Rebooting Human Immunology.

Recent progress in both conceptual and technological approaches to human immunology have rejuvenated a field that has long been in the shadow of the inbred mouse model. This is a healthy development both for the clinical relevance of immunology and for the fact that it is a way to gain access to the wealth of phenomenology in the many human diseases that involve the immune system. This is where we are likely to discover new immunological mechanisms and principals, especially those involving genetic heterogeneity or environmental influences that are difficult to model effectively in inbred mice. We also suggest that there are likely to be novel immunological mechanisms in long-lived, less fecund mammals such as human beings since they must remain healthy far longer than short-lived rodents in order for the species to survive.

Fluorescence imaging of large-scale neural ensemble dynamics.

Recent progress in fluorescence imaging allows neuroscientists to observe the dynamics of thousands of individual neurons, identified genetically or by their connectivity, across multiple brain areas and for extended durations in awake behaving mammals. We discuss advances in fluorescent indicators of neural activity, viral and genetic methods to express these indicators, chronic animal preparations for long-term imaging studies, and microscopes to monitor and manipulate the activity of large neural ensembles. Ca2+ imaging studies of neural activity can track brain area interactions and distributed information processing at cellular resolution. Across smaller spatial scales, high-speed voltage imaging reveals the distinctive spiking patterns and coding properties of targeted neuron types. Collectively, these innovations will propel studies of brain function and dovetail with ongoing neuroscience initiatives to identify new neuron types and develop widely applicable, non-human primate models. The optical toolkit's growing sophistication also suggests that "brain observatory" facilities would be useful open resources for future brain-imaging studies.

A genetically tractable jellyfish model for systems and evolutionary neuroscience.

Jellyfish are radially symmetric organisms without a brain that arose more than 500 million years ago. They achieve organismal behaviors through coordinated interactions between autonomously functioning body parts. Jellyfish neurons have been studied electrophysiologically, but not at the systems level. We introduce Clytia hemisphaerica as a transparent, genetically tractable jellyfish model for systems and evolutionary neuroscience. We generate stable F1 transgenic lines for cell-type-specific conditional ablation and whole-organism GCaMP imaging. Using these tools and computational analyses, we find that an apparently diffuse network of RFamide-expressing umbrellar neurons is functionally subdivided into a series of spatially localized subassemblies whose synchronous activation controls directional food transfer from the tentacles to the mouth. These data reveal an unanticipated degree of structured neural organization in this species. Clytia affords a platform for systems-level studies of neural function, behavior, and evolution within a clade of marine organisms with growing ecological and economic importance.

Two-component spike nanoparticle vaccine protects macaques from SARS-CoV-2 infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is continuing to disrupt personal lives, global healthcare systems, and economies. Hence, there is an urgent need for a vaccine that prevents viral infection, transmission, and disease. Here, we present a two-component protein-based nanoparticle vaccine that displays multiple copies of the SARS-CoV-2 spike protein. Immunization studies show that this vaccine induces potent neutralizing antibody responses in mice, rabbits, and cynomolgus macaques. The vaccine-induced immunity protects macaques against a high-dose challenge, resulting in strongly reduced viral infection and replication in the upper and lower airways. These nanoparticles are a promising vaccine candidate to curtail the SARS-CoV-2 pandemic.

Antidepressant drugs act by directly binding to TRKB neurotrophin receptors.

It is unclear how binding of antidepressant drugs to their targets gives rise to the clinical antidepressant effect. We discovered that the transmembrane domain of tyrosine kinase receptor 2 (TRKB), the brain-derived neurotrophic factor (BDNF) receptor that promotes neuronal plasticity and antidepressant responses, has a cholesterol-sensing function that mediates synaptic effects of cholesterol. We then found that both typical and fast-acting antidepressants directly bind to TRKB, thereby facilitating synaptic localization of TRKB and its activation by BDNF. Extensive computational approaches including atomistic molecular dynamics simulations revealed a binding site at the transmembrane region of TRKB dimers. Mutation of the TRKB antidepressant-binding motif impaired cellular, behavioral, and plasticity-promoting responses to antidepressants in vitro and in vivo. We suggest that binding to TRKB and allosteric facilitation of BDNF signaling is the common mechanism for antidepressant action, which may explain why typical antidepressants act slowly and how molecular effects of antidepressants are translated into clinical mood recovery.

Human hemato-lymphoid system mice: current use and future potential for medicine.

To directly study complex human hemato-lymphoid system physiology and respective system-associated diseases in vivo, human-to-mouse xenotransplantation models for human blood and blood-forming cells and organs have been developed over the past three decades. We here review the fundamental requirements and the remarkable progress made over the past few years in improving these systems, the current major achievements reached by use of these models, and the future challenges to more closely model and study human health and disease and to achieve predictive preclinical testing of both prevention measures and potential new therapies.

Establishing or Exaggerating Causality for the Gut Microbiome: Lessons from Human Microbiota-Associated Rodents.

Human diseases are increasingly linked with an altered or "dysbiotic" gut microbiota, but whether such changes are causal, consequential, or bystanders to disease is, for the most part, unresolved. Human microbiota-associated (HMA) rodents have become a cornerstone of microbiome science for addressing causal relationships between altered microbiomes and host pathology. In a systematic review, we found that 95% of published studies (36/38) on HMA rodents reported a transfer of pathological phenotypes to recipient animals, and many extrapolated the findings to make causal inferences to human diseases. We posit that this exceedingly high rate of inter-species transferable pathologies is implausible and overstates the role of the gut microbiome in human disease. We advocate for a more rigorous and critical approach for inferring causality to avoid false concepts and prevent unrealistic expectations that may undermine the credibility of microbiome science and delay its translation.

Restricted Clonality and Limited Germinal Center Reentry Characterize Memory B Cell Reactivation by Boosting.

Repeated exposure to pathogens or their antigens triggers anamnestic antibody responses that are higher in magnitude and affinity than the primary response. These involve reengagement of memory B cell (MBC) clones, the diversity and specificity of which determine the breadth and effectiveness of the ensuing antibody response. Using prime-boost models in mice, we find that secondary responses are characterized by a clonality bottleneck that restricts the engagement of the large diversity of MBC clones generated by priming. Rediversification of mutated MBCs is infrequent within secondary germinal centers (GCs), which instead consist predominantly of B cells without prior GC experience or detectable clonal expansion. Few MBC clones, generally derived from higher-affinity germline precursors, account for the majority of secondary antibody responses, while most primary-derived clonal diversity is not reengaged detectably by boosting. Understanding how to counter this bottleneck may improve our ability to elicit antibodies to non-immunodominant epitopes by vaccination.

ß-Arrestin-Biased Allosteric Modulator of NTSR1 Selectively Attenuates Addictive Behaviors.

Small molecule neurotensin receptor 1 (NTSR1) agonists have been pursued for more than 40 years as potential therapeutics for psychiatric disorders, including drug addiction. Clinical development of NTSR1 agonists has, however, been precluded by their severe side effects. NTSR1, a G protein-coupled receptor (GPCR), signals through the canonical activation of G proteins and engages ß-arrestins to mediate distinct cellular signaling events. Here, we characterize the allosteric NTSR1 modulator SBI-553. This small molecule not only acts as a ß-arrestin-biased agonist but also extends profound ß-arrestin bias to the endogenous ligand by selectively antagonizing G protein signaling. SBI-553 shows efficacy in animal models of psychostimulant abuse, including cocaine self-administration, without the side effects characteristic of balanced NTSR1 agonism. These findings indicate that NTSR1 G protein and ß-arrestin activation produce discrete and separable physiological effects, thus providing a strategy to develop safer GPCR-targeting therapeutics with more directed pharmacological action.

Decisions about dendritic cells: past, present, and future.

A properly functioning adaptive immune system signifies the best features of life. It is diverse beyond compare, tolerant without fail, and capable of behaving appropriately with a myriad of infections and other challenges. Dendritic cells are required to explain how this remarkable system is energized and directed. I frame this article in terms of the major decisions that my colleagues and I have made in dendritic cell science and some of the guiding themes at the time the decisions were made. As a result of progress worldwide, there is now evidence of a central role for dendritic cells in initiating antigen-specific immunity and tolerance. The in vivo distribution and development of a previously unrecognized white cell lineage is better understood, as is the importance of dendritic cell maturation to link innate and adaptive immunity in response to many stimuli. Our current focus is on antigen uptake receptors on dendritic cells. These receptors enable experiments involving selective targeting of antigens in situ and new approaches to vaccine design in preclinical and clinical systems.

The Genetics of Aging: A Vertebrate Perspective.

Aging negatively impacts vitality and health. Many genetic pathways that regulate aging were discovered in invertebrates. However, the genetics of aging is more complex in vertebrates because of their specialized systems. This Review discusses advances in the genetic regulation of aging in vertebrates from work in mice, humans, and organisms with exceptional lifespans. We highlight challenges for the future, including sex-dependent differences in lifespan and the interplay between genes and environment. We also discuss how the identification of reliable biomarkers of age and development of new vertebrate models can be leveraged for personalized interventions to counter aging and age-related diseases.

BMAL1-Driven Tissue Clocks Respond Independently to Light to Maintain Homeostasis.

Circadian rhythms control organismal physiology throughout the day. At the cellular level, clock regulation is established by a self-sustained Bmal1-dependent transcriptional oscillator network. However, it is still unclear how different tissues achieve a synchronized rhythmic physiology. That is, do they respond independently to environmental signals, or require interactions with each other to do so? We show that unexpectedly, light synchronizes the Bmal1-dependent circadian machinery in single tissues in the absence of Bmal1 in all other tissues. Strikingly, light-driven tissue autonomous clocks occur without rhythmic feeding behavior and are lost in constant darkness. Importantly, tissue-autonomous Bmal1 partially sustains homeostasis in otherwise arrhythmic and prematurely aging animals. Our results therefore support a two-branched model for the daily synchronization of tissues: an autonomous response branch, whereby light entrains circadian clocks without any commitment of other Bmal1-dependent clocks, and a memory branch using other Bmal1-dependent clocks to "remember" time in the absence of external cues.

Defining the Independence of the Liver Circadian Clock.

Mammals rely on a network of circadian clocks to control daily systemic metabolism and physiology. The central pacemaker in the suprachiasmatic nucleus (SCN) is considered hierarchically dominant over peripheral clocks, whose degree of independence, or tissue-level autonomy, has never been ascertained in vivo. Using arrhythmic Bmal1-null mice, we generated animals with reconstituted circadian expression of BMAL1 exclusively in the liver (Liver-RE). High-throughput transcriptomics and metabolomics show that the liver has independent circadian functions specific for metabolic processes such as the NAD+ salvage pathway and glycogen turnover. However, although BMAL1 occupies chromatin at most genomic targets in Liver-RE mice, circadian expression is restricted to ∼10% of normally rhythmic transcripts. Finally, rhythmic clock gene expression is lost in Liver-RE mice under constant darkness. Hence, full circadian function in the liver depends on signals emanating from other clocks, and light contributes to tissue-autonomous clock function.

Naturalizing mouse models for immunology.

Laboratory mice have provided invaluable insight into mammalian immune systems. Yet the immune phenotypes of mice bred and maintained in conventional laboratory conditions often differ from the immune phenotypes of wild mammals. Recent work to naturalize the environmental experience of inbred laboratory mice-to take them where the wild things are (to borrow a phrase from Maurice Sendak), via approaches such as construction of exposure histories, provision of fecal transplants or surrogate mothering by wild mice, and rewilding-is poised to expand understanding, complementing genetic and phylogenetic research on how natural selection has shaped mammalian immune systems while improving the translational potential of mouse research.

High-throughput phenotyping reveals expansive genetic and structural underpinnings of immune variation.

By developing a high-density murine immunophenotyping platform compatible with high-throughput genetic screening, we have established profound contributions of genetics and structure to immune variation (http://www.immunophenotype.org). Specifically, high-throughput phenotyping of 530 unique mouse gene knockouts identified 140 monogenic 'hits', of which most had no previous immunologic association. Furthermore, hits were collectively enriched in genes for which humans show poor tolerance to loss of function. The immunophenotyping platform also exposed dense correlation networks linking immune parameters with each other and with specific physiologic traits. Such linkages limit freedom of movement for individual immune parameters, thereby imposing genetically regulated 'immunologic structures', the integrity of which was associated with immunocompetence. Hence, we provide an expanded genetic resource and structural perspective for understanding and monitoring immune variation in health and disease.

Bedside Back to Bench: Building Bridges between Basic and Clinical Genomic Research.

Genome sequencing has revolutionized the diagnosis of genetic diseases. Close collaborations between basic scientists and clinical genomicists are now needed to link genetic variants with disease causation. To facilitate such collaborations, we recommend prioritizing clinically relevant genes for functional studies, developing reference variant-phenotype databases, adopting phenotype description standards, and promoting data sharing.

Specification of Physiologic and Disease States by Distinct Proteins and Protein Conformations.

Protein conformational states-from intrinsically disordered ensembles to amyloids that underlie the self-templating, infectious properties of prion-like proteins-have attracted much attention. Here, we highlight the diversity, including differences in biophysical properties, that drive distinct biological functions and pathologies among self-templating proteins. Advances in chemical genomics, gene editing, and model systems now permit deconstruction of the complex interplay between these protein states and the host factors that react to them. These methods reveal that conformational switches modulate normal and abnormal information transfer and that intimate relationships exist between the intrinsic function of proteins and the deleterious consequences of their misfolding.

Host-Microbe Co-metabolism Dictates Cancer Drug Efficacy in C. elegans.

Fluoropyrimidines are the first-line treatment for colorectal cancer, but their efficacy is highly variable between patients. We queried whether gut microbes, a known source of inter-individual variability, impacted drug efficacy. Combining two tractable genetic models, the bacterium E. coli and the nematode C. elegans, we performed three-way high-throughput screens that unraveled the complexity underlying host-microbe-drug interactions. We report that microbes can bolster or suppress the effects of fluoropyrimidines through metabolic drug interconversion involving bacterial vitamin B6, B9, and ribonucleotide metabolism. Also, disturbances in bacterial deoxynucleotide pools amplify 5-FU-induced autophagy and cell death in host cells, an effect regulated by the nucleoside diphosphate kinase ndk-1. Our data suggest a two-way bacterial mediation of fluoropyrimidine effects on host metabolism, which contributes to drug efficacy. These findings highlight the potential therapeutic power of manipulating intestinal microbiota to ensure host metabolic health and treat disease.

Regulated Intron Removal Integrates Motivational State and Experience.

Myriad experiences produce transient memory, yet, contingent on the internal state of the organism and the saliency of the experience, only some memories persist over time. How experience and internal state influence the duration of memory at the molecular level remains unknown. A self-assembled aggregated state of Drosophila Orb2A protein is required specifically for long-lasting memory. We report that in the adult fly brain the mRNA encoding Orb2A protein exists in an unspliced non-protein-coding form. The convergence of experience and internal drive transiently increases the spliced protein-coding Orb2A mRNA. A screen identified pasilla, the fly ortholog of mammalian Nova-1/2, as a mediator of Orb2A mRNA processing. A single-nucleotide substitution in the intronic region that reduces Pasilla binding and intron removal selectively impairs long-term memory. We posit that pasilla-mediated processing of unspliced Orb2A mRNA integrates experience and internal state to control Orb2A protein abundance and long-term memory formation.

Microbial Genetic Composition Tunes Host Longevity.

Homeostasis of the gut microbiota critically influences host health and aging. Developing genetically engineered probiotics holds great promise as a new therapeutic paradigm to promote healthy aging. Here, through screening 3,983 Escherichia coli mutants, we discovered that 29 bacterial genes, when deleted, increase longevity in the host Caenorhabditis elegans. A dozen of these bacterial mutants also protect the host from age-related progression of tumor growth and amyloid-beta accumulation. Mechanistically, we discovered that five bacterial mutants promote longevity through increased secretion of the polysaccharide colanic acid (CA), which regulates mitochondrial dynamics and unfolded protein response (UPRmt) in the host. Purified CA polymers are sufficient to promote longevity via ATFS-1, the host UPRmt-responsive transcription factor. Furthermore, the mitochondrial changes and longevity effects induced by CA are conserved across different species. Together, our results identified molecular targets for developing pro-longevity microbes and a bacterial metabolite acting on host mitochondria to promote longevity.