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Lack of TNF-α signaling through p55 makes the mice more susceptible to acute infection but does not alter state of latency and reactivation of HSV-1
Virus Research 244 (2018) 1–5
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Lack of TNF-α signaling through p55 makes the mice more susceptible to acute infection but does not alter state of latency and reactivation of HSV-1 Aditi Mohankrishnana, Rajesh Parmara, Vishakha Bhurania, Sarat Kumar Dalaia,b, a b
MARK
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Institute of Science, Nirma University, Ahmedabad, Gujarat, India Medical Virology Section, Laboratory of Clinical Investigation, NIAID, NIH, Bethesda, MD, USA
A R T I C L E I N F O
A B S T R A C T
Keywords: Herpes simplex virus Inflammatory cytokine Reactivation Prostaglandins
TNF-α has been shown to play an important role in pathogenesis and latency of HSV-1 infections. TNF-α signals through TNFR1 (p55) and TNFR2 (p75), and signaling through p55 generally results in gene activation leading to induction of inflammatory responses. Here, we studied the role of TNF-α signaling in latent virus reactivation in p55-knock out (KO) mouse model of ocular HSV-1 infection. We found that KO mice are more susceptible to HSV-1 infection compared to wild type C57Bl/6 mice. While the absence of TNFRI signaling enhanced the ganglion latent DNA content by two folds, there was no difference in the maintenance and reactivation of latent HSV-1. Strikingly, interfering with inflammatory responses through PGE2 synthesis by treating latently infected wild type mice with indomethacin (COX inhibitor) prior to UV-exposure prevented HSV-1 reactivation. These results suggest that reactivation of latent HSV-1 might result from the cumulative effects of a cascade of inflammatory cytokines including TNF-α.
1. Introduction Herpes Simplex Virus types 1 and 2 (HSV-1 and HSV-2) infection is initiated with viral replication at the site of entry followed by centripetal transit in sensory neurons to the trigeminal or dorsal root ganglia, where they establish life-long latency (Smith, 2012). Reactivation from latency occurs when a stimulus triggers renewed replication of virus with centrifugal spread of progeny virions along neuronal axons to the initial portal entry. Several studies have provided evidence that inflammation is associated with HSV infection (Pasieka et al., 2009). During acute infection, a cascade of cytokines namely, IL1, TNF-α, IFN, IL-6, MIP, MCP are synthesized (Pasieka et al., 2009). Similarly these cytokines are upregulated during reactivation of latent HSV induced by stress e.g., UV and hyperthermia (Huang et al., 2011; Keadle et al., 2000; Bryant-Hudson et al., 2014). TNF-α is one of the potent inflammatory cytokines. While TNF-α is shown to be protective, it has been reported to be involved in pathogenesis (Keadle et al., 2000; Bryant-Hudson et al., 2014) and enhancing HSV-1 reactivation (Walev et al., 1995; Minami et al., 2002). TNF signals through two receptors namely, TNFR1 (p55) and TNFR2 (p75). TNF and the closely related ligand lymphotoxin(LT) bind as homotrimers to p55 and p75 that are widely expressed on most cell types (Wallach et al., 1999). While p55 signaling induces gene activation leading to inflammatory and cytotoxic responses, p75 signaling is
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associated with thymocyte proliferation and T-cell activation (Wallach et al., 1999). Since inflammatory responses trigger latent HSV reactivation we wanted to test whether absence of TNF-α signaling through p55 would attenuate the state of HSV infection. In the present study we used p55 knockout mouse for HSV-1 infection. We showed that p55 deficient mice are more susceptible to acute HSV-1 infection compared to their parental wild type C57Bl/6 mice. Latently infected p55-/- mice harbor more HSV-1 genome although there was no difference in the rate of reactivation of latent virus. Treatment of latently infected WT mice with indomethacin (COX inhibitor) prior to UV exposure was found to prevent the reactivation of latent HSV-1 suggesting that TNF-α signaling through p55 may not be sufficient to reactivate latent HSV. 2. Materials and methods 2.1. Cells and virus HSV-1 strain McKrae was grown in Vero (African green monkey kidney) cells in EMEM: 199 medium (Quality Biologicals, Inc., Gaithersburg, Maryland) with 10% FBS (Life Technologies Gibco BRL, Gaithersburg, Maryland) and 1% Glutamine-Streptomycin-Penicillin (Life Technologies Gibco BRL). The McKrae strain was used because of its high neurovirulence compared with strains passaged multiple times
Corresponding author at: Institute of Science, Nirma University, Ahmedabad, Gujarat, India. E-mail address: [email protected] (S.K. Dalai).
https://doi.org/10.1016/j.virusres.2017.11.004 Received 6 July 2017; Received in revised form 28 September 2017; Accepted 2 November 2017 Available online 04 November 2017 0168-1702/ © 2017 Elsevier B.V. All rights reserved.
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in the laboratory that typically are attenuated. The virus dose chosen for inoculation was based on our earlier studies (LeBlanc et al., 1999a; Dalai et al., 2002), which showed that to study latency and reactivation, a sufficient quantity of input virus is needed.
250 mg/mouse) was orally fed to mice daily for four days. It was also provided in drinking water (20 mg/mouse) changing the bottle daily for four days. On day 3 post indomethacin treatment, mice were UV-exposed on both the eyes to reactivate HSV.
2.2. Animals and inoculations
2.7. Quantitative Real-Time PCR
Female C57Bl/6 (WT) and TNFRI-KO homozygous mice, 4–6 weeks old, were obtained from Jackson Laboratory (Bar Harbor, Maine), housed in American Association for Accreditation of Laboratory Animal Care, Int. accredited facilities, at the NIH and studied under an IACUCapproved animal research protocol. Mice were anesthetized with a 0.5 ml intraperitoneal (IP) injection of a mixture of ketamine/xylazine in PBS. Both corneas were scarified using a 25-gauge needle and 5 μl of virus inoculums were applied per eye for a total of 1 × 106 plaque forming units (PFU) per mouse. Control mice received 5 μl of PBS per sacrificed eye. Animals assigned to IgG treatment were given a single 0.5 ml dose of pooled human IgG (prepared for intravenous administration) (Abbott Labs, Chicago, IL) diluted 1:8 in PBS (3.75 mg IgG/ mouse) IP at 24 h after infection (LeBlanc et al., 1999b).
The protocol used to quantitate the latent viral genome copy number in ganglia was as reported (LeBlanc et al., 1999b). TG pairs from latently infected mice were dissected, separated, and rinsed three times each in PBS. DNA was isolated separately from each ganglion using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN) (Dalai et al., 2002) and pooled from both ganglia for each mouse. The number of copies of latent HSV-1 DNA was quantified by real-time fluorescence PCR using the Taqman System, ABI Prism 7700 Sequence Detector, (PE Applied Biosystems, Perkin-Elmer, Foster City, CA) with primers and probe specific for glycoprotein G of HSV-1 (gG1) (LeBlanc et al., 1999b). Each reaction of 100 ng TG DNA was done in triplicate and a standard curve ranging from 3 × 100 to 3 × 105 copies was generated from control plasmid with gG1 insert (cloned into PCRTM 2.1, Invitrogen).
2.3. Virus titers in trigeminal ganglia (TG) 2.8. Statistical analysis On days 2, 4, 6, 8, and 10 post-infection (p.i.), the TG were removed from mice using aseptic techniques and placed into separate tubes containing 1 ml of medium each. TGs were ground using a Tekmar Tissue homogenizer (VWR Scientific, McGaw Park, Illinois) and the homogenates were diluted serially and then plated in duplicate on Vero cell monolayers. After 1 h incubation at 37 °C on a rocking platform, cells were overlaid with media containing 0.5% pooled human IgG (Abbott Labs) and then placed in a humidified CO2 incubator at 37 °C for 48 h. Then, the dishes were stained with crystal violet and plaques were counted.
Analyses were done using JMP software from the SAS Institute (Cary, NC). Comparisons between the Kaplan-Meier survival estimates were done using the log-rank test. Nonparametric methods were done to analyze the data. Geometric means and one-way analysis of variance with log-transformed numbers were used to analyze quantitative PCR. The time of reactivation of samples were compared by the oneway Anova t-Test. 3. Results & discussion Under the influence of stressors like UV, hyperthermia, trauma, fever, menstruation, surgical resection, emotional stress, herpes virus is reactivated from latency (Huang et al., 2011; Buske-Kirschbaum et al., 2001). All these stimuli trigger syntheses of IL-1, IL-1Ra, IL-6, TNF-α, MIP, MCP, and RANTES and induce inflammatory responses in the host (Buske-Kirschbaum et al., 2001; Shimeld et al., 1999; Noisakran et al., 1998). IL-6 has been implicated in induction of HSV-1 reactivation (Kriesel et al., 1997). However, lack of IL-6 does not alter the status of reactivation in response to stress (LeBlanc et al., 1999b). Since TNF-α is a potent inflammatory cytokine and reported to be involved in pathogenesis and latency of HSV-infection, we wanted to test whether absence of its signaling would influence latency and reactivation of virus.
2.4. Survival of animals Groups of WT and KO mice (n = 10) were infected ocularly by bilateral corneal scarification with a range of HSV-1 doses 102 to 106 PFU each, and were observed daily for mortality up to day 14 post infection. 2.5. Explant Co-cultivation Animals that survived the acute HSV-1 infection were housed for more than 30 days p.i. Whole TG from both the groups of latently infected mice were aseptically harvested, and each pair of TG was placed onto separate Vero cell monolayers with media containing 0.1% N, N’hexamethylene-bis-acetamide (Sigma, St. Louis, MO) and 2% FBS. Explant cultures were kept in a 37 °C humidified CO2 incubator. The explants were checked daily for CPE, and they were carefully transferred onto fresh monolayer weekly, if necessary, to confirm cytopathy.
3.1. TNFRI (p55) deficient mice are more susceptible to acute HSV-1 infection TNFR1 (p55) and TNFR2 (p75) are two receptors for TNF-α p55 is the dominant functional one (Rothe et al., 1993). Deficiency of this receptor has been demonstrated to make mice more susceptible to intracellular pathogen (Rothe et al., 1993). For our study we obtained female p55 KO mouse and its parental wild type C57Bl/6 mouse, which have been shown to be susceptible to HSV-infection. To compare relative replication of virus in WT and p55 KO mice, acute titers of HSV-1 in TG tissue were acquired. We quantified the virus load in TG on day 2, 4, 6, 8, and 10 post infection (Fig. 1A). These tissues were harvested from groups of three mice each per treatment arm at each time point and the mean virus titers were determined. A potential increase (p = 0.69) of virus titer in p55 KO mice was observed suggesting that the deficiency of TNF-α signaling might make the mice more prone to HSV-1 infection. To define the role of p55 signaling on virus induced mortality, groups of 10 mice were infected by bilateral corneal scarification
2.6. In vivo reactivation The protocol used for inducing HSV reactivation in vivo was as reported (LeBlanc et al., 1999a). Mice were anesthetized, placed on top of a TM-20 transilluminator (UVP Inc., Upland, CA) and both eyes were exposed to UV light for 1 min (LeBlanc et al., 1999a; Dalai et al., 2002). Some infected animals were not exposed and served as “unstressed” controls. Forty-eight hours later, the pair of TG from each animal was homogenized; supernatant from centrifuged tissue homogenate was collected and plated on Vero cell monolayers. After an hour, supernatant was removed and the monolayer was thoroughly washed before adding fresh medium. Monolayers were incubated at 37 °C and checked daily for CPE. To test the effect of indomethacin on HSV-1 reactivation, we followed the same procedure except that mice were treated with indomethacin (Bratcher et al., 1993) as follows. Indomethacin (Sigma, 2
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Fig. 1. TNFRI (p55) deficient mice are more susceptible to acute HSV-1 infection. (A) Mice were infected with 106 pfu/mouse HSV-1 (McKrae) and administered IgG as described in Methods. On the indicated days, virus titers in TG were determined by plaquing on Vero cells. Each point represents the log value of the arithmetic mean and SD for three animals. (B) Groups of WT and TNFRI-KO mice (n = 10) were inoculated with 102–106 pfu HSV-1 (McKrae) using bilateral corneal scarification. Survival was monitored for the first 14 days post infection. The data shown represent one of the three independent experiments with similar results.
followed by inoculation with 102 to 106 pfu of HSV-1 (McKrae). Mortality was scored daily up to day 14 post infection (p.i.). As shown in Fig. 1B, 90–91% and 87–88% animals from both the groups of p55-/and WT mice survived at 102 and 103 pfu respectively. At 104 pfu, only 53.3% WT mice survived in contrast to 40% KO mice (P = 0.485). Only 55% WT and 17% KO mice survived HSV-1 induced death at 105 pfu (P = 0.008). At 106 pfu only 5% WT mice survived, whereas none of the KO mice survived beyond day11 p.i. (P = 0.72). A study reported by Lundberg, et al. suggests that p55-/- mice are equally resistant as their parental strain of C57Bl/6 mice inoculated with 3200 pfu HSV-1 (Lundberg et al., 2007). Our data is consistent with their findings when mice were inoculated with 102–103 pfu. In that study unlike ours, however, the resistance of p55-/- mice to acute infection with higher doses of virus was not tested. TNF-α is known to provide neuro-protection (Bruce et al., 1996). HSV-1 infected mice die of encephalitis. Since TNF-α plays a central role in innate immunity, the control of virus propagation at the primary entry site would have been impaired in p55-/- mice causing enhanced death seen in our study.
ganglia were harvested for DNA extraction and subsequent quantitative PCR. By comparing the quantitative PCR results to a standard curve of gG-1 plasmid DNA, we estimated the genome copy number in test samples (Fig. 2B). Though the difference in the latent DNA copy numbers between two groups was not highly significant (P = 0.025), there was two-fold rise in geometric mean copies of viral DNA in p55-/mice. Innate-immune responses followed by adaptive immune responses are mounted to check the viral replication during acute phase of viral infection (Ellison et al., 2000). Since p55-/- mice have defect in their hematopoietic system (Rebel et al., 1999) it is reasonable to assume that HSV-1 infection might not have been checked efficiently during the acute phase allowing more number of virus to establish latency. In some studies, it has been also shown that both the receptors p55 and p75 play an important role in providing protection against viral infection (Janet et al., 1997). Nevertheless, p55 but not p75 is demonstrated to be preferentially protecting the host from infections like toxoplasmosis (Martina et al., 1998). It is possible that the presence of p75 in absence of p55 might have minimal role in preventing HSV infection in our experimental setup.
3.2. Absence of TNFR1 signaling enhanced latent HSV infection 3.3. Indomethacin blocks UV-induced re-activation of latent HSV-1 HSV-1 establishes and maintains a latent infection in the TG and can be re-activated in vitro by explant co-cultivation. To detect any potential differences in relative rates of the establishment of latency and in vitro reactivation, an indicator of the quantity of latent virus, p55-/- and WT mice were sacrificed on day 30 p.i. and their TG was explanted for 11 days. 100% mice from both groups (WT, 8/8; p55-/-, 6/6) yielded virus (Fig. 2A) with no difference in time of reactivation (P = 0.62). Treatment with IgG did not entirely prevent establishment of HSV-1 latent infection. This observation indicates that inflammatory signals delivered by TNF-α through p55 may not affect the establishment of latency. However, the efficient reactivation does not imply that there are equivalent levels of latent HSV-1 genomes in the ganglia. It is possible that in the absence of p55 signaling there were different levels of latent virus, but the virus could still reactivate as long as some threshold quantity of latent virus genomes had been exceeded. To support this notion we determined the quantity of latent HSV-1 genomes in the TG by using real-time PCR (LeBlanc et al., 1999a; Dalai et al., 2002). Groups of eight to twelve WT and p55-/- mice latently infected with HSV-1 were sacrificed on day 79 p.i. and individual
Removal of the trigeminal ganglion and its co-cultivation ex vivo revealed equal rates of reactivation in the presence or absence of TNF-α signaling through p55. However, explant co-cultivation is maximal and non-physiologic stimulus to HSV-reactivation. Physiologic reactivation processes would ensure either spontaneous or stress-induced recurrence of infectious virus, as in humans with recurrent herpetic kerato-conjuctivitis. Since HSV does not reactivate spontaneously from mice, the role of p55 signaling in HSV-1 reactivation, and the impact of differential viral loads were explored by exposing the latently infected mice to UV irradiation (Dalai et al., 2002). After latency was established (day30 p.i.), the animals were exposed to UV-light for 1 min per eye (360 mJ/eye). Forty-eight hour later HSV-1 reactivation was tested in TG. 100% mice from both the groups yielded virus (Fig. 3A). There was no significant difference in the average time of reactivation (P = 0.17). These results suggest that TNF-α signaling through p55 may not play a dominant role in maintaining or reactivating latent herpes virus. Previously, we have shown that (LeBlanc et al., 1999b) absence of IL-6 does not influence the reactivation of HSV infection. In our 3
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Fig. 2. Absence of TNFR1 signaling enhanced latent HSV infection. (A) In vitro reactivation of HSV-1 by explant co-cultivation Each pair of TG from individual mouse was aseptically harvested from eight WT and six TNFRI-KO mice on day30 p.i and placed onto Vero cell monolayer with media containing 0.1% N, N’-hexamethylene-bis-acetamide. The explants were checked daily for CPE, and were carefully transferred onto fresh monolayer weekly, if necessary. (B) Quantitative determination of HSV-1 DNA loads in TG. DNA was extracted from individual ganglia from eight WT and TNFRI-KO mice day79 p.i. Each point represents the log value of the mean copy number for three replicate PCR determinations per pooled TG from each animal. The horizontal line along the width of each data ‘diamond’ indicates the combined mean HSV-1 DNA copy numbers for all eight mice per group. The shorter horizontal lines above and below this indicate the 95% confidence intervals around the mean.
kept under check due to the host immunity generated during the acute infection (Hoshino et al., 2007), induction of immune suppression by PGE2 at the site of latency might reactivate the latent virus. When the delivery of inflammatory signals by TNF-α through p55 is blocked, PGE2 release might not be adversely affected because of the inflammatory signals delivered through IL-1 and IL-6. Our preliminary data in IL-1 receptor deficient mice supports this notion (data not shown). Thus, recurrence of latent HSV in single inflammatory cytokine KO mouse would not be dramatically affected. The use of multiple inflammatory cytokine deficient mice or blocking the signaling of multiple inflammatory cytokines would be desirable to understand this phenomenon.
preliminary experiments, absence of IL-1R1 signaling does not seem to affect the HSV infection (Thomas et al., 1999). All these studies prompted us to look for a common pathway that might be responsible for inducing reactivation of HSV. Many stimuli including UV trigger syntheses of IL-1, IL-6, and TNF-α. All three of these pro-inflammatory cytokines induce cyclooxygenase 2 (COX-2) synthesis resulting PGE2 release (Kuwamoto et al., 2000). While PGE2 is known to induce proinflammatory responses, it also induces IL-10 and other anti-inflammatory cytokine causing immune suppression or immune-deviation (Shreedhar et al., 1998) that might favor the HSV reactivation. Upon treating mice with indomethacin, a broad inhibitor of cyclooxygenase (constitutive COX-1 and inducible COX-2) (Simmons et al., 2004) before exposing the eyes to UV irradiation, we found that HSV-1 reactivation was significantly prevented in mice treated with indomethacin compared to that of placebo control (P < 0.05) (Fig. 3B) (Walia et al., 2012; Higaki et al., 2009; Paul et al., 2013). It has been shown in guinea-pig model of HSV-2 genital infection that indomethacin treatment before UV-irradiation significantly reduces virus recurrence (Bratcher et al., 1993) supporting our hypothesis that prostaglandin plays a key role in the reactivation of latent HSV. Based on these experiments and our previous findings we propose the following. UV irradiation induces potent inflammatory cytokines namely, IL-1, IL-6, and TNF- α. All the three cytokines would activate COX-2 that triggers the release of PGE2. While latent HSV infection is
Conflict of interest The authors declare no commercial or financial conflict of interest. Acknowledgments Authors would like to acknowledge the contribution of Lesley Pesnicak and others from the laboratory of late Dr. Stephen E. Straus, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, NIH, Bethesda for assistance to SKD while he was as a visiting fellow.
Fig. 3. Indomethacin blocks UV-induced re-activation of latent HSV-1. (A) In-vivo reactivation of HSV-1 in latently infected mice. Following 48 h post-UV exposure, each pair of TG from latently infected WT or TNFRIKO mouse was harvested, ground, and supernatant was plated on Vero cell monolayer, incubated at 37 °C and checked daily for CPE. (B) Effect of indomethacin on UV-induced re-activation of HSV-1. Groups of nineteen latently infected WT mice were treated with indomethacin or PBS (placebo control) as described in methods. On day 3 post treatment, mice were UV-exposed on both the eyes, and 48 h later TG were harvested and checked for virus reactivation.
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Lack of TNF-α signaling through p55 makes the mice more susceptible to acute infection but does not alter state of latency and reactivation of HSV-1 Aditi Mohankrishnana, Rajesh Parmara, Vishakha Bhurania, Sarat Kumar Dalaia,b, a b
MARK
⁎
Institute of Science, Nirma University, Ahmedabad, Gujarat, India Medical Virology Section, Laboratory of Clinical Investigation, NIAID, NIH, Bethesda, MD, USA
A R T I C L E I N F O
A B S T R A C T
Keywords: Herpes simplex virus Inflammatory cytokine Reactivation Prostaglandins
TNF-α has been shown to play an important role in pathogenesis and latency of HSV-1 infections. TNF-α signals through TNFR1 (p55) and TNFR2 (p75), and signaling through p55 generally results in gene activation leading to induction of inflammatory responses. Here, we studied the role of TNF-α signaling in latent virus reactivation in p55-knock out (KO) mouse model of ocular HSV-1 infection. We found that KO mice are more susceptible to HSV-1 infection compared to wild type C57Bl/6 mice. While the absence of TNFRI signaling enhanced the ganglion latent DNA content by two folds, there was no difference in the maintenance and reactivation of latent HSV-1. Strikingly, interfering with inflammatory responses through PGE2 synthesis by treating latently infected wild type mice with indomethacin (COX inhibitor) prior to UV-exposure prevented HSV-1 reactivation. These results suggest that reactivation of latent HSV-1 might result from the cumulative effects of a cascade of inflammatory cytokines including TNF-α.
1. Introduction Herpes Simplex Virus types 1 and 2 (HSV-1 and HSV-2) infection is initiated with viral replication at the site of entry followed by centripetal transit in sensory neurons to the trigeminal or dorsal root ganglia, where they establish life-long latency (Smith, 2012). Reactivation from latency occurs when a stimulus triggers renewed replication of virus with centrifugal spread of progeny virions along neuronal axons to the initial portal entry. Several studies have provided evidence that inflammation is associated with HSV infection (Pasieka et al., 2009). During acute infection, a cascade of cytokines namely, IL1, TNF-α, IFN, IL-6, MIP, MCP are synthesized (Pasieka et al., 2009). Similarly these cytokines are upregulated during reactivation of latent HSV induced by stress e.g., UV and hyperthermia (Huang et al., 2011; Keadle et al., 2000; Bryant-Hudson et al., 2014). TNF-α is one of the potent inflammatory cytokines. While TNF-α is shown to be protective, it has been reported to be involved in pathogenesis (Keadle et al., 2000; Bryant-Hudson et al., 2014) and enhancing HSV-1 reactivation (Walev et al., 1995; Minami et al., 2002). TNF signals through two receptors namely, TNFR1 (p55) and TNFR2 (p75). TNF and the closely related ligand lymphotoxin(LT) bind as homotrimers to p55 and p75 that are widely expressed on most cell types (Wallach et al., 1999). While p55 signaling induces gene activation leading to inflammatory and cytotoxic responses, p75 signaling is
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associated with thymocyte proliferation and T-cell activation (Wallach et al., 1999). Since inflammatory responses trigger latent HSV reactivation we wanted to test whether absence of TNF-α signaling through p55 would attenuate the state of HSV infection. In the present study we used p55 knockout mouse for HSV-1 infection. We showed that p55 deficient mice are more susceptible to acute HSV-1 infection compared to their parental wild type C57Bl/6 mice. Latently infected p55-/- mice harbor more HSV-1 genome although there was no difference in the rate of reactivation of latent virus. Treatment of latently infected WT mice with indomethacin (COX inhibitor) prior to UV exposure was found to prevent the reactivation of latent HSV-1 suggesting that TNF-α signaling through p55 may not be sufficient to reactivate latent HSV. 2. Materials and methods 2.1. Cells and virus HSV-1 strain McKrae was grown in Vero (African green monkey kidney) cells in EMEM: 199 medium (Quality Biologicals, Inc., Gaithersburg, Maryland) with 10% FBS (Life Technologies Gibco BRL, Gaithersburg, Maryland) and 1% Glutamine-Streptomycin-Penicillin (Life Technologies Gibco BRL). The McKrae strain was used because of its high neurovirulence compared with strains passaged multiple times
Corresponding author at: Institute of Science, Nirma University, Ahmedabad, Gujarat, India. E-mail address: [email protected] (S.K. Dalai).
https://doi.org/10.1016/j.virusres.2017.11.004 Received 6 July 2017; Received in revised form 28 September 2017; Accepted 2 November 2017 Available online 04 November 2017 0168-1702/ © 2017 Elsevier B.V. All rights reserved.
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in the laboratory that typically are attenuated. The virus dose chosen for inoculation was based on our earlier studies (LeBlanc et al., 1999a; Dalai et al., 2002), which showed that to study latency and reactivation, a sufficient quantity of input virus is needed.
250 mg/mouse) was orally fed to mice daily for four days. It was also provided in drinking water (20 mg/mouse) changing the bottle daily for four days. On day 3 post indomethacin treatment, mice were UV-exposed on both the eyes to reactivate HSV.
2.2. Animals and inoculations
2.7. Quantitative Real-Time PCR
Female C57Bl/6 (WT) and TNFRI-KO homozygous mice, 4–6 weeks old, were obtained from Jackson Laboratory (Bar Harbor, Maine), housed in American Association for Accreditation of Laboratory Animal Care, Int. accredited facilities, at the NIH and studied under an IACUCapproved animal research protocol. Mice were anesthetized with a 0.5 ml intraperitoneal (IP) injection of a mixture of ketamine/xylazine in PBS. Both corneas were scarified using a 25-gauge needle and 5 μl of virus inoculums were applied per eye for a total of 1 × 106 plaque forming units (PFU) per mouse. Control mice received 5 μl of PBS per sacrificed eye. Animals assigned to IgG treatment were given a single 0.5 ml dose of pooled human IgG (prepared for intravenous administration) (Abbott Labs, Chicago, IL) diluted 1:8 in PBS (3.75 mg IgG/ mouse) IP at 24 h after infection (LeBlanc et al., 1999b).
The protocol used to quantitate the latent viral genome copy number in ganglia was as reported (LeBlanc et al., 1999b). TG pairs from latently infected mice were dissected, separated, and rinsed three times each in PBS. DNA was isolated separately from each ganglion using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN) (Dalai et al., 2002) and pooled from both ganglia for each mouse. The number of copies of latent HSV-1 DNA was quantified by real-time fluorescence PCR using the Taqman System, ABI Prism 7700 Sequence Detector, (PE Applied Biosystems, Perkin-Elmer, Foster City, CA) with primers and probe specific for glycoprotein G of HSV-1 (gG1) (LeBlanc et al., 1999b). Each reaction of 100 ng TG DNA was done in triplicate and a standard curve ranging from 3 × 100 to 3 × 105 copies was generated from control plasmid with gG1 insert (cloned into PCRTM 2.1, Invitrogen).
2.3. Virus titers in trigeminal ganglia (TG) 2.8. Statistical analysis On days 2, 4, 6, 8, and 10 post-infection (p.i.), the TG were removed from mice using aseptic techniques and placed into separate tubes containing 1 ml of medium each. TGs were ground using a Tekmar Tissue homogenizer (VWR Scientific, McGaw Park, Illinois) and the homogenates were diluted serially and then plated in duplicate on Vero cell monolayers. After 1 h incubation at 37 °C on a rocking platform, cells were overlaid with media containing 0.5% pooled human IgG (Abbott Labs) and then placed in a humidified CO2 incubator at 37 °C for 48 h. Then, the dishes were stained with crystal violet and plaques were counted.
Analyses were done using JMP software from the SAS Institute (Cary, NC). Comparisons between the Kaplan-Meier survival estimates were done using the log-rank test. Nonparametric methods were done to analyze the data. Geometric means and one-way analysis of variance with log-transformed numbers were used to analyze quantitative PCR. The time of reactivation of samples were compared by the oneway Anova t-Test. 3. Results & discussion Under the influence of stressors like UV, hyperthermia, trauma, fever, menstruation, surgical resection, emotional stress, herpes virus is reactivated from latency (Huang et al., 2011; Buske-Kirschbaum et al., 2001). All these stimuli trigger syntheses of IL-1, IL-1Ra, IL-6, TNF-α, MIP, MCP, and RANTES and induce inflammatory responses in the host (Buske-Kirschbaum et al., 2001; Shimeld et al., 1999; Noisakran et al., 1998). IL-6 has been implicated in induction of HSV-1 reactivation (Kriesel et al., 1997). However, lack of IL-6 does not alter the status of reactivation in response to stress (LeBlanc et al., 1999b). Since TNF-α is a potent inflammatory cytokine and reported to be involved in pathogenesis and latency of HSV-infection, we wanted to test whether absence of its signaling would influence latency and reactivation of virus.
2.4. Survival of animals Groups of WT and KO mice (n = 10) were infected ocularly by bilateral corneal scarification with a range of HSV-1 doses 102 to 106 PFU each, and were observed daily for mortality up to day 14 post infection. 2.5. Explant Co-cultivation Animals that survived the acute HSV-1 infection were housed for more than 30 days p.i. Whole TG from both the groups of latently infected mice were aseptically harvested, and each pair of TG was placed onto separate Vero cell monolayers with media containing 0.1% N, N’hexamethylene-bis-acetamide (Sigma, St. Louis, MO) and 2% FBS. Explant cultures were kept in a 37 °C humidified CO2 incubator. The explants were checked daily for CPE, and they were carefully transferred onto fresh monolayer weekly, if necessary, to confirm cytopathy.
3.1. TNFRI (p55) deficient mice are more susceptible to acute HSV-1 infection TNFR1 (p55) and TNFR2 (p75) are two receptors for TNF-α p55 is the dominant functional one (Rothe et al., 1993). Deficiency of this receptor has been demonstrated to make mice more susceptible to intracellular pathogen (Rothe et al., 1993). For our study we obtained female p55 KO mouse and its parental wild type C57Bl/6 mouse, which have been shown to be susceptible to HSV-infection. To compare relative replication of virus in WT and p55 KO mice, acute titers of HSV-1 in TG tissue were acquired. We quantified the virus load in TG on day 2, 4, 6, 8, and 10 post infection (Fig. 1A). These tissues were harvested from groups of three mice each per treatment arm at each time point and the mean virus titers were determined. A potential increase (p = 0.69) of virus titer in p55 KO mice was observed suggesting that the deficiency of TNF-α signaling might make the mice more prone to HSV-1 infection. To define the role of p55 signaling on virus induced mortality, groups of 10 mice were infected by bilateral corneal scarification
2.6. In vivo reactivation The protocol used for inducing HSV reactivation in vivo was as reported (LeBlanc et al., 1999a). Mice were anesthetized, placed on top of a TM-20 transilluminator (UVP Inc., Upland, CA) and both eyes were exposed to UV light for 1 min (LeBlanc et al., 1999a; Dalai et al., 2002). Some infected animals were not exposed and served as “unstressed” controls. Forty-eight hours later, the pair of TG from each animal was homogenized; supernatant from centrifuged tissue homogenate was collected and plated on Vero cell monolayers. After an hour, supernatant was removed and the monolayer was thoroughly washed before adding fresh medium. Monolayers were incubated at 37 °C and checked daily for CPE. To test the effect of indomethacin on HSV-1 reactivation, we followed the same procedure except that mice were treated with indomethacin (Bratcher et al., 1993) as follows. Indomethacin (Sigma, 2
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Fig. 1. TNFRI (p55) deficient mice are more susceptible to acute HSV-1 infection. (A) Mice were infected with 106 pfu/mouse HSV-1 (McKrae) and administered IgG as described in Methods. On the indicated days, virus titers in TG were determined by plaquing on Vero cells. Each point represents the log value of the arithmetic mean and SD for three animals. (B) Groups of WT and TNFRI-KO mice (n = 10) were inoculated with 102–106 pfu HSV-1 (McKrae) using bilateral corneal scarification. Survival was monitored for the first 14 days post infection. The data shown represent one of the three independent experiments with similar results.
followed by inoculation with 102 to 106 pfu of HSV-1 (McKrae). Mortality was scored daily up to day 14 post infection (p.i.). As shown in Fig. 1B, 90–91% and 87–88% animals from both the groups of p55-/and WT mice survived at 102 and 103 pfu respectively. At 104 pfu, only 53.3% WT mice survived in contrast to 40% KO mice (P = 0.485). Only 55% WT and 17% KO mice survived HSV-1 induced death at 105 pfu (P = 0.008). At 106 pfu only 5% WT mice survived, whereas none of the KO mice survived beyond day11 p.i. (P = 0.72). A study reported by Lundberg, et al. suggests that p55-/- mice are equally resistant as their parental strain of C57Bl/6 mice inoculated with 3200 pfu HSV-1 (Lundberg et al., 2007). Our data is consistent with their findings when mice were inoculated with 102–103 pfu. In that study unlike ours, however, the resistance of p55-/- mice to acute infection with higher doses of virus was not tested. TNF-α is known to provide neuro-protection (Bruce et al., 1996). HSV-1 infected mice die of encephalitis. Since TNF-α plays a central role in innate immunity, the control of virus propagation at the primary entry site would have been impaired in p55-/- mice causing enhanced death seen in our study.
ganglia were harvested for DNA extraction and subsequent quantitative PCR. By comparing the quantitative PCR results to a standard curve of gG-1 plasmid DNA, we estimated the genome copy number in test samples (Fig. 2B). Though the difference in the latent DNA copy numbers between two groups was not highly significant (P = 0.025), there was two-fold rise in geometric mean copies of viral DNA in p55-/mice. Innate-immune responses followed by adaptive immune responses are mounted to check the viral replication during acute phase of viral infection (Ellison et al., 2000). Since p55-/- mice have defect in their hematopoietic system (Rebel et al., 1999) it is reasonable to assume that HSV-1 infection might not have been checked efficiently during the acute phase allowing more number of virus to establish latency. In some studies, it has been also shown that both the receptors p55 and p75 play an important role in providing protection against viral infection (Janet et al., 1997). Nevertheless, p55 but not p75 is demonstrated to be preferentially protecting the host from infections like toxoplasmosis (Martina et al., 1998). It is possible that the presence of p75 in absence of p55 might have minimal role in preventing HSV infection in our experimental setup.
3.2. Absence of TNFR1 signaling enhanced latent HSV infection 3.3. Indomethacin blocks UV-induced re-activation of latent HSV-1 HSV-1 establishes and maintains a latent infection in the TG and can be re-activated in vitro by explant co-cultivation. To detect any potential differences in relative rates of the establishment of latency and in vitro reactivation, an indicator of the quantity of latent virus, p55-/- and WT mice were sacrificed on day 30 p.i. and their TG was explanted for 11 days. 100% mice from both groups (WT, 8/8; p55-/-, 6/6) yielded virus (Fig. 2A) with no difference in time of reactivation (P = 0.62). Treatment with IgG did not entirely prevent establishment of HSV-1 latent infection. This observation indicates that inflammatory signals delivered by TNF-α through p55 may not affect the establishment of latency. However, the efficient reactivation does not imply that there are equivalent levels of latent HSV-1 genomes in the ganglia. It is possible that in the absence of p55 signaling there were different levels of latent virus, but the virus could still reactivate as long as some threshold quantity of latent virus genomes had been exceeded. To support this notion we determined the quantity of latent HSV-1 genomes in the TG by using real-time PCR (LeBlanc et al., 1999a; Dalai et al., 2002). Groups of eight to twelve WT and p55-/- mice latently infected with HSV-1 were sacrificed on day 79 p.i. and individual
Removal of the trigeminal ganglion and its co-cultivation ex vivo revealed equal rates of reactivation in the presence or absence of TNF-α signaling through p55. However, explant co-cultivation is maximal and non-physiologic stimulus to HSV-reactivation. Physiologic reactivation processes would ensure either spontaneous or stress-induced recurrence of infectious virus, as in humans with recurrent herpetic kerato-conjuctivitis. Since HSV does not reactivate spontaneously from mice, the role of p55 signaling in HSV-1 reactivation, and the impact of differential viral loads were explored by exposing the latently infected mice to UV irradiation (Dalai et al., 2002). After latency was established (day30 p.i.), the animals were exposed to UV-light for 1 min per eye (360 mJ/eye). Forty-eight hour later HSV-1 reactivation was tested in TG. 100% mice from both the groups yielded virus (Fig. 3A). There was no significant difference in the average time of reactivation (P = 0.17). These results suggest that TNF-α signaling through p55 may not play a dominant role in maintaining or reactivating latent herpes virus. Previously, we have shown that (LeBlanc et al., 1999b) absence of IL-6 does not influence the reactivation of HSV infection. In our 3
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Fig. 2. Absence of TNFR1 signaling enhanced latent HSV infection. (A) In vitro reactivation of HSV-1 by explant co-cultivation Each pair of TG from individual mouse was aseptically harvested from eight WT and six TNFRI-KO mice on day30 p.i and placed onto Vero cell monolayer with media containing 0.1% N, N’-hexamethylene-bis-acetamide. The explants were checked daily for CPE, and were carefully transferred onto fresh monolayer weekly, if necessary. (B) Quantitative determination of HSV-1 DNA loads in TG. DNA was extracted from individual ganglia from eight WT and TNFRI-KO mice day79 p.i. Each point represents the log value of the mean copy number for three replicate PCR determinations per pooled TG from each animal. The horizontal line along the width of each data ‘diamond’ indicates the combined mean HSV-1 DNA copy numbers for all eight mice per group. The shorter horizontal lines above and below this indicate the 95% confidence intervals around the mean.
kept under check due to the host immunity generated during the acute infection (Hoshino et al., 2007), induction of immune suppression by PGE2 at the site of latency might reactivate the latent virus. When the delivery of inflammatory signals by TNF-α through p55 is blocked, PGE2 release might not be adversely affected because of the inflammatory signals delivered through IL-1 and IL-6. Our preliminary data in IL-1 receptor deficient mice supports this notion (data not shown). Thus, recurrence of latent HSV in single inflammatory cytokine KO mouse would not be dramatically affected. The use of multiple inflammatory cytokine deficient mice or blocking the signaling of multiple inflammatory cytokines would be desirable to understand this phenomenon.
preliminary experiments, absence of IL-1R1 signaling does not seem to affect the HSV infection (Thomas et al., 1999). All these studies prompted us to look for a common pathway that might be responsible for inducing reactivation of HSV. Many stimuli including UV trigger syntheses of IL-1, IL-6, and TNF-α. All three of these pro-inflammatory cytokines induce cyclooxygenase 2 (COX-2) synthesis resulting PGE2 release (Kuwamoto et al., 2000). While PGE2 is known to induce proinflammatory responses, it also induces IL-10 and other anti-inflammatory cytokine causing immune suppression or immune-deviation (Shreedhar et al., 1998) that might favor the HSV reactivation. Upon treating mice with indomethacin, a broad inhibitor of cyclooxygenase (constitutive COX-1 and inducible COX-2) (Simmons et al., 2004) before exposing the eyes to UV irradiation, we found that HSV-1 reactivation was significantly prevented in mice treated with indomethacin compared to that of placebo control (P < 0.05) (Fig. 3B) (Walia et al., 2012; Higaki et al., 2009; Paul et al., 2013). It has been shown in guinea-pig model of HSV-2 genital infection that indomethacin treatment before UV-irradiation significantly reduces virus recurrence (Bratcher et al., 1993) supporting our hypothesis that prostaglandin plays a key role in the reactivation of latent HSV. Based on these experiments and our previous findings we propose the following. UV irradiation induces potent inflammatory cytokines namely, IL-1, IL-6, and TNF- α. All the three cytokines would activate COX-2 that triggers the release of PGE2. While latent HSV infection is
Conflict of interest The authors declare no commercial or financial conflict of interest. Acknowledgments Authors would like to acknowledge the contribution of Lesley Pesnicak and others from the laboratory of late Dr. Stephen E. Straus, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, NIH, Bethesda for assistance to SKD while he was as a visiting fellow.
Fig. 3. Indomethacin blocks UV-induced re-activation of latent HSV-1. (A) In-vivo reactivation of HSV-1 in latently infected mice. Following 48 h post-UV exposure, each pair of TG from latently infected WT or TNFRIKO mouse was harvested, ground, and supernatant was plated on Vero cell monolayer, incubated at 37 °C and checked daily for CPE. (B) Effect of indomethacin on UV-induced re-activation of HSV-1. Groups of nineteen latently infected WT mice were treated with indomethacin or PBS (placebo control) as described in methods. On day 3 post treatment, mice were UV-exposed on both the eyes, and 48 h later TG were harvested and checked for virus reactivation.
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