Evgenia Dobrinskikh

A Crucial Role for Kupffer Cell-Derived Galectin-9 in Regulation of T Cell Immunity in Hepatitis C Infection

Approximately 200 million people throughout the world are infected with hepatitis C virus (HCV). One of the most striking features of HCV infection is its high propensity to establish persistence (∼70–80%) and progressive liver injury. Galectins are evolutionarily conserved glycan-binding proteins with diverse roles in innate and adaptive immune responses. Here, we demonstrate that galectin-9, the natural ligand for the T cell immunoglobulin domain and mucin domain protein 3 (Tim-3), circulates at very high levels in the serum and its hepatic expression (particularly on Kupffer cells) is significantly increased in patients with chronic HCV as compared to normal controls. Galectin-9 production from monocytes and macrophages is induced by IFN-γ, which has been shown to be elevated in chronic HCV infection. In turn, galectin-9 induces pro-inflammatory cytokines in liver-derived and peripheral mononuclear cells; galectin-9 also induces anti-inflammatory cytokines from peripheral but not hepatic mononuclear cells. Galectin-9 results in expansion of CD4+CD25+FoxP3+CD127low regulatory T cells, contraction of CD4+ effector T cells, and apoptosis of HCV-specific CTLs. In conclusion, galectin-9 production by Kupffer cells links the innate and adaptive immune response, providing a potential novel immunotherapeutic target in this common viral infection.

Control of creatine metabolism by HIF is an endogenous mechanism of barrier regulation in colitis

Significance Intestinal epithelial barrier dysregulation is a hallmark of inflammatory bowel diseases (IBDs). A central role for hypoxic signaling has been defined in barrier modulation during inflammation. We demonstrate that genes involved in creatine metabolism, the creatine kinases (CKs), are coordinately regulated by hypoxia-inducible transcription factors (HIFs) and that such regulation is critical to barrier function. Inhibition of the CK pathway abrogates apical junction assembly and barrier integrity. Dietary creatine supplementation profoundly attenuates the pathogenic course of mucosal inflammation in mouse colitis models. Moreover, we demonstrate altered expression of mitochondrial and cytosolic CK enzymes in IBD patient tissue. These findings highlight the fundamental contribution of creatine metabolism to intestinal mucosal function, homeostasis, and disease resolution. Mucosal surfaces of the lower gastrointestinal tract are subject to frequent, pronounced fluctuations in oxygen tension, particularly during inflammation. Adaptive responses to hypoxia are orchestrated largely by the hypoxia-inducible transcription factors (HIFs). As HIF-1α and HIF-2α are coexpressed in mucosal epithelia that constitute the barrier between the lumen and the underlying immune milieu, we sought to define the discrete contribution of HIF-1 and HIF-2 transactivation pathways to intestinal epithelial cell homeostasis. The present study identifies creatine kinases (CKs), key metabolic enzymes for rapid ATP generation via the phosphocreatine–creatine kinase (PCr/CK) system, as a unique gene family that is coordinately regulated by HIF. Cytosolic CKs are expressed in a HIF-2–dependent manner in vitro and localize to apical intestinal epithelial cell adherens junctions, where they are critical for junction assembly and epithelial integrity. Supplementation with dietary creatine markedly ameliorated both disease severity and inflammatory responses in colitis models. Further, enzymes of the PCr/CK metabolic shuttle demonstrate dysregulated mucosal expression in a subset of ulcerative colitis and Crohn disease patients. These findings establish a role for HIF-regulated CK in epithelial homeostasis and reveal a fundamental link between cellular bioenergetics and mucosal barrier.

Hepatitis C virus infection induces autocrine interferon signaling by human liver endothelial cells and release of exosomes, which inhibits viral replication.

BACKGROUND & AIMS Liver sinusoidal endothelial cells (LSECs) make up a large proportion of the nonparenchymal cells in the liver. LSECs are involved in induction of immune tolerance, but little is known about their functions during hepatitis C virus (HCV) infection. METHODS Primary human LSECs (HLSECs) and immortalized liver endothelial cells (TMNK-1) were exposed to various forms of HCV, including full-length transmitted/founder virus, sucrose-purified Japanese fulminant hepatitis-1 (JFH-1), a virus encoding a luciferase reporter, and the HCV-specific pathogen-associated molecular pattern molecules. Cells were analyzed by confocal immunofluorescence, immunohistochemical, and polymerase chain reaction assays. RESULTS HLSECs internalized HCV, independent of cell-cell contacts; HCV RNA was translated but not replicated. Through pattern recognition receptors (Toll-like receptor 7 and retinoic acid-inducible gene 1), HCV RNA induced consistent and broad transcription of multiple interferons (IFNs); supernatants from primary HLSECs transfected with HCV-specific pathogen-associated molecular pattern molecules increased induction of IFNs and IFN-stimulated genes in HLSECs. Recombinant type I and type III IFNs strongly up-regulated HLSEC transcription of IFN λ3 (IFNL3) and viperin (RSAD2), which inhibit replication of HCV. Compared with CD8(+) T cells, HLSECs suppressed HCV replication within Huh7.5.1 cells, also inducing IFN-stimulated genes in co-culture. Conditioned media from IFN-stimulated HLSECs induced expression of antiviral genes by uninfected primary human hepatocytes. Exosomes, derived from HLSECs after stimulation with either type I or type III IFNs, controlled HCV replication in a dose-dependent manner. CONCLUSIONS Cultured HLSECs produce factors that mediate immunity against HCV. HLSECs induce self-amplifying IFN-mediated responses and release of exosomes with antiviral activity.

SGLT2 Protein Expression Is Increased in Human Diabetic Nephropathy SGLT2 PROTEIN INHIBITION DECREASES RENAL LIPID ACCUMULATION, INFLAMMATION, AND THE DEVELOPMENT OF NEPHROPATHY IN DIABETIC MICE

There is very limited human renal sodium gradient-dependent glucose transporter protein (SGLT2) mRNA and protein expression data reported in the literature. The first aim of this study was to determine SGLT2 mRNA and protein levels in human and animal models of diabetic nephropathy. We have found that the expression of SGLT2 mRNA and protein is increased in renal biopsies from human subjects with diabetic nephropathy. This is in contrast to db-db mice that had no changes in renal SGLT2 protein expression. Furthermore, the effect of SGLT2 inhibition on renal lipid content and inflammation is not known. The second aim of this study was to determine the potential mechanisms of beneficial effects of SGLT2 inhibition in the progression of diabetic renal disease. We treated db/db mice with a selective SGLT2 inhibitor JNJ 39933673. We found that SGLT2 inhibition caused marked decreases in systolic blood pressure, kidney weight/body weight ratio, urinary albumin, and urinary thiobarbituric acid-reacting substances. SGLT2 inhibition prevented renal lipid accumulation via inhibition of carbohydrate-responsive element-binding protein-β, pyruvate kinase L, SCD-1, and DGAT1, key transcriptional factors and enzymes that mediate fatty acid and triglyceride synthesis. SGLT2 inhibition also prevented inflammation via inhibition of CD68 macrophage accumulation and expression of p65, TLR4, MCP-1, and osteopontin. These effects were associated with reduced mesangial expansion, accumulation of the extracellular matrix proteins fibronectin and type IV collagen, and loss of podocyte markers WT1 and synaptopodin, as determined by immunofluorescence microscopy. In summary, our study showed that SGLT2 inhibition modulates renal lipid metabolism and inflammation and prevents the development of nephropathy in db/db mice.

IFN-γ–Mediated Induction of an Apical IL-10 Receptor on Polarized Intestinal Epithelia

Cytokines secreted at sites of inflammation impact the onset, progression, and resolution of inflammation. In this article, we investigated potential proresolving mechanisms of IFN-γ in models of inflammatory bowel disease. Guided by initial microarray analysis, in vitro studies revealed that IFN-γ selectively induced the expression of IL-10R1 on intestinal epithelia. Further analysis revealed that IL-10R1 was expressed predominantly on the apical membrane of polarized epithelial cells. Receptor activation functionally induced canonical IL-10 target gene expression in epithelia, concomitant with enhanced barrier restitution. Furthermore, knockdown of IL-10R1 in intestinal epithelial cells results in impaired barrier function in vitro. Colonic tissue isolated from murine colitis revealed that levels of IL-10R1 and suppressor of cytokine signaling 3 were increased in the epithelium and coincided with increased tissue IFN-γ and IL-10 cytokines. In parallel, studies showed that treatment of mice with rIFN-γ was sufficient to drive expression of IL-10R1 in the colonic epithelium. Studies of dextran sodium sulfate colitis in intestinal epithelial-specific IL-10R1–null mice revealed a remarkable increase in disease susceptibility associated with increased intestinal permeability. Together, these results provide novel insight into the crucial and underappreciated role of epithelial IL-10 signaling in the maintenance and restitution of epithelial barrier and of the temporal regulation of these pathways by IFN-γ.

HIF-dependent regulation of claudin-1 is central to intestinal epithelial tight junction integrity

This study demonstrates a critical link between hypoxia-inducible factor (HIF) and claudin-1 (CLDN1). HIF1β-deficient intestinal epithelial cells develop abnormal tight junction (TJ) structure and have striking barrier defects. CLDN1 is an HIF target gene, and overexpression of CLDN1 in HIF1β-deficient cells restores TJ structure and function.

Human podocytes perform polarized, caveolae-dependent albumin endocytosis

The renal glomerulus forms a selective filtration barrier that allows the passage of water, ions, and small solutes into the urinary space while restricting the passage of cells and macromolecules. The three layers of the glomerular filtration barrier include the vascular endothelium, glomerular basement membrane (GBM), and podocyte epithelium. Podocytes are capable of internalizing albumin and are hypothesized to clear proteins that traverse the GBM. The present study followed the fate of FITC-labeled albumin to establish the mechanisms of albumin endocytosis and processing by podocytes. Confocal imaging and total internal reflection fluorescence microscopy of immortalized human podocytes showed FITC-albumin endocytosis occurred preferentially across the basal membrane. Inhibition of clathrin-mediated endocytosis and caveolae-mediated endocytosis demonstrated that the majority of FITC-albumin entered podocytes through caveolae. Once internalized, FITC-albumin colocalized with EEA1 and LAMP1, endocytic markers, and with the neonatal Fc receptor, a marker for transcytosis. After preloading podocytes with FITC-albumin, the majority of loaded FITC-albumin was lost over the subsequent 60 min of incubation. A portion of the loss of albumin occurred via lysosomal degradation as pretreatment with leupeptin, a lysosomal protease inhibitor, partially inhibited the loss of FITC-albumin. Consistent with transcytosis of albumin, preloaded podocytes also progressively released FITC-albumin into the extracellular media. These studies confirm the ability of podocytes to endocytose albumin and provide mechanistic insight into cellular mechanisms and fates of albumin handling in podocytes.

G Protein-Coupled Bile Acid Receptor TGR5 Activation Inhibits Kidney Disease in Obesity and Diabetes

Obesity and diabetes mellitus are the leading causes of renal disease. In this study, we determined the regulation and role of the G protein-coupled bile acid receptor TGR5, previously shown to be regulated by high glucose and/or fatty acids, in obesity-related glomerulopathy (ORG) and diabetic nephropathy (DN). Treatment of diabetic db/db mice with the selective TGR5 agonist INT-777 decreased proteinuria, podocyte injury, mesangial expansion, fibrosis, and CD68 macrophage infiltration in the kidney. INT-777 also induced renal expression of master regulators of mitochondrial biogenesis, inhibitors of oxidative stress, and inducers of fatty acid β-oxidation, including sirtuin 1 (SIRT1), sirtuin 3 (SIRT3), and Nrf-1. Increased activity of SIRT3 was evidenced by normalization of the increased acetylation of mitochondrial superoxide dismutase 2 (SOD2) and isocitrate dehydrogenase 2 (IDH2) observed in untreated db/db mice. Accordingly, INT-777 decreased mitochondrial H2O2 generation and increased the activity of SOD2, which associated with decreased urinary levels of H2O2 and thiobarbituric acid reactive substances. Furthermore, INT-777 decreased renal lipid accumulation. INT-777 also prevented kidney disease in mice with diet-induced obesity. In human podocytes cultured with high glucose, INT-777 induced mitochondrial biogenesis, decreased oxidative stress, and increased fatty acid β-oxidation. Compared with normal kidney biopsy specimens, kidney specimens from patients with established ORG or DN expressed significantly less TGR5 mRNA, and levels inversely correlated with disease progression. Our results indicate that TGR5 activation induces mitochondrial biogenesis and prevents renal oxidative stress and lipid accumulation, establishing a role for TGR5 in inhibiting kidney disease in obesity and diabetes.

NHE3 regulatory factor 1 (NHERF1) modulates intestinal sodium-dependent phosphate transporter (NaPi-2b) expression in apical microvilli.

Background: The type 2b sodium-dependent phosphate co-transporter (NaPi-2b) is the main mediator of intestinal active Pi absorption. Results: NaPi-2b interacts with the PDZ domain of NHE3 regulatory factor 1 (NHERF1). Conclusion: NHERF1 is an important regulator of NaPi-2b apical membrane targeting in response to a low Pi diet. Significance: Understanding of NaPi-2b adaptive mechanisms can help to design new therapies against hypo- and hyperphosphatemic disorders. Pi uptake in the small intestine occurs predominantly through the NaPi-2b (SLC34a2) co-transporter. NaPi-2b is regulated by changes in dietary Pi but the mechanisms underlying this regulation are largely undetermined. Sequence analyses show NaPi-2b has a PDZ binding motif at its C terminus. Immunofluorescence imaging shows NaPi-2b and two PDZ domain containing proteins, NHERF1 and PDZK1, are expressed in the apical microvillar domain of rat small intestine enterocytes. Co-immunoprecipitation studies in rat enterocytes show that NHERF1 associates with NaPi-2b but not PDZK1. In HEK co-expression studies, GFP-NaPi-2b co-precipitates with FLAG-NHERF1. This interaction is markedly diminished when the C-terminal four amino acids are truncated from NaPi-2b. FLIM-FRET analyses using tagged proteins in CACO-2BBE cells show a distinct phasor shift between NaPi-2b and NHERF1 but not between NaPi-2b and the PDZK1 pair. This shift demonstrates that NaPi-2b and NHERF1 reside within 10 nm of each other. NHERF1−/− mice, but not PDZK1−/− mice, had a diminished adaptation of NaPi-2b expression in response to a low Pi diet. Together these studies demonstrate that NHERF1 associates with NaPi-2b in enterocytes and regulates NaPi-2b adaptation.

Podocytes degrade endocytosed albumin primarily in lysosomes.

Albuminuria is a strong, independent predictor of chronic kidney disease progression. We hypothesize that podocyte processing of albumin via the lysosome may be an important determinant of podocyte injury and loss. A human urine derived podocyte-like epithelial cell (HUPEC) line was used for in vitro experiments. Albumin uptake was quantified by Western blot after loading HUPECs with fluorescein-labeled (FITC) albumin. Co-localization of albumin with lysosomes was determined by confocal microscopy. Albumin degradation was measured by quantifying FITC-albumin abundance in HUPEC lysates by Western blot. Degradation experiments were repeated using HUPECs treated with chloroquine, a lysosome inhibitor, or MG-132, a proteasome inhibitor. Lysosome activity was measured by fluorescence recovery after photo bleaching (FRAP). Cytokine production was measured by ELISA. Cell death was determined by trypan blue staining. In vivo, staining with lysosome-associated membrane protein-1 (LAMP-1) was performed on tissue from a Denys-Drash trangenic mouse model of nephrotic syndrome. HUPECs endocytosed albumin, which co-localized with lysosomes. Choloroquine, but not MG-132, inhibited albumin degradation, indicating that degradation occurs in lysosomes. Cathepsin B activity, measured by FRAP, significantly decreased in HUPECs exposed to albumin (12.5% of activity in controls) and chloroquine (12.8%), and declined further with exposure to albumin plus chloroquine (8.2%, p<0.05). Cytokine production and cell death were significantly increased in HUPECs exposed to albumin and chloroquine alone, and these effects were potentiated by exposure to albumin plus chloroquine. Compared to wild-type mice, glomerular staining of LAMP-1 was significantly increased in Denys-Drash mice and appeared to be most prominent in podocytes. These data suggest lysosomes are involved in the processing of endocytosed albumin in podocytes, and lysosomal dysfunction may contribute to podocyte injury and glomerulosclerosis in albuminuric diseases. Modifiers of lysosomal activity may have therapeutic potential in slowing the progression of glomerulosclerosis by enhancing the ability of podocytes to process and degrade albumin.