In this study, we explored HCV–HIV-1 interactions and the superimposition of the opiate drug morphine on host immune responses, including the production of free radicals and proinflammatory cytokines. While production of reactive nitrogen and oxygen species (RNS and ROS, respectively) is a component of innate defense against viral infection, their overproduction can be deleterious. Moreover, although morphine can alter HIV-1 entry into brain cells (19
), less is known about its effects in mono-infected or HCV- and HIV-1 coinfected hepatocytes. Using hepatocytes and in vitro
replication models of HCV, we present the following evidence: (i) that HCV induced the production of ROS, NO, and 3-NT and the proinflammatory cytokines TNF-α, IL-6, and, to a lesser extent, RANTES; (ii) that HIV-1 entered Huh7.5.1 cells via the HIV-1 coreceptors, CXCR4 and CCR5, in a CD4-independent manner, and coexposure with HIV-1 caused significant increases in HCV-induced oxyradical production and proinflammatory cytokine release; (iii) that morphine increased HIV-1 infectivity, and in combination with HIV-1 and HCV, morphine further enhanced virally induced inflammatory responses; and (iv) that the antioxidant NAC was effective in reversing ROS induction but was unable to attenuate HCV protein levels or virally induced cytokine release, while inhibiting the ubiquitin-proteasome system significantly attenuated viral protein production and host cytokine release. The last finding suggests that alterations in HCV pathogenesis are mediated by the host proteasome system through actions involving NF-κB.
While we find some hepatic cells are infected with HIV-1, many of the responses evoked by HIV-1 in liver parenchyma may be bystander effects (40
). The present study did not determine whether the infection with both viruses is in the same cell, different cells, or both. In fact, coinfection increases apoptotic death in Huh7.5.1 cells (23
), suggesting that coinfected cells are short-lived. Moreover, the extent to which coinfection in Huh7.5.1 hepatic cells, under artificial conditions, translates into cytopathic changes in human liver disease is uncertain. Though an appreciation of the cell and molecular responses of coinfected hepatocytes is fundamental toward understanding cellular pathogenesis, understanding the coordinated histopathologic response to dual infection is essential for managing the comorbidity therapeutically. As in the brain, we speculate that the collective hepatocyte inflammatory response to HIV-1 originates from a small percentage of infected macrophages/Kupffer cells and perhaps a smaller proportion of hepatocytes but is amplified by reverberant inflammatory responses of large numbers of uninfected macrophages and hepatocytes through paracrine and autocrine feedback.
In Huh-8 hepatoma cells containing a subgenomic HCV replicon, morphine was previously shown to increase the expression of HCV transcripts (30
). Furthermore, morphine withdrawal was found to exacerbate HCV replicon expression above levels seen with morphine alone, suggesting complex relationships between opiate drug exposure and HCV replication (66
). Morphine is also reported to facilitate HCV replication in human hepatocytes by inhibiting intrahepatic alpha interferon (IFN-α) expression (29
). Not only does heroin and morphine metabolism tax hepatocyte resources, but opioid receptors, including the μ-opioid receptor, are also widely distributed on lymphocytes, macrophages (including Kupffer cells [42
]), and hepatocytes, indicating that opioids can affect hepatic signaling (68
). Thus, opiates have the potential to directly regulate opioid receptor expression on the hepatocytes themselves (4
). The μ-opioid receptor, CCR5, and CXCR4 are G-protein-coupled receptors (GPCRs) (11
); they can undergo heterologous cross-desensitization and may even interact directly at the molecular level to form heterodimers (11
). Thus, exposure to opioids may affect a variety of functions in liver, including hepatocyte metabolism and function, while modifying immune-hepatocyte interactions, such as inhibiting cellular immune responses, modifying chemotactic responses of immune cells (37
), and inducing the expression of CCR5 receptors on immunocytes or hepatocytes (39
Our results corroborate the findings of other investigators that human hepatocytes and hepatocytic cell lines can be infected with HIV-1 (3
). In contrast, some human hepatoma cell lines lack both CD4 and CXCR4 at the cell surface and are not productively infected with X4-tropic HIV-1 strains (16
). This discrepancy could relate to methodological differences used to detect cell surface receptors or the loss of or altered HIV-1 coreceptor expression due to multiple passages of the cell lines. Our studies included the necessary controls, such as the HIV-1 entry inhibitors maraviroc and AMD3100, to permit discrimination between de novo
viral particle production and viral recycling. Furthermore, recent studies by Lin and coworkers examined direct virus-virus interactions and showed that X4- and R5-tropic HIV-1 strains can infect Huh7.5.1 cells and, additionally, demonstrated that HIV-1 or HIV-1 proteins can enhance HCV JFH1 replication (23
). In the present study, HCV infection significantly increased ROS and RNS, and these reactive products were further elevated by exposure to HIV-1 proteins or by coinfection with HIV-1. This substantiates the findings of other investigators that HCV, as well as HCV core, NS3, and NS5 proteins, increases ROS production, which may contribute to increases in viral replication (12
). The expression of TNF-α and its cognate receptors increases in various models of inflammatory liver injury, including HCV infection, presumably as part of innate immune defenses (20
). However, coinfection with HIV-1 caused a decrease in HCV-induced IL-6 production, suggesting that in cases where infection with both viruses intensifies TNF-α and RANTES release, HIV-1 can exert an additional role by suppressing some aspects of immune function in an attempt to protect itself from host challenges while exacerbating the infection.
Interestingly, morphine alone minimally affected the cellular response to HCV; however, in combination with HIV-1 proteins or R5-tropic HIV-1SF162
isolates, morphine significantly increased RANTES. Although RANTES has been shown to suppress R5 HIV-1 entry and replication in vitro
, RANTES has competing roles in the nervous system, where it has been demonstrated to recruit inflammatory macrophages and escalate reactive gliosis in an experimental model of HIV-1 encephalitis (14
). Moreover, at high concentrations, RANTES was shown to both activate the host immune response and enhance HIV-1 infection in vitro
). In fact, overexpression of RANTES reportedly exacerbates rabies virus pathogenicity by causing a persistent high level of expression of other chemokines, excessive infiltration, and accumulation of inflammatory cells in the nervous system and augmenting blood-brain barrier permeability (73
). This suggests that overexpression of some chemokines such as RANTES, although potentially important in controlling viral infection, may not always be beneficial to the host. In fact, we propose that the imbalances in homeostatic, host defense responses created by multiple infections and compounded by injection drug use are sufficiently complex that it is not feasible to predict whether the increases or decreases in a particular cytokine described in the present study are beneficial or detrimental without additional experiments.
Collectively, the results indicate that NF-κB regulates HIV-1 Tat, gp120, and/or morphine-induced inflammation and HCV expression and suggests that phosphorylation of p65 mediates key aspects of the exacerbated pathology caused by opiate exposure in HCV- and HIV-1-coinfected liver cells. Blocking proteasome function, which prevents NF-κB activation by impeding dissociation of IκBα from NF-κB p65/p50 subunit complexes, provided additional support for NF-κB and p65 involvement. Thus, HIV-1 proteins alone or in combination with morphine preferentially target the p65 subunit of the transcription factor, leading to the activation of cytokines and chemokines through modification of this protein in hepatocytes. Interestingly, a potent antioxidant, NAC, exacerbated the release of inflammatory cytokines. We speculate that HCV hijacks the cell, and the normal hepatic response to ROS, which typically signals inflammation, is overridden. Accompanying the increases in cytokine production with NAC exposure, there were reductions in p65 phosphorylation compared to controls (D and E).
Indeed, few studies thus far have examined virus-virus interactions in combination with opiate drug abuse because of the inherent complexities of modeling each disease. However, despite the complexity of the interactions, the present study reveals some potential common sites of HCV, HIV-1, and opiate convergence that might be targeted therapeutically. For example, our findings indicate that inhibiting the proteasome markedly reduced TNF-α and RANTES release and decreased HCV NS3 protein levels, irrespective of viral and/or morphine insults, while inhibiting ROS could paradoxically increase the production of some cytokines while decreasing HCV core protein levels. Further studies are needed to elucidate whether the decreased viral protein levels correlate with inhibition of HCV since proteasome inhibitors can have complex effects on HCV pathogenesis (46
). Understanding how opioids exacerbate the pathology and complications of HIV-1 and HCV coexposure by temporally distorting the production of proinflammatory cytokines or by sustaining or desynchronizing anti-HCV factors should improve our knowledge and ability to treat current and recovering HCV-infected and, especially, HCV/HIV-1-coinfected IDUs.