Nicola F. Fletcher, Rupesh Sutaria, Juandy Jo, Amy Barnes, Miroslava Blahova, Luke W. Meredith, Francois-Loic Cosset, Stuart M. Curbishley, David H. Adams, Antonio Bertoletti, and Jane A. McKeating.
Hepatology 59: 1320-30. 2014.
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The chronic HCV-infected liver shows a significant increase in total macrophage numbers that most likely represents a local proliferation of Kupffer cells and infiltration of bone-marrow-derived monocytes; however, their role in the HCV lifecycle is poorly understood. We found that activation of blood-derived and intra-hepatic macrophages with a panel of TLR agonists induce soluble mediators that promote HCV entry into polarized hepatoma cells and identified TNF-α as the major cytokine involved in this process. The ability of TNF-α to promote the permissivity of polarized hepatoma cells was not limited to HCV but was also seen with lentiviral pseudotypes expressing Lassa, measles, and VSV-G glycoproteins. These studies identify a new pathway for HCV to exploit inflammatory responses in the chronically infected liver to promote infection that may contribute to viral persistence.
TNF-α plays a key role in regulating cell differentiation, proliferation, innate and adaptive immune responses, and is expressed by a variety of immune cells. Our studies uncover a new role for TNF-α and IL-1β in promoting HCV infection of polarized hepatocytes and so may provide new therapeutic targets for antiviral therapy.
Macrophages are critical components of the innate immune response in the liver. Chronic hepatitis C is associated with immune infiltration and the infected liver shows a significant increase in total macrophage numbers; however, their role in the viral life cycle is poorly understood. Activation of blood-derived and intrahepatic macrophages with a panel of Toll-like receptor agonists induce soluble mediators that promote hepatitis C virus (HCV) entry into polarized hepatoma cells. We identified tumor necrosis factor a (TNF-α) as the major cytokine involved in this process. Importantly, this effect was not limited to HCV; TNF-α increased the permissivity of hepatoma cells to infection by Lassa, measles and vesicular stomatitis pseudoviruses. TNF-α induced a relocalization of tight junction protein occludin and increased the lateral diffusion speed of HCV receptor tetraspanin CD81 in polarized HepG2 cells, providing a mechanism for their increased permissivity to support HCV entry. High concentrations of HCV particles could stimulate macrophages to express TNF-α, providing a direct mechanism for the virus to promote infection. Conclusion: This study shows a new role for TNF-α to increase virus entry and highlights the potential for HCV to exploit existing innate immune responses in the liver to promote de novo infection events.
Figure 1: Conditioned media from LPS-stimulated macrophages promotes HCV entry.
To assess the role of macrophages in the HCV lifecycle, we cultured peripheral blood-derived CD14+ monocytes and stimulated them either with a combination of IFN-γ and TNF-α to generate M1-macrophages, with IL-4 to generate M2-macrophages, or with lipopolysaccharide (LPS). DCs were isolated from the same donor and stimulated with LPS to simulate the liver microenvironment. The culture medium was collected from each culture after 24 h and assessed for its effect on HCV pseudoparticle (HCVpp) infection of polarized HepG2.CD81 cells. The media collected from the LPS-stimulated macrophages significantly increased HCVpp infection whereas none of the other 3 media had any effect (A). Increasing the amount of LPS added to the macrophages (0.1-10 μg/mL) increased HCVpp infection, whereas simply adding LPS to the HepG2.CD81 culture had no effect (B). Cytokine array profiling identified TNF-α, and to a lower degree IL-1β, as uniquely elevated in the media collected from the LPS stimulated macrophages.
Figure 2. TNF-α is the major factor secreted from activated macrophages that promotes HCV infection.
IL-1β and TNF-α increased HCVpp (strain H77) infection of polarized HepG2.CD81 cells, whereas IFN-γ reduced infection in a dose-dependent manner (A). Anti TNF-α and anti-IL-1β neutralizing antibodies inhibited the effect of recombinant TNF-α and IL-1β on HCVpp infection (B). Importantly, preincubation of the LPS-stimulated macrophage conditioned medium with anti TNF-α reduced HCVpp infection to a comparable level observed in untreated hepatoma cells. In contrast, LPS-stimulated DCs secrete both TNF-α and IFN-γ which may explain their modest effect on HCVpp entry (Fig. 1), suggesting a balance between the antiviral activity of IFN-γ and proviral effect of TNF-α. Pretreating polarized hepatoma cells with TNF-α for 1 hour prior to inoculating with virus led to a significant increase in infection; however, this was not seen with a longer preincubation time of 24 hours, suggesting a limited response time for the cells and/or a reversible phenotype (C). These studies uncover a new role for activated macrophages expressed TNF-α in promoting HCV entry into polarized hepatoma cells.
Figure 3: TNF-α Promotes Entry of Diverse Viruses Into Polarized Hepatocytes.
To determine whether the proviral effect of TNF-α is specific for HCV, we generated pseudoparticles bearing the surface glycoproteins of Lassa, Measles, or Vesicular Stomatitis Virus (VSV). HepG2 polarization restricted the infection of all pseudoparticles tested (A). To determine whether TNF-α increased the number of infected cells or the viral burden per cell, we generated VSV-G pseudoparticles expressing a fluorescent reporter protein. Flow cytometry revealed that TNF-α increased both the number of infected cells (57% ± 4.2% versus 65% ± 1.8%) and their fluorescent intensity (B). Conditioned medium collected from LPS-stimulated macrophages increased the permissivity of HepG2.CD81 cells to all pseudoparticles in a TNF-dependent manner (C). These results highlight a role for TNF-α in enhancing the ability of polarized HepG2 cells to support infection by diverse viruses.
Figure 4: Effect of TNF-α on HepG2 permeability, occludin localization, and CD81 lateral diffusion.
Polarized HepG2.CD81 cells express the tight junction protein occludin at bile canalicular "ring" structures, indicating a complex hepatocellular polarity. Treatment of these cells with TNF-α, IL-1β or conditioned medium collected from LPS-stimulated macrophages induced a redistribution of occludin to the basolateral membrane (A). These observations lead us to conclude that these cytokines act to disrupt tight junctions. We have previously shown that polarization reduced viral receptor CD81 and HCVpp diffusion at the basal HepG2 membrane (Harris, 2013), which may explain their reduced ability to support infection. Measurement of basal membrane diffusion by FRAP showed that TNF-α induced an increase in the CD81 diffusion coefficient (B). These data highlight a role for TNF-α in regulating hepatoma tight junction and CD81 dynamics, providing an explanation for the increase in viral permissivity.
The constant stimulation of hepatic TLR4 by gut-derived LPS the liver is thought to induce tolerance mechanisms to limit hyperactivation of the immune system. However, recent studies demonstrate a loss of TLR tolerance in macrophages from chronic hepatitis B and C-infected patients, suggesting an association between LPS-induced macrophage activation and progression to endstage liver disease. It is interesting to note that both alcohol and HIV coinfection are associated with increased levels of plasma LPS. Our demonstration that LPS stimulates Kupffer cells to promote HCV infection provides a potential explanation for how these comorbidities may augment HCV infection and ensuing liver disease.