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1.  IL-33 induces nuocytes and modulates liver injury in viral hepatitis 
Molecules containing damage-associated molecular patterns (DAMP) play an important role in many pathogenic processes. Our aim was to investigate the role of IL-33, a DAMP molecule, in adenovirus (Ad)-induced liver inflammation. Ad-infected mice exhibited a steadily increased IL-33 and its receptor ST2 expression in the liver during the first week of the infection. Treatment of exogenous IL-33 resulted in a great decrease in the serum alanine aminotransferase (ALT) levels and the number of Councilman bodies in the liver. Attenuated liver injury by IL-33 correlated with an increase in T regulatory (Treg) cells but with a decrease in macrophages, dendritic cells and NK cells in the liver. IL-33 enhanced both type 1 (IL-2 and IFN-γ) and type 2 (IL-5 and IL-13) immune responses in infected mice. However, IL-33 inhibited TNF-α expression in hepatic T cells and macrophages, and significantly reduced TNF-α levels in the liver. We found that in addition to its direct effects, IL-33 strongly induced novel nuocytes in the livers and spleens of infected mice. When co-cultured with nuocytes, hepatic T cells and macrophages expressed lower levels of TNF-α. The IL-33-treated mice also demonstrated a slight delay, but no significant impairment, in eliminating an intrahepatic infection with Ad. In conclusion, this study reveals that IL-33 acts as a potent immune stimulator and a hepatoprotective cytokine in acute viral hepatitis. Its direct immunoregulatory functions and ability to induce novel nuocytes further suggest to us that it may be a potentially promising therapeutic candidate for the management of viral hepatitis.
doi:10.4049/jimmunol.1300117
PMCID: PMC3660413  PMID: 23630360
2.  Early IL-17 production by intrahepatic T cells is important for adaptive immune responses in viral hepatitis 
This study was conducted to examine the interactions among the innate and adaptive immune components of the liver parenchyma during acute viral hepatitis. Mice were i.v. infected with a recombinant adenovirus, and within the first 24 h of infection, we found a transient, but significant, accumulation of IL-17 and IL-23 in the liver. In vivo neutralization of these interleukins alleviated the liver injury. Further investigations showed that IL-17 neutralization halted the intrahepatic accumulation of CTL and Th1 cells. A majority of the IL-17-producing cells in the liver were γδ T cells. Additionally, intrahepatic IL-17+ γδ T cells, but not the IFN-γ+ ones, preferentially expressed IL-7Rα (CD127) on their surface, which coincided with an elevation of hepatocyte-derived IL-7 at 12 h post-infection. IL-7Rα blockade in vivo severely impeded the expansion of IL-17-producing cells following viral infection. In vitro, IL-7 synergized with IL-23 and directly stimulated IL-17 production from γδ T cells in response to TCRγδ stimulation. Finally, type I interferon (IFN-I) signaling was found to be critical for hepatic IL-7 induction. Collectively, these results showed that the IFN-I/IL-7/IL-17 cascade was important in priming T cell responses in the liver. Moreover, the highly coordinated cross talk among hepatocytes and innate and adaptive immune cells played a critical role in antiviral immunity in hepatitis.
doi:10.4049/jimmunol.1201970
PMCID: PMC3538895  PMID: 23233727
3.  IFNα/β and autophagy 
Autophagy  2011;7(11):1394-1396.
Hepatitis C virus (HCV) infects approximately 130 million people worldwide. The clinical sequelae of this chronic disease include cirrhosis, functional failure and carcinoma of the liver. HCV induces autophagy, a fundamental cellular process for maintaining homeostasis and mediating innate immune response, and also inhibits autophagic protein degradation and suppresses antiviral immunity. In addition to this ploy, the HCV serine protease composed of the viral nonstructural proteins 3/4A (NS3/4A) can enzymatically digest two cellular proteins, mitochondria-associated antiviral signaling protein (MAVS) and toll/interleukin-1 receptor domain containing adaptor inducing IFNβ (TRIF). Since these two proteins are the adaptor molecules in the retinoic acid-inducible gene I (RIG-I) and TLR3 pathways, respectively, their cleavage has been suggested as a pivotal mechanism by which HCV blunts the IFNα/β signaling and antiviral responses. Thus far, how HCV perturbs autophagy and copes with IFNα/β in the liver remains unclear.
doi:10.4161/auto.7.11.17514
PMCID: PMC3359484  PMID: 21997372
autophagy; hepatitis C virus; liver disease; NS3/4A protease; RIG-I; Type I interferon; TLR3; transgenic mouse
4.  Differential, Type I Interferon-Mediated Autophagic Trafficking of Hepatitis C Virus Proteins in Mouse Liver 
Gastroenterology  2011;141(2):674-685.e6.
BACKGROUND & AIMS
The hepatitis C virus (HCV) serine protease NS3/4A can cleave mitochondria-associated, anti-viral signaling protein (MAVS) and block retinoic acid-inducible gene I–mediated interferon (IFN) responses. Although this mechanism is thought to have an important role in HCV-mediated innate immunosuppression, its significance in viral persistence is not clear.
METHODS
We generated transgenic mice that express the HCV NS3/4A proteins specifically in the liver and challenged the animals with a recombinant vesicular stomatitis virus (VSV), a synthetic HCV genome, IFN-α, or IFN-β. We evaluated the effects of HCV serine protease on the innate immune responses and their interactions.
RESULTS
Expression of HCV NS3/4A resulted in cleavage of intrahepatic MAVS; challenge of transgenic mice with VSV or a synthetic HCV genome induced strong, type I IFN-mediated responses that were not significantly lower than those of control mice. Different challenge agents induced production of different ratios of IFN-α and -β, resulting in different autophagic responses and vesicular trafficking patterns of endoplasmic reticulum- and mitochondria-associated viral proteins. IFN-β promoted degradation of the viral proteins by the autolysosome. Variant isoforms of MAVS were associated with distinct, type I IFN-mediated autophagic responses; these responses have a role in trafficking of viral components to endosomal compartments that contain toll-like receptor -3.
CONCLUSIONS
IFN-β-mediates a distinct autophagic mechanism of anti-viral host defense. MAVS have an important role in type I IFN-induced autophagic trafficking of viral proteins.
doi:10.1053/j.gastro.2011.04.060
PMCID: PMC3152629  PMID: 21683701
Autophagy; TLR3; liver disease; RIG-I
5.  Parenchymal Expression of CD40 Exacerbates Adenovirus-Induced Hepatitis 
Hepatology (Baltimore, Md.)  2011;53(5):1455-1467.
The healthy adult human liver expresses low levels of MHC II and undetectable levels of immune co-stimulatory molecules. However, high levels of MHC class II, CD40 and B7 family molecules are expressed in the activated Kupffer cells and hepatocytes of patients having viral hepatitis. The precise role of these molecules in viral clearance and immune-mediated liver injury is not well understood. We hypothesize that parenchymal CD40 expression enhances T-cell recruitment and effector functions, which may facilitate viral clearance and alleviate liver injury. To test this hypothesis, we generated novel, liver-specific, conditional CD40 transgenic mice, and challenged them i.v. with recombinant replication-deficient adenovirus carrying Cre recombinase (AdCre). Wild-type mice infected with AdCre developed a relatively mild course of viral hepatitis and recovered spontaneously. CD40 expression in the liver of transgenic animals, however, resulted in CD80 and CD86 expression. Dysregulation of population dynamics and effector functions of intrahepatic lymphocytes results in severe lymphocytic infiltration, apoptosis, necroinflammation, and serum alanine transferase (ALT) elevation in a dose-dependent fashion. To our surprise, an early expansion followed by a contraction of intrahepatic lymphocytes, especially CD8+ and NK cells, accompanied by increased granzyme B and IFN-γ production, did not lead to a faster viral clearance in CD40 transgenic mice. Conclusion: Our results demonstrated that hepatic CD40 expression does not accelerate adenoviral clearance, but rather exacerbates liver injury. This study unveils a previously unknown deleterious effect of hepatic CD40 in adenovirus-induced liver inflammation.
doi:10.1002/hep.24270
PMCID: PMC3082591  PMID: 21360722
liver; animal models; T lymphocytes and co-stimulation
6.  Aberrant transcription and post-transcriptional processing of hepatitis C virus non-structural genes in transgenic mice 
Transgenic research  2011;20(6):1273-1284.
Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease worldwide. Since several aspects of the infection remain unresolved, there is a pressing need for a convenient animal model that can mimic the clinical disease and aid the evaluation of treatment strategies. Although some success has been achieved in transgenic approaches for development of rodent models of HCV, transgenic expression of the complete HCV polyprotein or an entire set of the viral non-structural (NS) proteins continues to be a serious challenge. Using northern blot and 5′ rapid amplification of cDNA ends (RACE), we unraveled two possible mechanisms that can impede HCV NS transgene expression in the mouse liver. Several truncated transcripts are produced from alternate transcription start sites along the HCV NS sequence within the murine environment, in vivo. Translation of these shorter transcripts is blocked either by the positioning of a contextual stop codon or through a shift in the reading frame. In addition, the complete NS transcript undergoes trans-splicing through 5′ recombination with a non-transgene-derived, spliced leader sequence that appends a potential stop codon upstream of the translation start. These findings thus demonstrate that HCV NS-derived transgenes are subject to aberrant transcriptional initiation and post-transcriptional processing in the nucleus of a mouse host. Strategies to prevent such aberrant transcription start/RNA processing might be key to the development of a successful HCV transgenic mouse model.
doi:10.1007/s11248-011-9494-x
PMCID: PMC3188684  PMID: 21347690
Hepatitis C virus; Non-structural proteins; Transgenic mouse; Aberrant transcription; Trans-splicing
7.  CD4+ CD25+ Regulatory T Cells Restrain Pathogenic Responses during Leishmania amazonensis Infection1 
Although activation of CD4+ T cells mediates pathogenesis in Leishmania amazonensis (La)-infected mice, these susceptible mice do not develop a polarized Th2 response, suggesting a unique mechanism of disease susceptibility. To understand how Th cell activities are regulated, we examined the frequency and phenotypes of regulatory T (Treg) cells. At 1–3 wk of infection, relatively high percentages of CD4+CD25+CD86+ T cells, as well as high levels of FoxP3, TGF-β1, and IL-10RI transcripts, were detected in the skin and draining lymph nodes, indicating local accumulation of Treg cells. Lesion-derived, IL-10-producing CD4+CD25+ cells effectively suppressed proliferation and cytokine (IL-2 and IFN-γ) production of CD4+CD25− effector cells. Adoptive transfer of lesion-derived CD4+CD25+ cells to syngeneic, naive C57BL/6 mice before infection significantly reduced disease development. To further validate the beneficial role of Treg cells in La infection, we adoptively transferred CD25+ T cell-depleted splenocytes (derived from naive mice) into RAG1-/- mice. This transfer rendered RAG1-/- mice more susceptible to La infection than the mice receiving control splenocytes. The beneficial effect of Treg cells was transitory and correlated with decreased activation of IFN-γ-producing effector T cells. This study uncovers an intriguing role of Treg cells in restraining pathogenic responses during nonhealing Leishmania infection and emphasizes a balance between Treg and Th1-like effector cells in determining the outcome of New World cutaneous leishmaniasis.
PMCID: PMC2812412  PMID: 15905558
8.  Cre-Estrogen Receptor-Mediated Hepatitis C Virus Structural Protein Expression in Mice 
Journal of virological methods  2007;146(1-2):5-13.
Hepatocyte apoptosis is an important feature of liver injury in hepatitis C virus (HCV) infection. However, the mechanism of apoptosis and consequences on disease progression in vivo have not been investigated fully in part due to the lack of adequate small animal models. In this study, transgenic (tg) mice were produced that express conditionally HCV structural proteins (core, E1, E2 and p7) in the liver following Cre-mediated DNA recombination. Using a novel Cre-estrogen receptor fusion protein (Cre-ER) induction strategy, tamoxifen was injected intraperitoneally (i.p.), which induced Cre nuclear translocation, transgene recombination and HCV protein expression in the liver. Hepatic expression of HCV core and envelope proteins resulted in increased hepatocyte apoptosis, detected by the TUNEL assay, between 7 and 33 days after induction. These results were confirmed by the presence of increased levels of apoptosis-associated cytokeratin 18 (CK18) in the sera of the same animals. The presence of cleaved caspase-3 and elevated levels of CHOP/GADD153 in the liver suggests an endoplasmic reticulum (ER) stress-associated apoptosis mechanism. This study suggests an in vivo correlation between HCV structural protein expression, ER stress and hepatocyte apoptosis, implicating a potentially important mechanism of HCV pathogenesis.
doi:10.1016/j.jviromet.2007.05.025
PMCID: PMC2104783  PMID: 17628708
viral hepatitis; transgenic mouse; animal model and liver injury
9.  Parenchymal Expression of CD86/B7.2 Contributes to Hepatitis C Virus-Related Liver Injury 
Journal of Virology  2005;79(16):10730-10739.
Hepatitis C virus (HCV) infection is a major global health problem. Hepatic expression of immune costimulatory signaling molecules (e.g., B7) is known to be associated with ongoing liver injury in hepatitis C patients. However, due to the general lack of viral culture systems and adequate animal models, the function of these molecules in disease pathogenesis is poorly understood. To investigate the role of CD86 in HCV-related liver injury, we developed two transgenic mouse lineages with inducible expression of HCV structural proteins and constitutive expression of the costimulatory molecule CD86/B7.2 in the liver. Using a hydrodynamic-based, nonviral delivery protocol, we induced HCV transgene expression in the livers of HCV and CD86 single- and double-transgenic mice. We found that hepatic CD86 expression resulted in increased activation of and cytokine production (e.g., interleukin-2 and gamma interferon) by CD4+ T cells and that the retention of these cells was associated with more pronounced necroinflammatory lesions in the liver. Taken together, these data suggest that augmented, parenchymal antigen presentation conferred by hepatocyte CD86 expression alters homeostasis and effector functions of CD4+ T cells and contributes to liver injury. This study provides an additional rationale for exploring immunomodulation-based therapies that could reduce disease progression in individuals with chronic HCV infection.
doi:10.1128/JVI.79.16.10730-10739.2005
PMCID: PMC1182684  PMID: 16051865
10.  Sand Fly Saliva Enhances Leishmania amazonensis Infection by Modulating Interleukin-10 Production  
Infection and Immunity  2004;72(3):1240-1247.
After transmission through the bite of female sand flies, Leishmania spp. can cause a broad spectrum of disease manifestations collectively known as leishmaniases. L. amazonensis is endemic in South America, where it causes cutaneous, diffuse cutaneous, and visceral leishmaniasis. In this study, we have provided evidence that salivary gland extracts (SGE) of Lutzomyia longipalpis enhances L. amazonensis infection. BALB/c mice infected intradermally in the ear with 105 metacyclic promastigotes of L. amazonensis together with SGE (equivalent to 0.5 gland) showed an early onset of disease and larger lesions that contained ∼3-log-units more parasites than did controls. To determine the potential mechanism underlying this enhancement, we assessed cytokine production via reverse transcriptase PCR and enzyme-linked immunosorbent assay. Mice coinjected with parasites and SGE displayed higher levels of interleukin-10 (IL-10) mRNA in the ear tissues, as well as higher levels of IL-10 in supernatants of restimulated draining lymph node (LN) cells, than did controls. Flow cytometric analysis revealed high frequencies of IL-10-producing CD4+ and CD8+ T cells in the draining LN of mice coinjected with the parasite and SGE. In addition, we examined bone marrow derived-macrophage cultures and detected increased IL-10 but decreased nitric oxide (NO) production in cells exposed to SGE prior to infection with L. amazonensis. Together, these results imply that the sand fly saliva facilitates Leishmania evasion of the host immune system by modulating IL-10 production.
doi:10.1128/IAI.72.3.1240-1247.2004
PMCID: PMC356033  PMID: 14977924
11.  Impaired Expression of Inflammatory Cytokines and Chemokines at Early Stages of Infection with Leishmania amazonensis  
Infection and Immunity  2003;71(8):4278-4288.
Infection of mice with Leishmania major results in disease progression or resolution, largely depending on the genetic backgrounds of the mouse strains. Infection with Leishmania amazonensis, on the other hand, causes progressive cutaneous lesions in most inbred strains of mice. We hypothesized that deficient activation of early immune responses contributes to the pathogenesis in L. amazonensis-infected mice. To distinguish early molecular events that determine the outcome of Leishmania infections, we examined cytokine gene expression in C57BL/6 mice infected with either L. amazonensis or L. major (a healing model). After 2 to 4 weeks, L. amazonensis-infected mice had significantly delayed and depressed expression of inflammatory cytokines (interleukin-12 [IL-12], gamma interferon, IL-1α, IL-1β), CC chemokines (CC chemokine ligand 3 [CCL3]/macrophage inflammatory protein 1α [MIP-1α], CCL4/MIP-1β, CCL5/RANTES, MIP-2), and chemokine receptors (CCR1, CCR2, CCR5) in foot tissues and draining lymph nodes compared to the expression in L. major-infected controls. These findings correlated with defective T-cell responsiveness to parasite stimulation in vivo and in vitro. Adoptive transfer of L. amazonensis-specific Th1 cells prior to infection overcame the immune defects of the animals, leading to complete control of the disease. Studies with gene knockout mice suggested that IL-10, but not IL-4, contributed partially to compromised immunity in L. amazonensis-infected hosts. The data suggest that there is impairment in multiple immune functions at early stages of infection with L. amazonensis parasites and provide a compelling rationale to explore immune augmentation as an intervention in American cutaneous leishmaniasis.
doi:10.1128/IAI.71.8.4278-4288.2003
PMCID: PMC166010  PMID: 12874303
12.  Hepatitis C Virus Core and Envelope Proteins Do Not Suppress the Host's Ability To Clear a Hepatic Viral Infection 
Journal of Virology  2001;75(24):11992-11998.
Several hepatitis C virus (HCV) proteins have been shown in vitro to interact with host cellular components that are involved in immune regulation. However, there is a paucity of data supporting the relevance of these observations to the in vivo situation. To test the hypothesis that such an interaction suppresses immune responses, we studied a line of transgenic C57BL/6 mice that express the HCV core and envelope proteins in the liver. The potential effects of these proteins on the hepatic immune response were evaluated by challenging these mice with a hepatotropic adenovirus. Both transgenic and nontransgenic mice developed similar courses of infection and cleared the virus from the liver by 28 days postinfection. Both groups of mice mounted similar immunoglobulin G (IgG), IgG2a, interleukin-2, and tumor necrosis factor alpha responses against the virus. Additionally, BALB/c mice were able to clear infection with recombinant adenovirus that does or does not express the HCV core and envelope 1 proteins in the same manner. These data suggest that HCV core and envelope proteins do not inhibit the hepatic antiviral mechanisms in these murine experimental systems and thus favor a model in which HCV circumvents host responses through a mechanism that does not involve general suppression of intrahepatic immune responses.
doi:10.1128/JVI.75.24.11992-11998.2001
PMCID: PMC116094  PMID: 11711589

Results 1-12 (12)