[Aging-associated Cyst Formation and Fibrosis].

Cysts are abnormal fluid-filled sacs found in various human organs, including the liver. Liver cysts can be associated with known causes such as parasite infections and gene mutations, or simply aging. Among these causes, simple liver cysts are often found in elderly people. While they are generally benign, they may occasionally grow but rarely shrink with age, indicating their clear association with aging. However, the mechanism behind the formation of simple liver cysts has not been thoroughly investigated. Recently, we have generated transgenic mice that specifically overexpress fibroblast growth factor (FGF)18 in hepatocytes. These mice exhibit severe liver fibrosis without inflammation and spontaneously develop liver cysts that grow with age. Our findings suggest that simple liver cysts can be induced by fibrosis accompanied by sterile inflammation or injury, whereas fibrosis accompanied by severe inflammation or injury may lead to cirrhosis. We also discuss the detrimental effects of disease- and aging-associated fibrosis in various organs, such as the heart, lungs, and kidneys. Additionally, we provide a brief summary of the two currently approved anti-fibrotic drugs for idiopathic pulmonary fibrosis, nintedanib and pirfenidone, as well as their possibility of future expansion of application toward other fibrotic diseases.

Necroptosis and Its Involvement in Various Diseases.

Necroptosis is a regulated form of cell death involved in the development of various pathological conditions. In contrast to apoptosis, plasma membrane rupture (PMR) occurs in cells in the relatively early stage of necroptosis; therefore, necroptosis induces a strong inflammatory response. Stimuli, including tumor necrosis factor (TNF), interferon (IFN)α/ß, lipopolysaccharide, polyI:C, and viral infection, induce the formation of necrosomes that lead to membrane rupture and the release of intracellular contents, termed danger-associated molecular patterns (DAMPs). DAMPs are the collective term for molecules that normally reside in the cytoplasm or nucleus in living cells without inducing inflammation but induce strong inflammatory responses when released outside cells. Recent studies have provided a better understanding of the mechanisms underlying PMR and the release of DAMPs. Moreover, necroptosis is involved in various pathological conditions, and mutations in necroptosis-related genes can cause hereditary autoinflammatory syndromes. Thus, manipulating necroptosis signaling pathways may be useful for treating diseases involving necroptosis.

Fibroblast growth factor 18 stimulates the proliferation of hepatic stellate cells, thereby inducing liver fibrosis.

Liver fibrosis results from chronic liver injury triggered by factors such as viral infection, excess alcohol intake, and lipid accumulation. However, the mechanisms underlying liver fibrosis are not fully understood. Here, we demonstrate that the expression of fibroblast growth factor 18 (Fgf18) is elevated in mouse livers following the induction of chronic liver fibrosis models. Deletion of Fgf18 in hepatocytes attenuates liver fibrosis; conversely, overexpression of Fgf18 promotes liver fibrosis. Single-cell RNA sequencing reveals that overexpression of Fgf18 in hepatocytes results in an increase in the number of Lrat+ hepatic stellate cells (HSCs), thereby inducing fibrosis. Mechanistically, FGF18 stimulates the proliferation of HSCs by inducing the expression of Ccnd1. Moreover, the expression of FGF18 is correlated with the expression of profibrotic genes, such as COL1A1 and ACTA2, in human liver biopsy samples. Thus, FGF18 promotes liver fibrosis and could serve as a therapeutic target to treat liver fibrosis.

Automatic Temperature Parameter Tuning for Reinforcement Learning Using Path Integral Policy Improvement.

In this article, we propose a novel variant of path integral policy improvement with covariance matrix adaptation ( [Formula: see text] - [Formula: see text] ), which is a reinforcement learning (RL) algorithm that aims to optimize a parameterized policy for the continuous behavior of robots. [Formula: see text] - [Formula: see text] has a hyperparameter called the temperature parameter, and its value is critical for performance; however, little research has been conducted on it and the existing method still contains a tunable parameter, which can be critical to performance. Therefore, tuning by trial and error is necessary in the existing method. Moreover, we show that there is a problem setting that cannot be learned by the existing method. The proposed method solves both problems by automatically adjusting the temperature parameter for each update. We confirmed the effectiveness of the proposed method using numerical tests.

A high-sensitivity ELISA for detection of human FGF18 in culture supernatants from tumor cell lines.

Fibroblast growth factor 18 (FGF18) is elevated in several human cancers, such as gastrointestinal and ovarian cancers, and stimulates the proliferation of tumor cells. This suggests that FGF18 may be a promising candidate biomarker in cancer patients. However, the lack of a high-sensitivity enzyme-linked immunosorbent assay (ELISA) does not permit testing of this possibility. In this study, we generated monoclonal antibodies against human FGF18 and developed a high-sensitivity ELISA to measure human FGF18 at concentrations as low as 10 pg/mL. Of the eight tumor cell lines investigated, we detected human FGF18 in culture supernatants from four tumor cell lines, including HeLa, OVCAR-3, BxPC-3, and SW620 cells, albeit the production levels were relatively low in the latter two cell lines. Moreover, the in-house ELISA could detect murine FGF18 in sera from mice overexpressing murine Fgf18 in hepatocytes, although the sensitivity in detecting murine FGF18 was relatively low. This FGF18 ELISA could be a valuable tool to validate FGF18 as a potential biomarker for cancer patients and to test the contribution of FGF18 for various disease models invivo and in vitro.

Interleukin 11 confers resistance to dextran sulfate sodium-induced colitis in mice.

Intestinal homeostasis is tightly regulated by epithelial cells, leukocytes, and stromal cells, and its dysregulation is associated with inflammatory bowel diseases. Interleukin (IL)-11, a member of the IL-6 family of cytokines, is produced by inflammatory fibroblasts during acute colitis. However, the role of IL-11 in the development of colitis is still unclear. Herein, we showed that IL-11 ameliorated DSS-induced acute colitis in mouse models. We found that deletion of Il11ra1 or Il11 rendered mice highly susceptible to DSS-induced colitis compared to the respective control mice. The number of apoptotic epithelial cells was increased in DSS-treated Il11ra1- or Il11-deficient mice. Moreover, we showed that IL-11 production was regulated by reactive oxygen species (ROS) produced by lysozyme M-positive myeloid cells. These findings indicate that fibroblast-produced IL-11 plays an important role in protecting the mucosal epithelium in acute colitis. Myeloid cell-derived ROS contribute to the attenuation of colitis through the production of IL-11.

The transcription factor ATF3 switches cell death from apoptosis to necroptosis in hepatic steatosis in male mice.

Hepatocellular death increases with hepatic steatosis aggravation, although its regulation remains unclear. Here we show that hepatic steatosis aggravation shifts the hepatocellular death mode from apoptosis to necroptosis, causing increased hepatocellular death. Our results reveal that the transcription factor ATF3 acts as a master regulator in this shift by inducing expression of RIPK3, a regulator of necroptosis. In severe hepatic steatosis, after partial hepatectomy, hepatic ATF3-deficient or -overexpressing mice display decreased or increased RIPK3 expression and necroptosis, respectively. In cultured hepatocytes, ATF3 changes TNFα-dependent cell death mode from apoptosis to necroptosis, as revealed by live-cell imaging. In non-alcoholic steatohepatitis (NASH) mice, hepatic ATF3 deficiency suppresses RIPK3 expression and hepatocellular death. In human NASH, hepatocellular damage is correlated with the frequency of hepatocytes expressing ATF3 or RIPK3, which overlap frequently. ATF3-dependent RIPK3 induction, causing a modal shift of hepatocellular death, can be a therapeutic target for steatosis-induced liver damage, including NASH.

Generation of transgenic mice expressing a FRET biosensor, SMART, that responds to necroptosis.

Necroptosis is a regulated form of cell death involved in various pathological conditions, including ischemic reperfusion injuries, virus infections, and drug-induced tissue injuries. However, it is not fully understood when and where necroptosis occurs in vivo. We previously generated a Forster resonance energy transfer (FRET) biosensor, termed SMART (the sensor for MLKL activation by RIPK3 based on FRET), which monitors conformational changes of MLKL along with progression of necroptosis in human and murine cell lines in vitro. Here, we generate transgenic (Tg) mice that express the SMART biosensor in various tissues. The FRET ratio is increased in necroptosis, but not apoptosis or pyroptosis, in primary cells. Moreover, the FRET signals are elevated in renal tubular cells of cisplatin-treated SMART Tg mice compared to untreated SMART Tg mice. Together, SMART Tg mice may provide a valuable tool for monitoring necroptosis in different types of cells in vitro and in vivo.

Semaphorin 4D induces articular cartilage destruction and inflammation in joints by transcriptionally reprogramming chondrocytes.

Proinflammatory cytokines play critical roles in the pathogenesis of joint diseases. Using a mass spectrometry-based cloning approach, we identified Semaphorin 4D (Sema4D) as an inflammatory cytokine that directly promoted cartilage destruction. Sema4d-deficient mice showed less cartilage destruction than wild-type mice in a model of rheumatoid arthritis. Sema4D induced a proinflammatory response in mouse articular chondrocytes characterized by the induction of proteolytic enzymes that degrade cartilage, such as matrix metalloproteinases (MMPs) and aggrecanases. The activation of Mmp13 and Mmp3 expression in articular chondrocytes by Sema4D did not depend on RhoA, a GTPase that mediates Sema4D-induced cytoskeletal rearrangements. Instead, it required NF-κB signaling and Ras-MEK-Erk1/2 signaling downstream of the receptors Plexin-B2 and c-Met and depended on the transcription factors IκBζ and C/EBPδ. Genetic and pharmacological blockade of these Sema4D signaling pathways inhibited MMP induction in chondrocytes and cartilage destruction in femoral head organ culture. Our results reveal a mechanism by which Sema4D signaling promotes cartilage destruction.

Lewis glycosphingolipids as critical determinants of TRAIL sensitivity in cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cancer cell death and contributes to tumor rejection by cytotoxic lymphocytes in cancer immunosurveillance and immunotherapy. TRAIL and TRAIL receptor agonists have garnered wide popularity as promising agents for cancer therapy. We previously demonstrated that the loss of fucosylation in cancer cells impairs TRAIL sensitivity; however, the precise structures of the fucosylated glycans that regulate TRAIL sensitivity and their carrier molecules remain elusive. Herein, we observed that Lewis glycans among various fucosylated glycans positively regulate TRAIL-induced cell death. Specifically, Lewis glycans on lacto/neolacto glycosphingolipids, but not glycoproteins including TRAIL receptors, enhanced TRAIL-induced formation of the cytosolic caspase 8 complex, without affecting the formation of the membranous receptor complex. Furthermore, type I Lewis glycan expression in colon cancer cell lines and patient-derived cancer organoids was positively correlated with TRAIL sensitivity. These findings provide novel insights into the regulatory mechanism of TRAIL-induced cell death and facilitate the identification of novel predictive biomarkers for TRAIL-related cancer therapies in future.

IκBζ controls IL-17-triggered gene expression program in intestinal epithelial cells that restricts colonization of SFB and prevents Th17-associated pathologies.

Control of gut microbes is crucial for not only local defense in the intestine but also proper systemic immune responses. Although intestinal epithelial cells (IECs) play important roles in cytokine-mediated control of enterobacteria, the underlying mechanisms are not fully understood. Here we show that deletion of IκBζ in IECs in mice leads to dysbiosis with marked expansion of segmented filamentous bacteria (SFB), thereby enhancing Th17 cell development and exacerbating inflammatory diseases. Mechanistically, the IκBζ deficiency results in decrease in the number of Paneth cells and impairment in expression of IL-17-inducible genes involved in IgA production. The decrease in Paneth cells is caused by aberrant activation of IFN-γ signaling and a failure of IL-17-dependent recovery from IFN-γ-induced damage. Thus, the IL-17R-IκBζ axis in IECs contributes to the maintenance of intestinal homeostasis by serving as a key component in a regulatory loop between the gut microbiota and immune cells.

Proscillaridin A Sensitizes Human Colon Cancer Cells to TRAIL-Induced Cell Death.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic cytokine that induces cancer cell death by binding to TRAIL receptors. Because of its selective cytotoxicity toward cancer cells, TRAIL therapeutics, such as recombinant TRAIL and agonistic antibodies targeting TRAIL receptors, have garnered attention as promising cancer treatment agents. However, many cancer cells acquire resistance to TRAIL-induced cell death. To overcome this issue, we searched for agents to sensitize cancer cells to TRAIL-induced cell death by screening a small-molecule chemical library consisting of diverse compounds. We identified a cardiac glycoside, proscillaridin A, as the most effective TRAIL sensitizer in colon cancer cells. Proscillaridin A synergistically enhanced TRAIL-induced cell death in TRAIL-sensitive and -resistant colon cancer cells. Additionally, proscillaridin A enhanced cell death in cells treated with TRAIL and TRAIL sensitizer, the second mitochondria-derived activator of caspase mimetic. Proscillaridin A upregulated TRAIL receptor expression, while downregulating the levels of the anti-cell death molecules, cellular FADD-like IL-1ß converting enzyme-like inhibitor protein and Mcl1, in a cell type-dependent manner. Furthermore, proscillaridin A enhanced TRAIL-induced cell death partly via O-glycosylation. Taken together, our findings suggest that proscillaridin A is a promising agent that enhances the anti-cancer efficacy of TRAIL therapeutics.

Regulation of the release of damage-associated molecular patterns from necroptotic cells.

Damage-associated molecular patterns (DAMPs) are molecules within living cells that are released when cell membranes are ruptured. Although DAMPs have physiological functions inside the cell, once DAMPs are released extracellularly, they elicit various biological responses, including inflammation, proliferation, tissue damage, and tissue repair, in a context-dependent manner. In past decades, it was assumed that the release of DAMPs was induced by a membrane rupture, caused by passive ATP depletion, or by chemical or mechanical damage to the membrane. However, that concept has been challenged by recent advancements in understanding the regulation of cell death. Necroptosis is a form of regulated cell death, where cells show necrotic morphology. Necroptosis is triggered by death receptors, toll-like receptors, and some viral infections. The membrane rupture is executed by the mixed lineage-like kinase domain-like pseudokinase (MLKL), which forms oligomers that translocate to the plasma membrane during necroptosis. Although the causal relationship between MLKL function and membrane rupture has been extensively investigated, the detailed molecular mechanisms by which oligomerized MLKL induces membrane rupture are not fully understood. This review summarizes recent advances in understanding how MLKL regulates DAMP release and new technologies for visualizing DAMP release at single-cell resolution.

The scaffold-dependent function of RIPK1 in dendritic cells promotes injury-induced colitis.

Receptor interacting protein kinase 1 (RIPK1) is a cytosolic multidomain protein that controls cell life and death. While RIPK1 promotes cell death through its kinase activity, it also functions as a scaffold protein to promote cell survival by inhibiting FADD-caspase 8-dependent apoptosis and RIPK3-MLKL-dependent necroptosis. This pro-survival function is highlighted by excess cell death and perinatal lethality in Ripk1-/- mice. Recently, loss of function mutation of RIPK1 was found in patients with immunodeficiency and inflammatory bowel diseases. Hematopoietic stem cell transplantation restored not only immunodeficiency but also intestinal inflammatory pathology, indicating that RIPK1 in hematopoietic cells is critical to maintain intestinal immune homeostasis. Here, we generated dendritic cell (DC)-specific Ripk1-/- mice in a genetic background with loss of RIPK1 kinase activity and found that the mice developed spontaneous colonic inflammation characterized by increased neutrophil and Ly6C+ monocytes. In addition, these mice were highly resistant to injury-induced colitis. The increased colonic inflammation and the resistance to colitis were restored by dual inactivation of RIPK3 and FADD, but not by inhibition of RIPK3, MLKL, or ZBP1 alone. Altogether, these results reveal a scaffold activity-dependent role of RIPK1 in DC-mediated maintenance of colonic immune homeostasis.

Time-Lapse Imaging of Necroptosis and DAMP Release at Single-Cell Resolution.

Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like protein (MLKL). Necroptotic cells release a variety of cellular and nuclear factors, referred to as danger-associated molecular patterns (DAMPs). We recently developed a förster resonance energy transfer (FRET) biosensor, termed SMART (a sensor for MLKL activation based on FRET). SMART comprises a fragment of MLKL, and it monitors necroptosis, but not apoptosis or necrosis. We performed live-cell imaging for secretion activity (LCI-S) to observe the release of high-mobility group box 1 (HMGB1) from necroptotic cells at single-cell resolution. Moreover, we combined SMART and LCI-S imaging techniques and found two different modes of HMGB1 release from necroptotic cells. Thus, SMART and LCI-S are valuable tools for investigating intimate cross talk between necroptosis and DAMP release at single-cell resolution.

Interleukin-11-expressing fibroblasts have a unique gene signature correlated with poor prognosis of colorectal cancer.

Interleukin (IL)-11 is a member of the IL-6 family of cytokines and is involved in multiple cellular responses, including tumor development. However, the origin and functions of IL-11-producing (IL-11+) cells are not fully understood. To characterize IL-11+ cells in vivo, we generate Il11 reporter mice. IL-11+ cells appear in the colon in murine tumor and acute colitis models. Il11ra1 or Il11 deletion attenuates the development of colitis-associated colorectal cancer. IL-11+ cells express fibroblast markers and genes associated with cell proliferation and tissue repair. IL-11 induces the activation of colonic fibroblasts and epithelial cells through phosphorylation of STAT3. Human cancer database analysis reveals that the expression of genes enriched in IL-11+ fibroblasts is elevated in human colorectal cancer and correlated with reduced recurrence-free survival. IL-11+ fibroblasts activate both tumor cells and fibroblasts via secretion of IL-11, thereby constituting a feed-forward loop between tumor cells and fibroblasts in the tumor microenvironment.

MIND bomb 2 prevents RIPK1 kinase activity-dependent and -independent apoptosis through ubiquitylation of cFLIPL.

Mind bomb 2 (MIB2) is an E3 ligase involved in Notch signalling and attenuates TNF-induced apoptosis through ubiquitylation of receptor-interacting protein kinase 1 (RIPK1) and cylindromatosis. Here we show that MIB2 bound and conjugated K48- and K63-linked polyubiquitin chains to a long-form of cellular FLICE-inhibitory protein (cFLIPL), a catalytically inactive homologue of caspase 8. Deletion of MIB2 did not impair the TNF-induced complex I formation that mediates NF-κB activation but significantly enhanced formation of cytosolic death-inducing signalling complex II. TNF-induced RIPK1 Ser166 phosphorylation, a hallmark of RIPK1 death-inducing activity, was enhanced in MIB2 knockout cells, as was RIPK1 kinase activity-dependent and -independent apoptosis. Moreover, RIPK1 kinase activity-independent apoptosis was induced in cells expressing cFLIPL mutants lacking MIB2-dependent ubiquitylation. Together, these results suggest that MIB2 suppresses both RIPK1 kinase activity-dependent and -independent apoptosis, through suppression of RIPK1 kinase activity and ubiquitylation of cFLIPL, respectively.

Regulation of T cell differentiation by the AP-1 transcription factor JunB.

JunB, a component of the activator protein-1 (AP-1) transcription factor, is known to exhibit an important role in bone formation and bone marrow cell proliferation. During T helper type 2 (Th2) cell differentiation, JunB contributes to the regulation of interleukin (IL)-4 expression, and AP-1 and nuclear factor of activated T cell (NFAT) constitute a heteromer and contribute to IL-2 production. However, the role of JunB in other T cells has not been investigated. In 2017, it was revealed that JunB, in collaboration with basic leucine zipper ATF-like transcription factor (BATF), regulates the expression of Th17-related genes. Furthermore, JunB was found to play an important role in regulatory T (Treg) cell differentiation, contributing to CD25 expression and IL-2 production. IL-2 is a T cell activator and has been shown as a necessary factor for Treg proliferation. Here, we review the role of JunB in T cells based on basic research data and discuss the potential for its clinical applications.

Identification of the hallmarks of necroptosis and ferroptosis by transmission electron microscopy.

Apoptosis is the prototype for a regulated form of cell death, but recent studies have revealed other types of regulated forms of cell death, including necroptosis and ferroptosis. The molecular mechanisms underlying the execution of these processes have been intensively investigated, yet the hallmarks of their morphology are not fully understood. Here, we report that electron lucent cytoplasm was a common feature of both necroptosis and ferroptosis, which was consistent with cytoplasmic vacuolization due to a defect in the cytoplasmic membrane integrity. Notably, the perinuclear space was dilated in necroptosis, but such dilation did not occur in ferroptosis. Cells undergoing ferroptosis, but not necroptosis, exhibited an electron lucent nucleus. We previously reported that one of the nuclear danger-associated molecular patterns (DAMPs), high mobility group box (HMGB)1, is rapidly released from the nucleus to the extracellular spaces of cells undergoing necroptosis through the ruptured nuclear and cytoplasmic membrane. Via time-lapse imaging of cells stably expressing HMGB1 fused to a fluorescence protein, we found that HMGB1 was also released from the nucleus to the cytosol, and then eventually released into the extracellular spaces in cells undergoing ferroptosis. Thus, nuclear membrane damage was induced prior to cytoplasmic membrane rupture in ferroptosis. Thus, dilation of the perinuclear space and an electron lucent nucleus may be the hallmarks of necroptosis and ferroptosis, respectively.

Identification of a phosphorylation site on Ulk1 required for genotoxic stress-induced alternative autophagy.

Alternative autophagy is an autophagy-related protein 5 (Atg5)-independent type of macroautophagy. Unc51-like kinase 1 (Ulk1) is an essential initiator not only for Atg5-dependent canonical autophagy but also for alternative autophagy. However, the mechanism as to how Ulk1 differentially regulates both types of autophagy has remained unclear. In this study, we identify a phosphorylation site of Ulk1 at Ser746, which is phosphorylated during genotoxic stress-induced alternative autophagy. Phospho-Ulk1746 localizes exclusively on the Golgi and is required for alternative autophagy, but not canonical autophagy. We also identify receptor-interacting protein kinase 3 (RIPK3) as the kinase responsible for genotoxic stress-induced Ulk1746 phosphorylation, because RIPK3 interacts with and phosphorylates Ulk1 at Ser746, and loss of RIPK3 abolishes Ulk1746 phosphorylation. These findings indicate that RIPK3-dependent Ulk1746 phosphorylation on the Golgi plays a pivotal role in genotoxic stress-induced alternative autophagy.