions and electrostatic charge to chemically position the substrate while shielding the reaction from solvent, this host has been shown to provide enhanced reaction rates that approach the performance of natural biocatalysts (4–10). Moreover, aqueous solvation of the substrate, host, and encapsulated solvent also play an important role in the whole catalytic cycle. In particular, the driving forces that release water from the nanocage host to favor the direct binding with the substrate is thought to be a critical factor in successful catalysis, but is challenging to probe directly (7, 8, 11–14).In both natural and artificial nanometer-sized environments, confined water displays uniquely modified structure and dynamics with respect to the bulk liquid (15–18). Recently, these modified properties were also found to have significant implications for the mechanism and energetics of reactions taking place in confined water with respect to those observed in bulk aqueous solution (19–21). In a pioneering study on supramolecular assemblies, Cram and collaborators (22) concluded that the interior of those cages is a “new and unique phase of matter” for the incarcerated guests. In more recent studies, it was postulated that, similar to graphitic and zeolite nanopores (23, 24), confined water within supramolecular host cavities is organized in stable small clusters [(H₂O)_n, with n = 8 to 19] that are different from gas phase water clusters (25). In these studies, the hydrogen-bonded water clusters were reported to be mostly ice- or clathrate-like by X-ray and neutron diffraction in the solid state at both ambient and cryogenic temperatures (26–32). However, to the best of our knowledge, such investigations have not characterized the Ga₄L₆¹²⁻ supramolecular tetrahedral assembly in the liquid state near room temperature and pressure, where the [Ga₄L₆]¹²⁻ capsule can perform catalytic reactions (6, 8, 9).Here, we use terahertz (THz) absorption spectroscopy and ab initio molecular dynamics (AIMD) to characterize low-frequency vibrations and structural organization of water in the nanoconfined environment. THz is ideally suited to probe the intermolecular collective dynamics of the water hydrogen bond (HB) network with extremely high sensitivity, as illustrated for different phases of water (33–38), and for aqueous solutions of salts, osmolytes, alcohols, and amino acids (36, 39–42). The THz spectra of the water inside the nanocage has been quantitatively reproduced with AIMD, allowing us to confidently characterize the water network in the cage in order to provide a more complete dynamical, structural, and thermodynamic picture. We have determined that the spectroscopic signature of the confined water in the nanocage is a dynamically arrested state whose structure bears none of the features of water at any alternate thermodynamic state point such as pressurized liquid or ice. Our experimental and theoretical study provides insight into the role played by encapsulated water in supramolecular catalysis, creating a low entropy and low enthalpy water droplet readily displaced by a catalytic substrate.