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.
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.
autophagy; hepatitis C virus; liver disease; NS3/4A protease; RIG-I; Type I interferon; TLR3; transgenic mouse
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.
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.
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.
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.
Autophagy; TLR3; liver disease; RIG-I
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.
liver; animal models; T lymphocytes and co-stimulation
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.
Hepatitis C virus; Non-structural proteins; Transgenic mouse; Aberrant transcription; Trans-splicing
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.
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.
viral hepatitis; transgenic mouse; animal model and liver injury
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.
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.
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.
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.