TTR exon-humanized mouse optimal for verifying new therapies for FAP.

Familial amyloidotic polyneuropathy (FAP) is caused by a mutation in the transthyretin (TTR) gene. In addition, deposition of wild-type TTR can cause senile systemic amyloidosis (SSA). To date, we have produced several transgenic mouse models for FAP and SSA by introducing TTR genes with different promoters or mutations. However, mouse TTR can associate with human TTR to produce hybrid tetramers in transgenic mice. Thus, these transgenic mice cannot be used to test the efficacy of a new therapy. In this study, we attempted to construct an optimized mouse model to verify a new therapy. The TTR gene consists of 4 exons and 3 introns. We prepared two gRNAs, one for the exon 1 and the other for exon 4, and a single donor vector carrying the whole TTR gene in which mouse exons were replaced with human exons. Using these vectors, we produced a TTR exon-humanized mouse with human exons and mouse introns using genome editing technology. These TTR exon-humanized mice showed normal TTR expression patterns in terms of serum TTR level and spatial specificity. These TTR exon-humanized mice will be useful for devising new treatment methods for FAP, including gene therapy.

SOX9 contributes to the progression of ductular reaction for the protection from chronic liver injury.

The transcription factor sex-determining region Y-box 9 (SOX9) is a biliary epithelial marker ectopically expressed in hepatocytes (SOX9 + hepatocytes). SOX9 + hepatocytes are believed to function in ductular reaction (DR), recognized as an essential phenomenon related to liver regeneration; however, the functional role of SOX9 and clinical implications of SOX9 + hepatocytes in DR progression are unclear. Human and mouse liver samples were subjected to immunohistochemical and gene functional analyses to investigate the functional role of SOX9 and the clinical significance of SOX9 + hepatocytes. SOX9 + hepatocytes were observed in a bile duct ligation (BDL) mouse model. Forced Sox9 expression in mouse hepatocytes by hydrodynamic injection converted them into cholangiocyte-like cells. DR progression was slower in liver epithelium-specific Sox9-knockout BDL mice than in wild-type BDL mice. SOX9 + hepatocytes were also observed in rare pediatric liver disease biliary atresia (BA). In patients with BA who underwent liver transplantation (LT), the median number of SOX9 + hepatocytes at LT was significantly lower than that at Kasai portoenterostomy (KP) performed prior to LT (P < 0.001). The high SOX9 + hepatocyte group at KP demonstrated significantly better native liver survival rates than the low SOX9 + hepatocyte group at a cut-off of 390 cells/mm2 (P = 0.019, log-rank test). Ectopic expression of SOX9 in hepatocytes of chronically injured livers may exert protective effects in DR progression. To our knowledge, this is the first study showing that SOX9 + hepatocyte count at KP can be a promising biomarker to predict native liver survival after KP in patients with BA.

Topoisomerase IIIß Deficiency Induces Neuro-Behavioral Changes and Brain Connectivity Alterations in Mice.

Topoisomerase IIIß (Top3ß), the only dual-activity topoisomerase in mammals that can change topology of both DNA and RNA, is known to be associated with neurodevelopment and mental dysfunction in humans. However, there is no report showing clear associations of Top3ß with neuropsychiatric phenotypes in mice. Here, we investigated the effect of Top3ß on neuro-behavior using newly generated Top3ß deficient (Top3ß-/-) mice. We found that Top3ß-/- mice showed decreased anxiety and depression-like behaviors. The lack of Top3ß was also associated with changes in circadian rhythm. In addition, a clear expression of Top3ß was demonstrated in the central nervous system of mice. Positron emission tomography/computed tomography (PET/CT) analysis revealed significantly altered connectivity between many brain regions in Top3ß-/- mice, including the connectivity between the olfactory bulb and the cerebellum, the connectivity between the amygdala and the olfactory bulb, and the connectivity between the globus pallidus and the optic nerve. These connectivity alterations in brain regions are known to be linked to neurodevelopmental as well as psychiatric and behavioral disorders in humans. Therefore, we conclude that Top3ß is essential for normal brain function and behavior in mice and that Top3ß could be an interesting target to study neuropsychiatric disorders in humans.

Cathepsin B and D deficiency in the mouse pancreas induces impaired autophagy and chronic pancreatitis.

The major lysosomal proteases, Cathepsin B (CTSB), Cathepsin D (CTSD) and Cathepsin L (CTSL), are implicated in autophagic activity. To investigate the role of each cathepsin in the exocrine pancreas, we generated mice in which the pancreas was specifically deficient in Ctsb, Ctsd and Ctsl. Each of these gene knockout (KO) and Ctsb;Ctsl and Ctsd;Ctsl double-knockout (DKO) mice were almost normal. However, we found cytoplasmic degeneration in the pancreatic acinar cells of Ctsb;Ctsd DKO mice, similar to autophagy related 5 (Atg5) KO mice. LC3 and p62 (autophagy markers) showed remarkable accumulation and the numbers of autophagosomes and autolysosomes were increased in the pancreatic acinar cells of Ctsb;Ctsd DKO mice. Moreover, these Ctsb;Ctsd DKO mice also developed chronic pancreatitis (CP). Thus, we conclude that both Ctsb and Ctsd deficiency caused impaired autophagy in the pancreatic acinar cells, and induced CP in mice.

QKI is a critical pre-mRNA alternative splicing regulator of cardiac myofibrillogenesis and contractile function.

The RNA-binding protein QKI belongs to the hnRNP K-homology domain protein family, a well-known regulator of pre-mRNA alternative splicing and is associated with several neurodevelopmental disorders. Qki is found highly expressed in developing and adult hearts. By employing the human embryonic stem cell (hESC) to cardiomyocyte differentiation system and generating QKI-deficient hESCs (hESCs-QKIdel) using CRISPR/Cas9 gene editing technology, we analyze the physiological role of QKI in cardiomyocyte differentiation, maturation, and contractile function. hESCs-QKIdel largely maintain normal pluripotency and normal differentiation potential for the generation of early cardiogenic progenitors, but they fail to transition into functional cardiomyocytes. In this work, by using a series of transcriptomic, cell and biochemical analyses, and the Qki-deficient mouse model, we demonstrate that QKI is indispensable to cardiac sarcomerogenesis and cardiac function through its regulation of alternative splicing in genes involved in Z-disc formation and contractile physiology, suggesting that QKI is associated with the pathogenesis of certain forms of cardiomyopathies.

HNF1α controls glucagon secretion in pancreatic α-cells through modulation of SGLT1.

Hepatocyte nuclear factor 1α (HNF1α) is a transcription factor required for normal insulin secretion and maintenance of ß-cell number in the pancreas. HNF1α is also expressed in pancreatic α-cells, but its role in these cells is unknown. The aim of this study was to clarify the role of HNF1α in α-cells. Male Hnf1a+/- mice with a mixed background were backcrossed to outbred ICR mice. Glucose tolerance, glucagon and insulin secretion, islet histology, and gene expression were investigated in ICR Hnf1a-/- and Hnf1a+/+ mice. Regulation of Slc5a1 (encoding sodium glucose cotransporter 1 [SGLT1]) expression by HNF1α and the effect of SGLT1 inhibition on glucagon secretion were also explored. ICR Hnf1a-/- mice were glucose intolerant and exhibited impaired glucose-stimulated insulin secretion. The ß-cell area of ICR mice was decreased in Hnf1a-/- mice, but the α-cell area in the pancreas was similar between Hnf1a-/- and Hnf1a+/+ mice. Hnf1a-/- mice showed higher fasting glucagon levels and exhibited inadequate suppression of glucagon after glucose load. In addition, glucagon release in response to hypoglycemia was impaired in Hnf1a-/- mice, and glucagon secretion after 1.1 mM glucose administration, was also decreased in Hnf1a-/- islets. Slc5a1 expression was decreased in Hnf1a-/- islets, while HNF1α activated the Slc5a1 promoter in αTC1-6 cells. Inhibition of SGLT1 suppressed 1.1 mM glucose-stimulated glucagon secretion in islets and αTC1-6 cells, but SGLT1 inhibition had no additional inhibitory effect in HNF1α-deficient cells. Our findings indicate that HNF1α modulates glucagon secretion in α-cells through the regulation of Slc5a1.

CRISPR/Cas9-mediated gene correction in hemophilia B patient-derived iPSCs.

The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system is an efficient genome-editing tool that holds potential for gene therapy. Here, we report an application of this system for gene repair in hemophilia B (HB) using induced pluripotent stem cells (iPSCs). We prepared targeting plasmids with homology arms containing corrected sequences to repair an in-frame deletion in exon 2 of the factor IX (F9) gene and transfected patient-derived iPSCs with the Cas9 nuclease and a guide RNA expression vector. To validate the expression of corrected F9, we attempted to induce the differentiation of iPSCs toward hepatocyte-like cells (HLCs) in vitro. We successfully repaired a disease-causing mutation in HB in patient-derived iPSCs. The transcription product of corrected F9 was confirmed in HLCs differentiated from gene-corrected iPSCs. Although further research should be undertaken to obtain completely functional hepatocytes with secretion of coagulation factor IX, our study provides a proof-of-principle for HB gene therapy using the CRISPR/Cas9 system.

Pivotal role of inter-organ aspartate metabolism for treatment of mitochondrial aspartate-glutamate carrier 2 (citrin) deficiency, based on the mouse model.

Previous studies using citrin/mitochondrial glycerol-3-phosphate (G3P) dehydrogenase (mGPD) double-knockout mice have demonstrated that increased dietary protein reduces the extent of carbohydrate-induced hyperammonemia observed in these mice. This study aimed to further elucidate the mechanisms of this effect. Specific amino acids were initially found to decrease hepatic G3P, or increase aspartate or citrulline levels, in mGPD-knockout mice administered ethanol. Unexpectedly, oral glycine increased ammonia in addition to lowering G3P and increasing citrulline. Subsequently, simultaneous glycine-plus-sucrose (Gly + Suc) administration led to a more severe hyperammonemic state in double-KO mice compared to sucrose alone. Oral arginine, ornithine, aspartate, alanine, glutamate and medium-chain triglycerides all lowered blood ammonia following Gly + Suc administration, with combinations of ornithine-plus-aspartate (Orn + Asp) or ornithine-plus-alanine (Orn + Ala) suppressing levels similar to wild-type. Liver perfusion and portal vein-arterial amino acid differences suggest that oral aspartate, similar to alanine, likely activated ureagenesis from ammonia and lowered the cytosolic NADH/NAD+ ratio through conversion to alanine in the small intestine. In conclusion, Gly + Suc administration induces a more severe hyperammonemic state in double-KO mice that Orn + Asp or Orn + Ala both effectively suppress. Aspartate-to-alanine conversion in the small intestine allows for effective oral administration of either, demonstrating a pivotal role of inter-organ aspartate metabolism for the treatment of citrin deficiency.

Recessive mutation in CD2AP causes focal segmental glomerulosclerosis in humans and mice.

Although sequence variants in CD2-associated protein (CD2AP) have been identified in patients with focal segmental glomerulosclerosis (FSGS), definitive proof of causality in human disease is meager. By whole-exome sequencing, we identified a homozygous frame-shift mutation in CD2AP (p.S198fs) in three siblings born of consanguineous parents who developed childhood-onset FSGS and end stage renal disease. When the same frameshift mutation was introduced in mice by gene editing, the mice developed FSGS and kidney failure. These results provide conclusive evidence that homozygous mutation of CD2AP causes FSGS in humans.

Independent modes of disease repair by AIM protein distinguished in AIM-felinized mice.

Tissue macrophage-derived apoptosis inhibitor of macrophage (AIM, encoded by cd5l gene) is a circulating protein that has suppressive functions in a broad range of diseases including obesity, liver steatosis, hepatocellular carcinoma (HCC), and acute kidney injury (AKI). In healthy states, high levels of AIM circulate in the inactivated state by associating with the immunoglobulin M (IgM) pentamer in the blood, whereas during AKI, AIM dissociates from IgM and gains disease repair activity. Here, we assessed whether AIM activation via its release from IgM is required to ameliorate other diseases. To this end, we employed a mouse line in which mouse AIM was replaced with feline AIM (AIM-felinized mice). Because feline AIM rarely dissociates from IgM due to its extremely high binding affinity for IgM, these mice exhibited deficient AKI repair as in cats. When fed a high-fat diet (HFD), similar to AIM-deficient (AIM-/-) mice, AIM-felinized mice exhibited enhanced triacylglycerol deposition in visceral adipocytes and hepatocytes, resulting in more prominent obesity and fatty liver than in wild-type mice. In contrast, the incidence of HCC after a 1-year HFD was remarkably lower in AIM-felinized mice than in AIM-/- mice, suggesting that AIM produced by liver Kupffer macrophages might directly facilitate the elimination of HCC cells. Accordingly, the marked deposition of AIM accompanied by accumulation of Kupffer cells was obvious during HCC tumour development in AIM-felinized mice. Δsµ mice, which harbour almost no circulating AIM due to the lack of secreted IgM, showed a phenotype comparable with that of AIM-felinized mice in prevention of those diseases. Thus, blood AIM released from IgM contributes to suppression of obesity and fatty liver as in AKI, whereas macrophage-derived noncirculating AIM mainly prevents HCC development. Our study depicted two different modes of disease prevention/repair facilitated by AIM, which could be the basis for HCC therapy that works by increasing AIM expression in macrophages.

Fryl deficiency is associated with defective kidney development and function in mice.

FRY like transcription coactivator ( Fryl) gene located on chromosome 5 is a paralog of FRY microtubule binding protein ( Fry) in vertebrates. It encodes a protein with unknown functions. Fryl gene is conserved in various species ranging from eukaryotes to human. Although there are several reports on functions of Fry gene, functions of Fryl gene remain unclear. A mouse line containing null mutation in Fryl gene by gene trapping was produced in this study for the first time. The survival and growth of Fryl-/- mice were observed. Fryl gene expression levels in mouse tissues were determined and histopathologic analyses were conducted. Most Fryl-/- mice died soon after birth. Rare Fryl-/- survivors showed growth retardation with significantly lower body weight compared to their littermate controls. Although they could breed, more than half of Fryl-/- survivors died of hydronephrosis before age 1. No abnormal histopathologic lesion was apparent in full-term embryo or adult tissues except the kidney. Abnormal lining cell layer detachments from walls of collecting and convoluted tubules in kidneys were apparent in Fryl-/- neonates and full-term embryos. Fryl gene was expressed in renal tubular tissues including the glomeruli and convoluted and collecting tubules. This indicates that defects in tubular systems are associated with Fryl functions and death of Fryl-/- neonates. Fryl protein is required for normal development and functional maintenance of kidney in mice. This is the first report of in vivo Fryl gene functions. Impact statement FRY like transcription coactivator ( Fryl) gene is conserved in various species ranging from eukaryotes to human. It expresses a protein with unknown function. We generated a Fryl gene mutant mouse line and found that most homozygous mice died soon after their birth. Rare Fryl-/- survivors showed growth retardation with significantly lower body weight compared to their littermate controls. Although they could breed, more than half of Fryl-/- survivors died of hydronephrosis before age 1. Full-term mutant embryos showed abnormal collecting and convoluted tubules in kidneys where Fryl gene was expressed. Collectively, these results indicate that Fryl protein is required for normal development and functional maintenance of kidney in mice. To the best of our knowledge, this is the first report on in vivo Fryl gene functions.

LPA5 signaling is involved in multiple sclerosis-mediated neuropathic pain in the cuprizone mouse model.

Lysophosphatidic acid (LPA) and LPA1 receptor signaling play a crucial role in the initiation of peripheral nerve injury-induced neuropathic pain through the alternation of pain-related genes/proteins expression and demyelination. However, LPA and its signaling in the brain are still poorly understood. In the present study, we revealed that the LPA5 receptor expression in corpus callosum elevated after the initiation of demyelination, and the hyperalgesia through Aδ-fibers following cuprizone-induced demyelination was mediated by LPA5 signaling. These data suggest that LPA5 signaling may play a key role in the mechanisms underlying neuropathic pain following demyelination in the brain.

Amyloid deposition in a mouse model humanized at the transthyretin and retinol-binding protein 4 loci.

Familial amyloidotic polyneuropathy is an autosomal dominant disorder caused by a point mutation in the transthyretin (TTR) gene. The process of TTR amyloidogenesis begins with rate-limiting dissociation of the TTR tetramer. Thus, the TTR stabilizers, such as Tafamidis and Diflunisal, are now in clinical trials. Mouse models will be useful to testing the efficacy of these drugs. Although several mouse models have been generated, they all express mouse Rbp4. Thus, human TTR associates with mouse RBP4, resulting in different kinetic and thermodynamic stability profiles of TTR tetramers. To overcome this problem, we previously produced humanized mouse strains at both the TTR and Rbp4 loci (Ttr hTTRVal30 , Ttr hTTRMet30 , and Rbp4 hRBP4 ). By mating these mice, we produced double-humanized mouse strains, Ttr hTTRVal30/hTTRVal30 :Rbp4 hRBP4/hRBP4 and Ttr hTTRVal30/Met30 :Rbp4 hRBP4/hRBP4 . We used conventional transgenic mouse strains on a wild-type (Ttr +/+ :Tg[6.0hTTRMet30]) or knockout Ttr background (Ttr-/-:Tg[6.0hTTRMet30]) as reference strains. The double-humanized mouse showed 1/25 of serum hTTR and 1/40 of serum hRBP4 levels. However, amyloid deposition was more pronounced in Ttr hTTRVal30/Met30 :Rbp4 hRBP4/hRBP4 than in conventional transgenic mouse strains. In addition, a similar amount of amyloid deposition was also observed in Ttr hTTRVal30/ hTTRVal30 :Rbp4 hRBP4/ hRBP4 mice that carried the wild-type human TTR gene. Furthermore, amyloid deposition was first observed in the sciatic nerve without any additional genetic change. In all strains, anti-TTR antibody-positive deposits were found in earlier age and at higher percentage than amyloid fibril deposition. In double-humanized mice, gel filtration analysis of serum revealed that most hTTR was free of hRBP4, suggesting importance of free TTR for amyloid deposition.

Co-precipitation molecules hemopexin and transferrin may be key molecules for fibrillogenesis in TTR V30M amyloidogenesis.

The disease model of familial amyloidotic polyneuropathy-7.2-hMet30 mice-manifests amyloid deposition that consists of a human amyloidogenic mutant transthyretin (TTR) (TTR V30M). Our previous study found amyloid deposits in 14 of 27 7.2-hMet30 mice at 21-24 months of age. In addition, non-fibrillar TTR deposits were found in amyloid-negative 7.2hMet30 mice. These results suggested that TTR amyloidogenesis required not only mutant TTR but also an additional factor (or factors) as an etiologic molecule. To determine the differences in serum proteome in amyloid-positive and amyloid-negative mice in the 7.2-hMet30 model, we used proteomic analyses and studied serum samples obtained from these mice. Hemopexin (HPX) and transferrin (Tf) were detected in the serum samples from amyloid-positive mice and were also found in amyloid deposits via immunohistochemistry, but serum samples from amyloid-negative mice did not contain HPX and Tf. These two proteins were also not detected in non-fibrillar TTR deposits. In addition, in silico analyses suggested that HPX and Tf facilitate destabilization of TTR secondary structures and misfolding of TTR. These results suggest that HPX and Tf may be associated with TTR amyloidogenesis after fibrillogenesis in vivo.

Deletion of GIRK2 subunit containing GIRK channels of neurons expressing dopamine transporter decrease immobility time on forced swimming in mice.

We previously reported that non-narcotic antitussives possessing inhibitory actions on G protein-coupled inwardly rectifying potassium (GIRK) channels have antidepressant-like effects in the forced swimming test in normal and adrenocoticotropic hormone (ACTH) treated rats. Furthermore, the antidepressant-like effects of the antitussives such as tipepidine were blocked by dopamine D1 receptor antagonist, and inhibitory actions on GIRK channels of dopamine neurons may be involved in the antidepressant-like effects of tipepidine. In this study, we generated GIRK2DATKO mice with Girk2/Kcnj6 conditional deletion and assessed depression-related behavior of the mice. The Cre/loxP system was used to selectively delete GIRK2 subunit containing GIRK channels in the neurons expressing dopamine transporter. First, deletion of GIRK2 subunits in the ventral tegmental area (VTA) neurons expressing dopamine transporters was confirmed by hisitochemically and electrophysiologically. In the mice, a significant decrease in the immobility time of forced swimming test was observed. Locomotor activity of the mice was not changed compared to that of GIRK2floxed mice, when tested in the open field. These results suggest that the antidepressant-like effect of antitussives such as tipepidine may be caused partly through the inhibitory actions on GIRK channels in the dopamine neurons.

CSP-1103 (CHF5074) stabilizes human transthyretin in healthy human subjects.

Hereditary amyloid polyneuropathy is a type of protein misfolding disease. Transthyretin (TTR) is a homotetrameric serum protein and TTR tetramer dissociation is the limiting step in amyloid fibril formation. Thus, prevention of TTR dissociation is a promising therapeutic approach and some TTR stabilizers have been approved for the treatment of TTR amyloidosis. CSP-1103 (CHF5074) is a non-steroidal anti-inflammatory derivative that lacks cyclooxygenase inhibitory activity. In vitro, CSP-1103 stabilizes the TTR tetramer by binding to the thyroxine (T4) binding site. We have previously shown that serum TTR levels were increased by oral CSP-1103 administration through stabilization of TTR tetramers in humanized mice at both the Ttr locus and the Rbp4 locus. To determine whether CSP-1103 stabilizes TTR tetramers in humans, multiple CSP-1103 oral doses were administered for two weeks to 48 healthy human volunteers in a double-blind, placebo-controlled, parallel-group study. CSP-1103 treatment stabilized TTR tetramers in a dose-dependent manner under normal or denaturing stress conditions, thereby increasing serum TTR levels. Preincubation of serum with CSP-1103 or diflunisal in vitro increased the TTR tetramer stability. Computer simulation analysis revealed that the binding affinities of CSP-1103 with TTR at pH 7.0 were similar to those of tafamidis, thus confirming that CSP-1103 has potent TTR-stabilizing activity.

Transgenic mouse model for imaging of ATF4 translational activation-related cellular stress responses in vivo.

Activating transcription factor 4 (ATF4) is a translationally activated protein that plays a role in cellular adaptation to several stresses. Because these stresses are associated with various diseases, the translational control of ATF4 needs to be evaluated from the physiological and pathological points of view. We have developed a transgenic mouse model to monitor the translational activation of ATF4 in response to cellular stress. By using this mouse model, we were able to detect nutrient starvation response, antivirus response, endoplasmic reticulum (ER) stress response, and oxidative stress in vitro and ex vivo, as well as in vivo. The reporter system introduced into our mouse model was also shown to work in a stress intensity-dependent manner and a stress duration-dependent manner. The mouse model is therefore a useful tool for imaging ATF4 translational activation at various levels, from cell cultures to whole bodies, and it has a range of useful applications in investigations on the physiological and pathological roles of ATF4-related stress and in the development of clinical drugs for treating ATF4-associated diseases.

Oral aversion to dietary sugar, ethanol and glycerol correlates with alterations in specific hepatic metabolites in a mouse model of human citrin deficiency.

Mice carrying simultaneous homozygous mutations in the genes encoding citrin, the mitochondrial aspartate-glutamate carrier 2 (AGC2) protein, and mitochondrial glycerol-3-phosphate dehydrogenase (mGPD), are a phenotypically representative model of human citrin (a.k.a., AGC2) deficiency. In this study, we investigated the voluntary oral intake and preference for sucrose, glycerol or ethanol solutions by wild-type, citrin (Ctrn)-knockout (KO), mGPD-KO, and Ctrn/mGPD double-KO mice; all substances that are known or suspected precipitating factors in the pathogenesis of human citrin deficiency. The double-KO mice showed clear suppressed intake of sucrose, consuming less with progressively higher concentrations compared to the other mice. Similar observations were made when glycerol or ethanol were given. The preference of Ctrn-KO and mGPD-KO mice varied with the different treatments; essentially no differences were observed for sucrose, while an intermediate intake or similar to that of the double-KO mice was observed for glycerol and ethanol. We next examined the hepatic glycerol 3-phosphate, citrate, citrulline, lysine, glutamate and adenine nucleotide levels following forced enteral administration of these solutions. A strong correlation between the simultaneous increased hepatic glycerol 3-phosphate and decreased ATP or total adenine nucleotide content and observed aversion of the mice during evaluation of their voluntary preferences was found. Overall, our results suggest that the aversion observed in the double-KO mice to these solutions is initiated and/or mediated by hepatic metabolic perturbations, resulting in a behavioral response to increased hepatic cytosolic NADH and a decreased cellular adenine nucleotide pool. These findings may underlie the dietary predilections observed in human citrin deficient patients.

Endothelial CD99 supports arrest of mouse neutrophils in venules and binds to neutrophil PILRs.

CD99 is a crucial regulator of the transmigration (diapedesis) of leukocytes through the blood vessel wall. Here, we report that CD99 acts at 2 different steps in the extravasation process. In agreement with previous antibody-blocking experiments, we found that CD99 gene inactivation caused neutrophil accumulation between venular endothelial cells and the basement membrane in the inflamed cremaster. Unexpectedly, we additionally found that leukocyte attachment to the luminal surface of the venular endothelium was impaired in the absence of CD99. Intravital video microscopy revealed that CD99 supported rapid chemokine-induced leukocyte arrest. Inhibition of leukocyte attachment and extravasation were both solely due to the absence of CD99 on endothelial cells, whereas CD99 on leukocytes was irrelevant. Therefore, we searched for heterophilic ligands of endothelial CD99 on neutrophils. We found that endothelial cells bind to the paired immunoglobulinlike receptors (PILRs) in a strictly CD99-dependent way. In addition, endothelial CD99 was coprecipitated with PILRs from neutrophils that adhered to endothelial cells. Furthermore, soluble CD99 carrying a transferable biotin tag could transfer this tag covalently to PILR when incubated with intact neutrophils. Binding of neutrophils under flow to a surface coated with P-selectin fragment crystallizable (Fc) and intercellular adhesion molecule 1 (ICAM-1) Fc became more shear resistant if CD99 Fc was coimmobilized. This increased shear resistance was lost if neutrophils were preincubated with anti-PILR antibodies. We concluded that endothelial CD99 promotes leukocyte attachment to endothelium in inflamed vessels by a heterophilic ligand. In addition, CD99 binds to PILRs on neutrophils, an interaction that leads to increased shear resistance of the neutrophil attachment to ICAM-1.

Inflammatory and anti-inflammatory states of adipose tissue in transgenic mice bearing a single TCR.

Obesity is accompanied by chronic, low-grade inflammation in adipose tissue, which is associated with insulin resistance and consequent multiple metabolic diseases. In addition to M1 macrophage infiltration, multiple involvements of adipose tissue T lymphocytes in the progression of inflammation have been highlighted recently. Here, we isolated a specific Vα5/Vß8.2 TCR-bearing T cell that accumulated in obese adipose tissue of mice, and generated transgenic mice expressing this TCR. Under lean conditions with a normal chow diet, CD4+FoxP3+ Treg cells and M2 macrophages increased in adipose tissue with ageing in wild-type mice, but not in transgenic mice. However, both mice exhibited no obvious adipose tissue inflammation such as the formation of crown-like structures (CLSs) of infiltrating macrophages. When fed a high-fat diet, the proportion of adipose tissue Treg cells was markedly small at a similar level in transgenic and wild-type mice. Both types of mice exhibited comparable inflammatory states in adipose tissue, including vast formation of macrophage CLSs, accompanied by insulin resistance. Together, our findings suggest that the absence of an increase in Treg cells and M2 macrophages is not sufficient to initiate inflammatory macrophage infiltration in lean adipose tissue and also provide a new view about the involvement of T cells in promoting obesity-associated inflammation.