Japan Institute for Advanced Dentistry

Systemic lupus erythematosus is a systemic autoimmune disease characterized by the production of autoantibodies and damage to multiple organs. Glomerulonephritis, a manifestation involving glomerular deposition of immune complexes and complement components, significantly contributes to disease morbidity. Although an endosomal single-stranded RNA sensor [Toll-like receptor 7 (TLR7)] is known to drive glomerulonephritis by promoting autoantibody production in B cells, the contribution of macrophage TLR7 responses to glomerulonephritis remains poorly understood. Here, we have examined Tlr7‒/‒ NZBWF1 (New Zealand Black/New Zealand White F1) mice and found that TLR7 deficiency ameliorates lupus nephritis by abolishing autoantibody production against RNA-associated antigens, C3 deposition, and macrophage accumulation in glomeruli. Furthermore, TLR7 signaling increased CD31 expression on glomerular endothelial cells and Ly6Clow macrophages but not on T and B cells, suggesting that CD31 mediates TLR7-dependent migration of monocytes into glomeruli. Compared to their splenic counterparts, glomerular macrophages produced IL-1β in a TLR7-dependent manner. In addition, single-cell RNA sequencing of glomerular macrophages revealed that TLR7 signaling induced expression of lupus-associated genes, including those encoding Chitinase 3 like 1, ferritin heavy chain 1, IKKε, and complement factor B (CfB). Although serum CfB did not increase in NZBWF1 mice, TLR7-dependent CfB protein expression was detected in glomerular macrophages. In addition, TLR7 signaling promoted C3 cleavage and deposition predominantly on mesangial cells. These findings suggest that TLR7 responses in glomerular macrophages accelerate the progression of glomerulonephritis in NZBWF1 mice.

Abstract Hepatic macrophages maintain liver homeostasis, but little is known about the signals that activate the hepatoprotective programs within macrophages. Here, we show that toll-like receptor 13 (TLR13), a sensor of bacterial 23S ribosomal RNA (rRNA), senses microbiome RNAs to drive tissue-protective responses in CD5Lhihepatic macrophages. Splenomegaly and hepatomegaly developed in the absence of the endosomal RNase, RNaseT2, via TLR13-dependent macrophage proliferation. Furthermore, TLR13 in hepatic Ly6Clomacrophages activated the transcription factors LXRα and MafB, leading to expression of tissue-clearance molecules, such as CD5L, C1qb, and Axl. Consequently,Rnaset2−/−mice developed resistance to acute liver injury caused by challenges with acetaminophen and lipopolysaccharide + D-galactosamine. TLR13 responses inRnaset2−/−mice were impaired by antibiotics, suggesting that TLR13 were activated by microbiome rRNAs, which was detected in the sera and hepatic macrophages. Repeated administration of wild-type mice with the TLR13 ligand, rather than other TLR ligands, selectively increased the number of Kupffer cells, which expressed immunoregulatory and tissue-clearance genes as hepatic macrophages inRnaset2−/−mice did. Our results suggest that microbiome ssRNA serves as an environmental cue for initiating tissue-protective TLR13 responses in hepatic macrophages.Graphical AbstractIn the absence of an endosomal RNase, RNase T2, microbiome RNAs circulating in the vasculature activate TLR13 in hepatic macrophages to drive hepatoprotective responses through expression of immunoregulatory and tissue-clearance molecules. Consequently, mice lacking RNase T2 are resistant against acute liver injuries caused by acetaminophen and LPS + D-galactosamine.

The small GTPase Rho and its effector Rho-kinase (ROCK) are involved in the pathogenesis of diabetic kidney disease. Accumulating evidence shows that renal dysfunction in diabetic patient is associated with abnormal fatty acid oxidation in the kidney. However, the interaction of ROCK and fatty acid oxidation in diabetic kidneys remains unclear. Extracellular flux analyzer demonstrated that deficiency of ROCK1, one of the ROCK isoforms, improves TGF-β-induced mitochondrial dysfunction of cultured mesangial cells. ROCK1 deficiency rescued TGF-β-induced downregulation of fatty acid oxidation mediators such as peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase 1A (CPT1A). An investigation of mechanisms underlying this observation revealed activated ROCK1 functions through the phosphorylation of AMPK and increased expression of PGC-1α. Furthermore, fluorescence histochemistry showed that ROCK1 deficiency suppresses ROS production as a result of mitochondrial damage due to abnormal fatty acid metabolism. Consistent with this result, ROCK1 knockout mice exhibited reduced albuminuria and histological abnormalities along with the recovery of impaired fatty acid utilization and mitochondrial fragmentation. These observations indicate that ROCK1 is a key player in the development of diabetic renal injury. Glomerular ROCK1 may be a potential therapeutic target for the treatment of diabetic kidney disease. Disclosure Y. Nagai: None. K. Matoba: Research Support; Self; Eli Lilly Japan K. K. K. Sekiguchi: None. R. Ukichi: None. Y. Takeda: None. T. Akamine: None. Y. Kanazawa: None. K. Utsunomiya: None. R. Nishimura: Speaker’s Bureau; Self; Abbott Japan Co., Ltd., Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly Japan K. K., Kissei Pharmaceutical Co., Ltd., Medtronic, MSD Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited.

Abstract Osaka Gas Co., Ltd., jointly with NTT DATA Mathematical Systems Inc., has developed unit‐commitment model for optimizing dispatch of power generation units. Main purpose of this development is to simulate the future world of electric power system in accordance with national energy policy aiming to introduce large amount of renewable energy as well as vitalized cross‐border power exchange via PX market to enhance economically‐efficient power system operation. The model incorporates not only constraints of supply‐demand balance but also constraints of operating reserves, regulation reserves, maximum CO2‐emissions, etc.

The aim was to investigate the local osteogenic effect of fluvastatin incorporated into a biodegradable gelatin-hydrogel (GH) scaffold. The GH scaffolds were prepared through crosslinking by ultraviolet irradiation followed by freeze-drying. Two circular defects were surgically created on fifteen-week-old male rats calvaria. All defects of each rat were randomly filled with two of three treatments, specifically: fluvastatin incorporated into a GH disk (Flu-GH), distilled water incorporated into a GH disk, and no treatment. New bone formation was quantitatively analyzed after 7, 14, and 28 days using a micro-computed tomography (micro-CT) system, and histologically observed. Evaluation by micro-CT revealed a significant difference in new bone formation among the three kinds of defect. A highly osteogenic effect was observed in the Flu-GH group. The results showed that the fluvastatin incorporated into a biodegradable GH scaffold promoted osteogenesis in rat calvarial bone, indicating its potential for bone regeneration.

Cbfa1 (Runx2) is a well recognized factor for osteoblast differentiation. However its role during odontogenesis is not well known. We examined the morphogenesis of tooth and gene expression of matrix protein in Cbfa1-knockout mice at ed 17.5 and day 0 of birth and compared them with mandible bone development. Incisor tooth germ showed morphological and functional differentiation of odontoblasts with expression of osteopontin and osteocalcin, whereas the mandible bone-forming site displayed lack of osteoblastic differentiation, and absence of osteopontin and osteocalcin expression. Stage-specific and cytodifferentiation differences demonstrated: incisor tooth germ progressed to the bell stage, whereas molar tooth germ showed maturational arrest at bud to cap stage. Present findings suggest that 1. Cbfa1 is associated with morphogenesis of teeth and matrix protein gene expression, 2. Compared to the incisor tooth germ, the molar tooth germ is more strongly subjected to control by Cbfa1, and 3. In Cbfa1-knockout mice, the odontoblast-like cells in the incisor and the spindle cells in the mandible forming region showed different patterns of gene expression of matrix proteins which are common to both teeth and bones.

Tumor suppressor genes are defined as genetic elements whose loss or mutational inactivation allows cells to display one or more phenotypes of neoplastic growth. Frequent deletion in a chromosomal region suggests existence of a candidate tumor suppressor gene. We analyzed Ch2q21-37.3 region by using 17 polymorphic microsatellite markers in 39 matched oral normal and cancer tissues. Loss of heterozygosity (LOH) was detected at least one location in 36 of 39 (92%) tumor tissues. High deletions were detected at microsatellite marker locations, D2S2304 (35%), D2S111 (40%), D2S155 (35%), D2S164 (29%), D2S125 (71%) and D2S140 (39%). Three frequently deleted regions at 2q22, 2q35-36 and 2q37.3 were observed. Chromosomal 2q22-37.3 region is highly populated with genes. Several candidate tumor suppressor genes in this region including such as ING5, CASP8, CASP10, PPP1R7 and BOK are located. We are currently analyzing inactivation mutations and mRNA expressions in oral squaomus cell carcinomas.

We recently reported the frequent deletion of chromosome 4q25 region in oral squamous cell carcinoma and discussed the Caspase-6 as a possible tumor suppressor gene in this region due to its near location to this hot spot. We this time examined the role of Caspase-6 in oral cancers. mRNA expression level, protein localization and gene mutation of Caspase-6 were analyzed. Reverse-transcription-PCR analysis demonstrated decreased expression in 40% and equal expression in 30% of Caspase-6 mRNA of primary tumors as compared with that matched normal samples. cDNA sequencing analysis of Caspase-6 did not reveal any somatic mutation. Caspase-6 protein localization was in similar proportion with its mRNA expression. Failure of Caspase-6 response in tumor samples as compared to normal samples could suggest Caspase-6 as a candidate tumor suppressor gene in a subset of oral cancer.

A candidate tumor suppressor gene, ING1 has been recently cloned and mapped to 13q33-34 region. We characterized the genomic structure of the human ING1 gene and detected somatic mutations of the ING1 gene in head and neck squamous cell carcinomas. 23 out of 34 informative cases (68%) of tumors showed loss of heterozygosity at chromosome 13q33-34, where the ING1 gene is located. By sequence analysis three exons and 2 introns of ING1 were identified. Mutation analysis of ING1 gene showed three missense and three silent changes.

It is well known that apatite cement causes inflammatory response if it is exposed to blood before setting. In this respect, the hemostatic procedure is very important. However, it has not been clarified how initial hemostasis affects the other basic properties of apatite cement. In the present study, the effect of initial hemostasis on the setting reaction was simulated by allowing the apatite cement paste to be hardened in an incubator for 1 to 30 minutes and then immersed in saline up to 7 days. We found faster transformation of apatite cement to apatitic mineral and higher mechanical strength of the set mass when the cement paste underwent a longer pre-hardening period. We also found that earlier exposure of apatite cement to saline resulted in a set mass with larger porosity. It is thought that the larger porosity of the cement is caused by the penetration of liquid into the cement paste, thus leading to lower mechanical strength and slower transformation of the apatite cement to apatitic mineral. We concluded, therefore, that hemostatic procedure is important not only to prevent inflammatory response but also to obtain a set mass with higher mechanical strength and faster transformation to apatitic mineral.