Annie J. Kruger

Leptin treatment confers clinical benefit at multiple stages of virally induced type 1 diabetes in BB rats

The adipokine, leptin, regulates blood glucose and the insulin secretory function of beta cells, while also modulating immune cell function. We hypothesized that the dual effects of leptin may prevent or suppress the autoreactive destruction of beta cells in a virally induced rodent model of type 1 diabetes. Nearly 100% of weanling BBDR rats treated with the combination of an innate immune system activator, polyinosinic:polycytidylic acid (pIC), and Kilham rat virus (KRV) become diabetic within a predictable time frame. We utilized this model to test the efficacy of leptin in preventing diabetes onset, remitting new onset disease, and preventing autoimmune recurrence in diabetic rats transplanted with syngeneic islet grafts. High doses of leptin delivered via an adenovirus vector (AdLeptin) or alzet pump prevented diabetes in>90% of rats treated with pIC+KRV. The serum hyperleptinemia generated by this treatment was associated with decreased body weight, decreased non-fasting serum insulin levels, and lack of islet insulitis in leptin-treated rats. In new onset diabetics, hyperleptinemia prevented rapid weight loss and diabetic ketoacidosis, and temporarily restored euglycemia. Leptin treatment also prolonged the survival of syngeneic islets transplanted into diabetic BBDR rats. In diverse therapeutic settings, we found leptin treatment to have significant beneficial effects in modulating virally induced diabetes. These findings merit further evaluation of leptin as a potential adjunct therapeutic agent for treatment of human type 1 diabetes.

Blockade of the Programmed Death-1 (PD1) Pathway Undermines Potent Genetic Protection from Type 1 Diabetes

Aims/Hypothesis Inhibition of PD1-PDL1 signaling in NOD mice accelerates onset of type 1 diabetes implicating this pathway in suppressing the emergence of pancreatic beta cell reactive T-cells. However, the molecular mechanism by which PD1 signaling protects from type 1 diabetes is not clear. We hypothesized that differential susceptibility of Idd mouse strains to type 1 diabetes when challenged with anti PDL1 will identify genomic loci that collaborate with PD1 signaling in suppressing type 1 diabetes. Methods Anti PDL1 was administered to NOD and various Idd mouse strains at 10 weeks of age and onset of disease was monitored by measuring blood glucose levels. Additionally, histological evaluation of the pancreas was performed to determine degree of insulitis. Statistical analysis of the data was performed using Log-Rank and Students t-test. Results Blockade of PDL1 rapidly precipitated type 1 diabetes in nearly all NOD Idd congenic strains tested, despite the fact that all are moderately (Idd5, Idd3 and Idd10/18) or highly (Idd3/10/18 and Idd9) protected from spontaneous type 1 diabetes by virtue of their protective Idd genes. Only the Idd3/5 strain, which is nearly 100% protected from spontaneous disease, remained normoglycemic following PDL1 blockade. Conclusions These results indicate that multiple Idd loci collaborate with PD1 signaling. Anti PDL1 treatment undermines a large portion of the genetic protection mediated by Idd genes in the NOD model of type 1 diabetes. Basal insulitis correlated with higher susceptibility to type 1 diabetes. These findings have important implications since the PD1 pathway is a target for immunotherapy.

The role of innate immune pathways in type 1 diabetes pathogenesis.

Purpose of reviewType 1 diabetes (T1D) is an autoimmune disease typically believed to result from malfunctions in adaptive immune response signaling which result in activation of self-reactive T cells. However, recent research has indicated components of the innate immune response as having a key role in the initiation of the autoimmune process of T1D. This review will highlight recent studies which examined the role of innate immune response signaling and the connections to T1D pathogenesis. Recent findingsInvestigations indicate that components of innate immunity, including inflammation and Toll-like receptor signaling, are involved in pancreatic islet infiltration and insulitis. Recent studies examining the role of viral infections in T1D development also implicate innate immune response signaling in disease pathogenesis. SummaryCurrent research indicates that components of innate immune response signaling are involved in the initiation of the autoimmune process which results in the eventual destruction of β cells during T1D pathogenesis. Continuing efforts by researchers to uncover the molecular pathways of innate immunity linked to T1D development could potentially lead to therapeutics capable of preventing and curing the autoimmune disease.

Haptoglobin as an early serum biomarker of virus-induced autoimmune type 1 diabetes in biobreeding diabetes resistant and LEW1.WR1 rats

Proteomic profiling of serum is a powerful technique to identify differentially expressed proteins that can serve as biomarkers predictive of disease onset. In this study, we utilized two-dimensional (2D) gel analysis followed by matrix-assisted-laser desorption/ionization time-of-flight mass spectrometry analysis to identify putative serum biomarkers for autoimmune type 1 diabetes (T1D) in biobreeding diabetes resistant (BBDR) rats induced to express the disease. Treatment with toll-like receptor 3 ligand, polyinosinic:polycytidilic acid (pIC), plus infection with Kilham rat virus (KRV), a rat parvovirus, results in nearly 100% of young BBDR rats becoming diabetic within 11–21 d. Sera collected from prediabetic rats at early time points following treatment with pIC + KRV were analyzed by 2D gel electrophoresis and compared with sera from control rats treated with phosphate-buffered saline, pIC alone or pIC + H1, a non-diabetogenic parvovirus. None of the latter three control treatments precipitates T1D. 2D gel analysis revealed that haptoglobin, an acute phase and hemoglobin scavenger protein, was differentially expressed in the sera of rats treated with pIC + KRV relative to control groups. These results were confirmed by Western blot and enzyme-linked immunosorbent assay studies, which further validated haptoglobin levels as being differentially increased in the sera of pIC + KRV-treated rats relative to controls during the first week following infection. Early elevations in serum haptoglobin were also observed in LEW1.WR1 rats that became diabetic following infection with rat cytomegalovirus. The identification and validation of haptoglobin as a putative serum biomarker for autoimmune T1D in rats now affords us the opportunity to test the validity of this protein as a biomarker for human T1D, particularly in those situations where viral infection is believed to precede the onset of disease.

TLR Agonists Prevent the Establishment of Allogeneic Hematopoietic Chimerism in Mice Treated with Costimulation Blockade

Activation of TLR4 by administration of LPS shortens the survival of skin allografts in mice treated with costimulation blockade through a CD8 T cell-dependent, MyD88-dependent, and type I IFN receptor-dependent pathway. The effect of TLR activation on the establishment of allogeneic hematopoietic chimerism in mice treated with costimulation blockade is not known. Using a costimulation blockade protocol based on a donor-specific transfusion (DST) and a short course of anti-CD154 mAb, we show that LPS administration at the time of DST matures host alloantigen-presenting dendritic cells, prevents the establishment of mixed allogeneic hematopoietic chimerism, and shortens survival of donor-specific skin allografts. LPS mediates its effects via a mechanism that involves both CD4+ and CD8+ T cells and results from signaling through either the MyD88 or the type I IFN receptor pathways. We also document that timing of LPS administration is critical, as injection of LPS 24 h before treatment with DST and anti-CD154 mAb does not prevent hematopoietic engraftment but administration the day after bone marrow transplantation does. We conclude that TLR4 activation prevents the induction of mixed allogeneic hematopoietic chimerism through type I IFN receptor and MyD88-dependent signaling, which leads to the up-regulation of costimulatory molecules on host APCs and the generation of alloreactive T cells. These data suggest that distinct but overlapping cellular and molecular mechanisms control the ability of TLR agonists to block tolerance induction to hematopoietic and skin allografts in mice treated with costimulation blockade.

Circulating Soluble CD163 is Associated with Steatohepatitis and Advanced Fibrosis in Nonalcoholic Fatty Liver Disease

OBJECTIVES:Soluble CD163 (sCD163), a marker of Kupffer cell activation detectable in serum, correlates with inflammation and fibrosis in chronic viral hepatitis, but its role in nonalcoholic fatty liver disease is unknown. We hypothesized that sCD163 would correlate with nonalcoholic fatty liver disease activity and fibrosis.METHODS:Liver biopsies and serum were obtained from 145 obese subjects undergoing gastric bypass surgery. Subjects were divided into four groups based on fibrosis stage and nonalcoholic fatty liver disease activity score (NAS); Group 1: F0, NAS=0; Group 2: F<2, 0<NAS<5; Group 3: NAS≥5, F<3; or Group 4: F≥3, any NAS. Serum sCD163 and the monocyte/macrophage marker sCD14 were measured by enzyme-linked immunosorbent assay. Relationships between sCD163, sCD14, fibrosis stage, and NAS were examined. Area under the receiver operating charateristic for the diagnosis of nonalcoholic steatohepatitis based on the Clinical Research Network definition was calculated.RESULTS:sCD163 increased with progressive liver histology, with lowest values in normal histology and highest levels in those with nonalcoholic steatohepatitis and advanced fibrosis (Group 1: 552 ng/ml, Group 2: 721 ng/ml, Group 3: 803 ng/ml, and Group 4:1,031; P=0.001). sCD14 also differed significantly across groups (Group 1: 1,877 ng/ml, Group 2: 1632 ng/ml, Group 3: 1,706 ng/ml, and Group 4: 2111; P=0.008, respectively). sCD163 correlated with steatosis grade (P<0.001), lobular inflammation (P=0.033), and hepatocyte ballooning (P<0.001). In a multivariable ordered logistic regression model, there was a significant association between every 100 ng/ml increase in sCD163 and higher fibrosis stage, with an odds ratio of 1.16 (95% confidence interval 1.02–1.31), P=0.020. The odds ratios of the association between every 100 ng/ml increase in sCD163 and higher NAS was 1.17 (95% confidence interval 1.04–1.32), P=0.010. A sCD163-based predictive score demonstrated an area under the receiver operating charateristic of 0.70 (95% confidence interval: 0.58–0.82) for the diagnosis of nonalcoholic steatohepatitis. Soluble CD14 did not correlate with fibrosis stage or NAS.CONCLUSIONS:In obese subjects, serum sCD163, but not sCD14, correlated with fibrosis stage and NAS. These data support a role for activated Kupffer cells in the pathogenesis of nonalcoholic steatohepatitis and fibrosis, and suggest potential clinical utility for assessment of sCD163 levels.

Exposure to human immunodeficiency virus/hepatitis C virus in hepatic and stellate cell lines reveals cooperative profibrotic transcriptional activation between viruses and cell types.

Human immunodeficiency virus (HIV)/hepatitis C virus (HCV) coinfection accelerates progressive liver fibrosis; however, the mechanisms remain poorly understood. HCV and HIV independently induce profibrogenic markers transforming growth factor beta‐1 (TGFβ1) (mediated by reactive oxygen species [ROS]) and nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NFκB) in hepatocytes and hepatic stellate cells in monoculture; however, they do not account for cellular crosstalk that naturally occurs. We created an in vitro coculture model and investigated the contributions of HIV and HCV to hepatic fibrogenesis. Green fluorescent protein reporter cell lines driven by functional ROS (antioxidant response elements), NFκB, and mothers against decapentaplegic homolog 3 (SMAD3) promoters were created in Huh7.5.1 and LX2 cells, using a transwell to generate cocultures. Reporter cell lines were exposed to HIV, HCV, or HIV/HCV. Activation of the 3 pathways was measured and compared according to infection status. Extracellular matrix products (collagen type 1 alpha 1 (CoL1A1) and tissue inhibitor of metalloproteinase 1 (TIMP1)) were also measured. Both HCV and HIV independently activated TGFβ1 signaling through ROS (antioxidant response elements), NFκB, and SMAD3 in both cell lines in coculture. Activation of these profibrotic pathways was additive following HIV/HCV coexposure. This was confirmed when examining CoL1A1 and TIMP1, where messenger RNA and protein levels were significantly higher in LX2 cells in coculture following HIV/HCV coexposure compared with either virus alone. In addition, expression of these profibrotic genes was significantly higher in the coculture model compared to either cell type in monoculture, suggesting an interaction and feedback mechanism between Huh7.5.1 and LX2 cells. Conclusion: HIV accentuates an HCV‐driven profibrogenic program in hepatocyte and hepatic stellate cell lines through ROS, NFκB, and TGFβ1 up‐regulation; coculture of hepatocyte and hepatic stellate cell lines significantly increased expression of CoL1A1 and TIMP1; and our novel coculture reporter cell model represents an efficient and more authentic system for studying transcriptional fibrosis responses and may provide important insights into hepatic fibrosis. (Hepatology 2016;64:1951‐1968).

IQGAP2 is a novel interferon-alpha antiviral effector gene acting non-conventionally through the NF-κB pathway

BACKGROUND & AIMS Type I interferons (IFN) provide the first line of defense against invading pathogens but its mechanism of action is still not well understood. Using unbiased genome-wide siRNA screens, we recently identified IQ-motif containing GTPase activating protein 2 (IQGAP2), a tumor suppressor predominantly expressed in the liver, as a novel gene putatively required for IFN antiviral response against hepatitis C virus (HCV) infection. Here we sought to characterize IQGAP2 role in IFN response. METHODS We used transient small interfering RNA knockdown strategy in hepatic cell lines highly permissive to JFH1 strain of HCV infection. RESULTS We found that IQGAP2 acts downstream of IFN binding to its receptor, and independently of the JAK-STAT pathway, by physically interacting with RelA (also known as p65), a subunit of the NF-κB transcription factor. Interestingly, our data reveal a mechanism distinct from the well-characterized role of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in IFN production. Indeed, IFN alone was sufficient to stimulate NF-κB-dependent transcription in the absence of viral infection. Finally, both IQGAP2 and RelA were required for the induction by IFN of a subset of IFN-stimulated genes (ISG) with known antiviral properties. CONCLUSIONS Our data identify a novel function for IQGAP2 in IFN antiviral response in hepatoma cells. We demonstrate the involvement of IQGAP2 in regulating ISG induction by IFN in an NF-κB-dependent manner. The IQGAP2 pathway may provide new targets for antiviral strategies in the liver, and may have a wider therapeutic implication in other disease pathogeneses driven by NF-κB activation. LAY SUMMARY In this study, we identify a novel mechanism of action of interferon involving the IQGAP2 protein and the NF-κB pathway that is ultimately protective against hepatitis C virus infection. This newly identified pathway functions independently of the well-known STAT pathway and may therefore provide new targets for antiviral strategies in the liver.

Prolonged cenicriviroc therapy reduces hepatic fibrosis despite steatohepatitis in a diet-induced mouse model of nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a progressive liver disease projected to become the leading cause of cirrhosis and liver transplantation in the next decade. Cenicriviroc (CVC), a dual chemokine receptor 2 and 5 antagonist, prevents macrophage trafficking and is under clinical investigation for the treatment of human NASH fibrosis. We assessed the efficacy and durability of short and prolonged CVC therapy in a diet‐induced mouse model of NASH, the choline deficient, L‐amino acid‐defined, high‐fat diet (CDAHFD) model. C57BL/6 mice received 4 or 14 weeks of standard chow or the CDAHFD. CVC (10 mg/kg/day and 30 mg/kg/day for 4 weeks and 20 mg/kg/day and 30 mg/kg/day for 14 weeks) was initiated simultaneously with the CDAHFD. At 4 and 14 weeks, livers were harvested for histology and flow cytometric analyses of intrahepatic immune cells. High‐dose CVC (30 mg/kg/day) therapy in CDAHFD mice for 4 or 14 weeks inhibited intrahepatic accumulation of Ly6Chigh bone marrow‐derived macrophages. Prolonged CVC therapy (14 weeks) yielded no significant differences in the total intrahepatic macrophage populations among treatment groups but increased the frequency of intrahepatic anti‐inflammatory macrophages in the high‐dose CVC group. Despite ongoing steatohepatitis, there was significantly less fibrosis in CDAHFD mice receiving high‐dose CVC for 14 weeks based on histologic and molecular markers, mirroring observations in human NASH CVC trials. CVC also directly inhibited the profibrotic gene signature of transforming growth factor‐β‐stimulated primary mouse hepatic stellate cells in vitro. Conclusion: CVC is a novel therapeutic agent that is associated with reduced fibrosis despite ongoing steatohepatitis. Its ability to alter intrahepatic macrophage populations and inhibit profibrogenic genes in hepatic stellate cells in NASH livers may contribute to its observed antifibrotic effect. (Hepatology Communications 2018;2:529‐545)

Macrophage Activation Marker Soluble CD163 Is a Dynamic Marker of Liver Fibrogenesis in Human Immunodeficiency Virus/Hepatitis C Virus Coinfection

Background Coinfection with human immunodeficiency virus (HIV) accelerates hepatitis C virus (HCV)-related liver fibrosis. Macrophages are triggered during both viral infections and are critical in liver inflammation/fibrogenesis. Liver fibrosis strongly associates with serum soluble CD163 (sCD163, a macrophage activation marker); comprehensive evaluation in HIV/HCV coinfection is lacking. Methods We retrospectively analyzed sCD163 (enzyme-linked immunosorbent assay) and hepatic CD163 (immunofluorescent CD163/CD68 costaining) in patients infected with HIV/HCV, HCV, or HIV, pre- and post-antiviral therapy. Results sCD163 was significantly higher in HIV/HCV compared to either monoinfection, and decreased following successful antiviral therapy, although did not fully normalize. In HIV/HCV, sCD163 was associated with necroinflammation, Ishak fibrosis scores, and noninvasive fibrosis scores. We observed a novel trend whereby sCD163 levels progressively increase with increasing Ishak fibrosis score, peaking at stage 4, above which levels plateaued. Periportal CD163+ macrophage frequency was also higher with increasing fibrosis score. When stratified by fibrosis stage, sCD163 levels were higher in HIV/HCV than HCV but only in individuals with mild to moderate fibrosis. Conclusions In HIV/HCV, increasing sCD163 levels accompanied periportal CD163+ macrophage enrichment in mild to moderate fibrosis, but not in established cirrhosis, suggesting that sCD163 is a dynamic biomarker of fibrogenesis rather than accumulated fibrosis. Our findings implicate HIV-related macrophage activation in accelerated fibrosis progression in HIV/HCV coinfection.