Antiviral Toll-like Receptor Signaling in Non-Parenchymal Liver Cells Is Restricted to TLR3.

The role of non-parenchymal liver cells as part of the hepatic, innate immune system in the defense against hepatotropic viruses is not well understood. Here, primary human Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells were isolated from liver tissue obtained after tumor resections or liver transplantations. Cells were stimulated with Toll-like receptor 1-9 ligands for 6-24 h. Non-parenchymal liver cells expressed and secreted inflammatory cytokines (IL6, TNF and IL10). Toll-like receptor- and cell type-specific downstream signals included the phosphorylation of NF-κB, AKT, JNK, p38 and ERK1/2. However, only supernatants of TLR3-activated Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells contained type I and type III interferons and mediated an antiviral activity in the interferon-sensitive subgenomic hepatitis C virus replicon system. The antiviral effect could not be neutralized by antibodies against IFNA, IFNB nor IFNL, but could be abrogated using an interferon alpha receptor 2-specific neutralization. Interestingly, TLR3 responsiveness was enhanced in liver sinusoidal endothelial cells isolated from hepatitis C virus-positive donors, compared to uninfected controls. In conclusion, non-parenchymal liver cells are potent activators of the hepatic immune system by mediating inflammatory responses. Furthermore, liver sinusoidal endothelial cells were identified to be hyperresponsive to viral stimuli in chronic hepatitis C virus infection.

Expanded Histopathology and Tropism of Ebola Virus in the Rhesus Macaque Model: Potential for Sexual Transmission, Altered Adrenomedullary Hormone Production, and Early Viral Replication in Liver.

The pathogenesis of Ebola virus disease (EVD) is still incomplete, in spite of the availability of a nonhuman primate modelfor more than 4 decades. To further investigate EVD pathogenesis, a natural history study was conducted using 27 Chinese-origin rhesus macaques. Of these, 24 macaques were exposed intramuscularly to Kikwit Ebola virus and euthanized at predetermined time points or when end-stage clinical disease criteria were met, and 3 sham-exposed macaques were euthanized on study day 0. This study showed for the first time that Ebola virus causes uterine cervicitis, vaginitis, posthitis, and medullary adrenalitis. Not only was Ebola virus detected in the interstitial stromal cells of the genital tract, but it was also present in the epididymal and seminal vesicular tubular epithelial cells, ectocervical and vaginal squamous epithelial cells, and seminal fluid. Furthermore, as early as day 3 after exposure, Ebola virus replicative intermediate RNA was detected in Kupffer cells and hepatocytes. These findings in the nonhuman model provide additional insight into potential sexual transmission, possible disruption of sympathetic hormone production, and early virus replication sites in human EVD patients.

MARCO+ lymphatic endothelial cells sequester arthritogenic alphaviruses to limit viremia and viral dissemination.

Viremia in the vertebrate host is a major determinant of arboviral reservoir competency, transmission efficiency, and disease severity. However, immune mechanisms that control arboviral viremia are poorly defined. Here, we identify critical roles for the scavenger receptor MARCO in controlling viremia during arthritogenic alphavirus infections in mice. Following subcutaneous inoculation, arthritogenic alphavirus particles drain via the lymph and are rapidly captured by MARCO+ lymphatic endothelial cells (LECs) in the draining lymph node (dLN), limiting viral spread to the bloodstream. Upon reaching the bloodstream, alphavirus particles are cleared from the circulation by MARCO-expressing Kupffer cells in the liver, limiting viremia and further viral dissemination. MARCO-mediated accumulation of alphavirus particles in the draining lymph node and liver is an important host defense mechanism as viremia and viral tissue burdens are elevated in MARCO-/- mice and disease is more severe. In contrast to prior studies implicating a key role for lymph node macrophages in limiting viral dissemination, these findings exemplify a previously unrecognized arbovirus-scavenging role for lymphatic endothelial cells and improve our mechanistic understanding of viremia control during arthritogenic alphavirus infection.

HCV Core Protein Induces Chemokine CCL2 and CXCL10 Expression Through NF-κB Signaling Pathway in Macrophages.

HCV core protein is the first structural protein synthesized during hepatitis C virus (HCV) infection and replication. It is released from virus infected liver cells and mediates multiple functions to affect host cell response. The innate immune response is the first line of defense against viral infection. After HCV infection, Kupffer cells (KCs) which are liver macrophages play an important role in host innate immune response. Kupffer cells act as phagocytes and release different cytokines and chemokines to counter viral infection and regulate inflammation and fibrosis in liver. Earlier, we have demonstrated that HCV core protein interacts with gC1qR and activates MAPK, NF-κB and PI3K/AKT pathways in macrophages. In this study, we explored the effect of HCV core protein on CCL2 and CXCL10 expression in macrophages and the signaling pathways involved. Upon silencing of gC1qR, we observed a significant decrease expression of CCL2 and CXCL10 in macrophages in the presence of HCV core protein. Inhibiting NF-κB pathway, but not P38, JNK, ERK and AKT pathways greatly reduced the expression of CCL2 and CXCL10. Therefore, our results indicate that interaction of HCV core protein with gC1qR could induce CCL2 and CXCL10 secretion in macrophages via NF-κB signaling pathway. These findings may shed light on the understanding of how leukocytes migrate into the liver and exaggerate host-derived immune responses and may provide novel therapeutic targets in HCV chronic inflammation.

Role of Kupffer Cells in Driving Hepatic Inflammation and Fibrosis in HIV Infection.

While the interactions between HIV and various liver cell populations have been explored, the relevance of these interactions when patients are well-controlled on ART is less clear. Therefore, we focus this perspective on HIV-related alterations that may drive hepatic inflammation and fibrosis in aviremic patients, with a focus on Kupffer cells and Hepatic Stellate Cells. Persistent CD4+ T cell depletion in the gut resulting in increased gut permeability has been postulated to play a role in systemic immune activation in HIV patients. The liver, with its unique location, remains the gatekeeper between the gut and the systemic circulation. The resident liver macrophage, Kupffer cell, is responsible for clearing and responding to these products. We propose that changes in Kupffer cell biology, in the context of HIV infection, creates a mileu that drives hepatic inflammation and fibrosis in response to microbial translocation. Targeting these pathways may be helpful in improving liver-related outcomes in HIV patients.

STING induces early IFN-ß in the liver and constrains myeloid cell-mediated dissemination of murine cytomegalovirus.

Cytomegalovirus is a DNA-encoded ß-herpesvirus that induces STING-dependent type 1 interferon responses in macrophages and uses myeloid cells as a vehicle for dissemination. Here we report that STING knockout mice are as resistant to murine cytomegalovirus (MCMV) infection as wild-type controls, whereas mice with a combined Toll-like receptor/RIG-I-like receptor/STING signaling deficiency do not mount type 1 interferon responses and succumb to the infection. Although STING alone is dispensable for survival, early IFN-ß induction in Kupffer cells is STING-dependent and controls early hepatic virus propagation. Infection experiments with an inducible reporter MCMV show that STING constrains MCMV replication in myeloid cells and limits viral dissemination via these cells. By contrast, restriction of viral dissemination from hepatocytes to other organs is independent of STING. Thus, during MCMV infection STING is involved in early IFN-ß induction in Kupffer cells and the restriction of viral dissemination via myeloid cells, whereas it is dispensable for survival.

Exploring Crimean-Congo Hemorrhagic Fever Virus-Induced Hepatic Injury Using Antibody-Mediated Type I Interferon Blockade in Mice.

Crimean-Congo hemorrhagic fever virus (CCHFV) can cause severe hepatic injury in humans. However, the mechanism(s) causing this damage is poorly characterized. CCHFV produces an acute disease, including liver damage, in mice lacking type I interferon (IFN-I) signaling due to either STAT-1 gene deletion or disruption of the IFN-I receptor 1 gene. Here, we explored CCHFV-induced liver pathogenesis in mice using an antibody to disrupt IFN-I signaling. When IFN-I blockade was induced within 24 h postexposure to CCHFV, mice developed severe disease with greater than 95% mortality by 6 days postexposure. In addition, we observed increased proinflammatory cytokines, chemoattractants, and liver enzymes in these mice. Extensive liver damage was evident by 4 days postexposure and was characterized by hepatocyte necrosis and the loss of CLEC4F-positive Kupffer cells. Similar experiments in CCHFV-exposed NOD-SCID-γ (NSG), Rag2-deficient, and perforin-deficient mice also demonstrated liver injury, suggesting that cytotoxic immune cells are dispensable for hepatic damage. Some apoptotic liver cells contained viral RNA, while other apoptotic liver cells were negative, suggesting that cell death occurred by both intrinsic and extrinsic mechanisms. Protein and transcriptional analysis of livers revealed that activation of tumor necrosis factor superfamily members occurred by day 4 postexposure, implicating these molecules as factors in liver cell death. These data provide insights into CCHFV-induced hepatic injury and demonstrate the utility of antibody-mediated IFN-I blockade in the study of CCHFV pathogenesis in mice.IMPORTANCE CCHFV is an important human pathogen that is both endemic and emerging throughout Asia, Africa, and Europe. A common feature of acute disease is liver injury ranging from mild to fulminant hepatic failure. The processes through which CCHFV induces severe liver injury are unclear, mostly due to the limitations of existing small-animal systems. The only small-animal model in which CCHFV consistently produces severe liver damage is mice lacking IFN-I signaling. In this study, we used antibody-mediated blockade of IFN-I signaling in mice to study CCHFV liver pathogenesis in various transgenic mouse systems. We found that liver injury did not depend on cytotoxic immune cells and observed extensive activation of death receptor signaling pathways in the liver during acute disease. Furthermore, acute CCHFV infection resulted in a nearly complete loss of Kupffer cells. Our model system provides insight into both the molecular and the cellular features of CCHFV hepatic injury.

Involvement of Pro-Inflammatory Macrophages in Liver Pathology of Pirital Virus-Infected Syrian Hamsters.

New World arenaviruses cause fatal hemorrhagic disease in South America. Pirital virus (PIRV), a mammarenavirus hosted by Alston’s cotton rat (Sigmodon alstoni), causes a disease in Syrian golden hamsters (Mesocricetus auratus) (biosafety level-3, BSL-3) that has many pathologic similarities to the South American hemorrhagic fevers (BSL-4) and, thus, is considered among the best small-animal models for human arenavirus disease. Here, we extend in greater detail previously described clinical and pathological findings in Syrian hamsters and provide evidence for a pro-inflammatory macrophage response during PIRV infection. The liver was the principal target organ of the disease, and signs of Kupffer cell involvement were identified in mortally infected hamster histopathology data. Differential expression analysis of liver mRNA revealed signatures of the pro-inflammatory response, hematologic dysregulation, interferon pathway and other host response pathways, including 17 key transcripts that were also reported in two non-human primate (NHP) arenavirus liver-infection models, representing both Old and New World mammarenavirus infections. Although antigen presentation may differ among rodent and NHP species, key hemostatic and innate immune-response components showed expression parallels. Signatures of pro-inflammatory macrophage involvement in PIRV-infected livers included enrichment of Ifng, Nfkb2, Stat1, Irf1, Klf6, Il1b, Cxcl10, and Cxcl11 transcripts. Together, these data indicate that pro-inflammatory macrophage M1 responses likely contribute to the pathogenesis of acute PIRV infection.

3D microfluidic liver cultures as a physiological preclinical tool for hepatitis B virus infection.

With more than 240 million people infected, hepatitis B virus (HBV) is a major health concern. The inability to mimic the complexity of the liver using cell lines and regular primary human hepatocyte (PHH) cultures pose significant limitations for studying host/pathogen interactions. Here, we describe a 3D microfluidic PHH system permissive to HBV infection, which can be maintained for at least 40 days. This system enables the recapitulation of all steps of the HBV life cycle, including the replication of patient-derived HBV and the maintenance of HBV cccDNA. We show that innate immune and cytokine responses following infection with HBV mimic those observed in HBV-infected patients, thus allowing the dissection of pathways important for immune evasion and validation of biomarkers. Additionally, we demonstrate that the co-culture of PHH with other non-parenchymal cells enables the identification of the cellular origin of immune effectors, thus providing a valuable preclinical platform for HBV research.

Contradictory intrahepatic immune responses activated in high-load hepatitis C virus livers compared with low-load livers.

We found a HLA class II histocompatibility antigen gene, DQ alpha 1 chain (HLA-DQA1), that was expressed more than 9-fold higher in high-load hepatitis C virus (HCV) livers than low-load HCV livers using transcriptomics of chronic HCV-infected livers. To further investigate this finding, we examined which cells were positive for HLA-DQA1 and what liver immune responses were different between HCV-high and -low livers. HLA-DQA1-positive cells were significantly increased in the HCV-high group, and most positive cells were identified as non-parenchymal sinusoid cells and lymphocytic infiltrates in the portal area. Parenchymal hepatocytes were negative for HLA-DQA1. HLA-DQA1-positive cells in the liver sinusoid were positive for CD68 (macrophages or Kupffer cells); those in the lymphocytic infiltrates were positive for CD20 (B cells) or CD3 (T cells). mRNA levels of antigen-presenting cell (APC) markers such as CD68 and CD11c were significantly upregulated in the HCV-high group and were correlated with HLA-DQA mRNA levels. CD8B mRNA (CD8+ T cells) was upregulated in both HCV-positive livers compared with HCV-negative livers, whereas CD154 mRNA (CD4+ T helper cell) was upregulated in the HCV-high group compared with the HCV-low group. The immune regulatory molecules FOXP3 mRNA (regulatory T cell, T reg) and programmed cell death ligand-1 (PD-L1) mRNA were significantly increased in the HCV-high group. HCV-high livers had two molecular immune responses: increased APC numbers and adaptive immunity and the induction of immune tolerance. The local hepatic imbalance of contradictory immune responses might be responsible for high HCV loads.

Male Syrian hamsters are more susceptible to intravenous infection with species C human adenoviruses than are females.

Recently, increasing attention has been focused on the influence of sex on the course of infectious diseases. Thus far, the best-documented examples point toward an immune-mediated mechanism: the generally stronger immune response in females can result in a faster clearance of the pathogen or, conversely, a more severe immune-mediated pathology. Here, we report that human species C adenoviruses replicate more and cause more pathology in male Syrian hamsters than in females. We also show that this sex disparity is not caused by a stronger immune response to the infection by the female hamsters. Rather, the liver of male hamsters is more susceptible to adenovirus infection: after intravenous injection, more hepatocytes become infected in male animals than in females. We hypothesize that Kupffer cells (hepatic tissue macrophages) of female animals are more active in sequestering circulating virions, and thus protect hepatocytes more efficiently than those of males.

Pathology in Permissive Syrian Hamsters after Infection with Species C Human Adenovirus (HAdV-C) Is the Result of Virus Replication: HAdV-C6 Replicates More and Causes More Pathology than HAdV-C5.

Syrian hamsters are permissive for the replication of species C human adenoviruses (HAdV-C). The virus replicates to high titers in the liver of these animals after intravenous infection, while respiratory infection results in virus replication in the lung. Here we show that two types belonging to species C, HAdV-C5 and HAdV-C6, replicate to significantly different extents and cause pathology with significantly different severities, with HAdV-C6 replicating better and inducing more severe and more widespread lesions. The virus burdens in the livers of HAdV-C6-infected hamsters are higher than the virus burdens in HAdV-C5-infected ones because more of the permissive hepatocytes get infected. Furthermore, when hamsters are infected intravenously with HAdV-C6, live, infectious virus can be isolated from the lung and the kidney, which is not seen with HAdV-C5. Similarly to mouse models, in hamsters, HAdV-C6 is sequestered by macrophages to a lesser degree than HAdV-C5. Depletion of Kupffer cells from the liver greatly increases the replication of HAdV-C5 in the liver, while it has only a modest effect on the replication of HAdV-C6. Elimination of Kupffer cells also dramatically increases the pathology induced by HAdV-C5. These findings indicate that in hamsters, pathology resulting from intravenous infection with adenoviruses is caused mostly by replication in hepatocytes and not by the abortive infection of Kupffer cells and the following cytokine storm.IMPORTANCE Immunocompromised human patients can develop severe, often lethal adenovirus infections. Respiratory adenovirus infection among military recruits is a serious problem, in some cases requiring hospitalization of the patient. Furthermore, adenovirus-based vectors are frequently used as experimental viral therapeutic agents. Thus, it is imperative that we investigate the pathogenesis of adenoviruses in a permissive animal model. Syrian hamsters are susceptible to infection with certain human adenoviruses, and the pathology accompanying these infections is similar to what is observed with adenovirus-infected human patients. We demonstrate that replication in permissive cells in a susceptible host animal is a major part of the mechanism by which systemic adenovirus infection induces pathology, as opposed to the chiefly immune-mediated pathology observed in nonsusceptible hosts. These findings support the use of compounds inhibiting adenovirus replication as a means to block adenovirus-induced pathology.

Farnesoid X Receptor in Mice Prevents Severe Liver Immunopathology During Lymphocytic Choriomeningitis Virus Infection.

BACKGROUND: Bile acids (BAs) are steroid molecules that are synthesized in the liver. In addition to their important role as a surfactant in solubilizing lipids and promoting the absorption of lipids in the gastrointestinal tract, they act as inflammagens. The role of BAs and their receptor farnesoid X receptor (FXR) during viral infection has not been studied in detail. METHODS: By using FXR-deficient mice, we investigated the role of bile acid receptor FXR during infection with lymphocytic choriomeningitis virus (LCMV). The importance of FXR in inducing IFN-I and monocytes proliferation were investigated and viral titers and T cell exhaustion were analyzed at different time points. RESULTS: This study shows that controlled levels of BAs activate FXR in hepatocytes and FXR in response upregulates the production of type I interferon. In turn, FXR maintains BAs within a balanced range to inhibit their toxic effects. The absence of FXR results in high levels of BAs, which inhibit the proliferation of monocytes and result in a defect in viral elimination, consequently leading to T cell exhaustion. CONCLUSION: We found that FXR contributes to IFN-I production in hepatocytes and balances BA levels to inhibit their toxic effects on monocytes.

Frontline Science: HIV infection of Kupffer cells results in an amplified proinflammatory response to LPS.

End-stage liver disease is a common cause of non-AIDS-related mortality in HIV+ patients, despite effective anti-retroviral therapies (ARTs). HIV-1 infection causes gut CD4 depletion and is thought to contribute to increased gut permeability, bacterial translocation, and immune activation. Microbial products drain from the gut into the liver via the portal vein where Kupffer cells (KCs), the resident liver macrophage, clear translocated microbial products. As bacterial translocation is implicated in fibrogenesis in HIV patients through unclear mechanisms, we tested the hypothesis that HIV infection of KCs alters their response to LPS in a TLR4-dependent manner. We showed that HIV-1 productively infected KCs, enhanced cell-surface TLR4 and CD14 expression, and increased IL-6 and TNF-α expression, which was blocked by a small molecule TLR4 inhibitor. Our study demonstrated that HIV infection sensitizes KCs to the proinflammatory effects of LPS in a TLR4-dependent manner. These findings suggest that HIV-1-infected KCs and their dysregulated innate immune response to LPS may play a role in hepatic inflammation and fibrosis and represent a novel target for therapy.

HBV inhibits LPS-induced NLRP3 inflammasome activation and IL-1ß production via suppressing the NF-κB pathway and ROS production.

BACKGROUND & AIMS: Hepatitis B virus (HBV) has developed strategies to evade immune responses. However, the mechanisms involved remain unclear. The NLRP3 inflammasome plays crucial roles in antiviral host defense and its downstream factor IL-1ß has been shown to inhibit HBV infection in vivo. This study aims to assess whether HBV can affect the NLRP3 inflammasome signaling pathways and shed light on the underlying mechanisms HBV utilizes to evade host innate immune responses. METHODS: HBV inhibition of the lipopolysaccharide (LPS)-induced NLRP3 inflammasome activation was evaluated by Western blot, quantitative RT-PCR, flow cytometry and immunofluorescence. RESULTS: Kupffer cells expressed significantly more NLRP3 and IL-1ß after LPS stimulation; whereas, chronic HBV infection suppressed LPS-induced NLRP3 and pro-IL-1ß expression as well as IL-1ß maturation. This inhibitory activity is mediated by HBeAg, and is involved in the inhibition of NF-κB signal pathway and reactive oxygen species (ROS) production. The inhibitory effect of HBeAg was confirmed in patients with chronic hepatitis B (CHB) and hepatocellular carcinoma by comparing the levels of IL-1ß and NLRP3-related proteins in para-carcinoma tissues from HBeAg-positive or negative patients. Moreover, chronic HBV infection increases the susceptibility of mice to S. typhimurium infection, possibly via inhibiting the NLRP3 inflammasome activation and IL-1ß production. CONCLUSIONS: HBeAg inhibits LPS-induced NLRP3 inflammasome activation and IL-1ß production via suppressing NF-κB pathway and ROS production. This finding provides a novel mechanism for HBV-mediated suppression of innate immune responses, and identifies new therapeutic targets for chronic HBV infection and related diseases. LAY SUMMARY: HBeAg suppresses LPS-induced NLRP3 inflammasome activation and IL-1ß production in two ways, one is to repress NLRP3 and pro-IL-1ß expression via inhibiting NF-κB phosphorylation, and the other is to repress caspase-1 activation and IL-1ß maturation via inhibiting ROS production. This effect contributes to the HBV persistence and immune tolerance.

Antagonism of RNase L Is Required for Murine Coronavirus Replication in Kupffer Cells and Liver Sinusoidal Endothelial Cells but Not in Hepatocytes.

Mouse hepatitis virus strain A59 infection of mice is a useful tool for studying virus-host interaction during hepatitis development. The NS2H126R mutant is attenuated in liver replication due to loss of phosphodiesterase activity, which the wild-type (WT) virus uses to block the 2',5'-oligoadenylate synthetase (OAS)-RNase L (RNase L) antiviral pathway. The activation of RNase L by NS2H126R is cell type dependent and correlates with high basal expression levels of OAS, as found in myeloid cells. We tested the hypothesis that the resident liver macrophages, Kupffer cells (KC), represent the cell type most likely to restrict NS2H126R and prevent hepatitis. As found previously, A59 and NS2H126R replicate similarly in hepatocytes and neither activates RNase L, as assessed by an rRNA degradation assay. In contrast, in KC, A59 exhibited a 100-fold-higher titer than NS2H126R and NS2H126R induced rRNA degradation. Interestingly, in liver sinusoidal endothelial cells (LSEC), the cells that form a barrier between blood and liver parenchymal cells, NS2H126R activates RNase L, which limits viral replication. Similar growth kinetics were observed for the two viruses in KC and LSEC from RNase L-/- mice, demonstrating that both use RNase L to limit NS2H126R replication. Depletion of KC by gadolinium(III) chloride or of LSEC by cyclophosphamide partially restores liver replication of NS2H126R, leading to hepatitis. Thus, during mouse hepatitis virus (MHV) infection, hepatitis, which damages the parenchyma, is prevented by RNase L activity in both KC and LSEC but not in hepatocytes. This may be explained by the undetectable levels of RNase L as well as by the OASs expressed in hepatocytes. IMPORTANCE: Mouse hepatitis virus infection of mice provides a useful tool for studying virus-host interactions during hepatitis development. The NS2H126R mutant is attenuated in liver replication due to loss of phosphodiesterase activity, by which the wild-type virus blocks the potent OAS-RNase L antiviral pathway. RNase L activation by NS2H126R is cell type dependent and correlates with high basal expression levels of OAS, as found in myeloid cells. We showed that the hepatocytes that comprise the liver parenchyma do not activate RNase L when infected with NS2H126R or restrict replication. However, both Kupffer cells (KC) (i.e., the liver-resident macrophages) and the liver sinusoidal endothelial cells (LSEC) which line the sinusoids activate RNase L in response to NS2H126R These data suggest that KC and LSEC prevent viral spread into the parenchyma, preventing hepatitis. Furthermore, hepatocytes express undetectable levels of OASs and RNase L, which likely explains the lack of RNase L activation during NS2H126R infection.

Organ system view of the hepatic innate immunity in HCV infection.

An orchestration of innate and adaptive immunity determines the infection outcome and whether the host achieves clearance or allows the pathogen to establish persistent infection. The robust activation of the innate immune response plays the most critical role in both limiting viral replication and halting the spread of the pathogen immediately after infection. The magnitude of innate immune activation is coupled with the efficient mounting of the adaptive immunity. Although immunity against HCV infection is known to be inadequate as most cases transitions to chronicity, approximately 25% of acute infection cases result in spontaneous clearance. The exact immune mechanisms that govern the infection outcome remain largely unknown; recent discoveries suggest that the innate immune system facilitates this event. Both infected hepatocytes and local innate immune cells trigger the front line defense program of the liver as well as the recruitment of diverse adaptive immune cells to the site of infection. Although hepatocyte is the target of HCV infection, nearly all cell types that exist in the liver are involved in the innate defense and contribute to the pathophysiology of hepatic inflammation. The main focus of this comprehensive review is to discuss the current knowledge on how each hepatic cell type contributes to the organ system level innate immunity against HCV infection as well as interplays with the viral evasion program. Furthermore, this review article also aims to synchronize the observations from both molecular biological studies and clinical studies with the ultimate goal of improving our understanding of HCV mediated hepatitis. J. Med. Virol. 88:2025-2037, 2016. © 2016 Wiley Periodicals, Inc.

Canine Circovirus 1 (CaCV-1) and Canine Parvovirus 2 (CPV-2): Recurrent Dual Infections in a Papillon Breeding Colony.

Recurrent outbreaks of sudden death and bloody diarrhea were reported in March 2013 and February 2014 in a breeding colony of Papillon dogs. During the first outbreak, 1 adult dog and 2 eight-month-old puppies died. During the second outbreak, 2 ten-week-old puppies died. One puppy from the first outbreak and 2 puppies from the second outbreak were examined at necropsy. Histologically, all 3 puppies had severe segmental crypt necrosis of the small intestine and marked lymphoid follicle depletion in the spleen and Peyer's patches. Real-time (RT) polymerase chain reaction (PCR) demonstrated abundant canine parvovirus (CPV-2) DNA (Ct<15) in the affected small intestine, and immunohistochemistry detected large amounts of CPV-2 antigen in intestinal crypt epithelium and Kupffer cells but few positive macrophages in lymphoid organs. All puppies had marked sinusoidal histiocytosis and multifocal granulomatous inflammation in mesenteric lymph nodes and spleen, prompting additional RT-PCR testing for canine circovirus 1 (CaCV-1). Very high levels of CaCV-1 DNA (Ct<13) were detected in small intestine, lymph nodes, and spleen. In situ hybridization for CaCV-1 detected rare positive nuclei of regenerating crypt epithelium but abundant amounts of CaCV-1 nucleic acid in the cytoplasm and nuclei of histiocytes in all lymphoid tissues, including granulomatous inflammatory foci and hepatic Kupffer cells. Significant levels of CaCV-1 DNA were detected in blood and serum (Ct as low as 13) but not feces from 3 surviving dogs at 2 months or 1 year after the outbreak, respectively. We hypothesize that CPV-2 infection predisposed dogs to CaCV-1 infection and ultimately resulted in more severe clinical disease.

IFN-α-Induced Downregulation of miR-221 in Dendritic Cells: Implications for HCV Pathogenesis and Treatment.

Although interferon (IFN)-α is known to exert immunomodulatory and antiproliferative effects on dendritic cells (DCs) through induction of protein-coding IFN-stimulated genes (ISGs), little is known about IFN-α-regulated miRNAs in DCs. Since several miRNAs are involved in regulating DC functions, it is important to investigate whether IFN-α's effects on DCs are mediated through miRNAs as well. In this study, we examined miRNA expression patterns in myeloid DCs (mDCs) and plasmacytoid DCs after exposing them to IFN-α. We report that IFN-α downregulates miR-221 in both DC subsets via inhibition of STAT3. We validated proapoptotic proteins BCL2L11 and CDKN1C as miR-221 targets suggesting that IFN-α can induce DC apoptosis via miR-221 downregulation. In addition, we validated another miR-221 target, SOCS1, which is known to be a negative regulator of JAK/STAT signaling. Consistent with this, miR-221 overexpression in mDCs enhanced the secretion of proinflammatory cytokines IL-6 and TNF-α. In peripheral blood mononuclear cells (PBMCs) of HIV-1/HCV co-infected individuals undergoing IFN-α-based treatment the baseline miR-221 expression was lower in non-responders compared with responders; and miR-221 expression directly correlated with DC frequency and IL-6/TNF-α secretion. In addition to PBMCs, we isolated total liver cells and kupffer cells from HCV-infected individuals and individuals with alcoholic cirrhosis. We found that both total liver cells and kupffer cells from HCV-infected individuals had significantly higher miR-221 levels compared with individuals with cirrhosis. Overall, we demonstrate that IFN-α exerts both antiproliferative and immunomodulatory effects on mDCs via miR-221 downregulation; and IFN-miR-221 axis can play important role in HCV pathogenesis and treatment.