Role reversals: non-canonical roles for immune and non-immune cells in the gut.

The intestine is home to an intertwined network of epithelial, immune, and neuronal cells as well as the microbiome, with implications for immunity, systemic metabolism, and behavior. While the complexity of this microenvironment has long since been acknowledged, recent technological advances have propelled our understanding to an unprecedented level. Notably, the microbiota and non-immune or structural cells have emerged as important conductors of intestinal immunity, and by contrast, cells of both the innate and adaptive immune systems have demonstrated non-canonical roles in tissue repair and metabolism. This review highlights recent works in the following two streams: non-immune cells of the intestine performing immunological functions; and traditional immune cells exhibiting non-immune functions in the gut.

Mouse skeletal muscle adaptations to different durations of treadmill exercise after the cessation of FOLFOX chemotherapy.

FOLFOX (5-fluorouracil, leucovorin, oxaliplatin) chemotherapy is a treatment for colorectal cancer that can induce persistent fatigue and metabolic dysfunction. Regular exercise after chemotherapy cessation is widely recommended for cancer patients and has been shown to improve fatigue resistance in mice. However, gaps remain in understanding whether the early systemic and skeletal muscle adaptations to regular exercise are altered by prior FOLFOX chemotherapy treatment. Furthermore, the effects of exercise duration on early metabolic and skeletal muscle transcriptional adaptations are not fully established. Purpose: Investigate the effects of prior FOLFOX chemotherapy treatment on the early adaptations to repeated short- or long-duration treadmill exercise, including the fasting regulation of circulating metabolic regulators, skeletal muscle COXIV activity and myokine/exerkine gene expression in male mice. Methods: Male C57BL6/J mice completed 4 cycles of FOLFOX or PBS and were allowed to recover for 4-weeks. Subsets of mice performed 14 sessions (6 d/wk, 18 m/min, 5% grade) of short- (10 min/d) or long-duration (55 min/d) treadmill exercise. Blood plasma and muscle tissues were collected 48-72 h after the last exercise bout for biochemical analyses. Results: Long-duration exercise increased fasting plasma osteocalcin, LIF, and IL-6 in healthy PBS mice, and these changes were ablated by prior FOLFOX treatment. Slow-oxidative soleus muscle COXIV activity increased in response to long-duration exercise in PBS mice, which was blocked by prior FOLFOX treatment. Fast-glycolytic plantaris muscle COXIV activity increased with short-duration exercise independent of FOLFOX administration. There was a main effect for long-duration exercise to increase fasting muscle IL-6 and COXIV mRNA expression independent of FOLFOX. FOLFOX administration reduced muscle IL-6, LIF, and BDNF mRNA expression irrespective of long-duration exercise. Interestingly, short-duration exercise suppressed the FOLXOX induction of muscle myostatin mRNA expression. Conclusion: FOLFOX attenuated early exercise adaptations related to fasting circulating osteocalcin, LIF, and IL-6. However, prior FOLFOX treatment did not alter the exercise adaptations of plantaris muscle COXIV activity and plasma adiponectin. An improved understanding of mechanisms underlying exercise adaptations after chemotherapy will provide the basis for successfully treating fatigue and metabolic dysfunction in cancer survivors.

A Roadmap for the Human Gut Cell Atlas.

The number of studies investigating the human gastrointestinal tract using various single-cell profiling methods has increased substantially in the past few years. Although this increase provides a unique opportunity for the generation of the first comprehensive Human Gut Cell Atlas (HGCA), there remains a range of major challenges ahead. Above all, the ultimate success will largely depend on a structured and coordinated approach that aligns global efforts undertaken by a large number of research groups. In this Roadmap, we discuss a comprehensive forward-thinking direction for the generation of the HGCA on behalf of the Gut Biological Network of the Human Cell Atlas. Based on the consensus opinion of experts from across the globe, we outline the main requirements for the first complete HGCA by summarizing existing data sets and highlighting anatomical regions and/or tissues with limited coverage. We provide recommendations for future studies and discuss key methodologies and the importance of integrating the healthy gut atlas with related diseases and gut organoids. Importantly, we critically overview the computational tools available and provide recommendations to overcome key challenges.

Activation of p53-regulated pro-survival signals and hypoxia-independent mitochondrial targeting of TIGAR by human papillomavirus E6 oncoproteins.

The high-risk subtype human papillomaviruses (hrHPVs) infect and oncogenically transform basal epidermal stem cells associated with the development of squamous-cell epithelial cancers. The viral E6 oncoprotein destabilizes the p53 tumor suppressor, inhibits p53 K120-acetylation by the Tat-interacting protein of 60 kDa (TIP60, or Kat5), and prevents p53-dependent apoptosis. Intriguingly, the p53 gene is infrequently mutated in HPV + cervical cancer clinical isolates which suggests a possible paradoxical role for this gatekeeper in viral carcinogenesis. Here, we demonstrate that E6 activates the TP53-induced glycolysis and apoptosis regulator (TIGAR) and protects cells against oncogene-induced oxidative genotoxicity. The E6 oncoprotein induces a Warburg-like stress response and activates PI3K/PI5P4K/AKT-signaling that phosphorylates the TIGAR on serine residues and induces its hypoxia-independent mitochondrial targeting in hrHPV-transformed cells. Primary HPV + cervical cancer tissues contain high levels of TIGAR, p53, and c-Myc and our xenograft studies have further shown that lentiviral-siRNA-knockdown of TIGAR expression inhibits hrHPV-induced tumorigenesis in vivo. These findings suggest the modulation of p53 pro-survival signals and the antioxidant functions of TIGAR could have key ancillary roles during HPV carcinogenesis.

Spatial and molecular profiling of the mononuclear phagocyte network in classic Hodgkin lymphoma.

Classic Hodgkin lymphoma (cHL) has a rich immune infiltrate, which is an intrinsic component of the neoplastic process. Malignant Hodgkin Reed-Sternberg cells (HRSCs) create an immunosuppressive microenvironment by the expression of regulatory molecules, preventing T-cell activation. It has also been demonstrated that mononuclear phagocytes (MNPs) in the vicinity of HRSCs express similar regulatory mechanisms in parallel, and their presence in tissue is associated with inferior patient outcomes. MNPs in cHL have hitherto been identified by a small number of canonical markers and are usually described as tumor-associated macrophages. The organization of MNP networks and interactions with HRSCs remains unexplored at high resolution. Here, we defined the global immune-cell composition of cHL and nonlymphoma lymph nodes, integrating data across single-cell RNA sequencing, spatial transcriptomics, and multiplexed immunofluorescence. We observed that MNPs comprise multiple subsets of monocytes, macrophages, and dendritic cells (DCs). Classical monocytes, macrophages and conventional DC2s were enriched in the vicinity of HRSCs, but plasmacytoid DCs and activated DCs were excluded. Unexpectedly, cDCs and monocytes expressed immunoregulatory checkpoints PD-L1, TIM-3, and the tryptophan-catabolizing protein IDO, at the same level as macrophages. Expression of these molecules increased with age. We also found that classical monocytes are important signaling hubs, potentially controlling the retention of cDC2 and ThExh via CCR1-, CCR4-, CCR5-, and CXCR3-dependent signaling. Enrichment of the cDC2-monocyte-macrophage network in diagnostic biopsies is associated with early treatment failure. These results reveal unanticipated complexity and spatial polarization within the MNP compartment, further demonstrating their potential roles in immune evasion by cHL.

Metabolic and cardiopulmonary impact of aquatic exercise and nutritional guidance for four individuals with chronic motor incomplete spinal cord injury: a case series.

BACKGROUND: Persons living with chronic spinal cord injury (SCI) demonstrate an increased risk of cardiovascular diseases.Purpose The aim of this report was to assess the cardiopulmonary and metabolic impact of prescribed aquatic exercise in combination with dietary guidance for four individuals experiencing chronic SCI. CASE DESCRIPTION: We measured peak oxygen consumption (peak VO2), resting energy expenditure (REE), weight, food logs, fasting glucose, insulin and glycated hemoglobin (HbA1C) in four men with incomplete SCI, aged 34 to 63 years. INTERVENTION: The men received a group aquatic exercise program three times per week for 10 weeks, and a weekly individual nutritional consultation by phone. OUTCOMES: Peak VO2 increased by 7.9% and 34.4% in participants #3 and #4 and decreased by 12% and 16.4% in #1 and #2. Glucose values decreased by 19.6% and 14.2% for #1 and #3, and increased by 9.3% for both #2 and #4. Body mass decreased by 9.9%, 3.0% and 5.7% for participants #1, #2 and #3, but demonstrated no change for participant #4. Dietary guidance and education produced positive changes, including reduced fat, carbohydrate, daily sugar, and average calorie intake. CONCLUSION: Moderate exercise with weekly nutritional guidance appeared to positively impact body mass and dietary selections with varied metabolic and cardiopulmonary results.

Redefining intestinal immunity with single-cell transcriptomics.

The intestinal immune system represents the largest collection of immune cells in the body and is continually exposed to antigens from food and the microbiota. Here we discuss the contribution of single-cell transcriptomics in shaping our understanding of this complex system. We consider the impact on resolving early intestine development, engagement with the neighbouring microbiota, diversity of intestinal immune cells, compartmentalisation within the intestines and interactions with non-immune cells. Finally, we offer a perspective on open questions about gut immunity that evolving single-cell technologies are well placed to address.

An Integrated Taxonomy for Monogenic Inflammatory Bowel Disease.

BACKGROUND & AIMS: Monogenic forms of inflammatory bowel disease (IBD) illustrate the essential roles of individual genes in pathways and networks safeguarding immune tolerance and gut homeostasis. METHODS: To build a taxonomy model, we assessed 165 disorders. Genes were prioritized based on penetrance of IBD and disease phenotypes were integrated with multi-omics datasets. Monogenic IBD genes were classified by (1) overlapping syndromic features, (2) response to hematopoietic stem cell transplantation, (3) bulk RNA-sequencing of 32 tissues, (4) single-cell RNA-sequencing of >50 cell subsets from the intestine of healthy individuals and patients with IBD (pediatric and adult), and (5) proteomes of 43 immune subsets. The model was validated by addition of newly identified monogenic IBD defects. As a proof-of-concept, we explore the intersection between immunometabolism and antimicrobial activity for a group of disorders (G6PC3/SLC37A4). RESULTS: Our quantitative integrated taxonomy defines the cellular landscape of monogenic IBD gene expression across 102 genes with high and moderate penetrance (81 in the model set and 21 genes in the validation set). We illustrate distinct cellular networks, highlight expression profiles across understudied cell types (e.g., CD8+ T cells, neutrophils, epithelial subsets, and endothelial cells) and define genotype-phenotype associations (perianal disease and defective antimicrobial activity). We illustrate processes and pathways shared across cellular compartments and phenotypic groups and highlight cellular immunometabolism with mammalian target of rapamycin activation as one of the converging pathways. There is an overlap of genes and enriched cell-specific expression between monogenic and polygenic IBD. CONCLUSION: Our taxonomy integrates genetic, clinical and multi-omic data; providing a basis for genomic diagnostics and testable hypotheses for disease functions and treatment responses.

A roadmap for the Human Developmental Cell Atlas.

The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development.

Cells of the human intestinal tract mapped across space and time.

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.

Single-Cell Sequencing of Developing Human Gut Reveals Transcriptional Links to Childhood Crohn's Disease.

Human gut development requires the orchestrated interaction of differentiating cell types. Here, we generate an in-depth single-cell map of the developing human intestine at 6-10 weeks post-conception. Our analysis reveals the transcriptional profile of cycling epithelial precursor cells; distinct from LGR5-expressing cells. We propose that these cells may contribute to differentiated cell subsets via the generation of LGR5-expressing stem cells and receive signals from surrounding mesenchymal cells. Furthermore, we draw parallels between the transcriptomes of ex vivo tissues and in vitro fetal organoids, revealing the maturation of organoid cultures in a dish. Lastly, we compare scRNA-seq profiles from pediatric Crohn's disease epithelium alongside matched healthy controls to reveal disease-associated changes in the epithelial composition. Contrasting these with the fetal profiles reveals the re-activation of fetal transcription factors in Crohn's disease. Our study provides a resource available at www.gutcellatlas.org, and underscores the importance of unraveling fetal development in understanding disease.

Transcriptome dynamics of CD4+ T cells during malaria maps gradual transit from effector to memory.

The dynamics of CD4+ T cell memory development remain to be examined at genome scale. In malaria-endemic regions, antimalarial chemoprevention protects long after its cessation and associates with effects on CD4+ T cells. We applied single-cell RNA sequencing and computational modelling to track memory development during Plasmodium infection and treatment. In the absence of central memory precursors, two trajectories developed as T helper 1 (TH1) and follicular helper T (TFH) transcriptomes contracted and partially coalesced over three weeks. Progeny of single clones populated TH1 and TFH trajectories, and fate-mapping suggested that there was minimal lineage plasticity. Relationships between TFH and central memory were revealed, with antimalarials modulating these responses and boosting TH1 recall. Finally, single-cell epigenomics confirmed that heterogeneity among effectors was partially reset in memory. Thus, the effector-to-memory transition in CD4+ T cells is gradual during malaria and is modulated by antiparasitic drugs. Graphical user interfaces are presented for examining gene-expression dynamics and gene-gene correlations ( http://haquelab.mdhs.unimelb.edu.au/cd4_memory/ ).

High-Resolution mRNA and Secretome Atlas of Human Enteroendocrine Cells.

Enteroendocrine cells (EECs) sense intestinal content and release hormones to regulate gastrointestinal activity, systemic metabolism, and food intake. Little is known about the molecular make-up of human EEC subtypes and the regulated secretion of individual hormones. Here, we describe an organoid-based platform for functional studies of human EECs. EEC formation is induced in vitro by transient expression of NEUROG3. A set of gut organoids was engineered in which the major hormones are fluorescently tagged. A single-cell mRNA atlas was generated for the different EEC subtypes, and their secreted products were recorded by mass-spectrometry. We note key differences to murine EECs, including hormones, sensory receptors, and transcription factors. Notably, several hormone-like molecules were identified. Inter-EEC communication is exemplified by secretin-induced GLP-1 secretion. Indeed, individual EEC subtypes carry receptors for various EEC hormones. This study provides a rich resource to study human EEC development and function.

Germs and germlines: how "public" B-cell clones evolve in the gut.

Chen et al. describe how B-cell clones observed in the gut of many different individuals (recurrent or "public" clonotypes) are shaped by the combined influences of common microbial antigens and underlying genomic recombination biases.

Malat1 Suppresses Immunity to Infection through Promoting Expression of Maf and IL-10 in Th Cells.

Despite extensive mapping of long noncoding RNAs in immune cells, their function in vivo remains poorly understood. In this study, we identify over 100 long noncoding RNAs that are differentially expressed within 24 h of Th1 cell activation. Among those, we show that suppression of Malat1 is a hallmark of CD4+ T cell activation, but its complete deletion results in more potent immune responses to infection. This is because Malat1-/- Th1 and Th2 cells express lower levels of the immunosuppressive cytokine IL-10. In vivo, the reduced CD4+ T cell IL-10 expression in Malat1-/- mice underpins enhanced immunity and pathogen clearance in experimental visceral leishmaniasis (Leishmania donovani) but more severe disease in a model of malaria (Plasmodium chabaudi chabaudi AS). Mechanistically, Malat1 regulates IL-10 through enhancing expression of Maf, a key transcriptional regulator of IL-10 Maf expression correlates with Malat1 in single Ag-specific Th cells from P. chabaudi chabaudi AS-infected mice and is downregulated in Malat1-/- Th1 and Th2 cells. The Malat1 RNA is responsible for these effects, as antisense oligonucleotide-mediated inhibition of Malat1 also suppresses Maf and IL-10 levels. Our results reveal that through promoting expression of the Maf/IL-10 axis in effector Th cells, Malat1 is a nonredundant regulator of mammalian immunity.

Immunology in the Era of Single-Cell Technologies.

Immune cells are characterized by diversity, specificity, plasticity, and adaptability-properties that enable them to contribute to homeostasis and respond specifically and dynamically to the many threats encountered by the body. Single-cell technologies, including the assessment of transcriptomics, genomics, and proteomics at the level of individual cells, are ideally suited to studying these properties of immune cells. In this review we discuss the benefits of adopting single-cell approaches in studying underappreciated qualities of immune cells and highlight examples where these technologies have been critical to advancing our understanding of the immune system in health and disease.

Distinct microbial and immune niches of the human colon.

Gastrointestinal microbiota and immune cells interact closely and display regional specificity; however, little is known about how these communities differ with location. Here, we simultaneously assess microbiota and single immune cells across the healthy, adult human colon, with paired characterization of immune cells in the mesenteric lymph nodes, to delineate colonic immune niches at steady state. We describe distinct helper T cell activation and migration profiles along the colon and characterize the transcriptional adaptation trajectory of regulatory T cells between lymphoid tissue and colon. Finally, we show increasing B cell accumulation, clonal expansion and mutational frequency from the cecum to the sigmoid colon and link this to the increasing number of reactive bacterial species.