Metabolic fitness of IgA+ plasma cells in the gut requires DOCK8.

Dedicator of cytokinesis 8 (DOCK8) mutations lead to a primary immunodeficiency associated with recurrent gastrointestinal infections and poor antibody responses but, paradoxically, heightened IgE to food antigens, suggesting that DOCK8 is central to immune homeostasis in the gut. Using Dock8-deficient mice, we found that DOCK8 was necessary for mucosal IgA production to multiple T cell-dependent antigens, including peanut and cholera toxin. Yet DOCK8 was not necessary in T cells for this phenotype. Instead, B cell-intrinsic DOCK8 was required for maintenance of antigen-specific IgA-secreting plasma cells (PCs) in the gut lamina propria. Unexpectedly, DOCK8 was not required for early B cell activation, migration, or IgA class switching. An unbiased interactome screen revealed novel protein partners involved in metabolism and apoptosis. Dock8-deficient IgA+ B cells had impaired cellular respiration and failed to engage glycolysis appropriately. These results demonstrate that maintenance of the IgA+ PC compartment requires DOCK8 and suggest that gut IgA+ PCs have unique metabolic requirements for long-term survival in the lamina propria.

Protocol for establishing primary human lung organoid-derived air-liquid interface cultures from cryopreserved human lung tissue.

Primary human lung organoid-derived air-liquid interface (ALI) cultures serve as a physiologically relevant model to study human airway epithelium in vitro. Here, we present a protocol for establishing these cultures from cryopreserved human lung tissue. We describe steps for lung tissue cryostorage, tissue dissociation, lung epithelial organoid generation, and ALI culture differentiation. We also include quality control steps and technical readouts for monitoring virus response. This protocol demonstrates severe acute respiratory syndrome coronavirus 2 infection in these cultures as an example of their utility. For complete details on the use and execution of this protocol, please refer to Diana Cadena Castaneda et al. (2023).1.

Harnessing large language models (LLMs) for candidate gene prioritization and selection.

BACKGROUND: Feature selection is a critical step for translating advances afforded by systems-scale molecular profiling into actionable clinical insights. While data-driven methods are commonly utilized for selecting candidate genes, knowledge-driven methods must contend with the challenge of efficiently sifting through extensive volumes of biomedical information. This work aimed to assess the utility of large language models (LLMs) for knowledge-driven gene prioritization and selection. METHODS: In this proof of concept, we focused on 11 blood transcriptional modules associated with an Erythroid cells signature. We evaluated four leading LLMs across multiple tasks. Next, we established a workflow leveraging LLMs. The steps consisted of: (1) Selecting one of the 11 modules; (2) Identifying functional convergences among constituent genes using the LLMs; (3) Scoring candidate genes across six criteria capturing the gene's biological and clinical relevance; (4) Prioritizing candidate genes and summarizing justifications; (5) Fact-checking justifications and identifying supporting references; (6) Selecting a top candidate gene based on validated scoring justifications; and (7) Factoring in transcriptome profiling data to finalize the selection of the top candidate gene. RESULTS: Of the four LLMs evaluated, OpenAI's GPT-4 and Anthropic's Claude demonstrated the best performance and were chosen for the implementation of the candidate gene prioritization and selection workflow. This workflow was run in parallel for each of the 11 erythroid cell modules by participants in a data mining workshop. Module M9.2 served as an illustrative use case. The 30 candidate genes forming this module were assessed, and the top five scoring genes were identified as BCL2L1, ALAS2, SLC4A1, CA1, and FECH. Researchers carefully fact-checked the summarized scoring justifications, after which the LLMs were prompted to select a top candidate based on this information. GPT-4 initially chose BCL2L1, while Claude selected ALAS2. When transcriptional profiling data from three reference datasets were provided for additional context, GPT-4 revised its initial choice to ALAS2, whereas Claude reaffirmed its original selection for this module. CONCLUSIONS: Taken together, our findings highlight the ability of LLMs to prioritize candidate genes with minimal human intervention. This suggests the potential of this technology to boost productivity, especially for tasks that require leveraging extensive biomedical knowledge.

Spatiotemporally organized immunomodulatory response to SARS-CoV-2 virus in primary human broncho-alveolar epithelia.

The COVID-19 pandemic continues to be a health crisis with major unmet medical needs. The early responses from airway epithelial cells, the first target of the virus regulating the progression toward severe disease, are not fully understood. Primary human air-liquid interface cultures representing the broncho-alveolar epithelia were used to study the kinetics and dynamics of SARS-CoV-2 variants infection. The infection measured by nucleoprotein expression, was a late event appearing between day 4-6 post infection for Wuhan-like virus. Other variants demonstrated increasingly accelerated timelines of infection. All variants triggered similar transcriptional signatures, an "early" inflammatory/immune signature preceding a "late" type I/III IFN, but differences in the quality and kinetics were found, consistent with the timing of nucleoprotein expression. Response to virus was spatially organized: CSF3 expression in basal cells and CCL20 in apical cells. Thus, SARS-CoV-2 virus triggers specific responses modulated over time to engage different arms of immune response.

Transcriptomic responses in the blood and sputum of cigarette smokers compared to e-cigarette vapers.

RATIONALE: Electronic (e)-cigarettes are popular among youth and cigarette smokers attempting to quit. Studies to date have focused on the utility of e-cigarettes as a smoking cessation tool, but the biological effects are largely unknown. OBJECTIVES: To identify transcriptomic differences in the blood and sputum of e-cigarette users compared to conventional cigarettes smokers and healthy controls and describe biological pathways affected by these tobacco products. METHODS: Cross-sectional analysis of whole blood and sputum RNA-sequencing data from 8 smokers, 9 e-cigarette users (e-cigs) and 4 controls. Weighted gene co-network analysis (WGCNA) identified gene module associations. Ingenuity Pathway Analysis (IPA) identified canonical pathways associated with tobacco products. MAIN RESULTS: In blood, a three-group comparison showed 16 differentially expressed genes (DEGs); pair-wise comparison showed 7 DEGs between e-cigs and controls, 35 DEGs between smokers and controls, and 13 DEGs between smokers and e-cigs. In sputum, 438 DEGs were in the three-group comparison. In pair-wise comparisons, there were 2 DEGs between e-cigs and controls, 270 DEGs between smokers and controls, and 468 DEGs between smokers and e-cigs. Only 2 genes in the smokers vs. control comparison overlapped between blood and sputum. Most gene modules identified through WGCNA associated with tobacco product exposures also were associated with cotinine and exhaled CO levels. IPA showed more canonical pathways altered by conventional cigarette smoking than by e-cigarette use. CONCLUSION: Cigarette smoking and e-cigarette use led to transcriptomic changes in both blood and sputum. However, conventional cigarettes induced much stronger transcriptomic responses in both compartments.

Spatiotemporally organized immunomodulatory response to SARS-CoV-2 virus in primary human broncho-alveolar epithelia.

The COVID-19 pandemic continues to be a health crisis with major unmet medical needs. The early responses from airway epithelial cells, the first target of the virus regulating the progression towards severe disease, are not fully understood. Primary human air-liquid interface cultures representing the broncho-alveolar epithelia were used to study the kinetics and dynamics of SARS-CoV-2 variants infection. The infection measured by nucleoprotein expression, was a late event appearing between day 4-6 post infection for Wuhan-like virus. Other variants demonstrated increasingly accelerated timelines of infection. All variants triggered similar transcriptional signatures, an "early" inflammatory/immune signature preceding a "late" type I/III IFN, but differences in the quality and kinetics were found, consistent with the timing of nucleoprotein expression. Response to virus was spatially organized: CSF3 expression in basal cells and CCL20 in apical cells. Thus, SARS-CoV-2 virus triggers specific responses modulated over time to engage different arms of immune response.

A genome-wide CRISPR activation screen identifies SCREEM a novel SNAI1 super-enhancer demarcated by eRNAs.

The genome is pervasively transcribed to produce a vast array of non-coding RNAs (ncRNAs). Long non-coding RNAs (lncRNAs) are transcripts of >200 nucleotides and are best known for their ability to regulate gene expression. Enhancer RNAs (eRNAs) are subclass of lncRNAs that are synthesized from enhancer regions and have also been shown to coordinate gene expression. The biological function and significance of most lncRNAs and eRNAs remain to be determined. Epithelial to mesenchymal transition (EMT) is a ubiquitous cellular process that occurs during cellular migration, homeostasis, fibrosis, and cancer-cell metastasis. EMT-transcription factors, such as SNAI1 induce a complex transcriptional program that coordinates the morphological and molecular changes associated with EMT. Such complex transcriptional programs are often subject to coordination by networks of ncRNAs and thus can be leveraged to identify novel functional ncRNA loci. Here, using a genome-wide CRISPR activation (CRISPRa) screen targeting ∼10,000 lncRNA loci we identified ncRNA loci that could either promote or attenuate EMT. We discovered a novel locus that we named SCREEM (SNAI1 cis-regulatory eRNAs expressed in monocytes). The SCREEM locus contained a cluster of eRNAs that when activated using CRISPRa induced expression of the neighboring gene SNAI1, driving concomitant EMT. However, the SCREEM eRNA transcripts themselves appeared dispensable for the induction of SNAI1 expression. Interestingly, the SCREEM eRNAs and SNAI1 were co-expressed in activated monocytes, where the SCREEM locus demarcated a monocyte-specific super-enhancer. These findings suggest a potential role for SNAI1 in monocytes. Exploration of the SCREEM-SNAI axis could reveal novel aspects of monocyte biology.

MYC regulates a pan-cancer network of co-expressed oncogenic splicing factors.

MYC is dysregulated in >50% of cancers, but direct targeting of MYC has been clinically unsuccessful. Targeting downstream MYC effector pathways represents an attractive alternative. MYC regulates alternative mRNA splicing, but the mechanistic links between MYC and the splicing machinery in cancer remain underexplored. Here, we identify a network of co-expressed splicing factors (SF-modules) in MYC-active breast tumors. Of these, one is a pan-cancer SF-module correlating with MYC activity across 33 tumor types. In mammary cell models, MYC activation leads to co-upregulation of pan-cancer module SFs and to changes in >4,000 splicing events. In breast cancer organoids, co-overexpression of the pan-cancer SF-module induces MYC-regulated splicing events and increases organoid size and invasiveness, while knockdown decreases organoid size. Finally, we uncover a MYC-activity pan-cancer splicing signature correlating with survival across tumor types. Our findings provide insight into the mechanisms of MYC-regulated splicing and for the development of therapeutics for MYC-driven tumors.

Bronchial epithelium epithelial-mesenchymal plasticity forms aberrant basaloid-like cells in vitro.

Although epithelial-mesenchymal transition (EMT) is a common feature of fibrotic lung disease, its role in fibrogenesis is controversial. Recently, aberrant basaloid cells were identified in fibrotic lung tissue as a novel epithelial cell type displaying a partial EMT phenotype. The developmental origin of these cells remains unknown. To elucidate the role of EMT in the development of aberrant basaloid cells from the bronchial epithelium, we mapped EMT-induced transcriptional changes at the population and single-cell levels. Human bronchial epithelial cells grown as submerged or air-liquid interface (ALI) cultures with or without EMT induction were analyzed by bulk and single-cell RNA-Sequencing. Comparison of submerged and ALI cultures revealed differential expression of 8,247 protein coding (PC) and 1,621 long noncoding RNA (lncRNA) genes and revealed epithelial cell-type-specific lncRNAs. Similarly, EMT induction in ALI cultures resulted in robust transcriptional reprogramming of 6,020 PC and 907 lncRNA genes. Although there was no evidence for fibroblast/myofibroblast conversion following EMT induction, cells displayed a partial EMT gene signature and an aberrant basaloid-like cell phenotype. The substantial transcriptional differences between submerged and ALI cultures highlight that care must be taken when interpreting data from submerged cultures. This work supports that lung epithelial EMT does not generate fibroblasts/myofibroblasts and confirms ALI cultures provide a physiologically relevant system to study aberrant basaloid-like cells and mechanisms of EMT. We provide a catalog of PC and lncRNA genes and an interactive browser (https://bronc-epi-in-vitro.cells.ucsc.edu/) of single-cell RNA-Seq data for further exploration of potential roles in the lung epithelium in health and lung disease.

Studying Tumor Angiogenesis and Cancer Invasion in a Three-Dimensional Vascularized Breast Cancer Micro-Environment.

Metastatic breast cancer is one of the deadliest forms of malignancy, primarily driven by its characteristic micro-environment comprising cancer cells interacting with stromal components. These interactions induce genetic and metabolic alterations creating a conducive environment for tumor growth. In this study, a physiologically relevant 3D vascularized breast cancer micro-environment is developed comprising of metastatic MDA-MB-231 cells and human umbilical vein endothelial cells loaded in human dermal fibroblasts laden fibrin, representing the tumor stroma. The matrix, as well as stromal cell density, impacts the transcriptional profile of genes involved in tumor angiogenesis and cancer invasion, which are hallmarks of cancer. Cancer-specific canonical pathways and activated upstream regulators are also identified by the differential gene expression signatures of these composite cultures. Additionally, a tumor-associated vascular bed of capillaries is established exhibiting dilated vessel diameters, representative of in vivo tumor physiology. Further, employing aspiration-assisted bioprinting, cancer-endothelial crosstalk, in the form of collective angiogenesis of tumor spheroids bioprinted at close proximity, is identified. Overall, this bottom-up approach of tumor micro-environment fabrication provides an insight into the potential of in vitro tumor models and enables the identification of novel therapeutic targets as a preclinical drug screening platform.

Poison Exon Splicing Regulates a Coordinated Network of SR Protein Expression during Differentiation and Tumorigenesis.

The RNA isoform repertoire is regulated by splicing factor (SF) expression, and alterations in SF levels are associated with disease. SFs contain ultraconserved poison exon (PE) sequences that exhibit greater identity across species than nearby coding exons, but their physiological role and molecular regulation is incompletely understood. We show that PEs in serine-arginine-rich (SR) proteins, a family of 14 essential SFs, are differentially spliced during induced pluripotent stem cell (iPSC) differentiation and in tumors versus normal tissues. We uncover an extensive cross-regulatory network of SR proteins controlling their expression via alternative splicing coupled to nonsense-mediated decay. We define sequences that regulate PE inclusion and protein expression of the oncogenic SF TRA2ß using an RNA-targeting CRISPR screen. We demonstrate location dependency of RS domain activity on regulation of TRA2ß-PE using CRISPR artificial SFs. Finally, we develop splice-switching antisense oligonucleotides to reverse the increased skipping of TRA2ß-PE detected in breast tumors, altering breast cancer cell viability, proliferation, and migration.

IL-10-Dependent Crosstalk between Murine Marginal Zone B Cells, Macrophages, and CD8α+ Dendritic Cells Promotes Listeria monocytogenes Infection.

Type 1 CD8α+ conventional dendritic cells (cDC1s) are required for CD8+ T cell priming but, paradoxically, promote splenic Listeria monocytogenes infection. Using mice with impaired cDC2 function, we ruled out a role for cDC2s in this process and instead discovered an interleukin-10 (IL-10)-dependent cellular crosstalk in the marginal zone (MZ) that promoted bacterial infection. Mice lacking the guanine nucleotide exchange factor DOCK8 or CD19 lost IL-10-producing MZ B cells and were resistant to Listeria. IL-10 increased intracellular Listeria in cDC1s indirectly by reducing inducible nitric oxide synthase expression early after infection and increasing intracellular Listeria in MZ metallophilic macrophages (MMMs). These MMMs trans-infected cDC1s, which, in turn, transported Listeria into the white pulp to prime CD8+ T cells. However, this also facilitated bacterial expansion. Therefore, IL-10-mediated crosstalk between B cells, macrophages, and cDC1s in the MZ promotes both Listeria infection and CD8+ T cell activation.

Experimental evolution of a fungal pathogen into a gut symbiont.

Gut microbes live in symbiosis with their hosts, but how mutualistic animal-microbe interactions emerge is not understood. By adaptively evolving the opportunistic fungal pathogen Candida albicans in the mouse gastrointestinal tract, we selected strains that not only had lost their main virulence program but also protected their new hosts against a variety of systemic infections. This protection was independent of adaptive immunity, arose as early as a single day postpriming, was dependent on increased innate cytokine responses, and was thus reminiscent of "trained immunity." Because both the microbe and its new host gain some advantages from their interaction, this experimental system might allow direct study of the evolutionary forces that govern the emergence of mutualism between a mammal and a fungus.

Advantages of meta-total RNA sequencing (MeTRS) over shotgun metagenomics and amplicon-based sequencing in the profiling of complex microbial communities.

Sequencing-based microbiome profiling aims at detecting and quantifying individual members of a microbial community in a culture-independent manner. While amplicon-based sequencing (ABS) of bacterial or fungal ribosomal DNA is the most widely used technology due to its low cost, it suffers from PCR amplification biases that hinder accurate representation of microbial population structures. Shotgun metagenomics (SMG) conversely allows unbiased microbiome profiling but requires high sequencing depth. Here we report the development of a meta-total RNA sequencing (MeTRS) method based on shotgun sequencing of total RNA and benchmark it on a human stool sample spiked in with known abundances of bacterial and fungal cells. MeTRS displayed the highest overall sensitivity and linearity for both bacteria and fungi, the greatest reproducibility compared to SMG and ABS, while requiring a ~20-fold lower sequencing depth than SMG. We therefore present MeTRS as a valuable alternative to existing technologies for large-scale profiling of complex microbiomes.

Migratory CD11b+ conventional dendritic cells induce T follicular helper cell-dependent antibody responses.

T follicular helper (Tfh) cells are a subset of CD4+ T cells that promote antibody production during vaccination. Conventional dendritic cells (cDCs) efficiently prime Tfh cells; however, conclusions regarding which cDC instructs Tfh cell differentiation have differed between recent studies. We found that these discrepancies might exist because of the unusual sites used for immunization in murine models, which differentially bias which DC subsets access antigen. We used intranasal immunization as a physiologically relevant route of exposure that delivers antigen to all tissue DC subsets. Using a combination of mice in which the function of individual DC subsets is impaired and different antigen formulations, we determined that CD11b+ migratory type 2 cDCs (cDC2s) are necessary and sufficient for Tfh induction. DC-specific deletion of the guanine nucleotide exchange factor DOCK8 resulted in an isolated loss of CD11b+ cDC2, but not CD103+ cDC1, migration to lung-draining lymph nodes. Impaired cDC2 migration or development in DC-specific Dock8 or Irf4 knockout mice, respectively, led to reduced Tfh cell priming, whereas loss of CD103+ cDC1s in Batf3-/- mice did not. Loss of cDC2-dependent Tfh cell priming impaired antibody-mediated protection from live influenza virus challenge. We show that migratory cDC2s uniquely carry antigen into the subanatomic regions of the lymph node where Tfh cell priming occurs-the T-B border. This work identifies the DC subset responsible for Tfh cell-dependent antibody responses, particularly when antigen dose is limiting or is encountered at a mucosal site, which could ultimately inform the formulation and delivery of vaccines.

The Transcriptional Response of Candida albicans to Weak Organic Acids, Carbon Source, and MIG1 Inactivation Unveils a Role for HGT16 in Mediating the Fungistatic Effect of Acetic Acid.

Candida albicans is a resident fungus of the human intestinal microflora. Commonly isolated at low abundance in healthy people, C. albicans outcompetes local microbiota during candidiasis episodes. Under normal conditions, members of the human gastrointestinal (GI) microbiota were shown to keep C. albicans colonization under control. By releasing weak organic acids (WOAs), bacteria are able to moderate yeast growth. This mechanism displays a synergistic effect in vitro with the absence of glucose in medium of culture, which underlines the complex interactions that C. albicans faces in its natural environment. Inactivation of the transcriptional regulator MIG1 in C. albicans results in a lack of sensitivity to this synergistic outcome. To decipher C. albicans transcriptional responses to glucose, WOAs, and the role of MIG1, we performed RNA sequencing (RNA-seq) on four biological replicates exposed to combinations of these three parameters. We were able to characterize the (i) glucose response, (ii) response to acetic and butyric acid, (iii) MIG1 regulation of C. albicans, and (iv) genes responsible for WOA resistance. We identified a group of six genes linked to WOA sensitivity in a glucose-MIG1-dependent manner and inactivated one of these genes, the putative glucose transporter HGT16, in a SC5314 wild-type background. As expected, the mutant displayed a partial complementation to WOA resistance in the absence of glucose. This result points toward a mechanism of WOA sensitivity in C. albicans involving membrane transporters, which could be exploited to control yeast colonization in human body niches.

A subpopulation of high IL-21-producing CD4(+) T cells in Peyer's Patches is induced by the microbiota and regulates germinal centers.

The production of IL-21 by T follicular helper (Tfh) cells is vital in driving the germinal centre reaction and high affinity antibody formation. However, the degree of Tfh cell heterogeneity and function is not fully understood. We used a novel IL-21eGFP reporter mouse strain to analyze the diversity and role of Tfh cells. Through the analysis of GFP expression in lymphoid organs of IL-21eGFP mice, we identified a subpopulation of GFP(+), high IL-21 producing Tfh cells present only in Peyer's Patches. GFP(+)Tfh cells were found to be polyclonal and related to GFP(-)Tfh cells of Peyer's Patches in TCR repertoire composition and overall gene expression. Studies on the mechanisms of induction of GFP(+)Tfh cells demonstrated that they required the intestinal microbiota and a diverse repertoire of CD4(+) T cells and B cells. Importantly, ablation of GFP(+) cells resulted in a reduced frequency of Peyer's Patches IgG1 and germinal center B cells in addition to small but significant shifts in gut microbiome composition. Our work highlights the diversity among IL-21 producing CD4(+) Tfh cells, and the interrelationship between the intestinal bacteria and Tfh cell responses in the gut.

ß-glucan Exposure on the Fungal Cell Wall Tightly Correlates with Competitive Fitness of Candida Species in the Mouse Gastrointestinal Tract.

Candida albicans is responsible for ~400,000 systemic fungal infections annually, with an associated mortality rate of 46-75%. The human gastrointestinal (GI) tract represents the largest natural reservoir of Candida species and is a major source of systemic fungal infections. However, the factors that control GI colonization by Candida species are not completely understood. We hypothesized that the fungal cell wall would play an important role in determining the competitive fitness of Candida species in the mammalian GI tract. To test this hypothesis, we generated a systematic collection of isogenic C. albicans cell wall mutants and measured their fitness in the mouse GI tract via quantitative competition assays. Whereas a large variation in competitive fitness was found among mutants, no correlation was observed between GI fitness and total levels of individual cell wall components. Similar results were obtained in a set of distantly-related Candida species, suggesting that total amounts of individual cell wall components do not determine the ability of fungi to colonize the GI tract. We then subjected this collection of Candida strains and species to an extensive quantitative phenotypic profiling in search for features that might be responsible for their differences in GI fitness, but found no association with the ability to grow in GI-mimicking and stressful environments or with in vitro and in vivo virulence. The most significant association with GI fitness was found to be the strength of signaling through the Dectin-1 receptor. Using a quantitative assay to measure the amount of exposed ß-glucan on the surface of fungal cells, we found this parameter, unlike total ß-glucan levels, to be strongly predictive of competitive fitness in the mouse GI tract. These data suggest that fungal cell wall architecture, more so than its crude composition, critically determines the ability of fungi to colonize the mammalian GI tract. In particular, recognition of exposed ß-glucan by Dectin-1 receptor appears to severely limit Candida GI fitness and hence represents a promising target to reduce fungal colonization in patients at risks of systemic candidiasis.

Gene Essentiality Is a Quantitative Property Linked to Cellular Evolvability.

Gene essentiality is typically determined by assessing the viability of the corresponding mutant cells, but this definition fails to account for the ability of cells to adaptively evolve to genetic perturbations. Here, we performed a stringent screen to assess the degree to which Saccharomyces cerevisiae cells can survive the deletion of ~1,000 individual "essential" genes and found that ~9% of these genetic perturbations could in fact be overcome by adaptive evolution. Our analyses uncovered a genome-wide gradient of gene essentiality, with certain essential cellular functions being more "evolvable" than others. Ploidy changes were prevalent among the evolved mutant strains, and aneuploidy of a specific chromosome was adaptive for a class of evolvable nucleoporin mutants. These data justify a quantitative redefinition of gene essentiality that incorporates both viability and evolvability of the corresponding mutant cells and will enable selection of therapeutic targets associated with lower risk of emergence of drug resistance.