The psychostimulant methamphetamine (METH) is an important drug of abuse in the United States and worldwide. It affects the central nervous system, causing short- and long-term deleterious health effects that include hyperthermia, chest pain, anxiety, aggressiveness, myocardial infarction, stroke, paranoia, and psychotic behavior.1,2 METH abuse is a persistent problem in teens and young adults, the members of society who are also particularly vulnerable to sexually transmitted infections (STIs), accounting for half of all new cases annually in the United States.3 The well-established link between METH abuse and increased incidence of STIs has historically been attributed to drug-induced increases in high-risk sexual risk behaviors.4 However, recent research suggests that METH produces biologic effects that may also contribute.4 Although the literature is limited and complicated by a lack of consistency in the METH dose and regimen used, in vitro, ex vivo, and in vivo studies suggest that METH can dysregulate normal host immune responses to pathogens.5 However, the impact of METH use at the time of exposure to an STI pathogen has not been examined.
Herpes simplex virus type 2 (HSV-2) causes genital herpes, an important STI worldwide. HSV-2 is typically transmitted via the genital tract where it infects and passes through the mucosal epithelium to establish a lifelong latent infection in the dorsal root ganglia (DRG) of the innervating nerves.6 Primary infection can be accompanied by painful vesiculoulcerative lesions.7 After resolution of the primary infection, latent virus periodically reactivates returning to the periphery to cause recurrent lesions, or to be shed into the genital tract in the absence of symptoms. This asymptomatic shedding of virus is believed to be the major source of genital herpes transmission to susceptible sex partners.8 In addition, previous infection with HSV-2 substantially increases the risk of subsequent HIV infection.9,10 This suggests that METH-induced alterations in the pathogenesis of genital herpes could also potentially affect susceptibility to HIV and other STIs.
In the studies described here, we used a well-established mouse model of genital HSV-2 infection to examine the impact of METH use at the time of exposure to the virus on susceptibility to vaginal infection, and the magnitude and duration of virus replication in the genital tract and innervating neuronal tissue. This research begins to elucidate the impact of METH on the normal cytokine responses to genital HSV-2 infection and allows for an evaluation of any association between METH-induced immune responses and disease pathogenesis.
MATERIALS AND METHODS
HSV-2 strains 186 and a thymidine kinase deficient strain 333 (HSV-2tk-) were prepared on Vero cell monolayers and stored in frozen state (−80°C) until used, as described previously.11
Female Swiss-Webster mice (Harlan Laboratories, Indianapolis, IN), 6 to 8 weeks old, were housed in Association for Assessment and Accreditation of Laboratory Animal Care approved quarters; all procedures were approved by the Institutional Animal Use and Care Committee of the University of Texas Medical Branch.
METH Treatment and Experimental Genital Herpes Disease
After acclimation to the vivarium for 7 days, mice were subcutaneously injected with 2 mg of medroxyprogesterone acetate (Upjohn, Don Mills, Ontario, Canada) to thin the genital epithelium, as previously described.12 Beginning 4 days later, mice were administered 10 mg/kg of METH dissolved in sterile saline (National Institute of Drug Abuse) subcutaneously in a final volume of 50 μL once daily for 5 days. Control animals were treated using the same schedule with 50 μL of saline. The mice were challenged with HSV-2 immediately after METH treatment on day 3 using previously described methods.13 Briefly, the vaginal vault was swabbed with a moistened calcium alginate tipped swab and 0.05-mL of inoculum containing 1 × 102 to 1 × 104 PFU HSV-2 strain 186 or 5 × 105 PFU HSV-2 strain 333tk- instilled into the vagina. To document infection, vaginal swab samples were collected from all animals on days 1 and 2 postinoculation, plated on susceptible cell monolayers, and incubated for 5 days. Animals for which either of these samples produce cytopathic effects characteristic of HSV-2 were defined as infected.11 For some studies, the virus titers in day 2 swab samples were determined by plaque assay, as previously described.12 The mice were examined daily until day 21 post virus inoculation for clinical signs of genital herpes disease (CSD), which includes cutaneous disease (hair loss and erythema on the perineum) and progression to more severe neurologic disease (urinary inconstance and hind limb paralysis). Mice progressing to severe neurologic involvement were euthanized and considered dead the following day for data analysis.
Vaginal Wall Thickness Assessment
To determine whether METH altered physical characteristics at the site of infection, mice were administered 2 mg of medroxyprogesterone acetate and treated with METH or saline as for virus challenge studies. They were euthanized on day 3 of treatment. Vaginal tracts were excised, formalin fixed, and hematoxylin and eosin stained. For each animal, a representative histology section was selected, a digital image generated, and epithelium thickness was measured using a calibrated measuring tool (ImageJ, National Institutes of Health, open source software, available at: http://rsb.info.nih.gov/ij/). Vaginal wall thickness was measured at 10 sites from the image using the same tool.
Detection and Quantification of Viral DNA in DRG
To determine the kinetics and magnitude of virus replication in DRG, METH- or saline-treated mice inoculated with HSV-2 were euthanized on days 3 and 5 post virus inoculation. The lumbar and sacral DRG were harvested, and viral DNA was quantified by an adaptation of our previously described methods.14 Briefly, DNA was extracted using the DNeasy DNAspin-column isolation kit (Qiagen, Valencia, CA) following manufacturer's instructions. Samples underwent quantitative real-time polymerase chain reaction using a CFX96 real-time system and associated reagents, including 1× iQ Supermix (Bio-Rad, Hercules, CA). HSV-2 DNA was quantified in 96-well plates using primers that target the HSV-2 glycoprotein B gene, with adaptations for real-time polymerase chain reaction analysis.15
Quantification of Cytokine Secretion in Sera and Genital Tract
To examine the impact of METH on local and systemic innate immune responses, vaginal lavages and sera were collected from mice treated with METH or saline and inoculated with HSV-2, as described earlier on days 1 and 2 post virus inoculation. Vaginal lavages were collected using a positive displacement pipette to instill and collect 25 μl of sterile saline 5 times. At the same time, blood was collected from the retro-orbital plexus and centrifuged to extract the sera. Recovered lavage fluid and serum were stored at −80°C, until cytokines were quantified using cytometric bead arrays with mouse cytokine 23-plex panels (Bio-Rad) according to the manufacturer's instructions.
Quantification of Interferon-α and -γ Production
To examine the impact of METH on interferon (IFN) production at the vaginal mucosal surface, mice were treated with METH or saline and inoculated with HSV-2, on days 1, 2, 3, and/or 5 post virus inoculation. Vaginal lavage samples were collected and stored, as described earlier. A VeriKine mouse IFN-α ELISA Kit (PBL Biomedical Laboratories, Piscataway, NJ) was used to quantify IFN-α levels according to the manufacturer's instructions. IFN-γ levels were measured using anti-IFN-γ as a capture antigen and compared with a standard reference antibody.
Fisher's exact test was used to compare all infection, disease, and outcome data. The Student's unpaired t test was used to compare group mean values. All reported P values are 2-tailed, and values <0.05 were considered to indicate statistical significance. All statistical analysis was performed using GraphPad Prism 5.0 for PC.
Selection of METH Dose and Treatment Regimen
In initial studies, we showed that the 5-day dosing regimen selected produced behavioral alterations that included overgrooming and hyperactivity but did not cause mortality. The behavioral changes began soon after METH administration and lasted for approximately 1 hour. Furthermore, the behavioral changes were seen after each treatment, indicating that the animals did not become acclimated during the 5 days.
Impact of METH on Susceptibility to HSV-2 Infection and Disease
We next examined whether METH would alter susceptibility to vaginal HSV-2 infection or CSD. For these studies, METH- or saline-treated animals were challenged with HSV-2 inocula ranging from 1 × 102 to 1 × 104 PFU. In 2 independent studies, there was no significant difference between METH-treated and control animals in the incidence of infection or the incidence of CSD in infected animals at any of the inocula tested (data not shown) indicating that METH did not increase susceptibility to infection or disease development. However, METH did alter the course of disease in infected animals. Figure 1 shows that the onset of CSD was significantly earlier in METH-treated animals (mean day of onset 4.8 ± 0.2 vs. 5.9 ± 0.2, P < 0.001). Further, the disease progressed to include neurologic signs of encephalitis more rapidly in METH-treated animals (mean day of death 9.6 ± 0.5 vs. 11.9 ± 0.8, P = 0.02).
METH Induced Higher Viral Load in the Vagina and DRG
Vaginal viral titers were determined on day 2 postinfection, the day of peak viral replication in the model. Viral titers were mildly increased in METH-treated animals, but the increase did not reach significance (mean log10 [PFU/mL], 5.0 ± 0.2 METH vs. 4.6 ± 0.2 control). We next examined the impact of METH on viral load in the DRG. Table 1 shows that on day 3 after vaginal virus challenge, HSV-2 DNA was detected in 24 of 35 METH-treated mice compared with only 18 of 35 control animals. Further, on day 3, for the animals in which viral DNA was detected, the viral load in METH-treated mice was significantly higher than in controls (P = 0.03). By day 5 postinoculation, HSV-2 DNA was detectable in 33 of 35 animals in both groups. At this time, the viral load in the METH-treated animals remained higher than in controls, although the difference was no longer significant (Table 1). Thus, our data showed that METH treatment resulted in earlier and greater replication of HSV-2 in the DRG.
METH Altered Vaginal Epithelium Thickness
Figure 2 shows vaginal wall thickness measurements in METH- and saline-treated mice at the time of vaginal challenge. The basal epithelium thickness did not differ between the 2 groups. However, the columnar epithelium was significantly (P < 0.0001) thinned in METH-treated mice (mean [μm], 22.3 ± 1.3) compared with saline controls (31.5 ± 1.3).
METH Alters Cytokine Production During an Infection
We also examined cytokine production in HSV-2-infected animals treated with METH both locally in the vaginal mucosa and systemically (Fig. 3A, B). On day 1 postinfection, interleukin (IL)-3, -6, and -9 were significantly (P = 0.003, 0.01, 0.04, respectively) increased in the vagina of METH-treated animals (Fig. 3A). On day 2 postinfection, the cytokine IL-12p40 and chemokine RANTES were significantly (P = 0.02, 0.003) elevated in the vagina, whereas IL-12p40 was the only cytokine to be significantly (P = 0.04) elevated in the serum on either day (Fig. 3B).
In addition, Figure 4 shows that IFN-α was detected in METH-treated mice but not in controls on day 1 postinfection, and IFN-γ production was significantly higher in METH-treated mice than controls on day 2 postinfection (P = 0.02).
In these studies, we examined the impact of METH treatment at the time of virus challenge on susceptibility to HSV-2 infection and the course of genital herpes disease in a mouse model. Female animals were used in these studies because women bear a disproportionate burden of STIs. We selected a METH treatment regimen designed to closely mimic a pattern of short binge drug use seen in METH abusers.1 The dose and regimen used were similar to that used previously in studies investigating the impact of another important drug of abuse, 3,4-methylenedioxymethamphetamine (MDMA).12 The METH regimen elicited behavioral changes in the mice after each drug administration, demonstrating that the animals did not become acclimated to treatment. METH treatment did not increase susceptibility to vaginal HSV-2 infection, a result that contrasts with that seen previously with MDMA.15 However, as we had seen previously with MDMA, infected METH-treated animals exhibited a significantly earlier onset of disease compared with saline-treated controls.
The accelerated onset of disease suggested that METH could be affecting normal innate immune responses to HSV-2. Therefore, we examined cytokine production both locally (vaginal mucosa) and systemically (serum). We found that METH had minimal impact on systemic responses, with IL-12p40 being the only cytokine in which there was a significant difference (an increase) compared with controls. The impact of METH in the vagina was greater, with levels of IL-12p40 and several other cytokines (IL-3, -6, and -9) and the chemokine RANTES being significantly increased. Vaginal HSV-2 infection in mice normally elicits a proinflammatory cytokine response with the development of type 1 immunity.16,17 It is interesting to note that both type-1 (IL-3, -12p40) and type-2 (IL-6, -9) cytokines were altered by METH treatment. Thus, our results show that METH dysregulated the normal cytokine response in the vagina rather than reduced cytokine levels or shifted from a type 1 to a type 2 response. These results are similar to those produced by MDMA, although the individual cytokines involved differed.12 In addition, MDMA treatment resulted in a more marked impact on systemic cytokine levels than what we have seen with METH.
IFN-α and -γ production is important in the control and clearance of HSV-2 from the vaginal mucosa in the mouse model.18 Consequently, we next examined the impact of METH on these important cytokines. Because IFN-α was not a part of the cytometric bead array panel, we used an enzyme-linked immunosorbent assay to measure both IFN-α and -γ. Although the earlier onset of CSD suggested an impaired IFN response, unexpectedly we found an earlier IFN-α response and an early and significantly increased IFN-γ response after HSV-2 infection in METH-treated mice. Interestingly, vaginal viral titers at this time were slightly higher in METH-treated mice, suggesting that the IFNs did not produce an effective antiviral response.
These results add to previous findings by others that METH alters normal immune responses.19–21 Unfortunately, differences between studies in dosing regimens, animal models used, and end points examined make it difficult to clearly define the effects of METH on immune responses. Our results indicate that METH is not simply immune suppressive, rather it dysregulates normal immune responses so that the immune system may be less effective in controlling infection.
Following HSV-2 infection at the genital epithelium, the virus enters innervating peripheral nerves and moves to the DRG to establish a latent infection. At the same time, some of the virus returns from the DRG to the periphery to produce CSD. The earlier onset of disease seen in METH-treated animals is suggestive of an alteration in the kinetics of this neural arc. Accordingly, we examined the impact of METH on the kinetics and magnitude of HSV-2 in the DRG and found both that viral DNA reached the DRG of METH-treated animals more rapidly and that there were higher levels of viral DNA in the DRG of METH-treated mice, indicating a sustained increase in viral replication during primary infection.
Our unexpected finding that the columnar epithelial outer layer of the vagina was significantly thinned in METH-treated animals at the time of virus challenge may contribute for virus being able to reach the DRG more rapidly and so led to the earlier onset of CSD. This finding suggests that the impact of METH on disease is not only due to alterations in the immune response.
This increased acute viral load in the DRG suggests the possibility that METH could also result in an increased latent viral infection. The magnitude of latent viral load has been correlated with both the frequency of recurrent disease and of viral shedding into the genital tract in the guinea pig model of genital herpes.22,23 Mice do not experience spontaneous recurrent disease or spontaneous virus shedding after vaginal HSV-2 infection, and therefore we were unable to determine the impact of METH on these end points. However, future studies in the guinea pig, to explore the impact of these end points, seem to be warranted. An increase in recurrent disease or in virus shedding would mean an increased likelihood of virus transmission to a susceptible partner, and therefore it might have important public health implications.
The mouse model of genital herpes is well suited to the study of the early events of disease after virus infection. However, as the infection progresses, there is a high incidence of mortality in mice due to HSV-2 spread to the brain, producing encephalitis. It is interesting to note that METH-treated mice experienced onset of CSD approximately 1 day earlier than controls, but death was approximately 2 days earlier. In studies with MDMA in the same mouse model, drug-treated animals also experienced approximately 1 day earlier onset of CSD; however, death was also approximately 1 day earlier than controls. Thus, METH seems to produce a greater sustained impact on the course of neurologic disease than was seen with MDMA. This finding may have important implications for other viral infections with central nervous system involvement.
In summary, we have extended our previous observations with MDMA to show that a second important drug of abuse, METH, also alters genital herpes disease in the mouse model. Our studies provide good evidence that drugs of abuse can affect STI infections, but that the effects of individual drugs vary. Further studies looking at other drugs of abuse and other STI pathogens seem to be warranted to more fully define the association between drug abuse and an increased burden of STIs.
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