TH2, allergy and group 2 innate lymphoid cells.

The initiation of type 2 immune responses by the epithelial cell-derived cytokines IL-25, IL-33 and TSLP has been an area of extensive research in the past decade. Such studies have led to the identification of a new innate lymphoid subset that produces the canonical type 2 cytokines IL-5, IL-9 and IL-13 in response to IL-25 and IL-33. These group 2 or type 2 innate lymphoid cells (ILC2 cells) represent a critical source of type 2 cytokines in vivo and serve an important role in orchestrating the type 2 response to helminths and allergens. Further characterization of ILC2 cell biology will enhance the understanding of type 2 responses and may identify new treatments for asthma, allergies and parasitic infections. Interactions between ILC2 cells and the adaptive immune system, as well as examination of potential roles for ILC2 cells in the maintenance of homeostasis, promise to be particularly fruitful areas of future research.

Intracellular calcium links milk stasis to lysosome-dependent cell death during early mammary gland involution.

Involution of the mammary gland after lactation is a dramatic example of coordinated cell death. Weaning causes distension of the alveolar structures due to the accumulation of milk, which, in turn, activates STAT3 and initiates a caspase-independent but lysosome-dependent cell death (LDCD) pathway. Although the importance of STAT3 and LDCD in early mammary involution is well established, it has not been entirely clear how milk stasis activates STAT3. In this report, we demonstrate that protein levels of the PMCA2 calcium pump are significantly downregulated within 2-4 h of experimental milk stasis. Reductions in PMCA2 expression correlate with an increase in cytoplasmic calcium in vivo as measured by multiphoton intravital imaging of GCaMP6f fluorescence. These events occur concomitant with the appearance of nuclear pSTAT3 expression but prior to significant activation of LDCD or its previously implicated mediators such as LIF, IL6, and TGFß3, all of which appear to be upregulated by increased intracellular calcium. We further demonstrate that increased intracellular calcium activates STAT3 by inducing degradation of its negative regulator, SOCS3. We also observed that milk stasis, loss of PMCA2 expression and increased intracellular calcium levels activate TFEB, an important regulator of lysosome biogenesis through a process involving inhibition of CDK4/6 and cell cycle progression. In summary, these data suggest that intracellular calcium serves as an important proximal biochemical signal linking milk stasis to STAT3 activation, increased lysosomal biogenesis, and lysosome-mediated cell death.

RFX4 is an intrinsic factor for neuronal differentiation through induction of proneural genes POU3F2 and NEUROD1.

Proneural genes play a crucial role in neuronal differentiation. However, our understanding of the regulatory mechanisms governing proneural genes during neuronal differentiation remains limited. RFX4, identified as a candidate regulator of proneural genes, has been reported to be associated with the development of neuropsychiatric disorders. To uncover the regulatory relationship, we utilized a combination of multi-omics data, including ATAC-seq, ChIP-seq, Hi-C, and RNA-seq, to identify RFX4 as an upstream regulator of proneural genes. We further validated the role of RFX4 using an in vitro model of neuronal differentiation with RFX4 knock-in and a CRISPR-Cas9 knock-out system. As a result, we found that RFX4 directly interacts with the promoters of POU3F2 and NEUROD1. Transcriptomic analysis revealed a set of genes associated with neuronal development, which are highly implicated in the development of neuropsychiatric disorders, including schizophrenia. Notably, ectopic expression of RFX4 can drive human embryonic stem cells toward a neuronal fate. Our results strongly indicate that RFX4 serves as a direct upstream regulator of proneural genes, a role that is essential for normal neuronal development. Impairments in RFX4 function could potentially be related to the development of various neuropsychiatric disorders. However, understanding the precise mechanisms by which the RFX4 gene influences the onset of neuropsychiatric disorders requires further investigation through human genetic studies.

SEPHS1 is dispensable for pluripotency maintenance but indispensable for cardiac differentiation in mouse embryonic stem cells.

Embryonic stem cells (ESCs) are derived from the inner cell mass of developing blastocysts, which have self-renewal ability and have the potential to develop or reconstitute into all embryonic lineages. Selenophosphate synthetase 1 (SEPHS1) is an essential protein in mouse early embryo development. However, the role of SEPHS1 in mouse ESCs remains to be elucidated. In this study, we generated Sephs1 KO ESCs and found that deficiency of SEPSH1 has little effect on pluripotency maintenance and proliferation. Notably, SEPHS1 deficiency impaired differentiation into three germ layers and gastruloid aggregation in vitro. RNA-seq analysis showed SEPHS1 is involved in cardiogenesis, verified by no beating signal in Sephs1 KO embryoid body at d10 and low expression of cardiac-related and contraction markers. Taken together, our results suggest that SPEHS1 is dispensable in ESC self-renewal, but indispensable in subsequent germ layer differentiation especially for functional cardiac lineage.

Caspase-10 affects the pathogenesis of primary biliary cholangitis by regulating inflammatory cell death.

Primary biliary cholangitis (PBC) is an autoimmune disease that involves chronic inflammation and injury to biliary epithelial cells. To identify critical genetic factor(s) in PBC patients, we performed whole-exome sequencing of five female siblings, including one unaffected and four affected sisters, in a multi-PBC family, and identified 61 rare heterozygote variants that segregated only within the affected sisters. Among them, we were particularly interested in caspase-10, for although several caspases are involved in cell death, inflammation and autoimmunity, caspase-10 is little known from this perspective. We generated caspase-10 knockout macrophages, and then investigated the obtained phenotypes in comparison to those of its structurally similar protein, caspase-8. Unlike caspase-8, caspase-10 does not play a role during differentiation into macrophages, but after differentiation, it regulates the process of inflammatory cell deaths such as necroptosis and pyroptosis more strongly. Interestingly, caspase-10 displays better protease activity than caspase-8 in the process of RIPK1 cleavage, and an enhanced ability to form a complex with RIPK1 and FADD in human macrophages. Higher inflammatory cell death affected the fibrotic response of hepatic stellate cells; this effect could be recovered by treatment with UDCA and OCA, which are currently approved for PBC patients. Our findings strongly indicate that the defective roles of caspase-10 in macrophages contribute to the pathogenesis of PBC, thereby suggesting a new therapeutic strategy for PBC treatment.

Oct-1 regulates IL-17 expression by directing interchromosomal associations in conjunction with CTCF in T cells.

Interchromosomal associations can regulate gene expression, but little is known about the molecular basis of such associations. In response to antigen stimulation, naive T cells can differentiate into Th1, Th2, and Th17 cells expressing IFN-γ, IL-4, and IL-17, respectively. We previously reported that in naive T cells, the IFN-γ locus is associated with the Th2 cytokine locus. Here we show that the Th2 locus additionally associates with the IL-17 locus. This association requires a DNase I hypersensitive region (RHS6) at the Th2 locus. RHS6 and the IL-17 promoter both bear Oct-1 binding sites. Deletion of either of these sites or Oct-1 gene impairs the association. Oct-1 and CTCF bind their cognate sites cooperatively, and CTCF deficiency similarly impairs the association. Finally, defects in the association lead to enhanced IL-17 induction. Collectively, our data indicate Th17 lineage differentiation is restrained by the Th2 locus via interchromosomal associations organized by Oct-1 and CTCF.

PLEKHA8P1 Promotes Tumor Progression and Indicates Poor Prognosis of Liver Cancer.

Hepatocellular carcinoma (HCC) records the second-lowest 5-year survival rate despite the avalanche of research into diagnosis and therapy. One of the major obstacles in treatment is chemoresistance to drugs such as 5-fluorouracil (5-FU), making identification and elucidation of chemoresistance regulators highly valuable. As the regulatory landscape grows to encompass non-coding genes such as long non-coding RNAs (lncRNAs), a relatively new class of lncRNA has emerged in the form of pseudogene-derived lncRNAs. Through bioinformatics analyses of the TCGA LIHC dataset, we have systematically identified pseudogenes of prognostic value. Initial experimental validation of selected pseudogene-derived lncRNA (PLEKHA8P1) and its parental gene (PLEKHA8), a well-studied transport protein in Golgi complex recently implicated as an oncogene in both colorectal and liver cancer, indicates that the pseudogene/parental gene pair promotes tumor progression and that their dysregulated expression levels affect 5-FU-induced chemoresistance in human HCC cell line FT3-7. Our study has thus confirmed cancer-related functions of PLEKHA8, and laid the groundwork for identification and validation of oncogenic pseudogene-derived lncRNA that shows potential as a novel therapeutic target in circumventing chemoresistance induced by 5-FU.

Promyelocytic Leukemia Proteins Regulate Fanconi Anemia Gene Expression.

Promyelocytic leukemia (PML) protein is the core component of subnuclear structures called PML nuclear bodies that are known to play important roles in cell survival, DNA damage responses, and DNA repair. Fanconi anemia (FA) proteins are required for repairing interstrand DNA crosslinks (ICLs). Here we report a novel role of PML proteins, regulating the ICL repair pathway. We found that depletion of the PML protein led to the significant reduction of damage-induced FANCD2 mono-ubiquitination and FANCD2 foci formation. Consistently, the cells treated with siRNA against PML showed enhanced sensitivity to a crosslinking agent, mitomycin C. Further studies showed that depletion of PML reduced the protein expression of FANCA, FANCG, and FANCD2 via reduced transcriptional activity. Interestingly, we observed that damage-induced CHK1 phosphorylation was severely impaired in cells with depleted PML, and we demonstrated that CHK1 regulates FANCA, FANCG, and FANCD2 transcription. Finally, we showed that inhibition of CHK1 phosphorylation further sensitized cancer cells to mitomycin C. Taken together, these findings suggest that the PML is critical for damage-induced CHK1 phosphorylation, which is important for FA gene expression and for repairing ICLs.

Constitutive Oxidative Stress by SEPHS1 Deficiency Induces Endothelial Cell Dysfunction.

The primary function of selenophosphate synthetase (SEPHS) is to catalyze the synthesis of selenophosphate that serves as a selenium donor during selenocysteine synthesis. In eukaryotes, there are two isoforms of SEPHS (SEPHS1 and SEPHS2). Between these two isoforms, only SEPHS2 is known to contain selenophosphate synthesis activity. To examine the function of SEPHS1 in endothelial cells, we introduced targeted null mutations to the gene for SEPHS1, Sephs1, in cultured mouse 2H11 endothelial cells. SEPHS1 deficiency in 2H11 cells resulted in the accumulation of superoxide and lipid peroxide, and reduction in nitric oxide. Superoxide accumulation in Sephs1-knockout 2H11 cells is due to the induction of xanthine oxidase and NADPH oxidase activity, and due to the decrease in superoxide dismutase 1 (SOD1) and 3 (SOD3). Superoxide accumulation in 2H11 cells also led to the inhibition of cell proliferation and angiogenic tube formation. Sephs1-knockout cells were arrested at G2/M phase and showed increased gamma H2AX foci. Angiogenic dysfunction in Sephs1-knockout cells is mediated by a reduction in nitric oxide and an increase in ROS. This study shows for the first time that superoxide was accumulated by SEPHS1 deficiency, leading to cell dysfunction through DNA damage and inhibition of cell proliferation.

Identification of Signaling Pathways for Early Embryonic Lethality and Developmental Retardation in Sephs1-/- Mice.

Selenophosphate synthetase 1 (SEPHS1) plays an essential role in cell growth and survival. However, the underlying molecular mechanisms remain unclear. In the present study, the pathways regulated by SEPHS1 during gastrulation were determined by bioinformatical analyses and experimental verification using systemic knockout mice targeting Sephs1. We found that the coagulation system and retinoic acid signaling were most highly affected by SEPHS1 deficiency throughout gastrulation. Gene expression patterns of altered embryo morphogenesis and inhibition of Wnt signaling were predicted with high probability at E6.5. These predictions were verified by structural abnormalities in the dermal layer of Sephs1-/- embryos. At E7.5, organogenesis and activation of prolactin signaling were predicted to be affected by Sephs1 knockout. Delay of head fold formation was observed in the Sephs1-/- embryos. At E8.5, gene expression associated with organ development and insulin-like growth hormone signaling that regulates organ growth during development was altered. Consistent with these observations, various morphological abnormalities of organs and axial rotation failure were observed. We also found that the gene sets related to redox homeostasis and apoptosis were gradually enriched in a time-dependent manner until E8.5. However, DNA damage and apoptosis markers were detected only when the Sephs1-/- embryos aged to E9.5. Our results suggest that SEPHS1 deficiency causes a gradual increase of oxidative stress which changes signaling pathways during gastrulation, and afterwards leads to apoptosis.

Myosin VI regulates gene pairing and transcriptional pause release in T cells.

Naive CD4 T cells differentiate into several effector lineages, which generate a stronger and more rapid response to previously encountered immunological challenges. Although effector function is a key feature of adaptive immunity, the molecular basis of this process is poorly understood. Here, we investigated the spatiotemporal regulation of cytokine gene expression in resting and restimulated effector T helper 1 (Th1) cells. We found that the Lymphotoxin (LT)/TNF alleles, which encode TNF-α, were closely juxtaposed shortly after T-cell receptor (TCR) engagement, when transcription factors are limiting. Allelic pairing required a nuclear myosin, myosin VI, which is rapidly recruited to the LT/TNF locus upon restimulation. Furthermore, transcription was paused at the TNF locus and other related genes in resting Th1 cells and released in a myosin VI-dependent manner following activation. We propose that homologous pairing and myosin VI-mediated transcriptional pause release account for the rapid and efficient expression of genes induced by an external stimulus.

AMCase is a crucial regulator of type 2 immune responses to inhaled house dust mites.

Chitinases are enzymes that cleave chitin, a component of the exoskeleton of many organisms including the house dust mite (HDM). Here we show that knockin mice expressing an enzymatically inactive acidic mammalian chitinase (AMCase), the dominant true chitinase in mouse lung, showed enhanced type 2 immune responses to inhaled HDM. We found that uncleaved chitin promoted the release of IL-33, whereas cleaved chitin could be phagocytosed and could induce the activation of caspase-1 and subsequent activation of caspase-7; this results in the resolution of type 2 immune responses, probably by promoting the inactivation of IL-33. These data suggest that AMCase is a crucial regulator of type 2 immune responses to inhaled chitin-containing aeroallergens.

Role of OCT-1 and partner proteins in T cell differentiation.

The understanding of CD4 T cell differentiation gives important insights into the control of immune responses against various pathogens and in autoimmune diseases. Naïve CD4 T cells become effector T cells in response to antigen stimulation in combination with various environmental cytokine stimuli. Several transcription factors and cis-regulatory regions have been identified to regulate epigenetic processes on chromatin, to allow the production of proper effector cytokines during CD4 T cell differentiation. OCT-1 (Pou2f1) is well known as a widely expressed transcription factor in most tissues and cells. Although the importance of OCT-1 has been emphasized during development and differentiation, its detailed molecular underpinning and precise role are poorly understood. Recently, a series of studies have reported that OCT-1 plays a critical role in CD4 T cells through regulating gene expression during differentiation and mediating long-range chromosomal interactions. In this review, we will describe the role of OCT-1 in CD4 T cell differentiation and discuss how this factor orchestrates the fate and function of CD4 effector T cells.

Macrophage C-type lectin is essential for phagosome maturation and acidification during Escherichia coli-induced peritonitis.

Sepsis is a life-threatening condition caused by an uncontrolled response to bacterial infection. Impaired bactericidal activity in the host is directly associated with severe sepsis; however, the underlying regulatory mechanism(s) is largely unknown. Here, we show that MCL (macrophage C-type lectin) plays a crucial role in killing bacteria during Escherichia coli-induced peritonitis. MCL-deficient mice with E. coli-induced sepsis showed lower survival rates and reduced bacterial clearance when compared with control mice, despite similar levels of proinflammatory cytokine production. Although the ability of macrophages from MCL-deficient mice to kill bacteria was impaired, they showed normal phagocytic activity and production of reactive oxygen species. In addition, MCL-deficient macrophages showed defective phagosome maturation and phagosomal acidification after E. coli infection. Taken together, these results indicate that MCL plays an important role in host defense against E. coli infection by promoting phagosome maturation and acidification, thereby providing new insight into the role of MCL during pathogenesis of sepsis and offering new therapeutic options.

Exogenous miRNA-146a Enhances the Therapeutic Efficacy of Human Mesenchymal Stem Cells by Increasing Vascular Endothelial Growth Factor Secretion in the Ischemia/Reperfusion-Injured Heart.

Adult stem cells have been studied as a promising therapeutic modality for the functional restoration of the damaged heart. In the present study, a strategy for enhancing the angiogenic efficacy of human mesenchymal stem cells (hMSCs) using micro-RNA was examined. We investigated whether micro-RNA-146a (miR-146a) influences the secretion of vascular endothelial growth factor (VEGF) and angiogenesis of MSCs. Our data indicated that miR-146a-transfected hMSCs (hMSCmiR-146a) decreased the expression of neurofibromin 2, an inhibitor of p21-activated kinase-1 (PAK1). miR-146a also increased the expression of Ras-related C3 botulinum toxin substrate 1 and PAK1, which are known to induce VEGF expression, and the formation of vascular branches was increased in hMSCmiR-146a compared to hMSCs treated with VEGF. VEGF and p-Akt were increased in hMSCmiR-146a. Furthermore, injection of hMSCmiR-146a after ischemia/reperfusion (I/R) injury led to a reduction of fibrosis area and increased VEGF expression, confirming the regenerative capacity such as reparative angiogenesis in the infarcted area. Cardiac functions in I/R injury were improved following injection of hMSCmiR-146a compared to the I/R group. Taken together, these data suggest that miR-146 is a novel microRNA that regulates VEGF expression, and its use may be an effective strategy for enhancing the therapeutic efficacy of hMSC transplantation into the I/R-injured heart.

Mycobacterial cord factor enhances migration of neutrophil-like HL-60 cells by prolonging AKT phosphorylation.

Trehalose 6,6'-dimycolate (TDM), or cord factor, is a crucial stimulus of immune responses during Mycobacterium tuberculosis infection. Although TDM has immuno-stimulatory properties, including adjuvant activity and the ability to induce granuloma formation, the mechanisms underlying these remain unknown. We hypothesized that TDM stimulates transendothelial migration of neutrophils, which are the first immune cells to infiltrate the tissue upon infection. In this study, it was shown that TDM enhances N-formylmethionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis and transendothelial movement by prolonging AKT phosphorylation in human neutrophils. TDM induced expression of macrophage-inducible C-type lectin, a receptor for TDM, and induced secretion of pro-inflammatory cytokines and chemokines in differentiated HL-60 cells. In 2- and 3-D neutrophil migration assays, TDM-stimulated neutrophils showed increased fMLP-induced chemotaxis and transendothelial migration. Interestingly, following fMLP stimulation of TDM-activated neutrophils, AKT, a crucial kinase for neutrophil polarization and chemotaxis, showed prolonged phosphorylation at serine 473. Taken together, these data suggest that TDM modulates transendothelial migration of neutrophils upon mycobacterial infection through prolonged AKT phosphorylation. AKT may therefore be a promising therapeutic target for enhancing immune responses to mycobacterial infection.

Anti-inflammatory mechanism of α-viniferin regulates lipopolysaccharide-induced release of proinflammatory mediators in BV2 microglial cells.

α-Viniferin is an oligostilbene of trimeric resveratrol and has anticancer activity; however, the molecular mechanism underlying the anti-inflammatory effects of α-viniferin has not been completely elucidated thus far. Therefore, we determined the mechanism by which α-viniferin regulates lipopolysaccharide (LPS)-induced expression of proinflammatory mediators in BV2 microglial cells. Treatment with α-viniferin isolated from Clematis mandshurica decreased LPS-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2). α-Viniferin also downregulated the LPS-induced expression of proinflammatory genes such as iNOS and COX-2 by suppressing the activity of nuclear factor kappa B (NF-κB) via dephosphorylation of Akt/PI3K. Treatment with a specific NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), indirectly showed that NF-κB is a crucial transcription factor for expression of these genes in the early stage of inflammation. Additionally, our results indicated that α-viniferin suppresses NO and PGE2 production in the late stage of inflammation through induction of heme oxygenase-1 (HO-1) regulated by nuclear factor erythroid 2-related factor (Nrf2). Taken together, our data indicate that α-viniferin suppresses the expression of proinflammatory genes iNOS and COX-2 in the early stage of inflammation by inhibiting the Akt/PI3K-dependent NF-κB activation and inhibits the production of proinflammatory mediators NO and PGE2 in the late stage by stimulating Nrf2-mediated HO-1 signaling pathway in LPS-stimulated BV2 microglial cells. These results suggest that α-viniferin may be a potential candidate to regulate LPS-induced inflammation.

Multifaceted roles of trained immunity in diverse pathological contexts.

Trained immunity, an innate immune response characterized by enhanced cellular responsiveness, exhibits a profound memory akin to adaptive immunity. This phenomenon involves intricate metabolic and epigenetic reprogramming triggered by stimuli such as ß-glucan and BCG, shaping innate immune memory. Following elucidation of the background on trained immunity, it is important to explore its multifaceted roles in various pathological contexts. In this review, we delve into the specific contributions of trained immunity in the intricate landscape of viral infections, tumorigenesis, and diverse inflammatory diseases, shedding light on its potential as a therapeutic target, and offering comprehensive understanding of its broader immunological implications.

Integrative analysis of circular RNA regulatory network in papillary thyroid carcinoma.

Papillary thyroid cancer (PTC) is the most common type of endocrine cancer worldwide. Generally, PTC has an excellent prognosis; however, lymph node metastases and recurrences occur frequently. Over the last decade, circular RNAs (circRNAs), a large class of noncoding RNAs (ncRNAs), have emerged as key regulators of various tumor progression pathways. Here, we aimed to identify novel circRNAs as PTC biomarkers. Differentially expressed circRNAs and mRNAs were analyzed using public datasets from the Gene Expression Omnibus and Cancer Genome Atlas. In addition, we screened for target miRNAs using online prediction databases. Based on these results, we established a circRNA-miRNA-mRNA regulatory network associated with PTC, in which protein-protein interaction networks led to the identification of hub genes. Functional enrichment and survival analyses were performed to gain insights into the biological mechanisms of circRNA involvement. As a result, we found that two circRNAs (hsa_circ_0041829 and has_circ_0092299), four miRNAs (miR-369, miR-486, miR-574, and miR-665), and nine hub genes (BBC3, E2F1, FYN, MAG, SDC1, SDC3, SNAP25, TK1, and TYMS) play significant roles in PTC progression. This study provides a novel framework for understanding the roles of circRNA-miRNA-mediated gene regulation in PTC. It also introduces potential therapeutic targets and prognostic biomarkers, which may serve as a basis for developing targeted therapeutic interventions for PTC.

Intracellular Calcium links Milk Stasis to Lysosome Dependent Cell Death by Activating a TGFß3/TFEB/STAT3 Pathway Early during Mammary Gland Involution.

Involution of the mammary gland after lactation is a dramatic example of coordinated cell death. Weaning causes distension of the alveolar structures due to the accumulation of milk, which, in turn, activates STAT3 and initiates a caspase-independent but lysosome-dependent cell death (LDCD) pathway. Although the importance of STAT3 and LDCD in early mammary involution is well established, it has not been entirely clear how milk stasis activates STAT3. In this report, we demonstrate that protein levels of the PMCA2 calcium pump are significantly downregulated within 2-4 hours of experimental milk stasis. Reductions in PMCA2 expression correlate with an increase in cytoplasmic calcium in vivo as measured by multiphoton intravital imaging of GCaMP6f fluorescence. These events occur concomitant with the appearance of nuclear pSTAT3 expression but prior to significant activation of LDCD or its previously implicated mediators such as LIF, IL6 and TGFß3, all of which appear to be upregulated by increased intracellular calcium. We also observed that milk stasis, loss of PMCA2 expression and increased intracellular calcium levels activate TFEB, an important regulator of lysosome biogenesis. This is the result of increased TGFß signaling and inhibition of cell cycle progression. Finally, we demonstrate that increased intracellular calcium activates STAT3 by inducing degradation of its negative regulator, SOCS3, a process which also appears to be mediated by TGFß signaling. In summary, these data suggest that intracellular calcium serves as an important proximal biochemical signal linking milk stasis to STAT3 activation, increased lysosomal biogenesis, and lysosome-mediated cell death.