High-mobility group box 1 fragment ameliorates chronic pancreatitis induced by caerulein in mice.

BACKGROUND: Chronic pancreatitis (CP) is a progressive disease characterized by pancreatic fibrosis for which effective treatment options are lacking. Mesenchymal stem cells (MSCs) have shown potential for fibrosis treatment but face limitations in clinical application. The high-mobility group box 1 (HMGB1) fragment mobilizes MSCs from bone marrow into the blood and has emerged as a promising therapeutic agent for tissue regeneration in various pathological conditions. The aim of this study was to investigate the potential therapeutic effects of systemic administration of the HMGB1 fragment in a mouse model of CP. METHODS: A caerulein-induced CP mouse model was used, and the HMGB1 fragment was administered by tail vein injection. Parameters such as body weight, pancreatic tissue damage, fibrosis, inflammatory cytokine expression, and collagen-related gene expression were evaluated using various assays, including immunohistochemistry, real-time PCR, serum analysis, and single-cell transcriptome analysis. And the migration of MSCs to the pancreas was evaluated using the parabiosis model. RESULTS: Administration of the HMGB1 fragment was associated with significant improvements in pancreatic tissue damage and fibrosis. It suppressed the expression of inflammatory cytokines and activated platelet-derived growth factor receptor-α+ MSCs, leading to their accumulation in the pancreas. The HMGB1 fragment also shifted gene expression patterns associated with pancreatic fibrosis toward those of the normal pancreas. Systemic administration of the HMGB1 fragment demonstrated therapeutic efficacy in attenuating pancreatic tissue damage and fibrosis in a CP mouse model. CONCLUSION: These findings highlight the potential of the HMGB1 fragment as a therapeutic target for the treatment of CP.

Genomic transcription factor binding site selection is edited by the chromatin remodeling factor CHD4.

Biologically precise enhancer licensing by lineage-determining transcription factors enables activation of transcripts appropriate to biological demand and prevents deleterious gene activation. This essential process is challenged by the millions of matches to most transcription factor binding motifs present in many eukaryotic genomes, leading to questions about how transcription factors achieve the exquisite specificity required. The importance of chromatin remodeling factors to enhancer activation is highlighted by their frequent mutation in developmental disorders and in cancer. Here, we determine the roles of CHD4 in enhancer licensing and maintenance in breast cancer cells and during cellular reprogramming. In unchallenged basal breast cancer cells, CHD4 modulates chromatin accessibility. Its depletion leads to redistribution of transcription factors to previously unoccupied sites. During cellular reprogramming induced by the pioneer factor GATA3, CHD4 activity is necessary to prevent inappropriate chromatin opening. Mechanistically, CHD4 promotes nucleosome positioning over GATA3 binding motifs to compete with transcription factor-DNA interaction. We propose that CHD4 acts as a chromatin proof-reading enzyme that prevents unnecessary gene expression by editing chromatin binding activities of transcription factors.

High-mobility group box-1 peptide ameliorates bronchopulmonary dysplasia by suppressing inflammation and fibrosis in a mouse model.

BACKGROUND: This study aimed to examine the effect of the HMGB1 peptide on Bronchopulmonary dysplasia (BPD)-related lung injury in a mouse model. RESULTS: HMGB1 peptide ameliorates lung injury by suppressing the release of inflammatory cytokines and decreasing soluble collagen levels in the lungs. Single-cell RNA sequencing showed that the peptide suppressed the hyperoxia-induced inflammatory signature in macrophages and the fibrotic signature in fibroblasts. These changes in the transcriptome were confirmed using protein assays. CONCLUSION: Systemic administration of HMGB1 peptide exerts anti-inflammatory and anti-fibrotic effects in a mouse model of BPD. This study provides a foundation for the development of new and effective therapies for BPD.

Genomic transcription factor binding site selection is edited by the chromatin remodeling factor CHD4.

Biologically precise enhancer licensing by lineage-determining transcription factors enables activation of transcripts appropriate to biological demand and prevents deleterious gene activation. This essential process is challenged by the millions of matches to most transcription factor binding motifs present in many eukaryotic genomes, leading to questions about how transcription factors achieve the exquisite specificity required. The importance of chromatin remodeling factors to enhancer activation is highlighted by their frequent mutation in developmental disorders and in cancer. Here we determine the roles of CHD4 to enhancer licensing and maintenance in breast cancer cells and during cellular reprogramming. In unchallenged basal breast cancer cells, CHD4 modulates chromatin accessibility at transcription factor binding sites; its depletion leads to altered motif scanning and redistribution of transcription factors to sites not previously occupied. During GATA3-mediated cellular reprogramming, CHD4 activity is necessary to prevent inappropriate chromatin opening and enhancer licensing. Mechanistically, CHD4 competes with transcription factor-DNA interaction by promoting nucleosome positioning over binding motifs. We propose that CHD4 acts as a chromatin proof-reading enzyme that prevents inappropriate gene expression by editing binding site selection by transcription factors.

Single-cell transcriptome analysis of a rat model of bilateral renal ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) causes massive tissue damage. Renal IRI is the most common type of acute renal injury, and the defects caused by it may progress to chronic kidney disease (CKD). Rodent models of renal IRI, with various patterns, have been used to study the treatment of human kidney injury. A rat model of bilateral IRI, in which the bilateral kidney blood vessels are clamped for 60 min, is widely used, inducing both acute and chronic kidney disease. However, the molecular mechanisms underlying the effects of bilateral IRI on kidney cells have not yet been fully elucidated. This study aimed to perform a whole-transcriptome analysis of the IRI kidney using single-cell RNA sequencing. We found renal parenchymal cells, including those from the proximal tubule, the loop of Henle, and distal tubules, to be damaged by IRI. In addition, we observed significant changes in macrophage population. Our study delineated the detailed cellular and molecular changes that occur in the rat model of bilateral IRI. Collectively, our data and analyses provided a foundation for understanding IRI-related kidney diseases in rat models.

Proteins That Read DNA Methylation.

Covalent modification of DNA via deposition of a methyl group at the 5' position on cytosine residues alters the chemical groups available for interaction in the major groove of DNA. This modification, thereby, alters the affinity and specificity of DNA-binding proteins; some of them favor interaction with methylated DNA, and others disfavor it. Molecular recognition of cytosine methylation by proteins often initiates sequential regulatory events that impact gene expression and chromatin structure. The known methyl-DNA-binding proteins have unique domains responsible for DNA methylation recognition: (1) the methyl-CpG-binding domain (MBD), (2) the SET- and RING finger-associated domain (SRA), and (3) some of TF families, such as the C2H2 zinc finger domain, basic helix-loop-helix (bHLH), basic leucine-zipper (bZIP), and homeodomain proteins. Structural analyses have revealed that each domain has a characteristic methylated DNA-binding pattern, and the difference in the recognition mechanisms renders the DNA methylation mark able to transmit complicated biological information. Recent genetic and genomic studies have revealed novel functions of methyl-DNA-binding proteins. These emerging data have also provided glimpses into how methyl-DNA-binding proteins possess unique features and, presumably, functions. In this chapter, we summarize structural and biochemical analyses elucidating the mechanisms for recognition of DNA methylation and correlate this information with emerging genomic and functional data.

Single-cell transcriptome analysis reveals cellular heterogeneity in mouse intra- and extra articular ligaments.

Ligaments are collagenous connective tissues that connect bones. Injury of knee ligaments, namely anterior cruciate ligament (ACL) and medial collateral ligament (MCL), is common in athletes. Both ligaments have important functions, but distinct regeneration capacities. The capacity for recovery after injury also diminishes with age. However, cellular heterogeneity in the ligaments remains unclear. Here, we profiled the transcriptional signatures of ACL and MCL cells in mice using single-cell RNA sequencing. These ligaments comprise three fibroblast types expressing Col22a1, Col12a1, or Col14a1, but have distinct localizations in the tissue. We found substantial heterogeneity in Col12a1- and Col14a1-positive cells between ACL and MCL. Gene Ontology analysis revealed that angiogenesis- and collagen regulation-related genes were specifically enriched in MCL cells. Furthermore, we identified age-related changes in cell composition and gene expression in the ligaments. This study delineates cellular heterogeneity in ligaments, serving as a foundation for identifying potential therapeutic targets for ligament injuries.

Plap-1 lineage tracing and single-cell transcriptomics reveal cellular dynamics in the periodontal ligament.

Periodontal tissue supports teeth in the alveolar bone socket via fibrous attachment of the periodontal ligament (PDL). The PDL contains periodontal fibroblasts and stem/progenitor cells, collectively known as PDL cells (PDLCs), on top of osteoblasts and cementoblasts on the surface of alveolar bone and cementum, respectively. However, the characteristics and lineage hierarchy of each cell type remain poorly defined. This study identified periodontal ligament associated protein-1 (Plap-1) as a PDL-specific extracellular matrix protein. We generated knock-in mice expressing CreERT2 and GFP specifically in Plap-1-positive PDLCs. Genetic lineage tracing confirmed the long-standing hypothesis that PDLCs differentiate into osteoblasts and cementoblasts. A PDL single-cell atlas defined cementoblasts and osteoblasts as Plap-1-Ibsp+Sparcl1+ and Plap-1-Ibsp+Col11a2+, respectively. Other populations, such as Nes+ mural cells, S100B+ Schwann cells, and other non-stromal cells, were also identified. RNA velocity analysis suggested that a Plap-1highLy6a+ cell population was the source of PDLCs. Lineage tracing of Plap-1+ PDLCs during periodontal injury showed periodontal tissue regeneration by PDLCs. Our study defines diverse cell populations in PDL and clarifies the role of PDLCs in periodontal tissue homeostasis and repair.

Synthesized HMGB1 peptide prevents the progression of inflammation, steatosis, fibrosis, and tumor occurrence in a non-alcoholic steatohepatitis mouse model.

AIM: Non-alcoholic steatohepatitis (NASH) with fibrosis eventually leads to cirrhosis and hepatocellular carcinoma. Thus, the development of therapies other than dietary restriction and exercise, particularly those that suppress steatosis and fibrosis of the liver and have a long-term beneficial effect, is necessary. We aimed to evaluate the therapeutic effects of the HMGB1 peptide synthesized from box A using the melanocortin-4 receptor-deficient (Mc4r-KO) NASH model mouse. METHODS: We performed short- and long-term administration of this peptide and evaluated the effects on steatosis, fibrosis, and carcinogenesis using Mc4r-KO mice. We also analyzed the direct effect of this peptide on macrophages and hepatic stellate cells in vitro and performed lipidomics and metabolomics techniques to evaluate the effect. RESULTS: Although this peptide did not show direct effects on macrophages and hepatic stellate cells in vitro, in the short-term administration model, we could confirm the reduction of liver damage, steatosis, and fibrosis progression. The results of lipidomics and metabolomics suggested that the peptide might ameliorate NASH by promoting lipolysis via the activation of fatty acid ß-oxidation and improving insulin resistance. In the long-term administration model, this peptide prevented progression to cirrhosis but retained the steatosis state, that is, the peptide prevents the progression to "burnt-out NASH." This peptide inhibited carcinogenesis by about one-third. CONCLUSION: This HMGB1 peptide can reduce liver damage, improve fibrosis and steatosis, and inhibit carcinogenesis, suggesting that the peptide would be a new treatment candidate for NASH and can contribute to the long-term prognosis for patients with NASH.

Single-cell transcriptome analysis of fractional CO2 laser efficiency in treating a mouse model of alopecia.

OBJECTIVES: Hair loss, including alopecia, is a common dermatological issue worldwide. At present, the application of fractional carbon dioxide (CO2 ) laser in the treatment of alopecia has been documented; however, the results vary between reports. These varying results may be due to the limited knowledge of cellular action in laser-irradiated skin. The objective of this study was to investigate the molecular and cellular mechanisms of laser treatment under effective conditions for hair cycle initiation. METHODS: A fractional CO2 laser was applied and optimized to initiate the hair cycle in a mouse model of alopecia. Several cellular markers were analyzed in the irradiated skin using immunofluorescence staining. Cellular populations and their comprehensive gene expression were analyzed using single-cell RNA sequencing and bioinformatics. RESULTS: The effective irradiation condition for initiating the hair cycle was found to be 15 mJ energy/spot, which generates approximately 500 µm depth columns, but does not penetrate the dermis, only reaching approximately 1 spot/mm2 . The proportion of macrophage clusters significantly increased upon irradiation, whereas the proportion of fibroblast clusters decreased. The macrophages strongly expressed C-C chemokine receptor type 2 (Ccr2), which is known to be a key signal for injury-induced hair growth. CONCLUSIONS: We found that fractional CO2 laser irradiation recruited Ccr2 positive macrophages, and induced hair regrowth in a mouse alopecia model. These findings may contribute to the development of stable and effective fractional laser irradiation conditions for human alopecia treatment.

A Novel Quantitative Evaluation of Bone Formation After Opening Wedge High Tibial Osteotomy Using Tomosynthesis.

This study aimed to establish and validate a novel evaluation method using digital tomosynthesis to quantify bone formation in the gap after opening wedge high tibial osteotomy (OW-HTO). We retrospectively analyzed bone formation in the gap in 22 patients who underwent OW-HTO using digital tomosynthesis at 1, 2, 3, 6, 9, and 12 months postoperatively. Bone formation was semi-quantitatively assessed using the modified van Hemert's score and density measurements on digital tomosynthesis images. The gap filling value (GFV) was calculated as the ratio of the intensities of the opening gap and the tibial shaft. In addition, the relationship between the modified van Hemert's score and GFV was evaluated. The reproducibility of GFV had an interclass correlation coefficient (ICC [1,2]) of 0.958 for intraobserver reliability and an ICC (2,1) of 0.975 for interobserver reliability. The GFV increased in a time-dependent manner and was moderately correlated with the modified van Hemert's score (r = 0.630, p < 0.001). The GFV plateaued at 6 months postoperatively. In addition, the GFV was higher in patients with a modified van Hemert's score of 2 than in patients with a modified van Hemert's score of 3 (p = 0.008). The GFVs obtained using digital tomosynthesis can be used to assess postoperative bone formation in the opening gap after OW-HTO with high accuracy and reproducibility.

Generation of a recessive dystrophic epidermolysis bullosa mouse model with patient-derived compound heterozygous mutations.

Recessive dystrophic epidermolysis bullosa (RDEB) is an intractable genetic disease of the skin caused by mutations in the COL7A1 gene. The majority of patients with RDEB harbor compound heterozygous mutations-two distinct mutations on each chromosome-without any apparent hotspots in the COL7A1 mutation pattern. This situation has made it challenging to establish a reliable RDEB mouse model with mutations that accurately mimic the genomic background of patients. Here, we established an RDEB mouse model harboring patient-type mutations in a compound heterozygous manner, using the CRISPR-based genome-editing technology i-GONAD. We selected two mutations, c.5818delC and E2857X, that have frequently been identified in cohorts of Japanese patients with RDEB. These mutations were introduced into the mouse genome at locations corresponding to those identified in patients. Mice homozygous for the 5818delC mutation developed severe RDEB-like phenotypes and died immediately after birth, whereas E2857X homozygous mice did not have a shortened lifespan compared to wild-type mice. Adult E2857X homozygous mice showed hair abnormalities, syndactyly, and nail dystrophy; these findings indicate that E2857X is indeed pathogenic in mice. Mice with the c.5818delC/E2857X compound heterozygous mutation presented an intermediate phenotype between the c.5818delC and E2857X homozygous mice. Single-cell RNA sequencing further clarified that the intrafollicular keratinocytes in c.5818delC/E2857X compound heterozygous mice exhibited abnormalities in cell cycle regulation. The proposed strategy to produce compound heterozygous mice, in addition to the established mouse line, will facilitate research on RDEB pathogenesis to develop a cure for this devastating disease.

Synthesized HMGB1 peptide attenuates liver inflammation and suppresses fibrosis in mice.

The liver has a high regenerative ability and can induce spontaneous regression of fibrosis when early liver damage occurs; however, these abilities are lost when chronic liver damage results in decompensated cirrhosis. Cell therapies, such as mesenchymal stem cell (MSC) and macrophage therapies, have attracted attention as potential strategies for mitigating liver fibrosis. Here, we evaluated the therapeutic effects of HMGB1 peptide synthesized from box A of high mobility group box 1 protein. Liver damage and fibrosis were evaluated using a carbon tetrachloride (CCl4)-induced cirrhosis mouse model. The effects of HMGB1 peptide against immune cells were evaluated by single-cell RNA-seq using liver tissues, and those against monocytes/macrophages were further evaluated by in vitro analyses. Administration of HMGB1 peptide did not elicit a rapid response within 36 h, but attenuated liver damage after 1 week and suppressed fibrosis after 2 weeks. Fibrosis regression developed over time, despite continuous liver damage, suggesting that administration of this peptide could induce fibrolysis. In vitro analyses could not confirm a direct effect of HMGB1 peptide against monocyte/macrophages. However, macrophages were the most affected immune cells in the liver, and the number of scar-associated macrophages (Trem2+Cd9+ cells) with anti-inflammatory markers increased in the liver following HMGB1 treatment, suggesting that indirect effects of monocytes/macrophages were important for therapeutic efficacy. Overall, we established a new concept for cell-free therapy using HMGB1 peptide for cirrhosis through the induction of anti-inflammatory macrophages.

Longitudinal Single-Cell Transcriptomics Reveals a Role for Serpina3n-Mediated Resolution of Inflammation in a Mouse Colitis Model.

BACKGROUND & AIMS: Proper resolution of inflammation is essential to maintaining homeostasis, which is important as a dysregulated inflammatory response has adverse consequences, even being regarded as a hallmark of cancer. However, our picture of dynamic changes during inflammation remains far from comprehensive. METHODS: Here we used single-cell transcriptomics to elucidate changes in distinct cell types and their interactions in a mouse model of chemically induced colitis. RESULTS: Our analysis highlights the stromal cell population of the colon functions as a hub with dynamically changing roles over time. Importantly, we found that Serpina3n, a serine protease inhibitor, is specifically expressed in stromal cell clusters as inflammation resolves, interacting with a potential target, elastase. Indeed, genetic ablation of the Serpina3n gene delays resolution of induced inflammation. Furthermore, systemic Serpina3n administration promoted the resolution of inflammation, ameliorating colitis symptoms. CONCLUSIONS: This study provides a comprehensive, single-cell understanding of cell-cell interactions during colorectal inflammation and reveals a potential therapeutic target that leverages inflammation resolution.

Controlled induction of immune tolerance by mesenchymal stem cells transferred by maternal microchimerism.

Feto-maternal immune tolerance is established during pregnancy; however, its mechanism and maintenance remain underexplored. Here, we investigated whether mesenchymal stem/stromal cells (MSCs) as non-inherited maternal antigens (NIMAs) transferred by maternal microchimerism could induce immune tolerance. We showed that MSCs had a potential equivalent to hematopoietic stem and progenitor cells (HSPCs) to induce immune tolerance and that MSCs were essential to induce tolerance to MSC-specific antigens. Furthermore, we demonstrated that MSCs as NIMAs transferred by maternal microchimerism could induce robust immune tolerance that can be further enhanced using a drug. Our data shed light on induction of immune tolerance and serve as a foundation to develop new therapies using maternally derived cells for autoimmune or genetic diseases.

Contribution of PDGFRα lineage cells in adult mouse hematopoiesis.

Platelet-derived growth factor receptor alpha (PDGFRα) is a dominant marker of mesodermal mesenchymal cells in mice. Previous studies demonstrated that PDGFRα-positive (PDGFRα+) mesodermal cells develop not only into mesenchymal cells but also into a subset of total hematopoietic cells (HCs) in the limited period during mouse embryogenesis. However, the precise characteristics of the PDGFRα lineage positive (PDGFRα Lin+) HCs in adult mouse hematopoiesis are largely unknown. In this study, we systematically evaluated the characteristics of PDGFRα Lin+ HCs in the bone marrow and peripheral blood using PDGFRα-CRE; ROSAtdTomato mice. Flow cytometry analysis revealed that PDGFRα Lin+ HCs accounted for approximately 20% of total HCs in both the bone marrow and peripheral blood in adult mice. Compositions of myeloid and lymphoid subpopulations among CD45+ mononuclear cells were almost identical in both PDGFRα Lin+ and PDGFRα Lin- cells. Single-cell RNA-sequencing analysis also demonstrated that the transcriptomic signatures of the PDGFRα Lin+ HCs in the peripheral blood largely overlapped with those of the PDGFRα Lin- HCs, suggesting equivalent functions of the PDGFRα Lin+ and PDGFRα Lin- HCs. Although pathophysiological activities of the PDGFRα Lin + HCs were not evaluated, our data clearly demonstrate a significant role of the PDGFRα Lin + HCs in physiological hematopoiesis in adult mice.

Cut-C: cleavage under tethered nuclease for conformational capture.

BACKGROUND: Deciphering the 3D structure of the genome is essential for elucidating the regulatory mechanisms of gene expression in detail. Existing methods, such as chromosome conformation capture (3C) and Hi-C have enabled the identification of novel aspects of chromatin structure. Further identification of protein-centric chromatin conformation is enabled by coupling the Hi-C procedure with a conventional chromatin immunoprecipitation assay. However, these methods are time-consuming and require independent methods for validation. RESULTS: To simultaneously identify protein-centric chromatin conformation and target protein localization, we have developed Cut-C, a method that combines antibody-mediated cleavage by tethered nuclease with chromosome conformation capture to identify chromatin interactions mediated by a protein of interest. Applying Cut-C to H3K4me3, a histone modification enriched at active gene promoters, we have successfully identified chromatin loops mediated by H3K4me3 along with the genome-wide distribution of H3K4me3. Cut-C also identified chromatin loops mediated by CTCF, validating the general applicability of the method. CONCLUSIONS: Cut-C identifies protein-centric chromatin conformations along with the genome-wide distribution of target proteins using simple procedures. The simplified protocol will improve the efficiency of analysing chromatin conformation using precious materials, such as clinical samples.