Type I interferons and the cytokine TNF cooperatively reprogram the macrophage epigenome to promote inflammatory activation.

Cross-regulation of Toll-like receptor (TLR) responses by cytokines is essential for effective host defense, avoidance of toxicity and homeostasis, but the underlying mechanisms are not well understood. Our comprehensive epigenomics approach to the analysis of human macrophages showed that the proinflammatory cytokines TNF and type I interferons induced transcriptional cascades that altered chromatin states to broadly reprogram responses induced by TLR4. TNF tolerized genes encoding inflammatory molecules to prevent toxicity while preserving the induction of genes encoding antiviral and metabolic molecules. Type I interferons potentiated the inflammatory function of TNF by priming chromatin to prevent the silencing of target genes of the transcription factor NF-κB that encode inflammatory molecules. The priming of chromatin enabled robust transcriptional responses to weak upstream signals. Similar chromatin regulation occurred in human diseases. Our findings reveal that signaling crosstalk between interferons and TNF is integrated at the level of chromatin to reprogram inflammatory responses, and identify previously unknown functions and mechanisms of action of these cytokines.

Interferon-γ Represses M2 Gene Expression in Human Macrophages by Disassembling Enhancers Bound by the Transcription Factor MAF.

Mechanisms by which interferon (IFN)-γ activates genes to promote macrophage activation are well studied, but little is known about mechanisms and functions of IFN-γ-mediated gene repression. We used an integrated transcriptomic and epigenomic approach to analyze chromatin accessibility, histone modifications, transcription-factor binding, and gene expression in IFN-γ-primed human macrophages. IFN-γ suppressed basal expression of genes corresponding to an "M2"-like homeostatic and reparative phenotype. IFN-γ repressed genes by suppressing the function of enhancers enriched for binding by transcription factor MAF. Mechanistically, IFN-γ disassembled a subset of enhancers by inducing coordinate suppression of binding by MAF, lineage-determining transcription factors, and chromatin accessibility. Genes associated with MAF-binding enhancers were suppressed in macrophages isolated from rheumatoid-arthritis patients, revealing a disease-associated signature of IFN-γ-mediated repression. These results identify enhancer inactivation and disassembly as a mechanism of IFN-γ-mediated gene repression and reveal that MAF regulates the macrophage enhancer landscape and is suppressed by IFN-γ to augment macrophage activation.

Prognostic Value of BUB1 for Predicting Non-Muscle-Invasive Bladder Cancer Progression.

Non-muscle-invasive bladder cancer (NMIBC) is a common disease with a high recurrence rate requiring lifetime surveillance. Although NMIBC is not life-threatening, it can progress to muscle-invasive bladder cancer (MIBC), a lethal form of the disease. The management of the two diseases differs, and patients with MIBC require aggressive treatments such as chemotherapy and radical cystectomy. NMIBC patients at a high risk of progression benefit from early immediate cystectomy. Thus, identifying concordant markers for accurate risk stratification is critical to predict the prognosis of NMIBC. Candidate genetic biomarkers associated with NMIBC prognosis were screened by RNA-sequencing of 24 tissue samples, including 16 NMIBC and eight normal controls, and by microarray analysis (GSE13507). Lastly, we selected and investigated a mitotic checkpoint serine/threonine kinase, BUB1, that regulates chromosome segregation during the cell cycle. BUB1 gene expression was tested in 86 NMIBC samples and 15 controls by real-time qPCR. The performance of BUB1 as a prognostic biomarker for NMIBC was validated in the internal Chungbuk cohort (GSE13507) and the external UROMOL cohort (E-MTAB-4321). BUB1 expression was higher in NMIBC patients than in normal controls (p < 0.05), and the overexpression of BUB1 was correlated with NMIBC progression (log-rank test, p = 0.007). In in vitro analyses, BUB1 promoted the proliferation of bladder cancer cells by accelerating the G2/M transition of the cell cycle. Conclusively, BUB1 modulates the G2/M transition to promote the proliferation of bladder cancer cells, suggesting that it could serve as a prognostic marker in NMIBC.

Vaccinia-based influenza vaccine overcomes previously induced immunodominance hierarchy for heterosubtypic protection.

Growing concerns about unpredictable influenza pandemics require a broadly protective vaccine against diverse influenza strains. One of the promising approaches was a T cell-based vaccine, but the narrow breadth of T-cell immunity due to the immunodominance hierarchy established by previous influenza infection and efficacy against only mild challenge condition are important hurdles to overcome. To model T-cell immunodominance hierarchy in humans in an experimental setting, influenza-primed C57BL/6 mice were chosen and boosted with a mixture of vaccinia recombinants, individually expressing consensus sequences from avian, swine, and human isolates of influenza internal proteins. As determined by IFN-γ ELISPOT and polyfunctional cytokine secretion, the vaccinia recombinants of influenza expanded the breadth of T-cell responses to include subdominant and even minor epitopes. Vaccine groups were successfully protected against 100 LD50 challenges with PR/8/34 and highly pathogenic avian influenza H5N1, which contained the identical dominant NP366 epitope. Interestingly, in challenge with pandemic A/Cal/04/2009 containing mutations in the dominant epitope, only the group vaccinated with rVV-NP + PA showed improved protection. Taken together, a vaccinia-based influenza vaccine expressing conserved internal proteins improved the breadth of influenza-specific T-cell immunity and provided heterosubtypic protection against immunologically close as well as distant influenza strains.

TMBIM6 deficiency leads to bone loss by accelerating osteoclastogenesis.

TMBIM6 is an endoplasmic reticulum (ER) protein that modulates various physiological and pathological processes, including metabolism and cancer. However, its involvement in bone remodeling has not been investigated. In this study, we demonstrate that TMBIM6 serves as a crucial negative regulator of osteoclast differentiation, a process essential for bone remodeling. Our investigation of Tmbim6-knockout mice revealed an osteoporotic phenotype, and knockdown of Tmbim6 inhibited the formation of multinucleated tartrate-resistant acid phosphatase-positive cells, which are characteristic of osteoclasts. Transcriptome and immunoblot analyses uncovered that TMBIM6 exerts its inhibitory effect on osteoclastogenesis by scavenging reactive oxygen species and preventing p65 nuclear localization. Additionally, TMBIM6 depletion was found to promote p65 localization to osteoclast-related gene promoters. Notably, treatment with N-acetyl cysteine, an antioxidant, impeded the osteoclastogenesis induced by TMBIM6-depleted cells, supporting the role of TMBIM6 in redox regulation. Furthermore, we discovered that TMBIM6 controls redox regulation via NRF2 signaling pathways. Our findings establish TMBIM6 as a critical regulator of osteoclastogenesis and suggest its potential as a therapeutic target for the treatment of osteoporosis.

Activation of OSM-STAT3 Epigenetically Regulates Tumor-Promoting Transcriptional Programs in Cervical Cancer.

Despite improvements in preventative strategies, such as regular screenings with Pap tests and human papillomavirus (HPV) tests as well as HPV vaccinations, effective treatment for advanced cervical cancer remains poor. Deregulation of STAT3 is an oncogenic factor that promotes tumorigenesis and epithelial-to-mesenchymal transition (EMT) in various cancers. Oncostatin M (OSM), a pleiotropic cytokine, induces STAT3 activation, exacerbating cervical cancer. However, the mechanism by which the OSM-STAT3 axis epigenetically regulates tumor-progression-related genes in cervical cancer is not well understood. Here, we show that OSM-mediated STAT3 activation promotes pro-tumorigenic gene expression programs, with chromatin remodeling in cervical cancer. Reanalysis of scRNA-seq data performed in cervical cancer uncovered an interaction between the oncostatin M receptor (OSMR) on tumor cells and OSM induced by tumor-associated macrophages (TAMs). Our gene expression profiling (bulk RNA-seq) shows that OSM-induced genes were involved in hypoxia, wound healing, and angiogenesis, which were significantly inhibited by SD-36, a STAT3-selective degrader. Additionally, ATAC-seq experiments revealed that STAT3 binding motifs were preferentially enriched in open chromatin regions of the OSM-STAT3-regulated genes. Among the 50 candidate genes that were regulated epigenetically through the OSM-STAT3 axis, we found that the expression levels of NDRG1, HK2, PLOD2, and NPC1 were significantly correlated with those of OSMR and STAT3 in three independent cervical cancer cohorts. Also, higher expression levels of these genes are significantly associated with poor prognosis in cervical cancer patients. Collectively, our findings demonstrate that the OSM-STAT3 signaling pathway regulates crucial transcriptomic programs through epigenetic changes and that selective inhibition of STAT3 may be a novel therapeutic strategy for patients with advanced cervical cancer.

Single-cell transcriptome analyses reveal distinct gene expression signatures of severe COVID-19 in the presence of clonal hematopoiesis.

Clonal hematopoiesis of indeterminate potential (CHIP), a common aging-related process that predisposes individuals to various inflammatory responses, has been reported to be associated with COVID-19 severity. However, the immunological signature and the exact gene expression program by which the presence of CHIP exerts its clinical impact on COVID-19 remain to be elucidated. In this study, we generated a single-cell transcriptome landscape of severe COVID-19 according to the presence of CHIP using peripheral blood mononuclear cells. Patients with CHIP exhibited a potent IFN-γ response in exacerbating inflammation, particularly in classical monocytes, compared to patients without CHIP. To dissect the regulatory mechanism of CHIP (+)-specific IFN-γ response gene expression in severe COVID-19, we identified DNMT3A CHIP mutation-dependent differentially methylated regions (DMRs) and annotated their putative target genes based on long-range chromatin interactions. We revealed that CHIP mutant-driven hypo-DMRs at poised cis-regulatory elements appear to facilitate the CHIP (+)-specific IFN-γ-mediated inflammatory immune response. Our results highlight that the presence of CHIP may increase the susceptibility to hyperinflammation through the reorganization of chromatin architecture, establishing a novel subgroup of severe COVID-19 patients.

Two Faces of Macrophages: Training and Tolerance.

Macrophages are present in almost all body tissues. They detect and quickly respond to "environmental signals" in the tissue. Macrophages have been associated with numerous beneficial roles, such as host defense, wound healing, and tissue regeneration; however, they have also been linked to the development of diverse illnesses, particularly cancers and autoimmune disorders. Complex signaling, epigenetic, and metabolic pathways drive macrophage training and tolerance. The induced intracellular program differs depending on the type of initial stimuli and the tissue microenvironment. Due to the essential roles of macrophages in homeostatic and their association with the pathogenesis of inflammatory diseases, recent studies have investigated the molecular mechanisms of macrophage training and tolerance. This review discusses the role of factors involved in macrophage training and tolerance, along with the current studies in human diseases.

Integrative Transcriptome Profiling Reveals SKA3 as a Novel Prognostic Marker in Non-Muscle Invasive Bladder Cancer.

Approximately 80% of all new bladder cancer patients are diagnosed with non-muscle invasive bladder cancer (NMIBC). However, approximately 15% of them progress to muscle-invasive bladder cancer (MIBC), for which prognosis is poor. The current study aimed to improve diagnostic accuracy associated with clinical outcomes in NMIBC patients. Nevertheless, it has been challenging to identify molecular biomarkers that accurately predict MIBC progression because this disease is complex and heterogeneous. Through integrative transcriptome profiling, we showed that high SKA3 expression is associated with poor clinical outcomes and MIBC progression. We performed RNA sequencing on human tumor tissues to identify candidate biomarkers in NMIBC. We then selected genes with prognostic significance by analyzing public datasets from multiple cohorts of bladder cancer patients. We found that SKA3 was associated with NMIBC pathophysiology and poor survival. We analyzed public single-cell RNA-sequencing (scRNA-seq) data for bladder cancer to dissect transcriptional tumor heterogeneity. SKA3 was expressed in an epithelial cell subpopulation expressing genes regulating the cell cycle. Knockdown experiments confirmed that SKA3 promotes bladder cancer cell proliferation by accelerating G2/M transition. Hence, SKA3 is a new prognostic marker for predicting NMIBC progression. Its inhibition could form part of a novel treatment lowering the probability of bladder cancer progression.

Epigenomic Analysis of RAD51 ChIP-seq Data Reveals cis-regulatory Elements Associated with Autophagy in Cancer Cell Lines.

RAD51 is a recombinase that plays a pivotal role in homologous recombination. Although the role of RAD51 in homologous recombination has been extensively studied, it is unclear whether RAD51 can be involved in gene regulation as a co-factor. In this study, we found evidence that RAD51 may contribute to the regulation of genes involved in the autophagy pathway with E-box proteins such as USF1, USF2, and/or MITF in GM12878, HepG2, K562, and MCF-7 cell lines. The canonical USF binding motif (CACGTG) was significantly identified at RAD51-bound cis-regulatory elements in all four cell lines. In addition, genome-wide USF1, USF2, and/or MITF-binding regions significantly coincided with the RAD51-associated cis-regulatory elements in the same cell line. Interestingly, the promoters of genes associated with the autophagy pathway, such as ATG3 and ATG5, were significantly occupied by RAD51 and regulated by RAD51 in HepG2 and MCF-7 cell lines. Taken together, these results unveiled a novel role of RAD51 and provided evidence that RAD51-associated cis-regulatory elements could possibly be involved in regulating autophagy-related genes with E-box binding proteins.

The KDM4B-CCAR1-MED1 axis is a critical regulator of osteoclast differentiation and bone homeostasis.

Bone undergoes a constant and continuous remodeling process that is tightly regulated by the coordinated and sequential actions of bone-resorbing osteoclasts and bone-forming osteoblasts. Recent studies have shown that histone demethylases are implicated in osteoblastogenesis; however, little is known about the role of histone demethylases in osteoclast formation. Here, we identified KDM4B as an epigenetic regulator of osteoclast differentiation. Knockdown of KDM4B significantly blocked the formation of tartrate-resistant acid phosphatase-positive multinucleated cells. Mice with myeloid-specific conditional knockout of KDM4B showed an osteopetrotic phenotype due to osteoclast deficiency. Biochemical analysis revealed that KDM4B physically and functionally associates with CCAR1 and MED1 in a complex. Using genome-wide chromatin immunoprecipitation (ChIP)-sequencing, we revealed that the KDM4B-CCAR1-MED1 complex is localized to the promoters of several osteoclast-related genes upon receptor activator of NF-κB ligand stimulation. We demonstrated that the KDM4B-CCAR1-MED1 signaling axis induces changes in chromatin structure (euchromatinization) near the promoters of osteoclast-related genes through H3K9 demethylation, leading to NF-κB p65 recruitment via a direct interaction between KDM4B and p65. Finally, small molecule inhibition of KDM4B activity impeded bone loss in an ovariectomized mouse model. Taken together, our findings establish KDM4B as a critical regulator of osteoclastogenesis, providing a potential therapeutic target for osteoporosis.

MEF2C regulates osteoclastogenesis and pathologic bone resorption via c-FOS.

Osteoporosis is a metabolic bone disease with dysregulated coupling between bone resorption and bone formation, which results in decreased bone mineral density. The MEF2C locus, which encodes the transcription factor MADS box transcription enhancer factor 2, polypeptide C (MEF2C), is strongly associated with adult osteoporosis and osteoporotic fractures. Although the role of MEF2C in bone and cartilage formation by osteoblasts, osteocytes, and chondrocytes has been studied, the role of MEF2C in osteoclasts, which mediate bone resorption, remains unclear. In this study, we identified MEF2C as a positive regulator of human and mouse osteoclast differentiation. While decreased MEF2C expression resulted in diminished osteoclastogenesis, ectopic expression of MEF2C enhanced osteoclast generation. Using transcriptomic and bioinformatic approaches, we found that MEF2C promotes the RANKL-mediated induction of the transcription factors c-FOS and NFATc1, which play a key role in osteoclastogenesis. Mechanistically, MEF2C binds to FOS regulatory regions to induce c-FOS expression, leading to the activation of NFATC1 and downstream osteoclastogenesis. Inducible deletion of Mef2c in mice resulted in increased bone mass under physiological conditions and protected mice from bone erosion by diminishing osteoclast formation in K/BxN serum induced arthritis, a murine model of inflammatory arthritis. Our findings reveal direct regulation of osteoclasts by MEF2C, thus adding osteoclasts as a cell type in which altered MEF2C expression or function can contribute to pathological bone remodeling.

A distal cis-regulatory element, CNS-9, controls NFAT1 and IRF4-mediated IL-10 gene activation in T helper cells.

IL-10 is a multifunctional cytokine that plays a critical role in maintaining the balance between immunity and tolerance. Previously, we identified proximal regulatory elements and alterations of chromatin structure in the IL-10 gene loci of Th1 and Th2 cells. We have now characterized a crucial cis-regulatory element, CNS-9, located 9kb upstream of the transcription start site in IL-10 gene loci. The CNS-9 region is highly conserved in vertebrate genomes, and contains clustered NFAT and IRF binding motifs. In vitro binding of NFAT1 and IRF4 to the CNS-9 region was observed by EMSA. Furthermore, Th2-preferential in vivo binding of NFAT1 and IRF4 to the CNS-9 region was observed by ChIP. Cyclosporine A treatment on wild type Th2 cells or Th2 cells derived from NFAT1 knockout (NFAT1(-/-)) mice showed significantly reduced trans-activity of CNS-9. The Th2 subset-specific enhancer activity of CNS-9 was upregulated synergistically by NFAT1 and its partner IRF4. Mutations in the binding sites for NFAT1 and IRF4 abrogated its enhancer activity of CNS-9. Collectively, our results establish crucial roles for enhancer element CNS-9, and NFAT1 and IRF4 that bind to it, for IL-10 expression in differential T helper subsets.

[Acute Suppurative Appendicitis Diagnosed by Acute Lower Gastrointestinal Hemorrhage].

A 49-year-old man visited the emergency room of Korea University Ansan Hospital with hematochezia starting the day before the visit. Recently, he was on anti-platelet medication due to hypertension. The patient had no definite symptoms other than hematochezia. Digital rectal exam was positive and laboratory tests showed severe anemia. Sigmoidoscopy was initiated and almost no fecal material was observed in the intestinal tract, allowing insertion into the cecum. Active bleeding from the appendiceal opening was noted. On abdominal CT, contrast enhancement was observed at the tip of the appendix. Under suspicion of acute appendicitis, we consulted with a surgeon. The patient underwent appendectomy with partial cecal resection. Pathologic examination revealed a diagnosis of appendix bleeding due to acute suppurative appendicitis. The patient had no further bleeding after surgery and was discharged in a stable state. Careful observation by the endoscopist is necessary for accurate diagnosis of lower gastrointestinal hemorrhage. Appendiceal hemorrhage is very rarely reported, but it has various pathophysiologies. CT scan is useful when appendiceal hemorrhage is confirmed by endoscopic findings. Surgical treatment was needed in almost all cases reported worldwide. If bleeding from the appendix is confirmed, surgical treatment should be considered for both therapeutic and diagnostic purposes.

IFN-γ selectively suppresses a subset of TLR4-activated genes and enhancers to potentiate macrophage activation.

Activation of macrophage proinflammatory and antimicrobial phenotypes is regulated by IFN-γ and LPS via synergistic induction of canonical, inflammatory NF-κB target genes. However, whether IFN-γ negatively regulates components of the LPS response, and how this may affect macrophage activation, is still unclear. Here we use combined transcriptomic and epigenomic approaches to find that IFN-γ selectively abrogates LPS-induced feedback and alters macrophage metabolic pathways by suppressing TLR4-mediated gene activation. In contrast to superinduction of inflammatory genes via enhancers that bind IRF1 and STAT1, IFN-γ represses target enhancers that bind STAT3. TLR4-activated but IFN-γ-suppressed enhancers comprise two subsets discernable by differential regulation of histone acetylation and recruitment of STAT3, CDK8 and cohesin. Our findings thus show that IFN-γ suppresses feedback inhibitory and metabolic components of TLR responses to enhance macrophage activation; they also provide insights for IFN-γ-mediated selective inhibition of TLR4-induced transcription. Such inhibition can contribute to severe and sustained inflammatory responses.

Effect of antioxidants on BPA-induced stress on sperm function in a mouse model.

In the past few years, bisphenol A, (BPA) an endocrine-disrupting chemical, has received increasing attention because of its detrimental health effects. There is ample evidence to support that BPA interferes with the reproductive health of humans and animals. In spermatozoa, BPA-induced adverse effects are mostly caused by increased oxidative stress. Using an in vitro experimental model, we examined whether antioxidants (glutathione, vitamin C, and vitamin E) have defensive effects against BPA-induced stress in spermatozoa. The results showed that antioxidants inhibit the overproduction of reactive oxygen species (basically cellular peroxides) and increase intracellular ATP levels, thereby preventing motility loss and abnormal acrosome reaction in BPA-exposed spermatozoa. In particular, glutathione and vitamin E reduced the protein kinase A-dependent tyrosine phosphorylation in spermatozoa and, thus, prevented the precocious acrosome reaction from occurring. Furthermore, we found that the compromised fertilisation and early embryo development mediated by BPA-exposed spermatozoa can be improved following their supplementation with glutathione and vitamin E. Based on these findings, we suggest that antioxidants reduce oxidative stress in BPA-exposed spermatozoa, thus preventing detrimental effects on their function and fertility.

Effect of Aminopeptidase N on functions and fertility of mouse spermatozoa in vitro.

Aminopeptidase N (APN) is defined as a multifunctional enzyme, which regulate cellular physiology of a wide variety of cells in human. Earlier studies reported that mammalian semen shares this common enzyme as a major protein of seminal plasma that has correlation with male fertility, while the regulatory mechanisms of APN in spermatozoa are still far from being well understood. Present study was designed to investigate the role of APN in biological and chemical functions of spermatozoa using an in vitro antagonistic approach. Results showed that lower APN activity in sperm culture medium significantly increased sperm motility and the percentage of high speed spermatozoa and decreased the percentage of slow speed spermatozoa after a dose dependent inhibitor treatment (10, 100, and 1000 µM leuhistin) on epididymal mouse spermatozoa in a capacitating media for 90 min. Both 100 µM and 1000 µM decreased APN activity, while only 1000 µM decreased cell viability and increased PKA activity significantly compared to control. Nonetheless capacitation status, acrosome reaction status, and lactate dehydrogenase activity were not affected. Intriguingly, the treatment affected embryonic development through decreasing tyrosine phosphorylation of proteins and increasing reactive oxygen species levels. Further in silico analysis revealed associated regulatory proteins, which have critical functional role for male fertility.

Dissection and function of autoimmunity-associated TNFAIP3 (A20) gene enhancers in humanized mouse models.

Enhancers regulate gene expression and have been linked with disease pathogenesis. Little is known about enhancers that regulate human disease-associated genes in primary cells relevant for pathogenesis. Here we use BAC transgenics and genome editing to dissect, in vivo and in primary immune cells, enhancers that regulate human TNFAIP3, which encodes A20 and is linked with autoimmune diseases. A20 expression is dependent on a topologically associating subdomain (sub-TAD) that harbors four enhancers, while another >20 enhancers in the A20 locus are redundant. This sub-TAD contains cell- and activation-specific enhancers, including an enhancer (termed TT>A) harboring a proposed causal SLE-associated SNV. Deletion of the sub-TAD or the TT>A enhancer results in enhanced inflammatory responses, autoantibody production, and inflammatory arthritis, thus establishing functional importance in vivo and linking enhancers with a specific disease phenotype. These findings provide insights into enhancers that regulate human A20 expression to prevent inflammatory pathology and autoimmunity.

Effect of endocrine disruptors on the ratio of X and Y chromosome-bearing live spermatozoa.

Although equal numbers of X and Y spermatozoa are produced during spermatogenesis, the sex chromosome ratio in ejaculated spermatozoa can be altered by exposure to endocrine-disrupting chemicals (EDCs), which can be reflected by altered sex ratios at birth. Here, we hypothesized EDCs affect sperm functions and viability of X and Y chromosome-bearing human spermatozoa. After exposure to genistein (Gen), bisphenol A (BPA), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), dibromochloropropane (DBCP), and diazinon (Diaz), we evaluated motility, viability, capacitation, and differential viability of X and Y spermatozoa. All EDCs tested altered sperm viability, motility, and capacitation. Interestingly, the Y/X ratio of live spermatozoa was significantly lower in sperm treated with TCDD, DBCP, and Diaz than control spermatozoa. Our results suggest that some of EDCs have larger effects on the viability of Y spermatozoa than X spermatozoa, implicating that a reduction in Y sperm viability may result in a female-biased sex ratio of offspring at birth.