👤 Ichiro Manabe

Methylprednisolone (mPSL) pulse therapy is an essential treatment for systemic lupus erythematosus (SLE); however, it carries a risk of osteonecrosis of the femoral head (ONFH). The pathogenesis of ONFH involves neutrophil extracellular trap (NET)-mediated microcirculation disorders. In BALB/c mice with imiquimod (IMQ)-induced lupus, mPSL pulse elevated serum levels of prenylcysteine oxidase 1 (PCYOX1), an enzyme that produces NET inducers hydrogen peroxide and farnesal, resulting in increased NETs in vivo. Although ischemia was observed in the femoral head, IMQ + mPSL-treated BALB/c mice did not develop ONFH. PCYOX1 is abundant in very-low-density lipoproteins. This study aimed to demonstrate that hyperlipidemia exacerbates NET-mediated microcirculation disorders and leads to ONFH development following mPSL pulse in lupus mice. To address this, ApoE mutant hyperlipidemic and BALB/c mice with IMQ-induced lupus received mPSL pulse. NET-forming neutrophils in peripheral blood were detected by flow cytometry. ONFH was assessed microscopically. As a result, IMQ + mPSL-treated ApoE mutant but not BALB/c mice developed ONFH, exhibiting higher levels of PCYOX1 and NET-forming neutrophils in circulation. In addition, NET-forming neutrophils accumulated in the vessels surrounding the femoral head, accompanied by osteocyte necrosis. This study demonstrated that mPSL pulse in lupus mice with hyperlipidemia enhanced PCYOX1 levels and NET formation, resulting in ONFH development, suggesting that hyperlipidemia may be a risk factor for ONFH following mPSL pulse therapy in SLE. Show less

Multisystem inflammatory syndrome in children (MIS-C) is a hyperinflammatory condition caused by recent infection with severe acute respiratory syndrome coronavirus 2, but the underlying immunological mechanisms driving this distinct syndrome are unknown. We utilized high-dimensional flow cytometry, cell-free (cf) DNA, and cytokine and chemokine profiling to identify mechanisms of critical illness distinguishing MIS-C from severe acute coronavirus disease 2019 (SAC). Compared to SAC, MIS-C patients demonstrated profound innate immune cell death and features of emergency myelopoiesis (EM), an understudied phenomenon observed in severe inflammation. EM signatures were characterized by fewer mature myeloid cells in the periphery and decreased expression of HLA-DR and CD86 on antigen-presenting cells. Interleukin 27 (IL-27), a cytokine known to drive hematopoietic stem cells toward EM, was increased in MIS-C, and correlated with immature cell signatures in MIS-C. Upon recovery, EM signatures decreased and IL-27 plasma levels returned to normal levels. Despite profound lymphopenia, we report a lack of cfDNA released by adaptive immune cells and increased CCR7 expression on T cells indicative of egress out of peripheral blood. Immune cell signatures of EM combined with elevated innate immune cell-derived cfDNA levels distinguish MIS-C from SAC in children and provide mechanistic insight into dysregulated immunity contributing toward MIS-C, offering potential diagnostic and therapeutic targets. Show less

Non-alcoholic steatohepatitis (NASH), characterized by chronic inflammation and fibrosis, is predicted to be the leading cause of cirrhosis and hepatocellular carcinoma (HCC) in the next decade. Although recent evidence suggests the importance of fibrosis as the strongest determinant of HCC development, the molecular mechanisms underlying NASH-induced carcinogenesis still remain unclear. Here we performed RNA sequencing analysis to compare gene expression profiles of activated fibroblasts prepared from two distinct liver fibrosis models: carbon tetrachloride-induced fibrosis as a model without obesity and HCC and genetically obese melanocortin 4 receptor-deficient (MC4R-KO) mice fed Western diet, which develop steatosis, NASH, and eventually HCC. Our data showed that activated fibroblasts exhibited distinct gene expression patterns in each etiology, and that the 'pathways in cancer' were selectively upregulated in the activated fibroblasts from MC4R-KO mice. The most upregulated gene in these pathways was fibroblast growth factor 9 (FGF9), which was induced by metabolic stress such as palmitate. FGF9 exerted anti-apoptotic and pro-migratory effects in fibroblasts and hepatoma cells in vitro and accelerated tumor growth in a subcutaneous xenograft model. This study reveals upregulation of cancer-associated gene expression in activated fibroblasts in NASH, which would contribute to the progression from NASH to HCC. Show less

DOCK180 is a guanine exchange factor of Rac1 originally identified as a protein bound to an SH3 domain of the Crk adaptor protein. DOCK180 induces tyrosine phosphorylation of p130(Cas), and recruits the Crk-p130(Cas) complex to focal adhesions. To understand the role of DOCK180 in cell adhesion and migration, we searched for DOCK180-binding proteins with a nano-LC/MS/MS system, and identified ANKRD28, a protein that contains twenty-six ankyrin domain repeats. Knockdown of ANKRD28 by RNA interference reduced the velocity of migration of HeLa cells, suggesting that this protein plays a physiologic role in the DOCK180-Rac1 signaling pathway. Furthermore, knockdown of ANKRD28 was found to alter the distribution of focal adhesion proteins such as Crk, paxillin, and p130(Cas). On the other hand, expression of ANKRD28, p130(Cas), Crk, and DOCK180 induced hyper-phosphorylation of p130(Cas), and impaired detachment of the cell membrane during migration. Consequently, cells expressing ANKRD28 exhibited multiple long cellular processes. ANKRD28 associated with DOCK180 in an SH3-dependent manner and competed with ELMO, another protein bound to the SH3 domain of DOCK180. In striking contrast to ANKRD28, overexpression of ELMO induced extensive lamellipodial protrusion around the entire circumference. These data suggest that ANKRD28 specifies the localization and the activity of the DOCK180-Rac1 pathway. Show less

The mRNA expression of GPRC5B, an orphan G protein-coupled receptor, is induced by retinoic acid (RA). Because RA plays critical roles in embryonic development, reproductive functions, metabolism and homeostasis, GPRC5B is also considered crucial in these physiological events. We investigated the changes in expression of GPRC5B and RA receptor (RAR) alpha mRNAs and immunohistochemical localization of their proteins in the murine placenta and yolk sac at 13.5, 15.5 and 17.5 days post coitus. Stable levels of GPRC5B and RARalpha mRNAs were detected in the placenta and yolk sac. In the placenta, GPRC5B was present in maternal and fetal vascular endothelial cells, stromal cells, fibroblast-like cells and glycogen cells. A strong reaction to RARalpha was detected in maternal and fetal vascular endothelial cells and stromal cells. The levels of GPRC5B and RARalpha proteins in maternal and fetal vascular endothelial cells decreased with gestation. In the yolk sac, GPRC5B and RARalpha proteins were detected in vascular endothelial cells, but their levels did not change during the gestation period. These findings indicate that GPRC5B is involved in RA-dependent morphogenesis/angiogenesis and regulation of extracellular matrix synthesis in the murine placenta and yolk sac. Show less