Department of Virology, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan
Correspondence
Hiroko Minagawa
hmina{at}virology.med.kyushu-u.ac.jp
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
MAIN TEXT |
---|
![]() ![]() ![]() ![]() |
---|
The reactivation of latent HSV-1 within the trigeminal ganglia (TG) causes recurrent debilitating keratitis. Studies of mouse TG latency models established that the presence of the HSV-1 genome within TG in vivo is associated with mononuclear infiltrates (Halford et al., 1996; Liu et al., 1996
; Shimeld et al., 1997
) and elevated production of proinflammatory cytokines, e.g. tumour necrosis factor (TNF), interleukin-6 (IL-6) and interferon-
(IFN-
) (Cantin et al., 1995
; Halford et al., 1996
; Liu et al., 1996
; Shimeld et al., 1997
, 1999
). The persistently elevated expression of cytokines during latency is attributed to infrequent spontaneous virus reactivation (Chen et al., 2000
; Feldman et al., 2002
; Halford et al., 1996
). These observations suggest that these proinflammatory cytokines are involved in the host defence response against HSV-1 reactivation. That the addition of syngeneic splenocytes to TG explant cultures significantly delays HSV-1 reactivation from latency (Noisakran & Carr, 1999
) further indicates that immunological mediators suppress HSV-1 reactivation.
TNF, while recognized as a critically important antiviral cytokine (Herbein & O'Brien, 2000; Rossol-Voth et al., 1991
; Wong & Goeddel, 1986
), has been blamed for unnecessary inflammation that can be harmful to the host, and thus has become a molecular target of anti-cytokine therapy. TNF antagonists (anti-TNF mAbs and etanercept, a dimeric molecule that contains two p75 TNF-receptor extracellular domains attached to the Fc portion of human IgG1) have been introduced to treat patients with rheumatoid arthritis and several other chronic inflammatory diseases (Fox, 2000
). These new therapies provide excellent control of inflammation, although they increase the risk of serious infections involving intracellular bacteria (Gardam et al., 2003
). Since HSV infection is common in patients of all ages, anti-TNF therapy may increase recurrent HSV diseases. The observation that clinical application of TNF antagonists is in fact associated with infectious complications urged us to study, in more depth, the effects of TNF on HSV infection, especially as a host defence factor. In this study, we analysed the effects of TNF deprivation on HSV-1 reactivation from latency in vitro, and examined HSV-1-infected TNF-knockout (TNF-ko) mice in vivo.
The HSV-1 7401H strain, a clinical isolate, was propagated and assayed on Vero cells grown in Eagle's MEM supplemented with 5 % calf serum and kanamycin (60 mg l-1) (Minagawa et al., 1997) before it was used as the challenge virus. For the corneal inoculation (Minagawa et al., 1997
), mice were anaesthetized with 50 mg pentobarbital sodium (kg body wt)-1. Both corneas were scarified with a 27-gauge needle, and inoculated with a 7 µl drop of virus suspension. All experimental procedures were approved by the institutional animal care and use committee. Mice were housed in the animal facility at our department under specific pathogen-free conditions.
We first examined the in-vitro HSV-1 reactivation from latently infected TG explant cultures (Fig. 1) after corneal inoculation. After confirming the former report (Noisakran & Carr, 1999
) that the addition of mononuclear cells from latently infected mouse spleen delays reactivation (data not shown), we suspected that cytokines, secreted by those cells, would be involved in the defence against HSV reactivation. We examined, therefore, the effects of neutralizing specific proinflammatory cytokines on HSV-1 reactivation from TG explants. In order to monitor TG explant cultures for reactivation, a 50 µl sample of the culture medium was taken from each and transferred to a well in a 24-well plate that contained indicator Vero cells and the appearance of cytopathic effect (CPE) on indicator cells was observed. The supernatant from each well of the TG cultures was sampled daily for the detection of infectious virus. As shown in Fig. 1
, depriving the explant of TNF accelerated reactivation; whereas the addition of anti-IL-6 neutralizing antibody to the explant medium delayed it, as well as ultimately reducing the reactivation frequency, as previously reported (Kriesel et al., 1997
). Differences between the following groups were statistically significant: anti-TNF-treated and the control, anti-IL-6-treated and the control (P values: 0·01 and 0·03, respectively; MantelCox log-rank test). Depletion of TNF from the TG explant co-cultivated with syngeneic mononuclear spleen cells also accelerated HSV-1 reactivation, while IL-6 depletion delayed and reduced reactivation (data not shown).
|
|
Since TNF plays a central role in innate immunity, the control of virus propagation at the primary entry site would be impaired in TNF-ko mice. Hence, we determined the infectious virus spread and propagation in the eyes, TG, and the brain during the primary phase of corneal infection. A group of three TNF-ko and three control B6 mice that had been inoculated with 5x105 p.f.u. of HSV-1 per eye was sacrificed each day on days 1, 3, 5, 8, 11 and 14 after inoculation. Eyes, TG and the brain of each mouse, as well as the liver, spleen, and the adrenal glands, were aseptically removed and assayed for infectious virus titres (Minagawa et al., 1997). As expected, infectious HSV-1 in the inoculated eyes (Fig. 3
A) and in the TG (Fig. 3B
) had higher titres and was detected for a more prolonged period in the TNF-ko group than in the control group. The brain from one TNF-ko mouse sacrificed 8 days after inoculation, was found to be positive for HSV-1 (10 p.f.u.). Infectious HSV-1 was not detected in any of the other brains tested. We did not detect any infectious HSV-1 in any of the organs mentioned below: the eyes, TG and the brain from TNF-ko mice or from control animals sacrificed 21 days after inoculation; the spleen, liver and adrenal glands from those TNF-ko mice that were found to be HSV-1-positive in the eyes and/or TG (i.e. those shown in Fig. 3
). The course of recovery from primary acute HSV-1 infection in the TNF-ko mice that survived did not differ greatly from that seen among the control mice.
|
TNF is required for appropriate immune responses against certain virus antigens (Kasahara et al., 2003; Trevejo et al., 2001
). In the case of this HSV-1 study, proper, albeit delayed, adaptive immune responses against the virus were induced, since there was no progressive persistent infection with continuous shedding of infectious virus (Fig. 3
) and latency was maintained. Furthermore, in infected TNF-ko mice, both humoral immune responses (i.e. serum neutralizing antibody) and cellular immune responses (i.e. circulating CD4+ and CD8+ T cells that produce IFN-
in response to HSV antigens) were at least as vigorous as those detected in the control mice (data not shown).
Since deprivation of TNF by neutralizing antibody induced HSV-1 reactivation in the explants (Fig. 1), presence of a small amount of TNF within the ganglion (Shimeld et al., 1997
) seems to play an important part in the initial host defence response against recurrent productive infection. As we did not remove infiltrating lymphocytes from the TG explants, the results of our in-vitro study (Fig. 1
) did not differentiate between direct and indirect effects of TNF on HSV-infected neurons. The latter indirect effects are likely to be caused by immune effectors under the control of TNF, such as non-cytocidal defensive procedures operated by a small number of T cells in each ganglion. The relative contribution of CD4+ T cells (Minagawa & Yanagi, 2000
) and CD8+ T cells (Liu et al., 2000
) in the abortion/control of productive recurrent infection (Ghiasi et al., 1999
) should be assessed in future studies.
TNF has long been known to exert synergistic antiviral effects together with IFNs (Schmitt et al., 1992; Wong & Goeddel, 1986
). In addition, significant contributions of proinflammatory cytokines other than TNF in HSV infections have been reported. IFN-
secreted by CD8+ T cells in TG has been shown to, at least partially, block HSV-1 reactivation from latency (Liu et al., 2001
). Minami et al. reported an increased reactivation rate (indicated by an increased rate of HSV DNA detected from the eye swab samples subjected to PCR) in both IFN-
-ko and TNF-ko mice (Minami et al., 2002
). Anti-IL-6 antibodies were shown to inhibit herpes simplex virus reactivation (Kriesel et al., 1997
), but in IL-6-ko mice, both latency and reactivation of HSV-1 were indistinguishable from controls (LeBlanc et al., 1999
). Since TNF itself induces IL-6 production, and as both TNF and IL-6 exert neurotropic effects in addition to proinflammatory activities (Hirano, 1998
; Zhang & Tracey, 1998
), the effects of these cytokines on virus latency/reactivation in the TG may be complex, involving both haematopoietic and neuronal cells within the ganglion. It is of note that one report has demonstrated an acceleration of virus reactivation by TNF (Walev et al., 1995
).
In conclusion, the present study indicated that TNF is a critical antiviral cytokine even in the presence of intact adaptive immunity, during both the primary and the reactivating phases of acute productive HSV-1 infection. Patients undergoing anti-TNF therapy should be followed closely for primary and recurrent HSV diseases.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
Chen, S. H., Garber, D. A., Schaffer, P. A., Knipe, D. M. & Coen, D. M. (2000). Persistent elevated expression of cytokine transcripts in ganglia latently infected with herpes simplex virus in the absence of ganglionic replication or reactivation. Virology 278, 207216.[CrossRef][Medline]
Feldman, L. T., Ellison, A. R., Voytek, C. C., Yang, L., Krause, P. & Margolis, T. P. (2002). Spontaneous molecular reactivation of herpes simplex virus type 1 latency in mice. Proc Natl Acad Sci U S A 99, 978983.
Fox, D. A. (2000). Cytokine blockade as a new strategy to treat rheumatoid arthritis: inhibition of tumor necrosis factor. Arch Intern Med 160, 437444.
Gardam, M. A., Keystone, E. C., Menzies, R., Manners, S., Skamene, E., Long, R. & Vinh, D. C. (2003). Anti-tumour necrosis factor agents and tuberculosis risk: mechanisms of action and clinical management. Lancet Infect Dis 3, 148155.[CrossRef][Medline]
Ghiasi, H., Perng, G., Nesburn, A. B. & Wechsler, S. L. (1999). Either a CD4+ or CD8+ T cell function is sufficient for clearance of infectious virus from trigeminal ganglia and establishment of herpes simplex virus type 1 latency in mice. Microb Pathog 27, 387394.[CrossRef][Medline]
Halford, W. P., Gebhardt, B. M. & Carr, D. J. (1996). Persistent cytokine expression in trigeminal ganglion latently infected with herpes simplex virus type 1. J Immunol 157, 35423549.[Abstract]
Herbein, G. & O'Brien, W. A. (2000). Tumor necrosis factor (TNF)-alpha and TNF receptors in viral pathogenesis. Proc Soc Exp Biol Med 223, 241257.
Hirano, T. (1998). Interleukin-6. In The Cytokine Handbook, 3rd edn, pp. 197228. Edited by A. Thomson. San Diego: Academic Press.
Jones, C. (2003). Herpes simplex virus type 1 and bovine herpesvirus 1 latency. Clin Microbiol Rev 16, 7995.
Kasahara, S., Ando, K. Saito, K. & 7 other authors (2003). Lack of tumor necrosis factor alpha induces impaired proliferation of hepatitis B virus-specific cytotoxic T lymphocytes. J Virol 77, 24692476.
Koelle, D. M. & Corey, L. (2003). Recent progress in herpes simplex virus immunobiology and vaccine research. Clin Microbiol Rev 16, 96113.
Kriesel, J. D., Gebhardt, B. M., Hill, J. M., Maulden, S. A., Hwang, I. P., Clinch, T. E., Cao, X., Spruance, S. L. & Araneo, B. A. (1997). Anti-interleukin-6 antibodies inhibit herpes simplex virus reactivation. J Infect Dis 175, 821827.[Medline]
LeBlanc, R. A., Pesnicak, L., Cabral, E. S., Godleski, M. & Straus, S. E. (1999). Lack of interleukin-6 (IL-6) enhances susceptibility to infection but does not alter latency or reactivation of herpes simplex virus type 1 in IL-6 knockout mice. J Virol 73, 81458151.
Liu, T., Tang, Q. & Hendricks, R. L. (1996). Inflammatory infiltration of the trigeminal ganglion after herpes simplex virus type 1 corneal infection. J Virol 70, 264271.[Abstract]
Liu, T., Khanna, K. M., Chen, X., Fink, D. J. & Hendricks, R. L. (2000). CD8+ T cells can block herpes simplex virus type 1 (HSV-1) reactivation from latency in sensory neurons. J Exp Med 191, 14591466.
Liu, T., Khanna, K. M., Carriere, B. N. & Hendricks, R. L. (2001). Gamma interferon can prevent herpes simplex virus type 1 reactivation from latency in sensory neurons. J Virol 75, 1117811184.
Minagawa, H. & Yanagi, Y. (2000). Latent herpes simplex virus-1 infection in SCID mice transferred with immune CD4+ T cells: a new model for latency. Arch Virol 145, 22592272.[CrossRef][Medline]
Minagawa, H., Tanaka, S., Toh, Y. & Mori, R. (1994). Detection of herpes simplex virus type 1-encoded RNA by polymerase chain reaction: different pattern of viral RNA detection in latently infected murine trigeminal ganglia following in vitro or in vivo reactivation. J Gen Virol 75, 647650.[Abstract]
Minagawa, H., Sakai, Y., Li, Y. Y., Ishibashi, T., Inomata, H. & Mori, R. (1997). Suppression of infectious virus spread and corneal opacification by the combined use of recombinant interferon beta and interleukin-10 following corneal infection with herpes simplex virus-1 in mice. Antiviral Res 36, 99105.[CrossRef][Medline]
Minami, M., Kita, M., Yan, X. Q., Yamamoto, T., Iida, T., Sekikawa, K., Iwakura, Y. & Imanishi, J. (2002). Role of IFN-gamma and tumor necrosis factor-alpha in herpes simplex virus type 1 infection. J Interferon Cytokine Res 22, 671676.[CrossRef][Medline]
Noisakran, S. & Carr, D. J. (1999). Lymphocytes delay kinetics of HSV-1 reactivation from in vitro explants of latent infected trigeminal ganglia. J Neuroimmunol 95, 126135.[CrossRef][Medline]
Pasparakis, M., Alexopoulou, L., Episkopou, V. & Kollias, G. (1996). Immune and inflammatory responses in TNF alpha-deficient mice: a critical requirement for TNF alpha in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response. J Exp Med 184, 13971411.[Abstract]
Rossol-Voth, R., Rossol, S., Schutt, K. H., Corridori, S., de Cian, W. & Falke, D. (1991). In vivo protective effect of tumour necrosis factor alpha against experimental infection with herpes simplex virus type 1. J Gen Virol 72, 143147.[Abstract]
Schmitt, D. A., Sasaki, H., Pollard, R. B. & Suzuki, F. (1992). Antiviral effects of recombinant human tumor necrosis factor-alpha in combination with natural interferon-beta in mice infected with herpes simplex virus type 1. Antiviral Res 19, 347352.[CrossRef][Medline]
Shimeld, C., Whiteland, J. L., Williams, N. A., Easty, D. L. & Hill, T. J. (1997). Cytokine production in the nervous system of mice during acute and latent infection with herpes simplex virus type 1. J Gen Virol 78, 33173325.[Abstract]
Shimeld, C., Easty, D. L. & Hill, T. J. (1999). Reactivation of herpes simplex virus type 1 in the mouse trigeminal ganglion: an in vivo study of virus antigen and cytokines. J Virol 73, 17671773.
Trevejo, J. M., Marino, M. W., Philpott, N., Josien, R., Richards, E. C., Elkon, K. B. & Falck-Pedersen, E. (2001). TNF-alpha-dependent maturation of local dendritic cells is critical for activating the adaptive immune response to virus infection. Proc Natl Acad Sci U S A 98, 1216212167.
Wagner, E. K. & Bloom, D. C. (1997). Experimental investigation of herpes simplex virus latency. Clin Microbiol Rev 10, 419443.[Abstract]
Walev, I., Podlech, J. & Falke, D. (1995). Enhancement by TNF-alpha of reactivation and replication of latent herpes simplex virus from trigeminal ganglia of mice. Arch Virol 140, 987992.[Medline]
White, D. W., Badovinac, V. P., Kollias, G. & Harty, J. T. (2000). Cutting edge: antilisterial activity of CD8+ T cells derived from TNF-deficient and TNF/perforin double-deficient mice. J Immunol 165, 59.
Whitley, R. J. (2001). Herpes simplex viruses. In Fields Virology, 4th edn, pp. 24612509. Edited by D. Knipe & P. Howley. Philadelphia: Lippincott Williams & Wilkins.
Wong, G. H. W. & Goeddel, D. V. (1986). Tumour necrosis factors and
inhibit virus replication and synergize with interferons. Nature 323, 819822.[Medline]
Zhang, M. & Tracey, K. J. (1998). Tumor necrosis factor. In The Cytokine Handbook, 3rd edn, pp. 517548. Edited by A. Thomson. San Diego: Academic Press.
Received 3 September 2003;
accepted 12 November 2003.