Sortilin regulates blood-brain barrier integrity.

Brain homeostasis depends on the existence of the blood-brain barrier (BBB). Despite decades of research, the factors and signalling pathways for modulating and maintaining BBB integrity are not fully elucidated. Here, we characterise the expression and function of the multifunctional receptor, sortilin, in the cells of the BBB, in vivo and in vitro. We show that sortilin acts as an important regulatory protein of the BBB's tightness. In rats lacking sortilin, the BBB was leaky, which correlated well with relocated distribution of the localisation of zonula occludens-1, VE-cadherin and ß-catenin junctional proteins. Furthermore, the absence of sortilin in brain endothelial cells resulted in decreased phosphorylation of Akt signalling protein and increased the level of phospho-ERK1/2. As a putative result of MAPK/ERK pathway activity, the junctions between the brain endothelial cells were disintegrated and the integrity of the BBB became compromised. The identified barrier differences between wild-type and Sort1-/- brain endothelial cells can pave the way for a better understanding of sortilin's role in the healthy and diseased BBB.

SCYL1 disease and liver transplantation diagnosed by reanalysis of exome sequencing and deletion/duplication analysis of SCYL1.

SCYL1 disease results from biallelic pathogenic variants in SCYL1. We report two new patients with severe hepatic phenotype requiring liver transplantation. Patient charts reviewed. DNA samples and skin fibroblasts were utilized. Literature was reviewed. 13-year-old boy and 9-year-old girl siblings had acute liver insufficiency and underwent living related donor liver transplantation in infancy with no genetic diagnosis. Both had tremor, global developmental delay, and cognitive dysfunction during their follow-up in the medical genetic clinic for diagnostic investigations after their liver transplantation. Exome sequencing identified a likely pathogenic variant (c.399delC; p.Asn133Lysfs*136) in SCYL1. Deletion/duplication analysis of SCYL1 identified deletions of exons 7-8 in Patient 1. Both variants were confirmed in Patient 2 and the diagnosis of SCYL1 disease was confirmed in both patients at the age of 13 and 9 years, respectively. SCYL1 protein was not expressed in both patients' fibroblast using western blot analysis. Sixteen patients with SCYL1 disease reported in the literature. Liver phenotype (n = 16), neurological phenotype (n = 13) and skeletal phenotype (n = 11) were present. Both siblings required liver transplantation in infancy and had variable phenotypes. Exome sequencing may miss the diagnosis and phenotyping of patients can help to diagnose patients.

Tristetraprolin regulates necroptosis during tonic Toll-like receptor 4 (TLR4) signaling in murine macrophages.

The necrosome is a protein complex required for signaling in cells that results in necroptosis, which is also dependent on tumor necrosis factor receptor (TNF-R) signaling. TNFα promotes necroptosis, and its expression is facilitated by mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2) but is inhibited by the RNA-binding protein tristetraprolin (TTP, encoded by the Zfp36 gene). We have stimulated murine macrophages from WT, MyD88-/-, Trif-/-, MyD88-/-Trif-/-, MK2-/-, and Zfp36-/- mice with graded doses of lipopolysaccharide (LPS) and various inhibitors to evaluate the role of various genes in Toll-like receptor 4 (TLR4)-induced necroptosis. Necrosome signaling, cytokine production, and cell death were evaluated by immunoblotting, ELISA, and cell death assays, respectively. We observed that during TLR4 signaling, necrosome activation is mediated through the adaptor proteins MyD88 and TRIF, and this is inhibited by MK2. In the absence of MK2-mediated necrosome activation, lipopolysaccharide-induced TNFα expression was drastically reduced, but MK2-deficient cells became highly sensitive to necroptosis even at low TNFα levels. In contrast, during tonic TLR4 signaling, WT cells did not undergo necroptosis, even when MK2 was disabled. Of note, necroptosis occurred only in the absence of TTP and was mediated by the expression of TNFα and activation of JUN N-terminal kinase (JNK). These results reveal that TTP plays an important role in inhibiting TNFα/JNK-induced necrosome signaling and resultant cytotoxicity.

The LC3-conjugation machinery specifies the loading of RNA-binding proteins into extracellular vesicles.

Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate that these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2) and LC3-dependent recruitment of factor associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.

A Population of Radio-Resistant Macrophages in the Deep Myenteric Plexus Contributes to Postoperative Ileus Via Toll-Like Receptor 3 Signaling.

Postoperative ileus (POI) is triggered by an innate immune response in the muscularis externa (ME) and is accompanied by bacterial translocation. Bacteria can trigger an innate immune response via toll-like receptor (TLR) activation, but the latter's contribution to POI has been disproved for several TLRs, including TLR2 and TLR4. Herein we investigated the role of double-stranded RNA detection via TLR3 and TIR-domain-containing adapter-inducing interferon-ß (TRIF) signaling pathway in POI. POI was induced by small bowel intestinal manipulation in wt, TRIF-/-, TLR3-/-, type I interferon receptor-/- and interferon-ß reporter mice, all on C57BL/6 background, and POI severity was quantified by gene expression analysis, gastrointestinal transit and leukocyte extravasation into the ME. TRIF/TLR3 deficiency reduced postoperative ME inflammation and prevented POI. With bone marrow transplantation, RNA-sequencing, flow cytometry and immunohistochemistry we revealed a distinct TLR3-expressing radio-resistant MHCIIhiCX3CR1- IBA-1+ resident macrophage population within the deep myenteric plexus. TLR3 deficiency in these cells, but not in MHCIIhiCX3CR1+ macrophages, reduced cytokine expression in POI. While this might not be an exclusive macrophage-privileged pathway, the TLR3/TRIF axis contributes to proinflammatory cytokine production in MHCIIhiCX3CR1- IBA-1+ macrophages during POI. Deficiency in TLR3/TRIF protects mice from POI. These data suggest that TLR3 antagonism may prevent POI in humans.

Mechanical stiffness of reconstituted actin patches correlates tightly with endocytosis efficiency.

Clathrin-mediated endocytosis involves the sequential assembly of more than 60 proteins at the plasma membrane. An important fraction of these proteins regulates the assembly of an actin-related protein 2/3 (Arp2/3)-branched actin network, which is essential to generate the force during membrane invagination. We performed, on wild-type (WT) yeast and mutant strains lacking putative actin crosslinkers, a side-by-side comparison of in vivo endocytic phenotypes and in vitro rigidity measurements of reconstituted actin patches. We found a clear correlation between softer actin networks and a decreased efficiency of endocytosis. Our observations support a chain-of-consequences model in which loss of actin crosslinking softens Arp2/3-branched actin networks, directly limiting the transmission of the force. Additionally, the lifetime of failed endocytic patches increases, leading to a larger number of patches and a reduced pool of polymerizable actin, which slows down actin assembly and further impairs endocytosis.

Toll-like receptors 2 and 7 mediate coagulation activation and coagulopathy in murine sepsis.

BACKGROUND: Sepsis is a life-threatening condition often manifested as marked inflammation and severe coagulopathy. Toll-like receptors (TLRs) play a pivotal role in inflammation, organ dysfunction and mortality in animal sepsis. OBJECTIVES: To investigate the role of TLR signaling in mediating sepsis-induced coagulopathy (SIC) in a mouse model. METHODS: Polymicrobial sepsis was created by cecal ligation and puncture (CLP) or fecal slurry peritoneal injection. To quantify global clotting function, two viscoelastic assays were performed with rotational thromboelastometry, and the results were presented as maximum clot firmness (MCF): (a) EXTEM to test tissue factor (TF)-initiated clot formation; and (b) FIBTEM to test EXTEM in the presence of a platelet inhibitor, cytochalasin D. Plasma coagulation factors were quantified with ELISA. TF gene expression and protein expression were determined with real-time quantitative reverse transcription PCR and flow cytometry, respectively. RESULTS: Between 4 and 24 hours after CLP surgery, wild-type mice showed significant MCF reduction in both EXTEM and FIBTEM tests. This was accompanied by marked thrombocytopenia and a significant increase in the levels of plasminogen activator inhibitor-1, plasma TF, and D-dimer. In comparison, TLR2-/- and TLR7-/- CLP mice showed preserved MCF and platelet counts, and near-normal plasma TF levels. Bone marrow-derived macrophages treated with a TLR2 agonist Pam3cys-Ser-(Lys)4 (Pam3cys) or a TLR7 agonist (R837) showed marked increases in TF gene expression and protein expression. MicroRNA-146a, a newly identified proinflammatory mediator that is upregulated during sepsis, induced TF production via a TLR7-dependent mechanism. CONCLUSIONS: Murine sepsis leads to an increased procoagulant response, thrombocytopenia, and global coagulopathy. TLR2 and TLR7 play an important role in procoagulant production and in SIC.

Hepatocyte sortilin 1 knockout and treatment with a sortilin 1 inhibitor reduced plasma cholesterol in Western diet-fed mice.

Sortilin 1 (Sort1) is a member of the Vps10p domain intracellular trafficking receptor family. Genetic variations of the SORT1 gene are strongly associated with plasma cholesterol levels in humans. Recent studies have linked Sort1 to regulation of cholesterol metabolism in hepatocytes and pro-inflammatory response in macrophages, but the tissue-specific roles of Sort1 in lipid metabolism have not been well defined. We developed Sort1 floxed mice and investigated the development of Western diet (WD)-induced steatosis, hepatic inflammatory response, and hyperlipidemia in hepatocyte Sort1 KO mice and myeloid cell Sort1 KO mice. Our findings suggest that hepatocyte Sort1 deficiency attenuated diet-induced hepatic steatosis and hypercholesterolemia in mice. In contrast, myeloid Sort1 deficiency did not reduce hepatic cytokine expression or plasma cholesterol levels, but exacerbated hepatic triglyceride accumulation in WD-fed mice. Finally, we showed that treating WD-fed mice with an orally bioavailable Sort1 inhibitor, AF38469, decreased plasma cholesterol and hepatic cytokine expression. AF38469 treatment did not affect diet-induced obesity or insulin resistance, but was associated with reduced hepatic VLDL secretion and higher hepatic cholesterol 7α-hydrolase expression in WD-fed mice. In conclusion, findings from this study suggest that Sort1 loss-of-function in hepatocytes contributes to lower plasma cholesterol, and pharmacological inhibition of Sort1 attenuates diet-induced hypercholesterolemia in mice.

Epsin deficiency promotes lymphangiogenesis through regulation of VEGFR3 degradation in diabetes.

Impaired lymphangiogenesis is a complication of chronic complex diseases, including diabetes. VEGF-C/VEGFR3 signaling promotes lymphangiogenesis, but how this pathway is affected in diabetes remains poorly understood. We previously demonstrated that loss of epsins 1 and 2 in lymphatic endothelial cells (LECs) prevented VEGF-C-induced VEGFR3 from endocytosis and degradation. Here, we report that diabetes attenuated VEGF-C-induced lymphangiogenesis in corneal micropocket and Matrigel plug assays in WT mice but not in mice with inducible lymphatic-specific deficiency of epsins 1 and 2 (LEC-iDKO). Consistently, LECs isolated from diabetic LEC-iDKO mice elevated in vitro proliferation, migration, and tube formation in response to VEGF-C over diabetic WT mice. Mechanistically, ROS produced in diabetes induced c-Src-dependent but VEGF-C-independent VEGFR3 phosphorylation, and upregulated epsins through the activation of transcription factor AP-1. Augmented epsins bound to and promoted degradation of newly synthesized VEGFR3 in the Golgi, resulting in reduced availability of VEGFR3 at the cell surface. Preclinically, the loss of lymphatic-specific epsins alleviated insufficient lymphangiogenesis and accelerated the resolution of tail edema in diabetic mice. Collectively, our studies indicate that inhibiting expression of epsins in diabetes protects VEGFR3 against degradation and ameliorates diabetes-triggered inhibition of lymphangiogenesis, thereby providing a novel potential therapeutic strategy to treat diabetic complications.

TRIF deficiency protects non-obese diabetic mice from type 1 diabetes by modulating the gut microbiota and dendritic cells.

The incidence of type 1 diabetes (T1D) is determined by both genetic and environmental factors. In recent years, the gut microbiota have been identified to be an important environmental factor that could modify diabetes susceptibility. We have previously shown that Myeloid differentiation primary response gene 88 (MyD88), a major adaptor protein downstream of most innate immune Toll-like receptor (TLR) signaling, is important for mediating diabetes susceptibility in the non-obese diabetic (NOD) mouse model of human T1D. Here we report the role of TIR-domain-containing adapter-inducing interferon-ß (TRIF) in T1D development, as TRIF is an important adaptor protein downstream of TLR3 and TLR4 signaling. We found that TRIF-deficient (TRIF-/-) NOD mice were protected from development of diabetes, but only when housed with TRIF-deficient (TRIF-/-) NOD mice. When housed with TRIF-sufficient wild type (WT, i.e., TRIF+/+) NOD mice, the mice developed diabetes. We further investigated the gut microbiota as a potential cause for the altered diabetes development. Interestingly, TRIF-/-NOD mice had a different microbiota composition compared to WT NOD mice, only if they were housed with TRIF-/-NOD mice. However, the composition of gut microbiota in the TRIF-/-NOD mice was indistinguishable from WT NOD mice, if they were housed with WT NOD mice. The difference in the gut microbiota in TRIF-/-NOD mice, due to cohousing, accorded with the diabetes development in TRIF-/-NOD mice. Comparing the gut microbiota in TRIF-/- and WT NOD mice, we identified changes in percentage of Sutterella, Rikenella and Turicibacter species. Moreover, bacteria from WT NOD mice induced significantly stronger inflammatory immune responses in vitro compared to those from TRIF-/-NOD mice. Further immunological analysis revealed impaired function of dendritic cells and reduced T cell activation and proliferation in TRIF-/-NOD mice. Our data show that TRIF-deficiency protects NOD mice from diabetes development through alteration of the gut microbiota and reduced immune cell activation; however, that protection is over-ridden upon exposure to WT NOD bacteria. Therefore exposure to different microbiota can modify disease susceptibility determined by genetic factors related to innate immunity.

Transcriptional control of intestinal cholesterol absorption, adipose energy expenditure and lipid handling by Sortilin.

The sorting receptor Sortilin functions in the regulation of glucose and lipid metabolism. Dysfunctional lipid uptake, storage, and metabolism contribute to several major human diseases including atherosclerosis and obesity. Sortilin associates with cardiovascular disease; however, the role of Sortilin in adipose tissue and lipid metabolism remains unclear. Here we show that in the low-density lipoprotein receptor-deficient (Ldlr-/-) atherosclerosis model, Sortilin deficiency (Sort1-/-) in female mice suppresses Niemann-Pick type C1-Like 1 (Npc1l1) mRNA levels, reduces body and white adipose tissue weight, and improves brown adipose tissue function partially via transcriptional downregulation of Krüppel-like factor 4 and Liver X receptor. Female Ldlr-/-Sort1-/- mice on a high-fat/cholesterol diet had elevated plasma Fibroblast growth factor 21 and Adiponectin, an adipokine that when reduced is associated with obesity and cardiovascular disease-related factors. Additionally, Sort1 deficiency suppressed cholesterol absorption in both female mice ex vivo intestinal tissue and human colon Caco-2 cells in a similar manner to treatment with the NPC1L1 inhibitor ezetimibe. Together our findings support a novel role of Sortilin in energy regulation and lipid homeostasis in female mice, which may be a potential therapeutic target for obesity and cardiovascular disease.

Monophosphoryl lipid A prevents impairment of medullary thick ascending limb [Formula: see text] absorption and improves plasma [Formula: see text] concentration in septic mice.

Metabolic acidosis is the most common acid-base disorder in septic patients and is associated with increased mortality. Previously, we demonstrated that sepsis induced by cecal ligation and puncture (CLP) impairs [Formula: see text] absorption in the medullary thick ascending limb (MTAL) by 1) decreasing the intrinsic [Formula: see text] absorptive capacity and 2) enhancing inhibition of [Formula: see text] absorption by LPS through upregulation of Toll-like receptor (TLR) 4 signaling. Both effects depend on ERK activation. Monophosphoryl lipid A (MPLA) is a detoxified TLR4 agonist that enhances innate antimicrobial immunity and improves survival following sepsis. Pretreatment of MTALs with MPLA in vitro prevents LPS inhibition of [Formula: see text] absorption. Here we examined whether pretreatment with MPLA would protect the MTAL against sepsis. Vehicle or MPLA was administered to mice 48 h before sham or CLP surgery, and MTALs were studied in vitro 18 h postsurgery. Pretreatment with MPLA prevented the effects of sepsis to decrease the basal [Formula: see text] absorption rate and enhance inhibition by LPS. These protective effects were mediated through MPLA stimulation of a Toll/IL-1 receptor domain-containing adaptor-inducing IFN-ß-(TRIF)-dependent phosphatidylinositol 3-kinase-Akt pathway that prevents sepsis- and LPS-induced ERK activation. The effects of MPLA to improve MTAL [Formula: see text] absorption were associated with marked improvement in plasma [Formula: see text] concentration, supporting a role for the kidneys in the pathogenesis of sepsis-induced metabolic acidosis. These studies support detoxified TLR4-based immunomodulators, such as MPLA, that enhance antimicrobial responses as a safe and effective approach to prevent or treat sepsis-induced renal tubule dysfunction and identify cell signaling pathways that can be targeted to preserve MTAL [Formula: see text] absorption and attenuate metabolic acidosis during sepsis.

Deficiency of Dab2 (Disabled Homolog 2) in Myeloid Cells Exacerbates Inflammation in Liver and Atherosclerotic Plaques in LDLR (Low-Density Lipoprotein Receptor)-Null Mice-Brief Report.

OBJECTIVE: Inflammatory macrophages promote the development of atherosclerosis. We have identified the adaptor protein Dab2 (disabled homolog 2) as a regulator of phenotypic polarization in macrophages. The absence of Dab2 in myeloid cells promotes an inflammatory phenotype, but the impact of myeloid Dab2 deficiency on atherosclerosis has not been shown. APPROACH AND RESULTS: To determine the role of myeloid Dab2 in atherosclerosis, Ldlr-/- mice were reconstituted with either Dab2-positive or Dab2-deficient bone marrow and fed a western diet. Consistent with our previous finding that Dab2 inhibits NFκB (nuclear factor κ-light-chain-enhancer of activated B cells) signaling in macrophages, Ldlr-/- mice reconstituted with Dab2-deficient bone marrow had increased systemic inflammation as evidenced by increased serum IL-6 (interleukin-6) levels and increased inflammatory cytokine expression levels in liver. Serum lipid levels were significantly lower in Ldlr-/- mice reconstituted with Dab2-deficient bone marrow, and further examination of livers from these mice revealed drastically increased inflammatory tissue damage and massive infiltration of immune cells. Surprisingly, the atherosclerotic lesion burden in Ldlr-/- mice reconstituted with Dab2-deficient bone marrow was decreased compared with Ldlr-/- mice reconstituted with wild-type bone marrow. Further analysis of aortic root sections revealed increased macrophage content and evidence of increased apoptosis in lesions from Ldlr-/- mice reconstituted with Dab2-deficient bone marrow but no difference in collagen or α-smooth muscle actin content. CONCLUSIONS: Dab2 deficiency in myeloid cells promotes inflammation in livers and atherosclerotic plaques in a mouse model of atherosclerosis. Nevertheless, decreased serum lipids as a result of massive inflammatory liver damage may preclude an appreciable increase in atherosclerotic lesion burden in mice reconstituted with Dab2-deficient bone marrow.

Innate immune adaptor TRIF deficiency accelerates disease progression of ALS mice with accumulation of aberrantly activated astrocytes.

There is compelling evidence that glial-immune interactions contribute to the progression of neurodegenerative diseases. The adaptive immune response has been implicated in disease processes of amyotrophic lateral sclerosis (ALS), but it remains unknown if innate immune signaling also contributes to ALS progression. Here we report that deficiency of the innate immune adaptor TIR domain-containing adaptor inducing interferon-ß (TRIF), which is essential for certain Toll-like receptor (TLR) signaling cascades, significantly shortens survival time and accelerates disease progression of ALS mice. While myeloid differentiation factor 88 (MyD88) is also a crucial adaptor for most TLR signaling pathways, MyD88 deficiency had only a marginal impact on disease course. Moreover, TRIF deficiency reduced the number of natural killer (NK), NK-T-lymphocytes, and CD8-T cells infiltrating into the spinal cord of ALS mice, but experimental modulation of these populations did not substantially influence survival time. Instead, we found that aberrantly activated astrocytes expressing Mac2, p62, and apoptotic markers were accumulated in the lesions of TRIF-deficient ALS mice, and that the number of aberrantly activated astrocytes was negatively correlated with survival time. These findings suggest that TRIF pathway plays an important role in protecting a microenvironment surrounding motor neurons by eliminating aberrantly activated astrocytes.

Differential role of MyD88 and TRIF signaling in myeloid cells in the pathogenesis of autoimmune diabetes.

Type 1 diabetes (T1D) is caused by the autoimmune destruction of the insulin-producing pancreatic beta cells. While the role of adaptive immunity has been extensively studied, the role of innate immune responses and particularly of Toll- like Receptor (TLR) signaling in T1D remains poorly understood. Here we show that myeloid cell-specific MyD88 deficiency considerably protected mice from the development of streptozotocin (STZ)-induced diabetes. The protective effect of MyD88 deficiency correlated with increased expression of the immunoregulatory enzyme indoleamine 2,3-dioxygenase (IDO) in pancreatic lymph nodes from STZ-treated mice and in bone marrow-derived dendritic cells (BMDC) stimulated with apoptotic cells. Mice with myeloid cell specific TIR-domain-containing adapter-inducing interferon-ß (TRIF) knockout showed a trend towards accelerated onset of STZ-induced diabetes, while TRIF deficiency resulted in reduced IDO expression in vivo and in vitro. Moreover, myeloid cell specific MyD88 deficiency delayed the onset of diabetes in Non-Obese Diabetic (NOD) mice, whereas TRIF deficiency had no effect. Taken together, these results identify MyD88 signaling in myeloid cells as a critical pathogenic factor in autoimmune diabetes, which is antagonized by TRIF-dependent responses. This differential function of MyD88 and TRIF depends at least in part on their opposite effects in regulating IDO expression in phagocytes exposed to apoptotic cells.

Tuning of in vivo cognate B-T cell interactions by Intersectin 2 is required for effective anti-viral B cell immunity.

Wiskott-Aldrich syndrome (WAS) is an immune pathology associated with mutations in WAS protein (WASp) or in WASp interacting protein (WIP). Together with the small GTPase Cdc42 and other effectors, these proteins participate in the remodelling of the actin network downstream of BCR engagement. Here we show that mice lacking the adaptor protein ITSN2, a G-nucleotide exchange factor (GEF) for Cdc42 that also interacts with WASp and WIP, exhibited increased mortality during primary infection, incomplete protection after Flu vaccination, reduced germinal centre formation and impaired antibody responses to vaccination. These defects were found, at least in part, to be intrinsic to the B cell compartment. In vivo, ITSN2 deficient B cells show a reduction in the expression of SLAM, CD84 or ICOSL that correlates with a diminished ability to form long term conjugates with T cells, to proliferate in vivo, and to differentiate into germinal centre cells. In conclusion, our study not only revealed a key role for ITSN2 as an important regulator of adaptive immune-response during vaccination and viral infection but it is also likely to contribute to a better understanding of human immune pathologies.

Implications of Sortilin in Lipid Metabolism and Lipid Disorder Diseases.

The present review provides a summary of recent evidence of sortilin expression, function, and regulation and its implications in lipid metabolism and development of lipid disorder diseases. As a member of the vacuolar protein sorting 10 protein (Vps10p) receptor family, sortilin mediates intracellular trafficking of diverse endogenous or exogenous protein substrates between the trans-Golgi network (TGN) and plasma membrane compartments. Recent studies reveal that sortilin regulates the expression of lipid genes, plasma lipid level, and the development of lipid disorder diseases. Sortilin promotes atherogenesis by regulating hepatic very low density lipoprotein (VLDL) secretion and plasma lipid level and subsequently macrophage lipid accumulation. Sortilin deficiency is caused by accelerated proteasome degradation under insulin resistance conditions and is thereby implicated in the hyperlipidemia of type 2 diabetes mellitus (T2DM). Sortilin facilitates hepatic cholesterol accumulation by inhibiting hepatic cholesterol catabolism, which promotes the development of nonalcoholic fatty liver disease (NAFLD). Sortilin plays an important role in lipid metabolism and represents a promising therapeutic target for lipid disorder diseases.

Generation of special autosomal dominant polycystic kidney disease iPSCs with the capability of functional kidney-like cell differentiation.

BACKGROUND: Human induced pluripotent stem cells (iPSCs) have been verified as a powerful cell model for the study of pathogenesis in hereditary disease. Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations of PKD or non-PKD genes. The pathogenesis of ADPKD remains unexplored because of the lack of a true human cell model. METHODS: Six ADPKD patients and four healthy individuals were recruited as donors of somatic cells from a Chinese ADPKD family without mutations of the PKD genes but carrying SAMSN1 gene deletion. The ADPKD-iPSCs were generated from somatic cells and were induced into kidney-like cells (KLCs) by a novel three-step method involving cytokines and renal epithelium growth medium. Furthermore, we analyzed functional properties of these KLCs by water transportation and albumin absorption assays. RESULTS: We successfully generated iPSCs from ADPKD patients and differentiated them into KLCs that showed morphological and functional characteristics of human kidney cells. Further, we also found that ADPKD-iPSC-KLCs had a significantly higher rate of apoptosis and a significantly lower capacity for water transportation and albumin absorption compared to healthy sibling-derived differentiated KLCs. Furthermore, knockdown of SAMSN1 in control iPSCs may attenuate differentiation and/or function of KLCs. CONCLUSIONS: These data show that we have created the first iPSCs established from ADPKD patients without mutations in the PKD genes, and suggest that the deletion mutation of SAMSN1 might be involved in the differentiation and/or function of KLCs. ADPKD-iPSC-KLCs can be used as a versatile model system for the study of kidney disease.

Intersectin-1s deficiency in pulmonary pathogenesis.

Intersectin-1s (ITSN-1s), a multidomain adaptor protein, plays a vital role in endocytosis, cytoskeleton rearrangement and cell signaling. Recent studies have demonstrated that deficiency of ITSN-1s is a crucial early event in pulmonary pathogenesis. In lung cancer, ITSN-1s deficiency impairs Eps8 ubiquitination and favors Eps8-mSos1 interaction which activates Rac1 leading to enhanced lung cancer cell proliferation, migration and metastasis. Restoring ITSN-1s deficiency in lung cancer cells facilitates cytoskeleton changes favoring mesenchymal to epithelial transformation and impairs lung cancer progression. ITSN-1s deficiency in acute lung injury leads to impaired endocytosis which leads to ubiquitination and degradation of growth factor receptors such as Alk5. This deficiency is counterbalanced by microparticles which, via paracrine effects, transfer Alk5/TGFßRII complex to non-apoptotic cells. In the presence of ITSN-1s deficiency, Alk5-restored cells signal via Erk1/2 MAPK pathway leading to restoration and repair of lung architecture. In inflammatory conditions such as pulmonary artery hypertension, ITSN-1s full length protein is cleaved by granzyme B into EHITSN and SH3A-EITSN fragments. The EHITSN fragment leads to pulmonary cell proliferation via activation of p38 MAPK and Elk-1/c-Fos signaling. In vivo, ITSN-1s deficient mice transduced with EHITSN plasmid develop pulmonary vascular obliteration and plexiform lesions consistent with pathological findings seen in severe pulmonary arterial hypertension. These novel findings have significantly contributed to understanding the mechanisms and pathogenesis involved in pulmonary pathology. As demonstrated in these studies, genetically modified ITSN-1s expression mouse models will be a valuable tool to further advance our understanding of pulmonary pathology and lead to novel targets for treating these conditions.

A Plant-Derived Nucleic Acid Reconciles Type I IFN and a Pyroptotic-like Event in Immunity against Respiratory Viruses.

Nucleic acids carrying pathogen-associated molecular patterns trigger innate immune responses and are used to activate host immunity. Although synthetic nucleic acids have been used for that purpose, they have shown limitations for in vivo and clinical applications. To address this issue, we tested a naturally occurring dsRNA extracted from rice bran (rb-dsRNA) and characterized it as a potent ligand of TLR3 and MDA5. In this study, intranasal administration of rb-dsRNA induced production of type I IFNs by alveolar macrophages and protected mice from morbidity and mortality resulting from respiratory virus infection, such as influenza A virus. This protection was completely absent in mice lacking both TRIF and MDA5, indicating the essential role of TLR3- and MDA5-dependent pathways. Interestingly, IFNAR1-deficient mice retained residual antiviral protection, which was abolished by pharmacological inhibition of caspase 1, but not IL-1ß signaling. In fact, rb-dsRNA activated caspase 1 via TRIF, resulting in the release of IL-1ß and LDH. In addition to the direct antiviral activity, rb-dsRNA modulated the immune cell population in the lungs by repopulating virus-depleted alveolar macrophages. Our data demonstrate that rb-dsRNA orchestrates IFN-dependent and -independent direct antiviral protection and that it is a potent immune stimulator modulating antiviral immunity in the lungs. These findings open doors to a range of precise immune-modulating studies and therapeutic options.