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Impaired endothelial cell (EC)–mediated angiogenesis contributes to critical limb ischemia in diabetic patients. The sonic hedgehog (SHH) pathway participates in angiogenesis but is repressed in hyperglycemia by obscure mechanisms. We investigated the orphan G protein–coupled receptor GPR39 on SHH pathway activation in ECs and ischemia-induced angiogenesis in animals with chronic hyperglycemia. Human aortic ECs from healthy and type 2 diabetic (T2D) donors were cultured in vitro. GPR39 mRNA expression was significantly elevated in T2D. The EC proliferation, migration, and tube formation were attenuated by adenovirus-mediated GPR39 overexpression (Ad-GPR39) or GPR39 agonist TC-G-1008 in vitro. The production of proangiogenic factors was reduced by Ad-GPR39. Conversely, human ECs transfected with GPR39 siRNA or the mouse aortic ECs isolated from GPR39 global knockout (GPR39 KO ) mice displayed enhanced migration and proliferation compared with their respective controls. GPR39 suppressed the basal and ligand-dependent activation of the SHH effector GLI1, leading to attenuated EC migration. Coimmunoprecipitation revealed that the GPR39 direct binding of the suppressor of fused (SUFU), the SHH pathway endogenous inhibitor, may achieve this. Furthermore, in ECs with GPR39 knockdown, the robust GLI1 activation and EC migration were abolished by SUFU overexpression. In a chronic diabetic model of diet-induced obesity (DIO) and low-dose streptozotocin (STZ)-induced hyperglycemia, the GPR39 KO mice demonstrated a faster pace of revascularization from hind limb ischemia and lower incidence of tissue necrosis than GPR39 wild-type (GPR39 WT ) counterparts. These findings have provided a conceptual framework for developing therapeutic tools that ablate or inhibit GPR39 for ischemic tissue repair under metabolic stress.

We present an approach to fabricate biological matrix composites made entirely from cultured plant cells. We utilize the cell’s innate ability to synthesize nanofibrillar cell walls, which serve as the composite’s fundamental building blocks. Following a controlled compression/dehydration process, the cells arrange into lamellar structures with hierarchical features. We demonstrate that the native cell wall nanofibrils tether adjacent cells together through fibrillar interlocking and intermolecular hydrogen bonding. These interactions facilitate intercellular adhesion and eliminate the need for other binders. Our fabrication process utilizes the entire plant cell, grown in an in vitro culture; requires no harsh chemical treatments or waste-generating extraction or selection processes; and leads to bulk biocomposites that can be produced in situ and biodegrade in soil. The final mechanical properties are comparable to commodity plastics and can be further modulated by introducing filler particles.

Significance Nerve terminals generally engage in two opposite and essential forms of synaptic plasticity (facilitation or depression) that play critical roles in learning and memory. While the molecular components of both types of terminals are similar with regards to vesicle fusion, much less is known about their molecular control of electrical signaling. Measurements of the electrical impulses (action potentials) underlying these two forms of plasticity have been difficult in small nerve terminals due to their size. In this study we deployed optical physiology measurements to overcome this size barrier. Here, we identify a unique mechanism (Kvβ1 subunit) that enables broadening of the presynaptic action potentials that selectively supports synaptic facilitation, but does not alter any other aspects of nerve terminal function., Analysis of the presynaptic action potential’s (APsyn) role in synaptic facilitation in hippocampal pyramidal neurons has been difficult due to size limitations of axons. We overcame these size barriers by combining high-resolution optical recordings of membrane potential, exocytosis, and Ca2+ in cultured hippocampal neurons. These recordings revealed a critical and selective role for Kv1 channel inactivation in synaptic facilitation of excitatory hippocampal neurons. Presynaptic Kv1 channel inactivation was mediated by the Kvβ1 subunit and had a surprisingly rapid onset that was readily apparent even in brief physiological stimulation paradigms including paired-pulse stimulation. Genetic depletion of Kvβ1 blocked all broadening of the APsyn during high-frequency stimulation and eliminated synaptic facilitation without altering the initial probability of vesicle release. Thus, using all quantitative optical measurements of presynaptic physiology, we reveal a critical role for presynaptic Kv channels in synaptic facilitation at presynaptic terminals of the hippocampus upstream of the exocytic machinery.

Significance In breast cancer, the chronically stiffening mechanical microenvironment promotes tumor growth/invasion. However, the molecular details and integration of cancer mechanotransduction signaling pathways are not well understood. We find that nontumorigenic breast epithelial cells are mechanically sensitive and respond to acute mechanical stimuli with the generation of ROS-stimulated calcium signaling in a microtubule-dependent manner. These results establish that epithelial cells conserve a mechanotransduction pathway from muscle and bone (X-ROS). Using common breast cancer genetic mutations, we further report that oncogene activation reduces X-ROS pathway mechanoresponsiveness at the level of ROS sensitivity. This definition of how oncogene activation disrupts highly conserved mechanotransduction signaling mechanisms could improve the understanding of altered tumor cell responses to the mechanical microenvironment and reveal new therapeutic opportunities., Changes in the mechanical microenvironment and mechanical signals are observed during tumor progression, malignant transformation, and metastasis. In this context, understanding the molecular details of mechanotransduction signaling may provide unique therapeutic targets. Here, we report that normal breast epithelial cells are mechanically sensitive, responding to transient mechanical stimuli through a two-part calcium signaling mechanism. We observed an immediate, robust rise in intracellular calcium (within seconds) followed by a persistent extracellular calcium influx (up to 30 min). This persistent calcium was sustained via microtubule-dependent mechanoactivation of NADPH oxidase 2 (NOX2)-generated reactive oxygen species (ROS), which acted on transient receptor potential cation channel subfamily M member 8 (TRPM8) channels to prolong calcium signaling. In contrast, the introduction of a constitutively active oncogenic KRas mutation inhibited the magnitude of initial calcium signaling and severely blunted persistent calcium influx. The identification that oncogenic KRas suppresses mechanically-induced calcium at the level of ROS provides a mechanism for how KRas could alter cell responses to tumor microenvironment mechanics and may reveal chemotherapeutic targets for cancer. Moreover, we find that expression changes in both NOX2 and TRPM8 mRNA predict poor clinical outcome in estrogen receptor (ER)-negative breast cancer patients, a population with limited available treatment options. The clinical and mechanistic data demonstrating disruption of this mechanically-activated calcium pathway in breast cancer patients and by KRas activation reveal signaling alterations that could influence cancer cell responses to the tumor mechanical microenvironment and impact patient survival.

Significance Chronic neurodegeneration, a major cause of the long-term disabilities that afflict survivors of traumatic brain injury (TBI), is linked to an increased risk for late-life neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease, vascular dementia, and chronic traumatic encephalopathy. Here, we report on the restoration of blood–brain barrier (BBB) structure and function by P7C3-A20 when administered 12 mo after TBI. This pharmacotherapy was associated with cessation of chronic neurodegeneration and recovery of normal cognitive function, benefits that persisted long after treatment cessation. Pharmacologic renewal of BBB integrity may thus provide a new treatment option for patients who have suffered a remote TBI, or other neurological conditions associated with BBB deterioration., Chronic neurodegeneration in survivors of traumatic brain injury (TBI) is a major cause of morbidity, with no effective therapies to mitigate this progressive and debilitating form of nerve cell death. Here, we report that pharmacologic restoration of the blood–brain barrier (BBB), 12 mo after murine TBI, is associated with arrested axonal neurodegeneration and cognitive recovery, benefits that persisted for months after treatment cessation. Recovery was achieved by 30 d of once-daily administration of P7C3-A20, a compound that stabilizes cellular energy levels. Four months after P7C3-A20, electron microscopy revealed full repair of TBI-induced breaks in cortical and hippocampal BBB endothelium. Immunohistochemical staining identified additional benefits of P7C3-A20, including restoration of normal BBB endothelium length, increased brain capillary pericyte density, increased expression of BBB tight junction proteins, reduced brain infiltration of immunoglobulin, and attenuated neuroinflammation. These changes were accompanied by cessation of TBI-induced chronic axonal degeneration. Specificity for P7C3-A20 action on the endothelium was confirmed by protection of cultured human brain microvascular endothelial cells from hydrogen peroxide-induced cell death, as well as preservation of BBB integrity in mice after exposure to toxic levels of lipopolysaccharide. P7C3-A20 also protected mice from BBB degradation after acute TBI. Collectively, our results provide insights into the pathophysiologic mechanisms behind chronic neurodegeneration after TBI, along with a putative treatment strategy. Because TBI increases the risks of other forms of neurodegeneration involving BBB deterioration (e.g., Alzheimer’s disease, Parkinson’s disease, vascular dementia, chronic traumatic encephalopathy), P7C3-A20 may have widespread clinical utility in the setting of neurodegenerative conditions.

Significance Cells generate a vast repertoire of lipid molecules whose functions are poorly understood. To investigate whether lipids can regulate cell fate decisions, we carried out a systematic lipidomic analysis and perturbation of lipid metabolism in cultured human epidermal keratinocytes, determining associations with the onset of differentiation. We identified individual lipid species that induced exit from the epidermal stem cell compartment. Our observations suggest that more research is warranted on the regulation of biological processes via lipid species, moving beyond the more conventional contribution of proteins and nucleic acids., While the lipids of the outer layers of mammalian epidermis and their contribution to barrier formation have been extensively described, the role of individual lipid species in the onset of keratinocyte differentiation remains unknown. A lipidomic analysis of primary human keratinocytes revealed accumulation of numerous lipid species during suspension-induced differentiation. A small interfering RNA screen of 258 lipid-modifying enzymes identified two genes that on knockdown induced epidermal differentiation: ELOVL1, encoding elongation of very long-chain fatty acids protein 1, and SLC27A1, encoding fatty acid transport protein 1. By intersecting lipidomic datasets from suspension-induced differentiation and knockdown keratinocytes, we pinpointed candidate bioactive lipid subspecies as differentiation regulators. Several of these—ceramides and glucosylceramides—induced differentiation when added to primary keratinocytes in culture. Our results reveal the potential of lipid subspecies to regulate exit from the epidermal stem cell compartment.

Significance Stem cells can be conceptualized as computational processors capable of sensing, processing, and converting environmental information (input) to yield a specific differentiation pathway (output). In this study, we employ a temperature-controlled polymer sheet actuator to interpret and transfer information, controlled by the material’s programming, to mesenchymal stem cells. The cell’s interpretation of mechanical, thermal, and biochemical signaling is shown to be dependent on the actuator’s activity, utilized to accelerate differentiation toward bone cells, further elucidating the role of microenvironmental parameters on mammalian cells. Our method provides a unique approach to processing two discrete stimuli into one biochemical signal, calcium ions, providing a basis for the logical control of the flow of biological signals and the design of cellular functions., Stem cells are capable of sensing and processing environmental inputs, converting this information to output a specific cell lineage through signaling cascades. Despite the combinatorial nature of mechanical, thermal, and biochemical signals, these stimuli have typically been decoupled and applied independently, requiring continuous regulation by controlling units. We employ a programmable polymer actuator sheet to autonomously synchronize thermal and mechanical signals applied to mesenchymal stem cells (MSCs). Using a grid on its underside, the shape change of polymer sheet, as well as cell morphology, calcium (Ca2+) influx, and focal adhesion assembly, could be visualized and quantified. This paper gives compelling evidence that the temperature sensing and mechanosensing of MSCs are interconnected via intracellular Ca2+. Up-regulated Ca2+ levels lead to a remarkable alteration of histone H3K9 acetylation and activation of osteogenic related genes. The interplay of physical, thermal, and biochemical signaling was utilized to accelerate the cell differentiation toward osteogenic lineage. The approach of programmable bioinstructivity provides a fundamental principle for functional biomaterials exhibiting multifaceted stimuli on differentiation programs. Technological impact is expected in the tissue engineering of periosteum for treating bone defects.

The cytoskeleton of eukaryotic cells is primarily composed of networks of filamentous proteins, F-actin, microtubules, and intermediate filaments. Interactions among the cytoskeletal components are important in determining cell structure and in regulating cell functions. For example, F-actin and microtubules work together to control cell shape and polarity, while the subcellular organization and transport of vimentin intermediate filament (VIF) networks depend on their interactions with microtubules. However, it is generally thought that F-actin and VIFs form two coexisting but separate networks that are independent due to observed differences in their spatial distribution and functions. In this paper, we present a closer investigation of both the structural and functional interplay between the F-actin and VIF cytoskeletal networks. We characterize the structure of VIFs and F-actin networks within the cell cortex using structured illumination microscopy and cryo-electron tomography. We find that VIFs and F-actin form an interpenetrating network (IPN) with interactions at multiple length scales, and VIFs are integral components of F-actin stress fibers. From measurements of recovery of cell contractility after transient stretching, we find that the IPN structure results in enhanced contractile forces and contributes to cell resilience. Studies of reconstituted networks and dynamic measurements in cells suggest direct and specific associations between VIFs and F-actin. From these results, we conclude that VIFs and F-actin work synergistically, both in their structure and in their function. These results profoundly alter our understanding of the contributions of the components of the cytoskeleton, particularly the interactions between intermediate filaments and F-actin.

Aortic valve stenosis (AVS) patients experience pathogenic valve leaflet stiffening due to excessive extracellular matrix (ECM) remodeling. Numerous microenvironmental cues influence pathogenic expression of ECM remodeling genes in tissue-resident valvular myofibroblasts, and the regulation of complex myofibroblast signaling networks depends on patient-specific extracellular factors. Here, we combined a manually curated myofibroblast signaling network with a data-driven transcription factor network to predict patient-specific myofibroblast gene expression signatures and drug responses. Using transcriptomic data from myofibroblasts cultured with AVS patient sera, we produced a large-scale, logic-gated differential equation model in which 11 biochemical and biomechanical signals were transduced via a network of 334 signaling and transcription reactions to accurately predict the expression of 27 fibrosis-related genes. Correlations were found between personalized model-predicted gene expression and AVS patient echocardiography data, suggesting links between fibrosis-related signaling and patient-specific AVS severity. Further, global network perturbation analyses revealed signaling molecules with the most influence over network-wide activity, including endothelin 1 (ET1), interleukin 6 (IL6), and transforming growth factor β (TGFβ), along with downstream mediators c-Jun N-terminal kinase (JNK), signal transducer and activator of transcription (STAT), and reactive oxygen species (ROS). Lastly, we performed virtual drug screening to identify patient-specific drug responses, which were experimentally validated via fibrotic gene expression measurements in valvular interstitial cells cultured with AVS patient sera and treated with or without bosentan-a clinically approved ET1 receptor inhibitor. In sum, our work advances the ability of computational approaches to provide a mechanistic basis for clinical decisions including patient stratification and personalized drug screening.

Significance In persons living with HIV-1 who start antiretroviral therapy, virus in the blood decreases rapidly to below the detection limit. The decrease occurs in two phases: a rapid initial decrease in the first weeks, followed by a second, slower phase occurring over the next few months. These decay processes are important because infected cells that remain may become part of the stable latent reservoir that prevents cure. The decay in virus levels in blood presumably reflects the loss of infected cells, but the relationship between the decay of free virus and of infected cells has been unclear. Here, we have analyzed this question using an assay that distinguishes between cells with intact and defective forms of the viral genome.

Significance Abdominal aortic aneurysm (AAA) is a common and severe disease with major genetic risk factors. In this study we generated enhancer-promoter contact data to identify regulatory elements in AAA-relevant cell types and identified changes in their predicted chromatin accessibility between AAA patients and controls. We integrated this information with disease-associated variants in regulatory elements and gene bodies to further understand the etiology and pathogenetic mechanisms of AAA. Our study combined whole-genome sequencing data with gene regulatory relations in disease-relevant cell types to reveal the important roles of the interleukin 6 pathway and ERG and KLF regulation in AAA pathogenesis., Abdominal aortic aneurysm (AAA) is a common degenerative cardiovascular disease whose pathobiology is not clearly understood. The cellular heterogeneity and cell-type-specific gene regulation of vascular cells in human AAA have not been well-characterized. Here, we performed analysis of whole-genome sequencing data in AAA patients versus controls with the aim of detecting disease-associated variants that may affect gene regulation in human aortic smooth muscle cells (AoSMC) and human aortic endothelial cells (HAEC), two cell types of high relevance to AAA disease. To support this analysis, we generated H3K27ac HiChIP data for these cell types and inferred cell-type-specific gene regulatory networks. We observed that AAA-associated variants were most enriched in regulatory regions in AoSMC, compared with HAEC and CD4+ cells. The cell-type-specific regulation defined by this HiChIP data supported the importance of ERG and the KLF family of transcription factors in AAA disease. The analysis of regulatory elements that contain noncoding variants and also are differentially open between AAA patients and controls revealed the significance of the interleukin-6-mediated signaling pathway. This finding was further validated by including information from the deleteriousness effect of nonsynonymous single-nucleotide variants in AAA patients and additional control data from the Medical Genome Reference Bank dataset. These results shed important insights into AAA pathogenesis and provide a model for cell-type-specific analysis of disease-associated variants.

Human phagocytes have key functions in the resolution of inflammation. Here, we assessed the role of the proposed 4S,5S-epoxy-resolvin intermediate in the biosynthesis of both resolvin D3 and resolvin D4. We found that human neutrophils converted this synthetic intermediate to resolvin D3 and resolvin D4. M2 macrophages transformed this labile epoxide intermediate to resolvin D4 and a previously unknown cysteinyl-resolvin isomer without appreciable amounts of resolvin D3. M2 macrophages play critical roles in the resolution of inflammation and in wound healing. Human M2 macrophages also converted leukotriene A(4) to lipoxins. The cysteinyl-resolvin isomer significantly accelerated tissue regeneration of surgically injured planaria. In a model of human granuloma formation, the cysteinyl-resolvin isomer significantly inhibited granuloma development by human peripheral blood leukocytes. Together, these results provide evidence for a human cell type–specific role of 4S,5S-epoxy-resolvin in the biosynthesis of resolvin D3 by neutrophils, resolvin D4 by both M2 macrophages and neutrophils, and a unique cysteinyl-resolvin isomer produced by M2 macrophages that carries potent biological activities in granuloma formation and tissue regeneration.

The particulate guanylyl cyclase A receptor (GC-A), via activation by its endogenous ligands atrial natriuretic peptide (ANP) and b-type natriuretic peptide (BNP), possesses beneficial biological properties such as blood pressure regulation, natriuresis, suppression of adverse remodeling, inhibition of the renin-angiotensin-aldosterone system, and favorable metabolic actions through the generation of its second messenger cyclic guanosine monophosphate (cGMP). Thus, the GC-A represents an important molecular therapeutic target for cardiovascular disease and its associated risk factors. However, a small molecule that is orally bioavailable and directly targets the GC-A to potentiate cGMP has yet to be discovered. Here, we performed a cell-based high-throughput screening campaign of the NIH Molecular Libraries Small Molecule Repository, and we successfully identified small molecule GC-A positive allosteric modulator (PAM) scaffolds. Further medicinal chemistry structure–activity relationship efforts of the lead scaffold resulted in the development of a GC-A PAM, MCUF-651, which enhanced ANP-mediated cGMP generation in human cardiac, renal, and fat cells and inhibited cardiomyocyte hypertrophy in vitro. Further, binding analysis confirmed MCUF-651 binds to GC-A and selectively enhances the binding of ANP to GC-A. Moreover, MCUF-651 is orally bioavailable in mice and enhances the ability of endogenous ANP and BNP, found in the plasma of normal subjects and patients with hypertension or heart failure, to generate GC-A–mediated cGMP ex vivo. In this work, we report the discovery and development of an oral, small molecule GC-A PAM that holds great potential as a therapeutic for cardiovascular, renal, and metabolic diseases.

Significance In hippocampal neurons, gene expression is triggered by electrical activity and Ca2+ entry via L-type Cav1.2 channels in a process called excitation–transcription coupling. We identified a domain on the voltage-gated K+ channel Kv2.1 that promotes the clustering of L-type Cav1.2 channels at endoplasmic reticulum–plasma membrane junctions in the soma of neurons. Importantly, we discovered by disrupting this domain that the Kv2.1-mediated clustering of Cav1.2 at this somatic microdomain is critical for depolarization-induced excitation–transcription coupling., In mammalian brain neurons, membrane depolarization leads to voltage-gated Ca2+ channel-mediated Ca2+ influx that triggers diverse cellular responses, including gene expression, in a process termed excitation–transcription coupling. Neuronal L-type Ca2+ channels, which have prominent populations on the soma and distal dendrites of hippocampal neurons, play a privileged role in excitation–transcription coupling. The voltage-gated K+ channel Kv2.1 organizes signaling complexes containing the L-type Ca2+ channel Cav1.2 at somatic endoplasmic reticulum–plasma membrane junctions. This leads to enhanced clustering of Cav1.2 channels, increasing their activity. However, the downstream consequences of the Kv2.1-mediated regulation of Cav1.2 localization and function on excitation–transcription coupling are not known. Here, we have identified a region between residues 478 to 486 of Kv2.1’s C terminus that mediates the Kv2.1-dependent clustering of Cav1.2. By disrupting this Ca2+ channel association domain with either mutations or with a cell-penetrating interfering peptide, we blocked the Kv2.1-mediated clustering of Cav1.2 at endoplasmic reticulum–plasma membrane junctions and the subsequent enhancement of its channel activity and somatic Ca2+ signals without affecting the clustering of Kv2.1. These interventions abolished the depolarization-induced and L-type Ca2+ channel-dependent phosphorylation of the transcription factor CREB and the subsequent expression of c-Fos in hippocampal neurons. Our findings support a model whereby the Kv2.1-Ca2+ channel association domain-mediated clustering of Cav1.2 channels imparts a mechanism to control somatic Ca2+ signals that couple neuronal excitation to gene expression.

Antigen (Ag) crosslinking of immunoglobulin E-receptor (IgE-FceRI) complexes in mast cells stimulates transmembrane (TM) signaling, requiring phosphorylation of the clustered FceRI by lipid-anchored Lyn tyrosine kinase. Previous studies showed that this stimulated coupling between Lyn and FceRI occurs in liquid ordered (Lo)-like nanodomains of the plasma membrane and that Lyn binds directly to cytosolic segments of FceRI that it initially phosphorylates for amplified activity. Net phosphorylation above a nonfunctional threshold is achieved in the stimulated state but not in the resting state, and current evidence supports the hypothesis that this relies on Ag crosslinking to disrupt a balance between Lyn and tyrosine phosphatase activities. However, the structural interactions that underlie the stimulation process remain poorly defined. This study evaluates the relative contributions and functional importance of different types of interactions leading to suprathreshold phosphorylation of Ag-crosslinked IgE-FceRI in live rat basophilic leukemia mast cells. Our high-precision diffusion measurements by imaging fluorescence correlation spectroscopy on multiple structural variants of Lyn and other lipid-anchored probes confirm subtle, stimulated stabilization of the Lo-like nanodomains in the membrane inner leaflet and concomitant sharpening of segregation from liquid disordered (Ld)-like regions. With other structural variants, we determine that lipid-based interactions are essential for access by Lyn, leading to phosphorylation of and protein-based binding to clustered FceRI. By contrast, TM tyrosine phosphatase, PTPα, is excluded from these regions due to its Ld-preference and steric exclusion of TM segments. Overall, we establish a synergy of lipid-based, protein-based, and steric interactions underlying functional TM signaling in mast cells.

Significance SARS-CoV-2 has had a crippling impact on human life globally. Vaccine development has been used as a first-line strategy for COVID-19 prevention and mitigation; however, small-molecule drugs are still vitally needed to extend treatment options. Traditional screening methods for identifying biologically active small molecules are sluggish and often sample an insufficient number of compounds to identify suitable hits. Here, we applied a screening method known as DNA-encoded chemistry technology (DEC-Tec) to screen billions of compounds against a critical viral protein, Mpro. In rapid fashion, we identified the compound CDD-1713 as a potent and selective Mpro inhibitor. This study illuminates DEC-Tec as a highly expeditious strategy toward generating small molecules against critical targets of infectious agents., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed more than 4 million humans globally, but there is no bona fide Food and Drug Administration–approved drug-like molecule to impede the COVID-19 pandemic. The sluggish pace of traditional therapeutic discovery is poorly suited to producing targeted treatments against rapidly evolving viruses. Here, we used an affinity-based screen of 4 billion DNA-encoded molecules en masse to identify a potent class of virus-specific inhibitors of the SARS-CoV-2 main protease (Mpro) without extensive and time-consuming medicinal chemistry. CDD-1714, the initial three-building-block screening hit (molecular weight [MW] = 542.5 g/mol), was a potent inhibitor (inhibition constant [Ki] = 20 nM). CDD-1713, a smaller two-building-block analog (MW = 353.3 g/mol) of CDD-1714, is a reversible covalent inhibitor of Mpro (Ki = 45 nM) that binds in the protease pocket, has specificity over human proteases, and shows in vitro efficacy in a SARS-CoV-2 infectivity model. Subsequently, key regions of CDD-1713 that were necessary for inhibitory activity were identified and a potent (Ki = 37 nM), smaller (MW = 323.4 g/mol), and metabolically more stable analog (CDD-1976) was generated. Thus, screening of DNA-encoded chemical libraries can accelerate the discovery of efficacious drug-like inhibitors of emerging viral disease targets.

Significance Nociceptors are sensory neurons that detect changes in the body’s internal and external milieu. Although occupying a primary role in signaling these changes to the nervous system, nociceptors also initiate neurogenic inflammation by sending antidromic signals back into the tissue. Because HMGB1 is a well-characterized endogenous mediator, which stimulates inflammation and is expressed by neurons, we reasoned HMGB1 release may be an important component of neurogenic inflammation. Here, by combining optogenetics, neuronal-specific ablation, nerve-injury, and inflammatory disease models, with direct assessment of inflammation and neuropathic pain, we show that nociceptor HMGB1 is required for an inflammatory response. These results provide direct evidence that nociceptor-related pain and inflammation can be prevented by targeting HMGB1., Inflammation, the body’s primary defensive response system to injury and infection, is triggered by molecular signatures of microbes and tissue injury. These molecules also stimulate specialized sensory neurons, termed nociceptors. Activation of nociceptors mediates inflammation through antidromic release of neuropeptides into infected or injured tissue, producing neurogenic inflammation. Because HMGB1 is an important inflammatory mediator that is synthesized by neurons, we reasoned nociceptor release of HMGB1 might be a component of the neuroinflammatory response. In support of this possibility, we show here that transgenic nociceptors expressing channelrhodopsin-2 (ChR2) directly release HMGB1 in response to light stimulation. Additionally, HMGB1 expression in neurons was silenced by crossing synapsin-Cre (Syn-Cre) mice with floxed HMGB1 mice (HMGB1f/f). When these mice undergo sciatic nerve injury to activate neurogenic inflammation, they are protected from the development of cutaneous inflammation and allodynia as compared to wild-type controls. Syn-Cre/HMGB1fl/fl mice subjected to experimental collagen antibody–induced arthritis, a disease model in which nociceptor-dependent inflammation plays a significant pathological role, are protected from the development of allodynia and joint inflammation. Thus, nociceptor HMGB1 is required to mediate pain and inflammation during sciatic nerve injury and collagen antibody–induced arthritis.

Significance The heart autoregulates its contractile strength to maintain cardiac output when pumping blood against the mechanical afterload from vascular resistance. Increased afterload in cardiovascular diseases is associated with cardiac arrhythmias. However, the underlying mechanisms remain elusive. Here, we studied the afterload effect on electrophysiology by embedding single cardiomyocytes in a three-dimensional viscoelastic hydrogel to apply afterload during cell contraction. We found that afterload is acutely transduced via nitric oxide synthase 1 (NOS1) signaling to modulate multiple ion channels to prolong action potential, increase Ca2+ transient, and enhance contractility. However, higher afterload causes a discordant alternans that is arrhythmogenic. Hence, our findings reveal an afterload-induced mechano-chemo-electro-transduction pathway that closes feedback loops in cardiac excitation–contraction coupling to enable autoregulation of contractility., The heart pumps blood against the mechanical afterload from arterial resistance, and increased afterload may alter cardiac electrophysiology and contribute to life-threatening arrhythmias. However, the cellular and molecular mechanisms underlying mechanoelectric coupling in cardiomyocytes remain unclear. We developed an innovative patch-clamp-in-gel technology to embed cardiomyocytes in a three-dimensional (3D) viscoelastic hydrogel that imposes an afterload during regular myocyte contraction. Here, we investigated how afterload affects action potentials, ionic currents, intracellular Ca2+ transients, and cell contraction of adult rabbit ventricular cardiomyocytes. We found that afterload prolonged action potential duration (APD), increased transient outward K+ current, decreased inward rectifier K+ current, and increased L-type Ca2+ current. Increased Ca2+ entry caused enhanced Ca2+ transients and contractility. Moreover, elevated afterload led to discordant alternans in APD and Ca2+ transient. Ca2+ alternans persisted under action potential clamp, indicating that the alternans was Ca2+ dependent. Furthermore, all these afterload effects were significantly attenuated by inhibiting nitric oxide synthase 1 (NOS1). Taken together, our data reveal a mechano-chemo-electrotransduction (MCET) mechanism that acutely transduces afterload through NOS1–nitric oxide signaling to modulate the action potential, Ca2+ transient, and contractility. The MCET pathway provides a feedback loop in excitation–Ca2+ signaling–contraction coupling, enabling autoregulation of contractility in cardiomyocytes in response to afterload. This MCET mechanism is integral to the individual cardiomyocyte (and thus the heart) to intrinsically enhance its contractility in response to the load against which it has to do work. While this MCET is largely compensatory for physiological load changes, it may also increase susceptibility to arrhythmias under excessive pathological loading.

Chronic ultraviolet (UV) radiation exposure is the greatest risk factor for cutaneous squamous cell carcinoma (cSCC) development, and compromised immunity accelerates this risk. Having previously identified that epidermal Langerhans cells (LC) facilitate the expansion of UV-induced mutant keratinocytes (KC), we sought to more fully elucidate the immune pathways critical to cutaneous carcinogenesis and to identify potential targets of intervention. Herein, we reveal that chronic UV induces and LC enhance a local immune shift toward RORγt+ interleukin (IL)-22/IL-17A–producing cells that occurs in the presence or absence of T cells while identifying a distinct RORγt+ Sca-1+ CD103+ ICOS+ CD2(+/−) CCR6+ intracellular CD3+ cutaneous innate lymphoid cell type-3 (ILC3) population (uvILC3) that is associated with UV-induced mutant KC growth. We further show that mutant KC clone size is markedly reduced in the absence of RORγt+ lymphocytes or IL-22, both observed in association with expanding KC clones, and find that topical application of a RORγ/γt inhibitor during chronic UV exposure reduces local expression of IL-22 and IL-17A while markedly limiting mutant p53 KC clonal expansion. We implicate upstream Toll-like receptor signaling in driving this immune response to chronic UV exposure, as MyD88/Trif double-deficient mice also show substantially reduced p53 island number and size. These data elucidate key immune components of chronic UV–induced cutaneous carcinogenesis that might represent targets for skin cancer prevention.

Significance In the classic view, telomerase is silenced in terminally differentiated cells and its reactivation supports immortalization and unlimited growth of most cancers. Here, we determine the involvement of telomerase during replicative senescence in primary fibroblasts from mouse and human. In both cases, we find that cells that are unable to produce telomerase approach cellular senescence earlier and exhibit a significantly higher rate of malignant transformation than the control cells. Furthermore, an evident upregulation of telomerase expression is detected in wild-type control cells at the presenescence stage, which is accountable for the protection. In conclusion, this study suggests that telomerase has a previously underappreciated, protective role in buffering senescence stresses due to short, dysfunctional telomeres, thereby preventing malignant transformation., Telomerase is an enzymatic ribonucleoprotein complex that acts as a reverse transcriptase in the elongation of telomeres. Telomerase activity is well documented in embryonic stem cells and the vast majority of tumor cells, but its role in somatic cells remains to be understood. Here, we report an unexpected function of telomerase during cellular senescence and tumorigenesis. We crossed Tert heterozygous knockout mice (mTert+/−) for 26 generations, during which time there was progressive shortening of telomeres, and obtained primary skin fibroblasts from mTert+/+ and mTert−/− progeny of the 26th cross. As a consequence of insufficient telomerase activities in prior generations, both mTert+/+ and mTert−/− fibroblasts showed comparable and extremely short telomere length. However, mTert−/− cells approached cellular senescence faster and exhibited a significantly higher rate of malignant transformation than mTert+/+ cells. Furthermore, an evident up-regulation of telomerase reverse-transcriptase (TERT) expression was detected in mTert+/+ cells at the presenescence stage. Moreover, removal or down-regulation of TERT expression in mTert+/+ and human primary fibroblast cells via CRISPR/Cas9 or shRNA recapitulated mTert−/− phenotypes of accelerated senescence and transformation, and overexpression of TERT in mTert−/− cells rescued these phenotypes. Taking these data together, this study suggests that TERT has a previously underappreciated, protective role in buffering senescence stresses due to short, dysfunctional telomeres, and preventing malignant transformation.

Significance Nonalcoholic steatohepatitis (NASH) is an increasingly common disease characterized by liver steatosis and inflammation—with fibrosis being an important indicator of disease progression and severity—and is associated with reduced endothelial function and NO–soluble guanylate cyclase (sGC) signaling. Signaling downstream of NO can be restored using praliciguat, an sGC stimulator. Within the liver, stellate cells and myofibroblasts express sGC (unlike hepatocytes) and thus can be stimulated by praliciguat. Increased sGC activity inhibits fibrotic transformation and inflammatory responses in stellate cells potentially through AMPK and SMAD7. The effects on isolated stellate cells translate to human microtissues and in vivo models where treatment with praliciguat reduces inflammation, fibrosis, and steatosis. These preclinical results support further investigation of praliciguat as a potential therapy for NASH/fibrosis., Endothelial dysfunction and reduced nitric oxide (NO) signaling are a key element of the pathophysiology of nonalcoholic steatohepatitis (NASH). Stimulators of soluble guanylate cyclase (sGC) enhance NO signaling; have been shown preclinically to reduce inflammation, fibrosis, and steatosis; and thus have been proposed as potential therapies for NASH and fibrotic liver diseases. Praliciguat, an oral sGC stimulator with extensive distribution to the liver, was used to explore the role of this signaling pathway in NASH. We found that sGC is expressed in hepatic stellate cells and stellate-derived myofibroblasts, but not in hepatocytes. Praliciguat acted directly on isolated hepatic stellate cells to inhibit fibrotic and inflammatory signaling potentially through regulation of AMPK and SMAD7. Using in vivo microdialysis, we demonstrated stimulation of the NO–sGC pathway by praliciguat in both healthy and fibrotic livers. In preclinical models of NASH, praliciguat treatment was associated with lower levels of liver fibrosis and lower expression of fibrotic and inflammatory biomarkers. Praliciguat treatment lowered hepatic steatosis and plasma cholesterol levels. The antiinflammatory and antifibrotic effects of praliciguat were recapitulated in human microtissues in vitro. These data provide a plausible cellular basis for the mechanism of action of sGC stimulators and suggest the potential therapeutic utility of praliciguat in the treatment of NASH.

Significance Unlike all differentiated cells, pluripotent stem cells do not elicit a productive antiviral response when infected by a pathogen. This observation seems at odds with the importance of pluripotent stem cells given their absolute requirement for the development of life. Here we investigate why this antiviral response is not utilized in these unique cells. We find that the factors required to maintain pluripotency are incompatible with those involved in eliciting the canonical interferon-based response to virus infection., Upon virus infection, pluripotent stem cells neither induce nor respond to canonical type I interferons (IFN-I). To better understand this biology, we characterized induced pluripotent stem cells (iPSCs) as well as their differentiated parental or rederived counterparts. We confirmed that only iPSCs failed to respond to viral RNA, IFN-I, or viral infection. This lack of response could be phenocopied in fibroblasts with the expression of a reprogramming factor which repressed the capacity to induce canonical antiviral pathways. To ascertain the consequences of restoring the antiviral response in the context of pluripotency, we engineered a system to engage these defenses in iPSCs. Inducible expression of a recombinant virus-activated transcription factor resulted in the successful reconstitution of antiviral defenses through the direct up-regulation of IFN-I–stimulated genes. Induction of the antiviral signature in iPSCs, even for a short duration, resulted in the dysregulation of genes associated with all three germ layers despite maintaining pluripotency markers. Trilineage differentiation of these same cells showed that engagement of the antiviral defenses compromised ectoderm and endoderm formation and dysregulated the development of mesodermal sublineages. In all, these data suggest that the temporal induction of the antiviral response primes iPSCs away from pluripotency and induces numerous aberrant gene products upon differentiation. Together these results suggest that the IFN-I system and pluripotency may be incompatible with each other and thus explain why stem cells do not utilize the canonical antiviral system.

The cancer-free photosensitive trichothiodystrophy (PS-TTD) and the cancer-prone xeroderma pigmentosum (XP) are rare monogenic disorders that can arise from mutations in the same genes, namely ERCC2/XPD or ERCC3/XPB. Both XPD and XPB proteins belong to the 10-subunit complex transcription factor IIH (TFIIH) that plays a key role in transcription and nucleotide excision repair, the DNA repair pathway devoted to the removal of ultraviolet-induced DNA lesions. Compelling evidence suggests that mutations affecting the DNA repair activity of TFIIH are responsible for the pathological features of XP, whereas those also impairing transcription give rise to TTD. By adopting a relatives-based whole transcriptome sequencing approach followed by specific gene expression profiling in primary fibroblasts from a large cohort of TTD or XP cases with mutations in ERCC2/XPD gene, we identify the expression alterations specific for TTD primary dermal fibroblasts. While most of these transcription deregulations do not impact on the protein level, very low amounts of prostaglandin I(2) synthase (PTGIS) are found in TTD cells. PTGIS catalyzes the last step of prostaglandin I(2) synthesis, a potent vasodilator and inhibitor of platelet aggregation. Its reduction characterizes all TTD cases so far investigated, both the PS-TTD with mutations in TFIIH coding genes as well as the nonphotosensitive (NPS)-TTD. A severe impairment of TFIIH and RNA polymerase II recruitment on the PTGIS promoter is found in TTD but not in XP cells. Thus, PTGIS represents a biomarker that combines all PS- and NPS-TTD cases and distinguishes them from XP.

Significance Clinical studies have shown that defects in type I interferon (IFN) production or antibodies to IFN appear to correlate with severe COVID-19 infection. Here, we demonstrate that proinflammatory cytokines but not type I IFN are produced by hypoxic human monocytes. We show that hypoxia suppresses production of type I IFN but not nuclear factor-κB–dependent proinflammatory cytokines. We demonstrate that hypoxia-inducible factor-1α is a direct transcriptional suppressor of interferon regulatory factors, the transcriptional activators of type I IFN. These findings may aid in understanding and control of impaired IFN production by severe acute respiratory syndrome coronavirus 2 infection., Patients with severe COVID-19 infection exhibit a low level of oxygen in affected tissue and blood. To understand the pathophysiology of COVID-19 infection, it is therefore necessary to understand cell function during hypoxia. We investigated aspects of human monocyte activation under hypoxic conditions. HMGB1 is an alarmin released by stressed cells. Under normoxic conditions, HMGB1 activates interferon regulatory factor (IRF)5 and nuclear factor-κB in monocytes, leading to expression of type I interferon (IFN) and inflammatory cytokines including tumor necrosis factor α, and interleukin 1β, respectively. When hypoxic monocytes are activated by HMGB1, they produce proinflammatory cytokines but fail to produce type I IFN. Hypoxia-inducible factor-1α, induced by hypoxia, functions as a direct transcriptional repressor of IRF5 and IRF3. As hypoxia is a stressor that induces secretion of HMGB1 by epithelial cells, hypoxia establishes a microenvironment that favors monocyte production of inflammatory cytokines but not IFN. These findings have implications for the pathogenesis of COVID-19.

Genetic editing of induced pluripotent stem (iPS) cells represents a promising avenue for an HIV cure. However, certain challenges remain before bringing this approach to the clinic. Among them, in vivo engraftment of cells genetically edited in vitro needs to be achieved. In this study, CD34(+) cells derived in vitro from iPS cells genetically modified to carry the CCR5Δ32 mutant alleles did not engraft in humanized immunodeficient mice. However, the CD34(+) cells isolated from teratomas generated in vivo from these genetically edited iPS cells engrafted in all experiments. These CD34(+) cells also gave rise to peripheral blood mononuclear cells in the mice that, when inoculated with HIV in cell culture, were resistant to HIV R5-tropic isolates. This study indicates that teratomas can provide an environment that can help evaluate the engraftment potential of CD34(+) cells derived from the genetically modified iPS cells in vitro. The results further confirm the possibility of using genetically engineered iPS cells to derive engraftable hematopoietic stem cells resistant to HIV as an approach toward an HIV cure.

Significance Microbes shape human health and disease. Today, there are increasing correlations of gut microbes with specific diseases, but the underlying molecules and mechanism linking the two are not known. This stifles both the understanding of disease causation and the development of suitable treatments. We investigated a dozen patient isolates of Ruminococcus gnavus, a prevalent gut microbe linked to inflammatory bowel disease (IBD). We found that some isolates possess a protective capsule that encourages a symbiotic relationship with the host immune system, while others lack this protective capsule and elicit a robust inflammatory immune response. This work reveals a path by which R. gnavus could be connected to IBD and potential therapeutic interventions., Active inflammatory bowel disease (IBD) often coincides with increases of Ruminococcus gnavus, a gut microbe found in nearly everyone. It was not known how, or if, this correlation contributed to disease. We investigated clinical isolates of R. gnavus to identify molecular mechanisms that would link R. gnavus to inflammation. Here, we show that only some isolates of R. gnavus produce a capsular polysaccharide that promotes a tolerogenic immune response, whereas isolates lacking functional capsule biosynthetic genes elicit robust proinflammatory responses in vitro. Germ-free mice colonized with an isolate of R. gnavus lacking a capsule show increased measures of gut inflammation compared to those colonized with an encapsulated isolate in vivo. These observations in the context of our earlier identification of an inflammatory cell-wall polysaccharide reveal how some strains of R. gnavus could drive the inflammatory responses that characterize IBD.

Significance Through conducting quantitative proteomics upon Salmonella infection, we identified a SIK2 signaling network, implementing the kinase into a so far concealed biological function. Our data exposed SIK2 as a central orchestrator of an actin regulatory network, coordinating the stability of Salmonella-containing vacuole (SCV) and cellular actin assembly, in order to limit the acute phase of the infection. Most strikingly, SIK2 is not exclusively acting locally on actin assembly associated with the SCV but impacts the actin cytoskeleton architecture in its entirety upon Salmonella infection. Our work provides a mechanistic framework for how the actin cytoskeleton is regulated and how it helps to control an acute Salmonella infection., Salmonella is an intracellular pathogen of a substantial global health concern. In order to identify key players involved in Salmonella infection, we performed a global host phosphoproteome analysis subsequent to bacterial infection. Thereby, we identified the kinase SIK2 as a central component of the host defense machinery upon Salmonella infection. SIK2 depletion favors the escape of bacteria from the Salmonella-containing vacuole (SCV) and impairs Xenophagy, resulting in a hyperproliferative phenotype. Mechanistically, SIK2 associates with actin filaments under basal conditions; however, during bacterial infection, SIK2 is recruited to the SCV together with the elements of the actin polymerization machinery (Arp2/3 complex and Formins). Notably, SIK2 depletion results in a severe pathological cellular actin nucleation and polymerization defect upon Salmonella infection. We propose that SIK2 controls the formation of a protective SCV actin shield shortly after invasion and orchestrates the actin cytoskeleton architecture in its entirety to control an acute Salmonella infection after bacterial invasion.

The production of proinflammatory cytokines, particularly granulocyte-macrophage colony-stimulating factor (GM-CSF), by pathogenic CD4(+) T cells is central for mediating tissue injury in inflammatory and autoimmune diseases. However, the factors regulating the T cell pathogenic gene expression program remain unclear. Here, we investigated how the Ikaros transcription factor regulates the global gene expression and chromatin accessibility changes in murine T cells during Th17 polarization and after activation via the T cell receptor (TCR) and CD28. We found that, in both conditions, Ikaros represses the expression of genes from the pathogenic signature, particularly Csf2, which encodes GM-CSF. We show that, in TCR/CD28-activated T cells, Ikaros binds a critical enhancer downstream of Csf2 and is required to regulate chromatin accessibility at multiple regions across this locus. Genome-wide Ikaros binding is associated with more compact chromatin, notably at multiple sites containing NFκB or STAT5 target motifs, and STAT5 or NFκB inhibition prevents GM-CSF production in Ikaros-deficient cells. Importantly, Ikaros also limits GM-CSF production in TCR/CD28-activated human T cells. Our data therefore highlight a critical conserved transcriptional mechanism that antagonizes GM-CSF expression in T cells.

Significance Macrophages protect against microbial infection, in part by engulfing and killing invading microbes. Fungal pathogens such as Candida albicans are known to evade phagocytic killing by forming hyphae that are physically challenging to engulf because of their length. We now find that macrophages can respond by folding the hyphae of C. albicans (and other fungal species). Hyphal folding implies that immune cells can continue to apply mechanical force after their cargo has been internalized. The involvement of Dectin-1, β2-integrin, and actin–myosin polymerization provides initial mechanistic insight. Folding damages hyphae, inhibits their growth, and facilitates their complete engulfment. Therefore, hyphal folding represents an additional weapon in the immune cell armory that presumably contributes to fungal clearance., Innate immunity provides essential protection against life-threatening fungal infections. However, the outcomes of individual skirmishes between immune cells and fungal pathogens are not a foregone conclusion because some pathogens have evolved mechanisms to evade phagocytic recognition, engulfment, and killing. For example, Candida albicans can escape phagocytosis by activating cellular morphogenesis to form lengthy hyphae that are challenging to engulf. Through live imaging of C. albicans–macrophage interactions, we discovered that macrophages can counteract this by folding fungal hyphae. The folding of fungal hyphae is promoted by Dectin-1, β2-integrin, VASP, actin–myosin polymerization, and cell motility. Folding facilitates the complete engulfment of long hyphae in some cases and it inhibits hyphal growth, presumably tipping the balance toward successful fungal clearance.

Complement protein C3dg, a key linkage between innate and adaptive immunity, is capable of stimulating both humoral and cell-mediated immune responses, leading to considerable interest in its use as a molecular adjuvant. However, the potential of C3dg as an adjuvant is limited without ways of controllably assembling multiple copies of it into vaccine platforms. Here, we report a strategy to assemble C3dg into supramolecular nanofibers with excellent compositional control, using β-tail fusion tags. These assemblies were investigated as therapeutic active immunotherapies, which may offer advantages over existing biologics, particularly toward chronic inflammatory diseases. Supramolecular assemblies based on the Q11 peptide system containing β-tail-tagged C3dg, B cell epitopes from TNF, and the universal T cell epitope PADRE raised strong antibody responses against both TNF and C3dg, and prophylactic immunization with these materials significantly improved protection in a lethal TNF-mediated inflammation model. Additionally, in a murine model of psoriasis induced by imiquimod, the C3dg-adjuvanted nanofiber vaccine performed as well as anti-TNF monoclonal antibodies. Nanofibers containing only β-tail-C3dg and lacking the TNF B cell epitope also showed improvements in both models, suggesting that supramolecular C3dg, by itself, played an important therapeutic role. We observed that immunization with β-tail-C3dg caused the expansion of an autoreactive C3dg-specific T cell population, which may act to dampen the immune response, preventing excessive inflammation. These findings indicate that molecular assemblies displaying C3dg warrant further development as active immunotherapies.

Measles virus (MeV) is highly infectious by the respiratory route and remains an important cause of childhood mortality. However, the process by which MeV infection is efficiently established in the respiratory tract is controversial with suggestions that respiratory epithelial cells are not susceptible to infection from the apical mucosal surface. Therefore, it has been hypothesized that infection is initiated in lung macrophages or dendritic cells and that epithelial infection is subsequently established through the basolateral surface by infected lymphocytes. To better understand the process of respiratory tract initiation of MeV infection, primary differentiated respiratory epithelial cell cultures were established from rhesus macaque tracheal and nasal tissues. Infection of these cultures with MeV from the apical surface was more efficient than from the basolateral surface with shedding of viable MeV-producing multinucleated giant cell (MGC) syncytia from the surface. Despite presence of MGCs and infectious virus in supernatant fluids after apical infection, infected cells were not detected in the adherent epithelial sheet and transepithelial electrical resistance was maintained. After infection from the basolateral surface, epithelial damage and large clusters of MeV-positive cells were observed. Treatment with fusion inhibitory peptides showed that MeV production after apical infection was not dependent on infection of the basolateral surface. These results are consistent with the hypothesis that MeV infection is initiated by apical infection of respiratory epithelial cells with subsequent infection of lymphoid tissue and systemic spread.

Significance The cGAS-STING pathway is important for immune defense against infection and cancer. STING activation triggers multiple signaling cascades leading to activation of IRF3, NF-κB, and autophagy. By generating mice harboring mutations of STING that specifically inactivate different signaling cascades, we found that ablation of IRF3 activation, which is essential for the induction of type I interferons, was not sufficient to abolish the immune defense against virus infection and cancer in mouse models. Rather, impairing the ability of STING to recruit TBK1, which is important for activating both IRF3 and NF-κB, abolished the immune defense functions of STING. These results demonstrate that the recruitment of TBK1 to STING has functions that are broader than activating IRF3 and inducing type I interferons., The induction of type I interferons through the transcription factor interferon regulatory factor 3 (IRF3) is considered a major outcome of stimulator of interferon genes (STING) activation that drives immune responses against DNA viruses and tumors. However, STING activation can also trigger other downstream pathways such as nuclear factor κB (NF-κB) signaling and autophagy, and the roles of interferon (IFN)-independent functions of STING in infectious diseases or cancer are not well understood. Here, we generated a STING mouse strain with a mutation (S365A) that disrupts IRF3 binding and therefore type I interferon induction but not NF-κB activation or autophagy induction. We also generated STING mice with mutations that disrupt the recruitment of TANK-binding kinase 1 (TBK1), which is important for both IRF3 and NF-κB activation but not autophagy induction (L373A or ∆CTT, which lacks the C-terminal tail). The STING-S365A mutant mice, but not L373A or ∆CTT mice, were still resistant to herpes simplex virus 1 (HSV-1) infections and mounted an antitumor response after cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) treatment despite the absence of STING-induced interferons. These results demonstrate that STING can function independently of type I interferons and autophagy, and that TBK1 recruitment to STING is essential for antiviral and antitumor immunity.