The pathogenesis of IPF is not fully delineated, but a critical event may be ongoing injury of the lung epithelium. Chronic herpesvirus infection is a potential cause of epithelial cell dysfunction, either by causing direct epithelial injury via virus lytic replication or by altering cell phenotype via a latent infection that induces immune responses that promote abnormal repair and fibrosis. We found that chronic herpesvirus lung infection in a mouse biased toward a Th2-type response resulted in progressive pulmonary fibrosis. Using this animal model, we demonstrate that active MHV68 replication during the chronic phase of infection is required for the development of virus-induced lung fibrosis. This finding has significant implications when developing an antiviral strategy in patients with IPF and infected with herpesvirus, as current antiherpesvirus treatments control only viruses undergoing lytic but not latent infection.
Prevention of viral replication with the antiviral cidofovir in chronically infected mice, beginning on Day 45 postinfection, mediated virus clearance, decreased lung levels of proinflammatory and profibrotic cytokines, and had a dramatic effect of lung fibrosis. These findings were associated with prevention of mortality and improvement of the clinical disease. Histopathologic analyses of the lungs of MHV68-infected mice treated with cidofovir showed persistence of lymphocytic infiltrates that in the past we have shown to be B cells (17
). Although antiviral treatment is effective only against lytic forms of the virus, ongoing productive replication is essential for maintaining high levels of latently infected cells. Thus, we found that mice treated with cidofovir had a reduction in the number of copies of transcripts of the viral latent genes M2 and M11, as expected (data not shown). Studies showed that mice infected intranasally with MHV68 and treated with cidofovir from Day 2 postinfection established long-term infection in lung B cells but were unable to establish latency in the spleen. Similar results were obtained when mice were infected intranasally with a gene 50 stop. MHV68 gene 50 encodes Rta, the major trans
-activator of the lytic program (36
). The role of the persistently latent infected B cells in lung fibrosis is unclear. B cells have been found to confer a protective role against silica-induced lung fibrosis by the production of prostaglandin E2
). On the other hand, B-cell–deficient mice have markedly reduced collagen deposition in a model of liver fibrosis produced by chronic treatment with CCl4
It is known that some viral proteins expressed during latency can modify the virus-mediated pathology. For instance, mice infected with MHV76, a virus that is deficient in expression of the unique set of latent viral proteins M1 to M4, or mice infected with an MHV68 virus that does not express the M1 latent protein, do not develop splenomegalia or chronic pathology (40
). Preliminary studies with the M1 mutant MHV68 show that IFN-γR−/−
mice infected with this virus have acute pneumonitis but no lung and spleen fibrosis on Day 180 postinfection. Analyses to discern the mechanism of M1-mediated virus pathology are in progress. Expression of M2 viral latent protein down-regulates Stat1 and Stat2, resulting in inhibition of interferon-mediated transcriptional activation that might enhance the Th2 profibrotic responses (42
). M3 is a chemokine-binding protein that can regulate the chemotaxis of neutrophils, lymphocytes, and monocytes (43
). T-cell responses and macrophages have been implicated in the development of virus-mediated pathology. Finally, the absence of chronic arteritis is also observed in IFN-γR−/−
mice infected with an MHV68 deficient in the M11 viral gene. M11 is a bcl-2 homolog with antiapoptotic activity required for efficient reactivation from latency (46
). M11 prevents apoptosis induced either by expression of viral genes critical for ex vivo
reactivation or by proapoptotic host genes that come into play during ex vivo
Persistent lymphocytic infiltrates without fibrosis were also found in lungs of mice infected with the mutant MHV68, v-cyclin stop. This virus has the capacity to establish latency, but it is defective in reactivation from latency. Taken together, these results suggest that active lytic replication in the chronic phase of infection is a driving mechanism for the fibrogenic process. A common finding in animals treated with antiviral agent beginning on Day 45 and in v-cyclin stop MHV68–infected animals is the lack of macrophage recruitment and lack of expression of alternative activation markers. Studies show expression of markers of alternative macrophage activation in the lungs of patients with IPF (47
). Our experimental model shows a similar pattern of activation for alveolar macrophages in chronically infected animals (19
). Macrophages activated by the alternative pathway have been implicated in wound repair (24
). These macrophages have up-regulated arginase activity and high expression of chitinase-like lectins Ym1/2 as well as of TGF-β and extracellular matrix proteins including fibronectin. We demonstrate here that by controlling lung injury by antiviral treatment or diminution of virus reactivation from latency, Th2-mediated activation of macrophages is prevented, and pulmonary fibrosis as well. These data suggest that alternatively activated macrophages have an active role in the exaggerated reparative response to lung injury associated with fibrosis.
Another mediator of collagen deposition that is associated with Th2 responses is VEGF. A longitudinal study evaluating VEGF levels in the plasma of patients with IPF showed a direct correlation between VEGF levels and clinical and radiologic deterioration (33
). In our model of pulmonary fibrosis, we demonstrated that chronic γ-herpesvirus infection is associated with high levels of VEGF in the lung that diminished with control of lytic infection by antiviral treatment or by infection with the v-cyclin stop mutant virus. These results support the concept that lytic infection mediates up-regulation of VEGF expression. Similarly, enhancement of VEGF expression has been reported during EBV reactivation (48
). On the other hand, the maintenance of high levels of VEGF in mice receiving antiviral from Day 60 postinfection and the studies with the bleomycin lung fibrosis model eliminate a direct effect of cidofovir on VEGF expression regulation.
Finally, we analyzed the effectiveness of antiviral treatment in symptomatic mice undergoing viral replication as demonstrated by high copy numbers of gB transcripts, a product of lytic replication. This group of mice had high mortality that improved with the antiviral treatment. The control of viral replication was incomplete in these symptomatic animals, as well as in the asymptomatic animals, probably because a low dose of antiviral agent was used to avoid the nephrotoxicity of this compound. Pilot experiments conducted with 25 mg/kg of body weight resulted in 50% mortality after the first week of treatment. Antiviral treatment failed to reverse lung fibrosis and alternative activation of macrophages, although there was a significant reduction in the severity of the fibrosis. It is possible that increasing the dose of antiviral or adding IFN-γ in the therapeutic regimen could result in better control of virus replication.
No current therapies for IPF have been proven to alter lung fibrosis or survival. Corticosteroids, and immunosuppressive or cytotoxic agents, have not proven to be of benefit and have potentially serious toxicities (49
). Our data in the animal model demonstrate that antiviral therapy aimed at replicating virus can prevent disrepair and fibrosis in a susceptible host. We also show that antiviral treatment in herpesvirus-infected mice improves clinical disease and survival. It is possible that treatment of γ-herpesvirus infection in patients with IPF with associated viral infection might help to control the progression of the fibrotic process. Future studies in this mouse model will be required to determine the impact of combination therapies in ameliorating pulmonary fibrosis.
In summary, using agents that stop replication of the virus and a replication-defective virus, we show that lytic infection is an important mechanism for virus-induced fibrosis. Furthermore, our data support the notion that activation of alveolar macrophages by the alternative pathway is a critical partner in the development of virus-induced fibrosis. Finally, the potential therapeutic ramification of our study is that antiviral therapy in herpesvirus-infected patients with IPF may be effective.