Dietary Regulation of Immunity.

Integrated immunometabolic responses link dietary intake, energy utilization, and storage to immune regulation of tissue function and is therefore essential for the maintenance and restoration of homeostasis. Adipose-resident leukocytes have non-traditional immunological functions that regulate organismal metabolism by controlling insulin action, lipolysis, and mitochondrial respiration to control the usage of substrates for production of heat versus ATP. Energetically expensive vital functions such as immunological responses might have thus evolved to respond accordingly to dietary surplus and deficit of macronutrient intake. Here, we review the interaction of dietary intake of macronutrients and their metabolism with the immune system. We discuss immunometabolic checkpoints that promote healthspan and highlight how dietary fate and regulation of glucose, fat, and protein metabolism might affect immunity.

Ikaros Inhibits Group 3 Innate Lymphoid Cell Development and Function by Suppressing the Aryl Hydrocarbon Receptor Pathway.

Group 3 innate lymphoid cells (ILC3s) expressing the transcription factor (TF) RORγt are important for the defense and homeostasis of host intestinal tissues. The zinc finger TF Ikaros, encoded by Ikzf1, is essential for the development of RORγt(+) fetal lymphoid tissue inducer (LTi) cells and lymphoid organogenesis, but its role in postnatal ILC3s is unknown. Here, we show that small-intestinal ILC3s had lower Ikaros expression than ILC precursors and other ILC subsets. Ikaros inhibited ILC3s in a cell-intrinsic manner through zinc-finger-dependent inhibition of transcriptional activity of the aryl hydrocarbon receptor, a key regulator of ILC3 maintenance and function. Ablation of Ikzf1 in RORγt(+) ILC3s resulted in increased expansion and cytokine production of intestinal ILC3s and protection against infection and colitis. Therefore, in contrast to being required for LTi development, Ikaros inhibits postnatal ILC3 development and function to regulate gut immune responses at steady state and in disease.

Inflammasome-driven catecholamine catabolism in macrophages blunts lipolysis during ageing.

Catecholamine-induced lipolysis, the first step in the generation of energy substrates by the hydrolysis of triglycerides, declines with age. The defect in the mobilization of free fatty acids in the elderly is accompanied by increased visceral adiposity, lower exercise capacity, failure to maintain core body temperature during cold stress, and reduced ability to survive starvation. Although catecholamine signalling in adipocytes is normal in the elderly, how lipolysis is impaired in ageing remains unknown. Here we show that adipose tissue macrophages regulate the age-related reduction in adipocyte lipolysis in mice by lowering the bioavailability of noradrenaline. Unexpectedly, unbiased whole-transcriptome analyses of adipose macrophages revealed that ageing upregulates genes that control catecholamine degradation in an NLRP3 inflammasome-dependent manner. Deletion of NLRP3 in ageing restored catecholamine-induced lipolysis by downregulating growth differentiation factor-3 (GDF3) and monoamine oxidase A (MAOA) that is known to degrade noradrenaline. Consistent with this, deletion of GDF3 in inflammasome-activated macrophages improved lipolysis by decreasing levels of MAOA and caspase-1. Furthermore, inhibition of MAOA reversed the age-related reduction in noradrenaline concentration in adipose tissue, and restored lipolysis with increased levels of the key lipolytic enzymes adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL). Our study reveals that targeting neuro-immunometabolic signalling between the sympathetic nervous system and macrophages may offer new approaches to mitigate chronic inflammation-induced metabolic impairment and functional decline.

Characterization of TMC-1 in C. elegans sodium chemotaxis and sodium conditioned aversion.

BACKGROUND: While sodium is attractive at low and aversive at high concentrations in most studied species, including Caenorhabditis elegans, the molecular mechanisms behind transduction remain poorly understood. Additionally, past studies with C. elegans provide evidence that the nematode's innate behavior can be altered by previous experiences. Here we investigated the molecular aspects of both innate and conditioned responses to salts. Transmembrane channel-like 1 (tmc-1) has been suggested to encode a sodium-sensitive channel required for sodium chemosensation in C. elegans, but its specific role remains unclear. RESULTS: We report that TMC-1 is necessary for sodium attraction, but not aversion in the nematode. We show that TMC-1 contributes to the nematode's lithium induced attraction behavior, but not potassium or magnesium attraction thus clarifying the specificity of the response. In addition, we show that sodium conditioned aversion is dependent on TMC-1 and disrupts not only sodium induced attraction, but also lithium. CONCLUSIONS: These findings represent the first time a role for TMC-1 has been described in sodium and lithium attraction in vivo, as well as in sodium conditioned aversion. Together this clarifies TMC-1's importance in sodium hedonics and offer molecular insight into salt chemotaxis learning.

WHO implementation workshop on guidelines on procedures and data requirements for changes to approved biotherapeutic products, Seoul, Republic of Korea, 25-26 June 2019.

The first global workshop on implementation of the WHO guidelines on procedures and data requirements for changes to approved biotherapeutic products adopted by the WHO Expert Committee in 2018 was held in June 2019. The workshop participants recognized that the principles based on sound science and the potential for risk, as described in the WHO Guidelines on post-approval changes, which constitute the global standard for product life-cycle management are providing clarity and helping national regulatory authorities in establishing guidance while improving time-lines for an efficient regulation of products. Consequently, the regulatory situation for post-approval changes and guideline implementation is changing but there is a disparity between different countries. While the guidelines are gradually being implemented in some countries and also being considered in other countries, the need for regional workshops and further training on post-approval changes was a common theme reiterated by many participants. Given the complexities relating to post-approval changes in different regions/countries, there was a clear understanding among all participants that an efficient approach for product life-cycle management at a national level is needed to ensure faster availability of high standard, safe and efficacious medicines to patients as per the World Health Assembly Resolution 67.21.

Distinct Autocatalytic α- N-Methylating Precursors Expand the Borosin RiPP Family of Peptide Natural Products.

Backbone N-methylations impart several favorable characteristics to peptides including increased proteolytic stability and membrane permeability. Nonetheless, amide bond N-methylations incorporated as post-translational modifications are scarce in nature and were first demonstrated in 2017 for a single set of fungal metabolites. Here we expand on our previous discovery of iterative, autocatalytic α- N-methylating precursor proteins in the borosin family of ribosomally encoded peptide natural products. We identify over 50 putative pathways in a variety of ascomycete and basidiomycete fungi and functionally validate nearly a dozen new self-α- N-methylating catalysts. Significant differences in precursor size, architecture, and core peptide properties subdivide this new peptide family into three discrete structural types. Lastly, using targeted genomics, we link the biosynthetic origins of the potent antineoplastic gymnopeptides to the borosin natural product family. This work highlights the metabolic potential of fungi for ribosomally synthesized peptide natural products.

A critical review on ramucirumab in the treatment of advanced urothelial cancer.

Advanced urothelial cancer (UC) is a lethal disease despite current advances in systemic therapy, including platinum chemotherapy combinations and immune checkpoint inhibition. Tumor angiogenesis is involved in UC growth and metastatic progression. Proangiogenic signaling through the VEGFR is a key process in UC with prognostic significance. Targeting of VEGFR2 with the monoclonal antibody ramucirumab has been tested in various different tumor types. In this review, we discuss the development of the drug in the context of its preclinical and clinical use with a focus on UC. Improvements in our ability to predict responses and resistance are key for maximizing its efficacy and selecting the most appropriate combinations with other active agents.

Restriction of IL-22-producing T cell responses and differential regulation of regulatory T cell compartments by zinc finger transcription factor Ikaros.

Proper immune responses are needed to control pathogen infection at mucosal surfaces. IL-22-producing CD4(+) T cells play an important role in controlling bacterial infection in the gut; however, transcriptional regulation of these cells remains elusive. In this study, we show that mice with targeted deletion of the fourth DNA-binding zinc finger of the transcription factor Ikaros had increased IL-22-producing, but not IL-17-producing, CD4(+) T cells in the gut. Adoptive transfer of CD4(+) T cells from these Ikaros-mutant mice conferred enhanced mucosal immunity against Citrobacter rodentium infection. Despite an intact in vivo thymic-derived regulatory T cell (Treg) compartment in these Ikaros-mutant mice, TGF-ß, a cytokine well known for induction of Tregs, failed to induce Foxp3 expression in Ikaros-mutant CD4(+) T cells in vitro and, instead, promoted IL-22. Aberrant upregulation of IL-21 in CD4(+) T cells expressing mutant Ikaros was responsible, at least in part, for the enhanced IL-22 expression in a Stat3-dependent manner. Genetic analysis using compound mutations further demonstrated that the aryl hydrocarbon receptor, but not RORγt, was required for aberrant IL-22 expression by Ikaros-mutant CD4(+) T cells, whereas forced expression of Foxp3 was sufficient to inhibit this aberrant cytokine production. Together, our data identified new functions for Ikaros in maintaining mucosal immune homeostasis by restricting IL-22 production by CD4(+) T cells.

Discovery of Borosin Catalytic Strategies and Function through Bioinformatic Profiling.

Borosins are ribosomally synthesized and post-translationally modified peptides (RiPPs) containing backbone α-N-methylations. These modifications confer favorable pharmacokinetic properties including increased membrane permeability and resistance to proteolytic degradation. Previous studies have biochemically and bioinformatically explored several borosins, revealing (1) numerous domain architectures and (2) diverse core regions lacking conserved sequence elements. Due to these characteristics, large-scale computational identification of borosin biosynthetic genes remains challenging and often requires additional, time-intensive manual inspection. This work builds upon previous findings and updates the genome-mining tool RODEO to automatically evaluate borosin biosynthetic gene clusters (BGCs) and identify putative precursor peptides. Using the new RODEO module, we provide an updated analysis of borosin BGCs identified in the NCBI database. From our data set, we bioinformatically predict and experimentally characterize a new fused borosin domain architecture, in which the modified natural product core is encoded N-terminal to the methyltransferase domain. Additionally, we demonstrate that a borosin precursor peptide is a native substrate of shewasin A, a reported aspartyl peptidase with no previously identified substrates. Shewasin A requires post-translational modification of the leader peptide for proteolytic maturation, a feature not previously observed in RiPPs. Overall, this work provides a user-friendly and open-access tool for the analysis of borosin BGCs and we demonstrate its utility to uncover additional biosynthetic strategies within the borosin class of RiPPs.

Computationally guided exploration of borosin biosynthetic strategies.

Borosins are ribosomally synthesized and post-translationally modified peptides containing backbone α- N -methylations. Identification of borosin precursor peptides is difficult because (1) there are no conserved sequence elements among borosin precursor peptides and (2) the biosynthetic gene clusters contain numerous domain architectures and peptide fusions. To tackle this problem, we updated the genome mining tool RODEO to automatically evaluate putative borosin BGCs and identify precursor peptides. Enabled by the new borosin module, we analyzed all borosin BGCs found in available sequence data and assigned precursor peptides to previously orphan borosin methyltransferases. Additionally, we bioinformatically predict and experimentally characterize a new fused borosin domain architecture, in which the modified core is N-terminal to the methyltransferase domain. Finally, we demonstrate that a borosin precursor peptide is the native substrate of shewasin A, a previously characterized pepsin-like aspartic peptidase whose native biological function was unknown.

[WITHDRAWN] Activation of transsulfuration pathway to maintain cysteine is a thermogenic checkpoint for the conservation of energy.

The authors have requested that this preprint be removed from Research Square.

Reduction of SPARC protects mice against NLRP3 inflammasome activation and obesity.

The comprehensive assessment of long-term effects of reducing intake of energy (CALERIE-II; NCT00427193) clinical trial established that caloric restriction (CR) in humans lowers inflammation. The identity and mechanism of endogenous CR-mimetics that can be deployed to control obesity-associated inflammation and diseases are not well understood. Our studies have found that 2 years of 14% sustained CR in humans inhibits the expression of the matricellular protein, secreted protein acidic and rich in cysteine (SPARC), in adipose tissue. In mice, adipose tissue remodeling caused by weight loss through CR and low-protein diet feeding decreased, while high-fat diet-induced (HFD-induced) obesity increased SPARC expression in adipose tissue. Inducible SPARC downregulation in adult mice mimicked CR's effects on lowering adiposity by regulating energy expenditure. Deletion of SPARC in adipocytes was sufficient to protect mice against HFD-induced adiposity, chronic inflammation, and metabolic dysfunction. Mechanistically, SPARC activates the NLRP3 inflammasome at the priming step and downregulation of SPARC lowers macrophage inflammation in adipose tissue, while excess SPARC activated macrophages via JNK signaling. Collectively, reduction of adipocyte-derived SPARC confers CR-like metabolic and antiinflammatory benefits in obesity by serving as an immunometabolic checkpoint of inflammation.

Diverse Protein Architectures and α-N-Methylation Patterns Define Split Borosin RiPP Biosynthetic Gene Clusters.

Borosins are ribosomally synthesized and post-translationally modified peptides (RiPPs) with α-N-methylations installed on the peptide backbone that impart unique properties like proteolytic stability to these natural products. The borosin RiPP family was initially reported only in fungi until our recent discovery and characterization of a Type IV split borosin system in the metal-respiring bacterium Shewanella oneidensis. Here, we used hidden Markov models and sequence similarity networks to identify over 1600 putative pathways that show split borosin biosynthetic gene clusters are widespread in bacteria. Noteworthy differences in precursor and α-N-methyltransferase open reading frame sizes, architectures, and core peptide properties allow further subdivision of the borosin family into six additional discrete structural types, of which five have been validated in this study.

TaxiBGC: a Taxonomy-Guided Approach for Profiling Experimentally Characterized Microbial Biosynthetic Gene Clusters and Secondary Metabolite Production Potential in Metagenomes.

Biosynthetic gene clusters (BGCs) in microbial genomes encode bioactive secondary metabolites (SMs), which can play important roles in microbe-microbe and host-microbe interactions. Given the biological significance of SMs and the current profound interest in the metabolic functions of microbiomes, the unbiased identification of BGCs from high-throughput metagenomic data could offer novel insights into the complex chemical ecology of microbial communities. Currently available tools for predicting BGCs from shotgun metagenomes have several limitations, including the need for computationally demanding read assembly, predicting a narrow breadth of BGC classes, and not providing the SM product. To overcome these limitations, we developed taxonomy-guided identification of biosynthetic gene clusters (TaxiBGC), a command-line tool for predicting experimentally characterized BGCs (and inferring their known SMs) in metagenomes by first pinpointing the microbial species likely to harbor them. We benchmarked TaxiBGC on various simulated metagenomes, showing that our taxonomy-guided approach could predict BGCs with much-improved performance (mean F1 score, 0.56; mean PPV score, 0.80) compared with directly identifying BGCs by mapping sequencing reads onto the BGC genes (mean F1 score, 0.49; mean PPV score, 0.41). Next, by applying TaxiBGC on 2,650 metagenomes from the Human Microbiome Project and various case-control gut microbiome studies, we were able to associate BGCs (and their SMs) with different human body sites and with multiple diseases, including Crohn's disease and liver cirrhosis. In all, TaxiBGC provides an in silico platform to predict experimentally characterized BGCs and their SM production potential in metagenomic data while demonstrating important advantages over existing techniques. IMPORTANCE Currently available bioinformatics tools to identify BGCs from metagenomic sequencing data are limited in their predictive capability or ease of use to even computationally oriented researchers. We present an automated computational pipeline called TaxiBGC, which predicts experimentally characterized BGCs (and infers their known SMs) in shotgun metagenomes by first considering the microbial species source. Through rigorous benchmarking techniques on simulated metagenomes, we show that TaxiBGC provides a significant advantage over existing methods. When demonstrating TaxiBGC on thousands of human microbiome samples, we associate BGCs encoding bacteriocins with different human body sites and diseases, thereby elucidating a possible novel role of this antibiotic class in maintaining the stability of microbial ecosystems throughout the human body. Furthermore, we report for the first time gut microbial BGC associations shared among multiple pathologies. Ultimately, we expect our tool to facilitate future investigations into the chemical ecology of microbial communities across diverse niches and pathologies.

Aging Induces an Nlrp3 Inflammasome-Dependent Expansion of Adipose B Cells That Impairs Metabolic Homeostasis.

During aging, visceral adiposity is often associated with alterations in adipose tissue (AT) leukocytes, inflammation, and metabolic dysfunction. However, the contribution of AT B cells in immunometabolism during aging is unexplored. Here, we show that aging is associated with an expansion of a unique population of resident non-senescent aged adipose B cells (AABs) found in fat-associated lymphoid clusters (FALCs). AABs are transcriptionally distinct from splenic age-associated B cells (ABCs) and show greater expansion in female mice. Functionally, whole-body B cell depletion restores proper lipolysis and core body temperature maintenance during cold stress. Mechanistically, the age-induced FALC formation, AAB, and splenic ABC expansion is dependent on the Nlrp3 inflammasome. Furthermore, AABs express IL-1R, and inhibition of IL-1 signaling reduces their proliferation and increases lipolysis in aging. These data reveal that inhibiting Nlrp3-dependent B cell accumulation can be targeted to reverse metabolic impairment in aging AT.

Antibody-Drug Conjugates in Bladder Cancer.

Urothelial carcinoma (UC) is characterized by expression of a plethora of cell surface antigens, thus offering opportunities for specific therapeutic targeting with use of antibody-drug conjugates (ADCs). ADCs are structured from two major constituents, a monoclonal antibody (mAb) against a specific target and a cytotoxic drug connected via a linker molecule. Several ADCs are developed against different UC surface markers, but the ones at most advanced stages of development include sacituzumab govitecan (IMMU-132), enfortumab vedotin (ASG-22CE/ASG-22ME), ASG-15ME for advanced UC, and oportuzumab monatox (VB4-845) for early UC. Several new targets are identified and utilized for novel or existing ADC testing. The most promising ones include human epidermal growth factor receptor 2 (HER2) and members of the fibroblast growth factor receptor axis (FGF/FGFR). Positive preclinical and early clinical results are reported in many cases, thus the next step involves further improving efficacy and reducing toxicity as well as testing combination strategies with approved agents.

Energy Sparing Orexigenic Inflammation of Obesity.

The neuro-immune interactions that integrate host metabolism in health and disease are unclear. A new study by Valdearcos et al. (2017) describes how sensing of high-fat diet by microglia, brain's resident innate immune cells, recruits additional bone-marrow-derived myeloid cells into the hypothalamus to produce inflammation and cause weight gain.