Masataka Asagiri

NFAT and Osterix cooperatively regulate bone formation

Immunosuppressants are crucial in the prevention of detrimental immune reactions associated with allogenic organ transplantation, but they often cause adverse effects in a number of biological systems, including the skeletal system. Calcineurin inhibitors FK506 and cyclosporin A inhibit nuclear factor of activated T cells (NFAT) activity and induce strong immunosuppression. Among NFAT proteins, NFATc1 is crucial for the differentiation of bone-resorbing osteoclasts. Here we show FK506 administration induces the reduction of bone mass despite a blockade of osteoclast differentiation. This reduction is caused by severe impairment of bone formation, suggesting that NFAT transcription factors also have an important role in the transcriptional program of osteoblasts. In fact, bone formation is inhibited in Nfatc1- and Nfatc2-deficient cells as well as in FK506-treated osteoblasts. Overexpression of NFATc1 stimulates Osterix-dependent activation of the Col1a1 (encoding type I collagen) promoter, but not Runx2-dependent activation of the Bglap1 (encoding osteocalcin) promoter. NFAT and Osterix form a complex that binds to DNA, and this interaction is important for the transcriptional activity of Osterix. Thus, NFAT and Osterix cooperatively control osteoblastic bone formation. These results may provide important insight into the management of post-transplantation osteoporosis as well as a new strategy for promoting bone regeneration in osteopenic disease.

Cathepsin K-Dependent Toll-Like Receptor 9 Signaling Revealed in Experimental Arthritis

Cathepsin K was originally identified as an osteoclast-specific lysosomal protease, the inhibitor of which has been considered might have therapeutic potential. We show that inhibition of cathepsin K could potently suppress autoimmune inflammation of the joints as well as osteoclastic bone resorption in autoimmune arthritis. Furthermore, cathepsin K–/– mice were resistant to experimental autoimmune encephalomyelitis. Pharmacological inhibition or targeted disruption of cathepsin K resulted in defective Toll-like receptor 9 signaling in dendritic cells in response to unmethylated CpG DNA, which in turn led to attenuated induction of T helper 17 cells, without affecting the antigen-presenting ability of dendritic cells. These results suggest that cathepsin K plays an important role in the immune system and may serve as a valid therapeutic target in autoimmune diseases.

Origin of myofibroblasts in the fibrotic liver in mice

Significance Liver resident activated hepatic stellate cells (aHSCs), and activated portal fibroblasts (aPFs) are the major source of the fibrous scar in the liver. aPFs have been implicated in liver fibrosis caused by cholestatic liver injury, whereas fibrosis in hepatotoxic liver injury is attributed to aHSCs. However, the contribution of aPFs to cholestatic fibrosis is not well characterized because of difficulties in cell purification and the lack of identified aPF-specific markers. We have developed a novel flow cytometry-based method of aPFs purification from the nonparenchymal cell fraction of collagen-α1(I)-GFP mice and have identified potential aPF-specific markers. The goal of this study is to determine whether aPFs contribute to cholestatic liver fibrosis and identify the mechanism(s) of their activation. Hepatic myofibroblasts are activated in response to chronic liver injury of any etiology to produce a fibrous scar. Despite extensive studies, the origin of myofibroblasts in different types of fibrotic liver diseases is unresolved. To identify distinct populations of myofibroblasts and quantify their contribution to hepatic fibrosis of two different etiologies, collagen-α1(I)-GFP mice were subjected to hepatotoxic (carbon tetrachloride; CCl4) or cholestatic (bile duct ligation; BDL) liver injury. All myofibroblasts were purified by flow cytometry of GFP+ cells and then different subsets identified by phenotyping. Liver resident activated hepatic stellate cells (aHSCs) and activated portal fibroblasts (aPFs) are the major source (>95%) of fibrogenic myofibroblasts in these models of liver fibrosis in mice. As previously reported using other methodologies, hepatic stellate cells (HSCs) are the major source of myofibroblasts (>87%) in CCl4 liver injury. However, aPFs are a major source of myofibroblasts in cholestatic liver injury, contributing >70% of myofibroblasts at the onset of injury (5 d BDL). The relative contribution of aPFs decreases with progressive injury, as HSCs become activated and contribute to the myofibroblast population (14 and 20 d BDL). Unlike aHSCs, aPFs respond to stimulation with taurocholic acid and IL-25 by induction of collagen-α1(I) and IL-13, respectively. Furthermore, BDL-activated PFs express high levels of collagen type I and provide stimulatory signals to HSCs. Gene expression analysis identified several novel markers of aPFs, including a mesothelial-specific marker mesothelin. PFs may play a critical role in the pathogenesis of cholestatic liver fibrosis and, therefore, serve as an attractive target for antifibrotic therapy.

Contribution of Nuclear Factor of Activated T Cells c1 to the Transcriptional Control of Immunoreceptor Osteoclast-associated Receptor but Not Triggering Receptor Expressed by Myeloid Cells-2 during Osteoclastogenesis

Bone homeostasis depends on the coordination of osteoclastic bone resorption and osteoblastic bone formation. Receptor activator of NF-κB ligand (RANKL) induces osteoclast differentiation through activating a transcriptional program mediated by the key transcription factor nuclear factor of activated T cells (NFAT) c1. Immunoreceptors, including osteoclast-associated receptor (OSCAR) and triggering receptor expressed by myeloid cells (TREM)-2, constitute the co-stimulatory signals required for RANKL-mediated activation of calcium signaling, which leads to the activation of NFATc1. However, it remains unknown whether the expression of immunoreceptors are under the control of NFATc1. Here we demonstrate that the expression of OSCAR, but not that of TREM-2, is up-regulated during osteoclastogenesis and markedly suppressed by the calcineurin inhibitor FK506, suggesting that OSCAR is transcriptionally regulated by NFATc1. NFATc1 expression results in the activation of the OSCAR promoter, which was found to be further enhanced by co-expression of PU.1 and microphthalmia-associated transcription factor (MITF). We further provide evidence that NFATc1 specifically regulates OSCAR by chromatin immunoprecipitation assay and quantification of OSCAR and TREM-2 mRNA in NFATc1-/- cells. Thus, OSCAR but not TREM-2 is involved in the positive feedback loop of the immunoreceptor-NFATc1 pathway during osteoclastogenesis. Although several immunoreceptors have been identified as co-stimulatory molecules for RANKL, the expression and function are differentially regulated. These mechanisms, possibly together with the delicate regulation of their ligands on osteoblasts, may provide the exquisite machinery for the modulation of osteoclastogenesis in the maintenance of bone homeostasis.

Inhibition of RANKL-induced osteoclastogenesis by (-)-DHMEQ, a novel NF-κB inhibitor, through downregulation of NFATc1

(−)‐DHMEQ, a newly designed NF‐κB inhibitor, inhibited RANKL‐induced osteoclast differentiation in mouse BMMs through downregulation of the induction of NFATc1, an essential transcription factor of osteoclastogenesis.

Control of RelB during dendritic cell activation integrates canonical and noncanonical NF-κB pathways

The NF-κB protein RelB controls dendritic cell (DC) maturation and may be targeted therapeutically to manipulate T cell responses in disease. Here we report that RelB promoted DC activation not as the expected RelB-p52 effector of the noncanonical NF-κB pathway, but as a RelB-p50 dimer regulated by canonical IκBs, IκBα and IκBɛ. IκB control of RelB minimized spontaneous maturation but enabled rapid pathogen-responsive maturation. Computational modeling of the NF-κB signaling module identified control points of this unexpected cell type–specific regulation. Fibroblasts that we engineered accordingly showed DC-like RelB control. Canonical pathway control of RelB regulated pathogen-responsive gene expression programs. This work illustrates the potential utility of systems analyses in guiding the development of combination therapeutics for modulating DC-dependent T cell responses.

Necrostatin‐1 protects against reactive oxygen species (ROS)‐induced hepatotoxicity in acetaminophen‐induced acute liver failure

Excessive acetaminophen (APAP) use is one of the most common causes of acute liver failure. Various types of cell death in the damaged liver are linked to APAP‐induced hepatotoxicity, and, of these, necrotic cell death of hepatocytes has been shown to be involved in disease pathogenesis. Until recently, necrosis was commonly considered to be a random and unregulated form of cell death; however, recent studies have identified a previously unknown form of programmed necrosis called receptor‐interacting protein kinase (RIPK)‐dependent necrosis (or necroptosis), which is controlled by the kinases RIPK1 and RIPK3. Although RIPK‐dependent necrosis has been implicated in a variety of disease states, including atherosclerosis, myocardial organ damage, stroke, ischemia–reperfusion injury, pancreatitis, and inflammatory bowel disease. However its involvement in APAP‐induced hepatocyte necrosis remains elusive. Here, we showed that RIPK1 phosphorylation, which is a hallmark of RIPK‐dependent necrosis, was induced by APAP, and the expression pattern of RIPK1 and RIPK3 in the liver overlapped with that of CYP2E1, whose activity around the central vein area has been demonstrated to be critical for the development of APAP‐induced hepatic injury. Moreover, a RIPK1 inhibitor ameliorated APAP‐induced hepatotoxicity in an animal model, which was underscored by significant suppression of the release of hepatic enzymes and cytokine expression levels. RIPK1 inhibition decreased reactive oxygen species levels produced in APAP‐injured hepatocytes, whereas CYP2E1 expression and the depletion rate of total glutathione were unaffected. Of note, RIPK1 inhibition also conferred resistance to oxidative stress in hepatocytes. These data collectively demonstrated a RIPK‐dependent necrotic mechanism operates in the APAP‐injured liver and inhibition of this pathway may be beneficial for APAP‐induced fulminant hepatic failure.

HIF-1α-PDK1 axis-induced active glycolysis plays an essential role in macrophage migratory capacity

In severely hypoxic condition, HIF-1α-mediated induction of Pdk1 was found to regulate glucose oxidation by preventing the entry of pyruvate into the tricarboxylic cycle. Monocyte-derived macrophages, however, encounter a gradual decrease in oxygen availability during its migration process in inflammatory areas. Here we show that HIF-1α-PDK1-mediated metabolic changes occur in mild hypoxia, where mitochondrial cytochrome c oxidase activity is unimpaired, suggesting a mode of glycolytic reprogramming. In primary macrophages, PKM2, a glycolytic enzyme responsible for glycolytic ATP synthesis localizes in filopodia and lammelipodia, where ATP is rapidly consumed during actin remodelling processes. Remarkably, inhibition of glycolytic reprogramming with dichloroacetate significantly impairs macrophage migration in vitro and in vivo. Furthermore, inhibition of the macrophage HIF-1α-PDK1 axis suppresses systemic inflammation, suggesting a potential therapeutic approach for regulating inflammatory processes. Our findings thus demonstrate that adaptive responses in glucose metabolism contribute to macrophage migratory activity.

Hepatic stellate cells relay inflammation signaling from sinusoids to parenchyma in mouse models of immune-mediated hepatitis

Hepatic stellate cells (HSCs) constitute the liver sinusoid with Kupffer cells and liver sinusoidal endothelial cells. While the sinusoid functions as the gateway to liver inflammation, whether HSCs contribute to liver inflammation and, if so, how they exert such functions remain elusive. Here, we found that mouse as well as human HSCs expressed DP1 receptor for prostaglandin D2 selectively in the liver. Pharmacological stimulation of DP1 by BW245C, a DP1‐selective agonist, suppressed the activation of cultured HSCs by tumor necrosis factor‐α at least in part through down‐regulation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells signaling and inhibition of c‐Jun N‐terminal kinase phosphorylation. DP1 deficiency or BW245C administration in mice significantly enhanced or suppressed concanavalin A (ConA)–induced hepatitis, respectively. ConA injection induced tumor necrosis factor‐α and interferon‐γ expression in the sinusoid, which was suppressed by administration of BW245C. Coculture of spleen cells and liver nonparenchymal cells showed that ConA first activated spleen cells and that this activation led to activation of nonparenchymal cells to secondarily produce tumor necrosis factor‐α and interferon‐γ. Microarray analysis revealed ConA‐induced expression of endothelin‐1, tissue factor, and chemokines in the liver and inducible nitric oxide synthase in hepatocytes, resulting in flow stagnation, leukocyte adherence and migration to the parenchyma, and hepatocyte death. DP1 stimulation inhibits all these events in the liver. Therefore, HSCs mediate amplification of ConA‐induced liver inflammation in the sinusoid, causing direct and indirect hepatocyte injury, and DP1 stimulation inhibits this HSC activation. Conclusions: HSCs integrate cytokine‐mediated inflammatory responses in the sinusoids and relay them to the liver parenchyma, and these HSC actions are inhibited by DP1 stimulation. (Hepatology 2016;63:1325–1339)

Defective Regulation of CXCR2 Facilitates Neutrophil Release from Bone Marrow Causing Spontaneous Inflammation in Severely NF-κB–Deficient Mice

NF-κB is a major regulator of innate and adaptive immunity. Neutrophilic granulocytes (neutrophils) constitutively express RelA/p65 (Rela), c-Rel (Crel), and p50 (Nfκb1) but not p52 (Nfκb2) subunits. In this paper, we describe Crel−/−Nfκb1−/−Rela+/− mice that have the most severe genetic neutrophil NF-κB deficiency compatible with life, Rela−/− mice being embryonic lethal. Crel−/−Nfκb1−/−Rela+/− mice developed spontaneous dermal and intestinal inflammation associated with chronic neutrophilia, elevated CXCL1, and G-CSF. The bone marrow contained fewer nucleated cells and was enriched in myeloid progenitor cells. Neutrophilia was preserved when Crel−/−Nfκb1−/−Rela+/− bone marrow was transferred into wild-type mice, but mixed bone marrow chimeras receiving wild-type and Crel−/−Nfκb1−/−Rela+/− bone marrow showed normal circulating neutrophil numbers, excluding an intrinsic proliferation advantage. In mixed bone marrow chimeras, Crel−/−Nfκb1−/−Rela+/− neutrophils were preferentially mobilized from the bone marrow in response to CXCL1 injection, LPS-induced lung inflammation, and thioglycollate-induced peritonitis. Crel−/−Nfκb1−/−Rela+/− neutrophils expressed higher levels of the CXCL1 receptor CXCR2 both under resting and stimulated conditions and failed to downregulate CXCR2 during inflammation. Treatment with an anti-CXCR2 Ab abolished preferential mobilization of Crel−/−Nfκb1−/−Rela+/− neutrophils in peritonitis in mixed chimeric mice and neutrophilia in Crel−/−Nfκb1−/−Rela+/− mice. We conclude that severe NF-κB deficiency facilitates neutrophil mobilization, which causes elevated numbers of preactivated neutrophils in blood and tissues, leading to spontaneous inflammation. These neutrophil effects may limit the usefulness of global NF-κB inhibitors for the treatment of inflammatory diseases.