TIM-3 on myeloid cells promotes pulmonary inflammation through increased production of galectin-3.

T cell immunoglobulin and mucin-containing molecule 3 (TIM-3) exhibits unique, cell type- and context-dependent characteristics and functions. Here, we report that TIM-3 on myeloid cells plays essential roles in modulating lung inflammation. We found that myeloid cell-specific TIM-3 knock-in (FSF-TIM3/LysM-Cre+) mice have lower body weight and shorter lifespan than WT mice. Intriguingly, the lungs of FSF-TIM3/LysM-Cre+ mice display excessive inflammation and features of disease-associated pathology. We further revealed that galectin-3 levels are notably elevated in TIM-3-overexpressing lung-derived myeloid cells. Furthermore, both TIM-3 blockade and GB1107, a galectin-3 inhibitor, ameliorated lung inflammation in FSF-TIM3/LysM-Cre+/- mice. Using an LPS-induced lung inflammation model with myeloid cell-specific TIM-3 knock-out mice, we demonstrated the association of TIM-3 with both lung inflammation and galectin-3. Collectively, our findings suggest that myeloid TIM-3 is an important regulator in the lungs and that modulation of TIM-3 and galectin-3 could offer therapeutic benefits for inflammation-associated lung diseases.

Single-cell RNA sequencing of nc886, a non-coding RNA transcribed by RNA polymerase III, with a primer spike-in strategy.

Single-cell RNA sequencing (scRNA-seq) has emerged as a versatile tool in biology, enabling comprehensive genomic-level characterization of individual cells. Currently, most scRNA-seq methods generate barcoded cDNAs by capturing the polyA tails of mRNAs, which exclude many non-coding RNAs (ncRNAs), especially those transcribed by RNA polymerase III (Pol III). Although previously thought to be expressed constitutively, Pol III-transcribed ncRNAs are expressed variably in healthy and disease states and play important roles therein, necessitating their profiling at the single-cell level. In this study, we developed a measurement protocol for nc886 as a model case and initial step for scRNA-seq for Pol III-transcribed ncRNAs. Specifically, we spiked in an oligo-tagged nc886-specific primer during the polyA tail capture process for the 5'scRNA-seq. We then produced sequencing libraries for standard 5' gene expression and oligo-tagged nc886 separately, to accommodate different cDNA sizes and ensure undisturbed transcriptome analysis. We applied this protocol in three cell lines that express high, low, and zero levels of nc886. Our results show that the identification of oligo tags exhibited limited target specificity, and sequencing reads of nc886 enabled the correction of non-specific priming. These findings suggest that gene-specific primers (GSPs) can be employed to capture RNAs lacking a polyA tail, with subsequent sequence verification ensuring accurate gene expression counting. Moreover, we embarked on an analysis of differentially expressed genes in cell line sub-clusters with differential nc886 expression, demonstrating variations in gene expression phenotypes. Collectively, the primer spike-in strategy allows combined analysis of ncRNAs and gene expression phenotype.

Galectin-9 Mediates the Functions of Microglia in the Hypoxic Brain Tumor Microenvironment.

Galectin-9 is a multifaceted regulator of various pathophysiological processes that exerts positive or negative effects in a context-dependent manner. Here, we elucidated the distinctive functional properties of galectin-9 on myeloid cells within the brain tumor microenvironment. Galectin-9-expressing cells were abundant at the hypoxic tumor edge in the tumor-bearing ipsilateral hemisphere compared to the contralateral hemisphere in an intracranial mouse brain tumor model. Galectin-9 was highly expressed in microglia and macrophages in tumor-infiltrating cells. In primary glia, both the expression and secretion of galectin-9 were influenced by tumors. Analysis of a human glioblastoma bulk RNA-sequencing dataset and a single-cell RNA-sequencing dataset from a murine glioma model revealed a correlation between galectin-9 expression and glial cell activation. Notably, the galectin-9high microglial subset was functionally distinct from the galectin-9neg/low subset in the brain tumor microenvironment. Galectin-9high microglia exhibited properties of inflammatory activation and higher rates of cell death, whereas galectin-9neg/low microglia displayed a superior phagocytic ability against brain tumor cells. Blockade of galectin-9 suppressed tumor growth and altered the activity of glial and T cells in a mouse glioma model. Additionally, glial galectin-9 expression was regulated by Hif-2α in the hypoxic brain tumor microenvironment. Myeloid-specific Hif-2α deficiency led to attenuated tumor progression. Together, these findings reveal that galectin-9 on myeloid cells is an immunoregulator and putative therapeutic target in brain tumors.

Adenovirus expressing nc886, an anti-interferon and anti-apoptotic non-coding RNA, is an improved gene delivery vector.

Recombinant adenovirus (rAdV) vector is the most promising vehicle to deliver an exogenous gene into target cells and is preferred for gene therapy. Exogenous gene expression from rAdV is often too inefficient to induce phenotypic changes and the amount of administered rAdV must be very high to achieve a therapeutic dose. However, it is often hampered because a high dose of rAdV is likely to induce cytotoxicity by activating immune responses. nc886, a 102-nucleotide non-coding RNA that is transcribed by RNA polymerase III, acts as an immune suppressor and a facilitator of AdV entry into the nucleus. Therefore, in this study, we have constructed an rAdV expressing nc886 (AdV:nc886) to explore whether AdV:nc886 overcomes the aforementioned drawbacks of conventional rAdV vectors. When infected into mouse cell lines and mice, AdV:nc886 expresses a sufficient amount of nc886, which suppresses the induction of interferon-stimulated genes and apoptotic pathways triggered by AdV infection. As a result, AdV:nc886 is less cytotoxic and produces more rAdV-delivered gene products, compared with the parental rAdV vector lacking nc886. In conclusion, this study demonstrates that the nc886-expressing rAdV could become a superior gene delivery vehicle with greater safety and higher efficiency for in vivo gene therapy.

Erratum: Inhibition of Chitinase-3-like-1 by K284-6111 Reduces Atopic Skin Inflammation via Repressing Lactoferrin.

[This corrects the article e22 in vol. 21, PMID: 34277112.].

Anti-chitinase-3-like 1 antibody attenuated atopic dermatitis-like skin inflammation through inhibition of STAT3-dependent CXCL8 expression.

BACKGROUND AND PURPOSE: Chitinase-3-like 1 (CHI3L1) causes skin inflammation in the progression of atopic dermatitis. We investigated if anti-CHI3L1 antibody could prevent the development of atopic dermatitis and its mechanisms of action. EXPERIMENTAL APPROACH: The effect of CHI3L1 antibody on phthalic anhydride-induced atopic dermatitis animal model and in vitro reconstructed human skin (RHS) model were investigated. Expression and release of atopic dermatitis-related cytokines were determined using an enzyme-linked immunosorbent assay, and RT-qPCR, STAT3 and CXCL8 signalling were measured by western blotting. KEY RESULTS: Anti-CHI3L1 antibody suppressed phthalic anhydride-induced epidermal thickening, clinical score, IgE level and infiltration of inflammatory cells, and reduced phthalic anhydride-induced inflammatory cytokines concentration. In addition, CHI3L1 antibody treatment inhibited the expression of STAT3 activity in phthalic anhydride-treated skin. It was also confirmed that CHI3L1 antibody treatment alleviated atopic dermatitis-related inflammation in the RHS model. The inhibitory effects of CHI3L1 antibody was similar or more effective compared with that of the IL-4 antibody. We further found that CHI3L1 is associated with CXCL8 by protein-association network analysis. siRNA of CHI3L1 blocked the mRNA levels of CHI3L1, IL-1ß, IL-4, CXCL8, TSLP, and the expression of CHI3L1 and p-STAT, and the level of CXCL8, whereas recombinant level of CXCL8 was elevated. Moreover, siRNA of STAT3 reduced the mRNA level of these cytokines. CHI3L1 and p-STAT3 expression correlated with the reduced CXCL8 level in the RHS in vitro model. CONCLUSION AND IMPLICATIONS: Our data demonstrated that CHI3L1 antibody could be a promising effective therapeutic drug for atopic dermatitis.

Inhibition of Amyloid-ß (Aß)-Induced Cognitive Impairment and Neuroinflammation in CHI3L1 Knockout Mice through Downregulation of ERK-PTX3 Pathway.

Several clinical studies reported that the elevated expression of Chitinase-3-like 1 (CHI3L1) was observed in patients suffering from a wide range of diseases: cancer, metabolic, and neurological diseases. However, the role of CHI3L1 in AD is still unclear. Our previous study demonstrated that 2-({3-[2-(1-Cyclohexen-1-yl)ethyl]-6,7-dimethoxy-4-oxo-3,4-dihydro-2-quinazolinyl}culfanyl)-N-(4-ethylphenyl)butanamide, a CHI3L1 inhibiting compound, alleviates memory and cognitive impairment and inhibits neuroinflammation in AD mouse models. In this study, we studied the detailed correlation of CHI3L1 and AD using serum from AD patients and using CHI3L1 knockout (KO) mice with Aß infusion (300 pmol/day, 14 days). Serum levels of CHI3L1 were significantly elevated in patients with AD compared to normal subjects, and receiver operating characteristic (ROC) analysis data based on serum analysis suggested that CHI3L1 could be a significant diagnostic reference for AD. To reveal the role of CHI3L1 in AD, we investigated the CHI3L1 deficiency effect on memory impairment in Aß-infused mice and microglial BV-2 cells. In CHI3L1 KO mice, Aß infusion resulted in lower levels of memory dysfunction and neuroinflammation compared to that of WT mice. CHI3L1 deficiency selectively inhibited phosphorylation of ERK and IκB as well as inhibition of neuroinflammation-related factors in vivo and in vitro. On the other hand, treatment with recombinant CHI3L1 increased neuroinflammation-related factors and promoted phosphorylation of IκB except for ERK in vitro. Web-based gene network analysis and our results showed that CHI3L1 is closely correlated with PTX3. Moreover, in AD patients, we found that serum levels of PTX3 were correlated with serum levels of CHI3L1 by Spearman correlation analysis. These results suggest that CHI3L1 deficiency could inhibit AD development by blocking the ERK-dependent PTX3 pathway.

Integrative transcriptomic and genomic analyses unveil the IFI16 variants and expression as MASLD progression markers.

BACKGROUND AND AIMS: Metabolic dysfunction-associated steatotic liver disease (MASLD) encompasses a broad and continuous spectrum of liver diseases ranging from fatty liver to steatohepatitis. The intricate interactions of genetic, epigenetic, and environmental factors in the development and progression of MASLD remain elusive. Here, we aimed to achieve an integrative understanding of the genomic and transcriptomic alterations throughout the progression of MASLD. APPROACH AND RESULTS: RNA-Seq profiling (n = 146) and whole-exome sequencing (n = 132) of MASLD liver tissue samples identified 3 transcriptomic subtypes (G1-G3) of MASLD, which were characterized by stepwise pathological and molecular progression of the disease. Macrophage-driven inflammatory activities were identified as a key feature for differentiating these subtypes. This subtype-discriminating macrophage interplay was significantly associated with both the expression and genetic variation of the dsDNA sensor IFI16 (rs6940, A>T, T779S), establishing it as a fundamental molecular factor in MASLD progression. The in vitro dsDNA-IFI16 binding experiments and structural modeling revealed that the IFI16 variant exhibited increased stability and stronger dsDNA binding affinity compared to the wild-type. Further downstream investigation suggested that the IFI16 variant exacerbated DNA sensing-mediated inflammatory signals through mitochondrial dysfunction-related signaling of the IFI16-PYCARD-CASP1 pathway. CONCLUSIONS: This study unveils a comprehensive understanding of MASLD progression through transcriptomic classification, highlighting the crucial roles of IFI16 variants. Targeting the IFI16-PYCARD-CASP1 pathway may pave the way for the development of novel diagnostics and therapeutics for MASLD.

Identification of signature gene set as highly accurate determination of metabolic dysfunction-associated steatotic liver disease progression.

BACKGROUND/AIMS: Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by fat accumulation in the liver. MASLD encompasses both steatosis and MASH. Since MASH can lead to cirrhosis and liver cancer, steatosis and MASH must be distinguished during patient treatment. Here, we investigate the genomes, epigenomes, and transcriptomes of MASLD patients to identify signature gene set for more accurate tracking of MASLD progression. METHODS: Biopsy-tissue and blood samples from patients with 134 MASLD, comprising 60 steatosis and 74 MASH patients were performed omics analysis. SVM learning algorithm were used to calculate most predictive features. Linear regression was applied to find signature gene set that distinguish the stage of MASLD and to validate their application into independent cohort of MASLD. RESULTS: After performing WGS, WES, WGBS, and total RNA-seq on 134 biopsy samples from confirmed MASLD patients, we provided 1,955 MASLD-associated features, out of 3,176 somatic variant callings, 58 DMRs, and 1,393 DEGs that track MASLD progression. Then, we used a SVM learning algorithm to analyze the data and select the most predictive features. Using linear regression, we identified a signature gene set capable of differentiating the various stages of MASLD and verified it in different independent cohorts of MASLD and a liver cancer cohort. CONCLUSION: We identified a signature gene set (i.e., CAPG, HYAL3, WIPI1, TREM2, SPP1, and RNASE6) with strong potential as a panel of diagnostic genes of MASLD-associated disease.

nc886, an RNA Polymerase III-Transcribed Noncoding RNA Whose Expression Is Dynamic and Regulated by Intriguing Mechanisms.

nc886 is a medium-sized non-coding RNA that is transcribed by RNA polymerase III (Pol III) and plays diverse roles in tumorigenesis, innate immunity, and other cellular processes. Although Pol III-transcribed ncRNAs were previously thought to be expressed constitutively, this concept is evolving, and nc886 is the most notable example. The transcription of nc886 in a cell, as well as in human individuals, is controlled by multiple mechanisms, including its promoter CpG DNA methylation and transcription factor activity. Additionally, the RNA instability of nc886 contributes to its highly variable steady-state expression levels in a given situation. This comprehensive review discusses nc886's variable expression in physiological and pathological conditions and critically examines the regulatory factors that determine its expression levels.

Generation and analysis of whole-genome sequencing data in human mammary epithelial cells.

Breast cancer is the most common cancer worldwide, and advanced breast cancer with metastases is incurable mainly with currently available therapies. Therefore, it is essential to understand molecular characteristics during the progression of breast carcinogenesis. Here, we report a dataset of whole genomes from the human mammary epithelial cell system derived from a reduction mammoplasty specimen. This system comprises pre-stasis 184D cells, considered normal, and seven cell lines along cancer progression series that are immortalized or additionally acquired anchorage-independent growth. Our analysis of the whole-genome sequencing (WGS) data indicates that those seven cancer progression series cells have somatic mutations whose number ranges from 8,393 to 39,564 (with an average of 30,591) compared to 184D cells. These WGS data and our mutation analysis will provide helpful information to identify driver mutations and elucidate molecular mechanisms for breast carcinogenesis.

Combined Toxicological Effects of Di (2-Ethylhexyl) Phthalate and UV-B Irradiation through Endoplasmic Reticulum Stress-Tight Junction Disruption in Human HaCaT Keratinocytes.

Di (2-ethylhexyl) phthalate (DEHP) is widely used as a plasticizer, and human exposure to DEHP is widespread and frequent. However, information about the combined effect of DEHP and ultraviolet (UV)-B on the skin are still limited. We investigated the cytotoxic effects of DEHP and UV-B on HaCaT keratinocytes and evaluated the related underlying mechanisms involving endoplasmic reticulum (ER) stress signals and the disruption of junction complexes as an effective target for skin inflammation. Our results revealed that co-treatment with DEHP and UV-B irradiation alleviated the cell cytotoxicity and markedly decreased X-box binding protein 1 (XBP1), endoplasmic reticulum oxidoreductase 1 alpha (Ero1α), and C/EBP homologous protein (CHOP) whereas a single dose of 40 mJ/cm2 UV-B generated mild ER stress to slightly less or similar levels as that seen with DEHP. DEHP was also shown to inhibit tight junctions (TJs) after UV-B irradiation, increased apoptosis by altering apoptotic gene Bax and stress kinases, JNK, and p38 MAPK. Furthermore, exposure of HaCaT cells to DEHP and UV-B irradiation resulted in the marked suppression of the nuclear factor kappa B (NF-κB)/p65 signaling pathway. Taken together, our data suggest that nontoxic DEHP and UV-B irradiation regulated ER stress and epidermal TJ disruption with the induction of apoptosis activation and the secretion of proinflammatory cytokines such as interleukin 1 beta (IL-1ß) and IL-6 in human keratinocytes. Further investigation is needed to confirm the mechanisms implicated in its toxicity and determine the effects of exposure to DEHP and UV-B irradiation on markers involved in this study.

Are We Studying Non-Coding RNAs Correctly? Lessons from nc886.

Non-coding RNAs (ncRNAs), such as microRNAs or long ncRNAs, have brought about a new paradigm in the regulation of gene expression. Sequencing technologies have detected transcripts with tremendous sensitivity and throughput and revealed that the majority of them lack protein-coding potential. Myriad articles have investigated numerous ncRNAs and many of them claim that ncRNAs play gene-regulatory roles. However, it is questionable whether all these articles draw conclusions through cautious gain- and loss-of function experiments whose design was reasonably based on an ncRNA's correct identity and features. In this review, these issues are discussed with a regulatory ncRNA, nc886, as an example case to represent cautions and guidelines when studying ncRNAs.

A host non-coding RNA, nc886, plays a pro-viral role by promoting virus trafficking to the nucleus.

Elucidation of the interplay between viruses and host cells is crucial for taming viruses to benefit human health. Cancer therapy using adenovirus, called oncolytic virotherapy, is a promising treatment option but is not robust in all patients. In addition, inefficient replication of human adenovirus in mouse hampered the development of an in vivo model for preclinical evaluation of therapeutically engineered adenovirus. nc886 is a human non-coding RNA that suppresses Protein Kinase R (PKR), an antiviral protein. In this study, we have found that nc886 greatly promotes adenoviral gene expression and replication. Remarkably, the stimulatory effect of nc886 is not dependent on its function to inhibit PKR. Rather, nc886 facilitates the nuclear entry of adenovirus via modulating the kinesin pathway. nc886 is not conserved in mouse and, when xenogeneically expressed in mouse cells, promotes adenovirus replication. Our investigation has discovered a novel mechanism of how a host ncRNA plays a pro-adenoviral role. Given that nc886 expression is silenced in a subset of cancer cells, our study highlights that oncolytic virotherapy might be inefficient in those cells. Furthermore, our findings open future possibilities of harnessing nc886 to improve the efficacy of oncolytic adenovirus and to construct nc886-expressing transgenic mice as an animal model.

G721-0282 Exerts Anxiolytic-Like Effects on Chronic Unpredictable Mild Stress in Mice Through Inhibition of Chitinase-3-Like 1-Mediated Neuroinflammation.

Chronic stress is thought to be a major contributor to the onset of mental disorders such as anxiety disorders. Several studies have demonstrated a correlation between anxiety state and neuroinflammation, but the detailed mechanism is unclear. Chitinase-3-like 1 (CHI3L1) is expressed in several chronic inflammatorily damaged tissues and is well known to play a major role in mediating inflammatory responses. In the present study, we investigated the anxiolytic-like effect of N-Allyl-2-[(6-butyl-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidin-5-yl)sulfanyl]acetamide (G721-0282), an inhibitor of CHI3L1, on mice treated with chronic unpredictable mild stress (CUMS), as well as the mechanism of its action. We examined the anxiolytic-like effect of G721-0282 by conducting several behavioral tests with oral administration of G721-0282 to CUMS-treated BALB/c male mice. We found that administration of G721-0282 relieves CUMS-induced anxiety. Anxiolytic-like effects of G721-0282 have been shown to be associated with decreased expressions of CUMS-induced inflammatory proteins and cytokines in the hippocampus. The CUMS-elevated levels of CHI3L1 and IGFBP3 were inhibited by treatment with G721-0282 in vivo and in vitro. However, CHI3L1 deficiency abolished the anti-inflammatory effects of G721-0282 in microglial BV-2 cells. These results suggest that G721-0282 could lower CUMS-induced anxiety like behaviors by regulating IGFBP3-mediated neuroinflammation via inhibition of CHI3L1.

A small molecule targeting CHI3L1 inhibits lung metastasis by blocking IL-13Rα2-mediated JNK-AP-1 signals.

Our previous big data analyses showed a high level of association between chitinase 3 like1 (CHI3L1) expression and lung tumor development. In the present study, we investigated whether a CHI3L1-inhibiting chemical, 2-({3-[2-(1-cyclohexen-1-yl)ethyl]-6,7-dimethoxy-4-oxo-3,4-dihydro-2-quinazolinyl}sulfanyl)-N-(4-ethylphenyl)butanamide (K284), could inhibit lung metastasis and studied its mechanism of action. We investigated the antitumor effect of K284 both in vitro and in vivo. K284 (0.5 mg·kg-1 body weight) significantly inhibited lung metastasis in in vivo models after injection of murine melanoma cells (B16F10) or adenocarcinomic human alveolar basal epithelial cells (A549). K284 significantly and concentration-dependently also inhibited cancer cell proliferation and migration in the A549 and H460 lung cancer cell lines. We found that the binding of K284 to the chitin-binding domain (CBD) of CHI3L1 prevented the binding of CHI3L1 to its receptor, interleukin-13 receptor subunit alpha-2 (IL-13Rα2), thereby suppressing the CHI3L1 signal. This blocking of the CHI3L1-IL-13Rα2 signal caused the inhibition of c-Jun N-terminal kinase (JNK)-activator protein 1 (AP-1) signals, resulting in the prevention of lung metastasis and cancer cell growth. Our data demonstrate that K284 may serve as a potential candidate anticancer compound targeting CHI3L1.

Editorial of Special Issue "Non-Coding RNAs in Pathogen-Host Interaction".

Clinical outcomes after pathologic infection are variable in infected individuals, ranging from no symptoms, moderate symptoms, hospitalization, and even death [...].

Digital Therapeutics: Exploring the Possibilities of Digital Intervention for Myopia.

Pediatric myopia is increasing globally and has become a major public health issue. However, the mechanism of pediatric myopia is still poorly understood, and there is no effective treatment to prevent its progression. Based on results from animal and clinical studies, certain neuronal-humoral factors (NHFs), such as IGF-1, dopamine, and cortisol may be involved in the progression of pediatric myopia. Digital therapeutics uses evidence-based software as therapeutic interventions and it has the potential to offer innovative treatment strategies for pediatric myopia beyond conventional treatment methods. In this perspective article, we introduce digital therapeutics SAT-001, a software algorithm that modulates the level of NHFs to reduce the progression of pediatric myopia. The proposed mechanism is based on a theoretical hypothesis derived from scientific research and clinical studies and will be further confirmed by evidence generated from clinical studies involving pediatric myopia.