Tumour-associated myeloid cells expressing IL-10R2/IL-22R1 as a potential biomarker for diagnosis and recurrence of pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a poor survival rate, largely due to the lack of early diagnosis. Although myeloid cells are crucial in the tumour microenvironment, whether their specific subset can be a biomarker of PDAC progression is unclear. METHODS: We analysed IL-22 receptor expression in PDAC and peripheral blood. Additionally, we analysed gene expression profiles of IL-10R2+/IL-22R1+ myeloid cells and the presence of these cells using single-cell RNA sequencing and murine orthotropic PDAC models, respectively, followed by examining the immunosuppressive function of IL-10R2+/IL-22R1+ myeloid cells. Finally, the correlation between IL-10R2 expression and PDAC progression was evaluated. RESULTS: IL-10R2+/IL-22R1+ myeloid cells were present in PDAC and peripheral blood. Blood IL-10R2+ myeloid cells displayed a gene expression signature associated with tumour-educated circulating monocytes. IL-10R2+/IL-22R1+ myeloid cells from human myeloid cell culture inhibited T cell proliferation. By mouse models for PDAC, we found a positive correlation between pancreatic tumour growth and increased blood IL-10R2+/IL-22R1+ myeloid cells. IL-10R2+/IL-22R1+ myeloid cells from an early phase of the PDAC model suppressed T cell proliferation and cytotoxicity. IL-10R2+ myeloid cells indicated tumour recurrence 130 days sooner than CA19-9 in post-pancreatectomy patients. CONCLUSIONS: IL-10R2+/IL-22R1+ myeloid cells in the peripheral blood might be an early marker of PDAC prognosis.

A dietary commensal microbe enhances antitumor immunity by activating tumor macrophages to sequester iron.

Innate immune cells generate a multifaceted antitumor immune response, including the conservation of essential nutrients such as iron. These cells can be modulated by commensal bacteria; however, identifying and understanding how this occurs is a challenge. Here we show that the food commensal Lactiplantibacillus plantarum IMB19 augments antitumor immunity in syngeneic and xenograft mouse tumor models. Its capsular heteropolysaccharide is the major effector molecule, functioning as a ligand for TLR2. In a two-pronged manner, it skews tumor-associated macrophages to a classically active phenotype, leading to generation of a sustained CD8+ T cell response, and triggers macrophage 'nutritional immunity' to deploy the high-affinity iron transporter lipocalin-2 for capturing and sequestering iron in the tumor microenvironment. This process induces a cycle of tumor cell death, epitope expansion and subsequent tumor clearance. Together these data indicate that food commensals might be identified and developed into 'oncobiotics' for a multi-layered approach to cancer therapy.

TM4SF19-mediated control of lysosomal activity in macrophages contributes to obesity-induced inflammation and metabolic dysfunction.

Adipose tissue (AT) adapts to overnutrition in a complex process, wherein specialized immune cells remove and replace dysfunctional and stressed adipocytes with new fat cells. Among immune cells recruited to AT, lipid-associated macrophages (LAMs) have emerged as key players in obesity and in diseases involving lipid stress and inflammation. Here, we show that LAMs selectively express transmembrane 4 L six family member 19 (TM4SF19), a lysosomal protein that represses acidification through its interaction with Vacuolar-ATPase. Inactivation of TM4SF19 elevates lysosomal acidification and accelerates the clearance of dying/dead adipocytes in vitro and in vivo. TM4SF19 deletion reduces the LAM accumulation and increases the proportion of restorative macrophages in AT of male mice fed a high-fat diet. Importantly, male mice lacking TM4SF19 adapt to high-fat feeding through adipocyte hyperplasia, rather than hypertrophy. This adaptation significantly improves local and systemic insulin sensitivity, and energy expenditure, offering a potential avenue to combat obesity-related metabolic dysfunction.

Baf-mediated transcriptional regulation of teashirt is essential for the development of neural progenitor cell lineages.

Accumulating evidence hints heterochromatin anchoring to the inner nuclear membrane as an upstream regulatory process of gene expression. Given that the formation of neural progenitor cell lineages and the subsequent maintenance of postmitotic neuronal cell identity critically rely on transcriptional regulation, it seems possible that the development of neuronal cells is influenced by cell type-specific and/or context-dependent programmed regulation of heterochromatin anchoring. Here, we explored this possibility by genetically disrupting the evolutionarily conserved barrier-to-autointegration factor (Baf) in the Drosophila nervous system. Through single-cell RNA sequencing, we demonstrated that Baf knockdown induces prominent transcriptomic changes, particularly in type I neuroblasts. Among the differentially expressed genes, our genetic analyses identified teashirt (tsh), a transcription factor that interacts with beta-catenin, to be closely associated with Baf knockdown-induced phenotypes that were suppressed by the overexpression of tsh or beta-catenin. We also found that Baf and tsh colocalized in a region adjacent to heterochromatin in type I NBs. Notably, the subnuclear localization pattern remained unchanged when one of these two proteins was knocked down, indicating that both proteins contribute to the anchoring of heterochromatin to the inner nuclear membrane. Overall, this study reveals that the Baf-mediated transcriptional regulation of teashirt is a novel molecular mechanism that regulates the development of neural progenitor cell lineages.

Fc-fused IL-7 provides broad antiviral effects against respiratory virus infections through IL-17A-producing pulmonary innate-like T cells.

Repeated pandemics caused by the influenza virus and severe acute respiratory syndrome coronavirus (SARS-CoV) have resulted in serious problems in global public health, emphasizing the need for broad-spectrum antiviral therapeutics against respiratory virus infections. Here, we show the protective effects of long-acting recombinant human interleukin-7 fused with hybrid Fc (rhIL-7-hyFc) against major respiratory viruses, including influenza virus, SARS-CoV-2, and respiratory syncytial virus. Administration of rhIL-7-hyFc in a therapeutic or prophylactic regimen induces substantial antiviral effects. During an influenza A virus (IAV) infection, rhIL-7-hyFc treatment increases pulmonary T cells composed of blood-derived interferon γ (IFNγ)+ conventional T cells and locally expanded IL-17A+ innate-like T cells. Single-cell RNA transcriptomics reveals that rhIL-7-hyFc upregulates antiviral genes in pulmonary T cells and induces clonal expansion of type 17 innate-like T cells. rhIL-7-hyFc-mediated disease prevention is dependent on IL-17A in both IAV- and SARS-CoV-2-infected mice. Collectively, we suggest that rhIL-7-hyFc can be used as a broadly active therapeutic for future respiratory virus pandemic.

Unique adipose tissue invariant natural killer T cell subpopulations control adipocyte turnover in mice.

Adipose tissue invariant natural killer T (iNKT) cells are a crucial cell type for adipose tissue homeostasis in obese animals. However, heterogeneity of adipose iNKT cells and their function in adipocyte turnover are not thoroughly understood. Here, we investigate transcriptional heterogeneity in adipose iNKT cells and their hierarchy using single-cell RNA sequencing in lean and obese mice. We report that distinct subpopulations of adipose iNKT cells modulate adipose tissue homeostasis through adipocyte death and birth. We identify KLRG1+ iNKT cells as a unique iNKT cell subpopulation in adipose tissue. Adoptive transfer experiments showed that KLRG1+ iNKT cells are selectively generated within adipose tissue microenvironment and differentiate into a CX3CR1+ cytotoxic subpopulation in obese mice. In addition, CX3CR1+ iNKT cells specifically kill enlarged and inflamed adipocytes and recruit macrophages through CCL5. Furthermore, adipose iNKT17 cells have the potential to secrete AREG, and AREG is involved in stimulating adipose stem cell proliferation. Collectively, our data suggest that each adipose iNKT cell subpopulation plays key roles in the control of adipocyte turnover via interaction with adipocytes, adipose stem cells, and macrophages in adipose tissue.

Impaired BCAA catabolism in adipose tissues promotes age-associated metabolic derangement.

Adipose tissues are central in controlling metabolic homeostasis and failure in their preservation is associated with age-related metabolic disorders. The exact role of mature adipocytes in this phenomenon remains elusive. Here we describe the role of adipose branched-chain amino acid (BCAA) catabolism in this process. We found that adipocyte-specific Crtc2 knockout protected mice from age-associated metabolic decline. Multiomics analysis revealed that BCAA catabolism was impaired in aged visceral adipose tissues, leading to the activation of mechanistic target of rapamycin complex (mTORC1) signaling and the resultant cellular senescence, which was restored by Crtc2 knockout in adipocytes. Using single-cell RNA sequencing analysis, we found that age-associated decline in adipogenic potential of visceral adipose tissues was reinstated by Crtc2 knockout, via the reduction of BCAA-mTORC1 senescence-associated secretory phenotype axis. Collectively, we propose that perturbation of BCAA catabolism by CRTC2 is critical in instigating age-associated remodeling of adipose tissue and the resultant metabolic decline in vivo.

Intermediate cells of in vitro cellular reprogramming and in vivo tissue regeneration require desmoplakin.

Amphibians and fish show considerable regeneration potential via dedifferentiation of somatic cells into blastemal cells. In terms of dedifferentiation, in vitro cellular reprogramming has been proposed to share common processes with in vivo tissue regeneration, although the details are elusive. Here, we identified the cytoskeletal linker protein desmoplakin (Dsp) as a common factor mediating both reprogramming and regeneration. Our analysis revealed that Dsp expression is elevated in distinct intermediate cells during in vitro reprogramming. Knockdown of Dsp impedes in vitro reprogramming into induced pluripotent stem cells and induced neural stem/progenitor cells as well as in vivo regeneration of zebrafish fins. Notably, reduced Dsp expression impairs formation of the intermediate cells during cellular reprogramming and tissue regeneration. These findings suggest that there is a Dsp-mediated evolutionary link between cellular reprogramming in mammals and tissue regeneration in lower vertebrates and that the intermediate cells may provide alternative approaches for mammalian regenerative therapy.

Gossypetin ameliorates 5xFAD spatial learning and memory through enhanced phagocytosis against Aß.

BACKGROUND: Microglia are the resident immune cells found in our brain. They have a critical role in brain maintenance. Microglia constantly scavenge various waste materials in the brain including damaged or apoptotic neurons and Aß. Through phagocytosis of Aß, microglia prevent the accumulation of Aß plaque in the brain. However, in Alzheimer's disease (AD) patients, chronic exposure to Aß makes microglia to become exhausted, which reduces their phagocytic activity against Aß. Since microglia play an important role in Aß clearance, enhancing microglial phagocytic activity against Aß is a promising target for AD treatment. Therefore, there is a great need for therapeutic candidate that enhances microglial Aß clearance while inhibiting microglia's pathogenic properties. METHODS: In vivo studies were conducted with 5xFAD AD model mice by treating gossypetin for 13 weeks through intragastric administration. Their spatial learning and memory were evaluated through behavior tests such as Y-maze and Morris Water Maze test. Hippocampus and cortex were acquired from the sacrificed mice, and they were used for histological and biochemical analysis. Also, mouse tissues were dissociated into single cells for single-cell RNA sequencing (scRNA-seq) analysis. Transcriptome of microglial population was analyzed. Mouse primary microglia and BV2 mouse microglial cell line were cultured and treated with fluorescent recombinant Aß to evaluate whether their phagocytic activity is affected by gossypetin. RESULTS: Gossypetin treatment improved the spatial learning and memory of 5xFAD by decreasing Aß deposition in the hippocampus and cortex of 5xFAD. Gossypetin induced transcriptomic modulations in various microglial subpopulations, including disease-associated microglia. Gossypetin enhanced phagocytic activity of microglia while decreasing their gliosis. Gossypetin also increased MHC II+ microglial population. CONCLUSIONS: Gossypetin showed protective effects against AD by enhancing microglial Aß phagocytosis. Gossypetin appears to be a novel promising therapeutic candidate against AD.

Dissecting chicken germ cell dynamics by combining a germ cell tracing transgenic chicken model with single-cell RNA sequencing.

Avian germ cells can be distinguished by certain characteristics during development. On the basis of these characteristics, germ cells can be used for germline transmission. However, the dynamic transcriptional landscape of avian germ cells during development is unknown. Here, we used a novel germ-cell-tracing method to monitor and isolate chicken germ cells at different stages of development. We targeted the deleted in azoospermia like (DAZL) gene, a germ-cell-specific marker, to integrate a green fluorescent protein (GFP) reporter gene without affecting endogenous DAZL expression. The resulting transgenic chickens (DAZL::GFP) were used to uncover the dynamic transcriptional landscape of avian germ cells. Single-cell RNA sequencing of 4,752 male and 13,028 female DAZL::GFP germ cells isolated from embryonic day E2.5 to 1 week post-hatch identified sex-specific developmental stages (4 stages in male and 5 stages in female) and trajectories (apoptosis and meiosis paths in female) of chicken germ cells. The male and female trajectories were characterized by a gradual acquisition of stage-specific transcription factor activities. We also identified evolutionary conserved and species-specific gene expression programs during both chicken and human germ-cell development. Collectively, these novel analyses provide mechanistic insights into chicken germ-cell development.

Homeostatic serum IgE is secreted by plasma cells in the thymus and enhances mast cell survival.

Increased serum levels of immunoglobulin E (IgE) is a risk factor for various diseases, including allergy and anaphylaxis. However, the source and ontogeny of B cells producing IgE under steady state conditions are not well defined. Here, we show plasma cells that develop in the thymus and potently secrete IgE and other immunoglobulins, including IgM, IgA, and IgG. The development of these IgE-secreting plasma cells are induced by IL-4 produced by invariant Natural Killer T cells, independent of CD1d-mediated interaction. Single-cell transcriptomics suggest the developmental landscape of thymic B cells, and the thymus supports development of transitional, mature, and memory B cells in addition to plasma cells. Furthermore, thymic plasma cells produce polyclonal antibodies without somatic hypermutation, indicating they develop via the extra-follicular pathway. Physiologically, thymic-derived IgEs increase the number of mast cells in the gut and skin, which correlates with the severity of anaphylaxis. Collectively, we define the ontogeny of thymic plasma cells and show that steady state thymus-derived IgEs regulate mast cell homeostasis, opening up new avenues for studying the genetic causes of allergic disorders.

Distinct properties of adipose stem cell subpopulations determine fat depot-specific characteristics.

In mammals, white adipose tissues are largely divided into visceral epididymal adipose tissue (EAT) and subcutaneous inguinal adipose tissue (IAT) with distinct metabolic properties. Although emerging evidence suggests that subpopulations of adipose stem cells (ASCs) would be important to explain fat depot differences, ASCs of two fat depots have not been comparatively investigated. Here, we characterized heterogeneous ASCs and examined the effects of intrinsic and tissue micro-environmental factors on distinct ASC features. We demonstrated that ASC subpopulations in EAT and IAT exhibited different molecular features with three adipogenic stages. ASC transplantation experiments revealed that intrinsic ASC features primarily determined their adipogenic potential. Upon obesogenic stimuli, EAT-specific SDC1+ ASCs promoted fibrotic remodeling, whereas IAT-specific CXCL14+ ASCs suppressed macrophage infiltration. Moreover, IAT-specific BST2high ASCs exhibited a high potential to become beige adipocytes. Collectively, our data broaden the understanding of ASCs with new insights into the origin of white fat depot differences.

Single-cell RNA sequencing identifies shared differentiation paths of mouse thymic innate T cells.

Invariant natural killer T (iNKT), mucosal-associated invariant T (MAIT), and γδ T cells are innate T cells that acquire memory phenotype in the thymus and share similar biological characteristics. However, how their effector differentiation is developmentally regulated is still unclear. Here, we identify analogous effector subsets of these three innate T cell types in the thymus that share transcriptional profiles. Using single-cell RNA sequencing, we show that iNKT, MAIT and γδ T cells mature via shared, branched differentiation rather than linear maturation or TCR-mediated instruction. Simultaneous TCR clonotyping analysis reveals that thymic maturation of all three types is accompanied by clonal selection and expansion. Analyses of mice deficient of TBET, GATA3 or RORγt and additional in vivo experiments corroborate the predicted differentiation paths, while human innate T cells from liver samples display similar features. Collectively, our data indicate that innate T cells share effector differentiation processes in the thymus.

Correction to: PiggyBac mutagenesis and exome sequencing identify genetic driver landscapes and potential therapeutic targets of EGFR-mutant gliomas.

An amendment to this paper has been published and can be accessed via the original article.

PiggyBac mutagenesis and exome sequencing identify genetic driver landscapes and potential therapeutic targets of EGFR-mutant gliomas.

BACKGROUND: Glioma is the most common intrinsic brain tumor and also occurs in the spinal cord. Activating EGFR mutations are common in IDH1 wild-type gliomas. However, the cooperative partners of EGFR driving gliomagenesis remain poorly understood. RESULTS: We explore EGFR-mutant glioma evolution in conditional mutant mice by whole-exome sequencing, transposon mutagenesis forward genetic screening, and transcriptomics. We show mutant EGFR is sufficient to initiate gliomagenesis in vivo, both in the brain and spinal cord. We identify significantly recurrent somatic alterations in these gliomas including mutant EGFR amplifications and Sub1, Trp53, and Tead2 loss-of-function mutations. Comprehensive functional characterization of 96 gliomas by genome-wide piggyBac insertional mutagenesis in vivo identifies 281 known and novel EGFR-cooperating driver genes, including Cdkn2a, Nf1, Spred1, and Nav3. Transcriptomics confirms transposon-mediated effects on expression of these genes. We validate the clinical relevance of new putative tumor suppressors by showing these are frequently altered in patients' gliomas, with prognostic implications. We discover shared and distinct driver mutations in brain and spinal gliomas and confirm in vivo differential tumor suppressive effects of Pten between these tumors. Functional validation with CRISPR-Cas9-induced mutations in novel genes Tead2, Spred1, and Nav3 demonstrates heightened EGFRvIII-glioma cell proliferation. Chemogenomic analysis of mutated glioma genes reveals potential drug targets, with several investigational drugs showing efficacy in vitro. CONCLUSION: Our work elucidates functional driver landscapes of EGFR-mutant gliomas, uncovering potential therapeutic strategies, and provides new tools for functional interrogation of gliomagenesis.

Discovery of new epigenomics-based biomarkers and the early diagnosis of neurodegenerative diseases.

Treatment options for many neurodegenerative diseases are limited due to the lack of early diagnostic procedures that allow timely delivery of therapeutic agents to affected neurons prior to cell death. While notable advances have been made in neurodegenerative disease biomarkers, whether or not the biomarkers discovered to date are useful for early diagnosis remains an open question. Additionally, the reliability of these biomarkers has been disappointing, due in part to the large dissimilarities between the tissues traditionally used to source biomarkers and primarily diseased neurons. In this article, we review the potential viability of atypical epigenetic and/or consequent transcriptional alterations (ETAs) as biomarkers of early-stage neurodegenerative disease, and present our perspectives on the discovery and practical use of such biomarkers in patient-derived neural samples using single-cell level analyses, thereby greatly enhancing the reliability of biomarker application.

Loss of IL-10 signaling in macrophages limits bacterial killing driven by prostaglandin E2.

Loss of IL-10 signaling in macrophages (Mφs) leads to inflammatory bowel disease (IBD). Induced pluripotent stem cells (iPSCs) were generated from an infantile-onset IBD patient lacking a functional IL10RB gene. Mφs differentiated from IL-10RB-/- iPSCs lacked IL-10RB mRNA expression, were unable to phosphorylate STAT3, and failed to reduce LPS induced inflammatory cytokines in the presence of exogenous IL-10. IL-10RB-/- Mφs exhibited a striking defect in their ability to kill Salmonella enterica serovar Typhimurium, which was rescuable after experimentally introducing functional copies of the IL10RB gene. Genes involved in synthesis and receptor pathways for eicosanoid prostaglandin E2 (PGE2) were more highly induced in IL-10RB-/- Mφs, and these Mφs produced higher amounts of PGE2 after LPS stimulation compared with controls. Furthermore, pharmacological inhibition of PGE2 synthesis and PGE2 receptor blockade enhanced bacterial killing in Mφs. These results identify a regulatory interaction between IL-10 and PGE2, dysregulation of which may drive aberrant Mφ activation and impaired host defense contributing to IBD pathogenesis.

A Genome-Wide Knockout Screen in Human Macrophages Identified Host Factors Modulating Salmonella Infection.

A genome-scale CRISPR knockout library screen of THP-1 human macrophages was performed to identify loss-of-function mutations conferring resistance to Salmonella uptake. The screen identified 183 candidate genes, from which 14 representative genes involved in actin dynamics (ACTR3, ARPC4, CAPZB, TOR3A, CYFIP2, CTTN, and NHLRC2), glycosaminoglycan metabolism (B3GNT1), receptor signaling (PDGFB and CD27), lipid raft formation (CLTCL1), calcium transport (ATP2A2 and ITPR3), and cholesterol metabolism (HMGCR) were analyzed further. For some of these pathways, known chemical inhibitors could replicate the Salmonella resistance phenotype, indicating their potential as targets for host-directed therapy. The screen indicated a role for the relatively uncharacterized gene NHLRC2 in both Salmonella invasion and macrophage differentiation. Upon differentiation, NHLRC2 mutant macrophages were hyperinflammatory and did not exhibit characteristics typical of macrophages, including atypical morphology and inability to interact and phagocytose bacteria/particles. Immunoprecipitation confirmed an interaction of NHLRC2 with FRYL, EIF2AK2, and KLHL13.IMPORTANCESalmonella exploits macrophages to gain access to the lymphatic system and bloodstream to lead to local and potentially systemic infections. With an increasing number of antibiotic-resistant isolates identified in humans, Salmonella infections have become major threats to public health. Therefore, there is an urgent need to identify alternative approaches to anti-infective therapy, including host-directed therapies. In this study, we used a simple genome-wide screen to identify 183 candidate host factors in macrophages that can confer resistance to Salmonella infection. These factors may be potential therapeutic targets against Salmonella infections.

Dissecting Cellular Heterogeneity Using Single-Cell RNA Sequencing.

Cell-to-cell variability in gene expression exists even in a homogeneous population of cells. Dissecting such cellular heterogeneity within a biological system is a prerequisite for understanding how a biological system is developed, homeo-statically regulated, and responds to external perturbations. Single-cell RNA sequencing (scRNA-seq) allows the quantitative and unbiased characterization of cellular heterogeneity by providing genome-wide molecular profiles from tens of thousands of individual cells. A major question in analyzing scRNA-seq data is how to account for the observed cell-to-cell variability. In this review, we provide an overview of scRNA-seq protocols, computational approaches for dissecting cellular heterogeneity, and future directions of single-cell transcriptomic analysis.

Mitogen-activated protein kinase phosphatase 2 regulates histone H3 phosphorylation via interaction with vaccinia-related kinase 1.

Mitogen-activated protein kinase phosphatase 2 (MKP2) is a member of the dual-specificity MKPs that regulate MAP kinase signaling. However, MKP2 functions are still largely unknown. In this study, we showed that MKP2 could regulate histone H3 phosphorylation under oxidative stress conditions. We found that MKP2 inhibited histone H3 phosphorylation by suppressing vaccinia-related kinase 1 (VRK1) activity. Moreover, this regulation was dependent on the selective interaction with VRK1, regardless of its phosphatase activity. The interaction between MKP2 and VRK1 mainly occurred in the chromatin, where histones are abundant. We also observed that the protein level of MKP2 and its interaction with histone H3 increased from G1 to M phase during the cell cycle, which is similar to the VRK1 profile. Furthermore, MKP2 specifically regulated the VRK1-mediated histone H3 phosphorylation at M phase. Taken together, these data suggest a novel function of MKP2 as a negative regulator of VRK1-mediated histone H3 phosphorylation.