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Keywords:

  • HSV-2;
  • tetranortriterpenoid;
  • cytokines;
  • immunomodulator

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. Conflict of Interest
  8. REFERENCES

The limonoid 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM) isolated from leaf extracts of Melia azedarach L, has potent antiherpetic effect in epithelial cells. Since Meliacine, the partially purified extract source of CDM, has therapeutic effect on murine genital herpes, the potential use of CDM as microbicide against herpetic infections was studied here. To determine the cytotoxic effect of CDM, the MTT assay and acridine orange staining of living cells were performed. The antiherpetic action of CDM was measured by plaque reduction assay, and the immunomodulatory effect was determined by measuring the cytokine production using a bioassay and ELISA method. The results presented here showed that CDM inhibited Herpes Simplex Virus type 2 (HSV-2) multiplication in Vero cells but did not affect its replication in macrophages which were not permissive to HSV infection. In macrophages, levels of TNF-α, IFN-γ, NO, IL-6 and IL-10 were increased by CDM used alone or in combination with HSV-2. Besides, CDM not only synergized TNF-α production combined with IFN-γ, but also prolonged its expression in time. Results indicate that CDM inhibits HSV-2 multiplication in epithelial cells and also increases cytokine production in macrophages, both important actions to the clearance of infecting virus in the mouse vagina. Copyright © 2013 John Wiley & Sons, Ltd.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. Conflict of Interest
  8. REFERENCES

Every year, there are an estimated 500,000 new cases of symptomatic first episode of Herpes Simplex Virus type 2 (HSV-2) infection (Herbst-Kralovetz and Pyles 2006). The impact of genital herpes as a public health threat is amplified because of its epidemiological synergy with human immunodeficiency virus (Corey et al., 2004). The growing incidence of genital HSV-2 infection in the population and the lack of an approved vaccine have led to evaluate approaches designed to protect the genital mucosa against infection. One promising advance to prevent or reduce sexually transmitted infections is the application of topical microbicides (Elias and Coggins, 1996; Herold et al., 1997), which may work inhibiting pathogen replication or enhancing local immune responses in the mucosa (Herold et al., 1997; Milligan et al., 1998; Maguire et al., 2001; Catalone et al., 2004).

Due to the raise in drug resistance to the conventionally used antimicrobials, there is an increasing need for new substances with antiviral activity. Since natural products are considered powerful sources of novel drug discovery and development, we have been working with a partially purified extract from leaves of Melia azedarach L., Meliacine (MA) (Andrei et al., 1988), which has a broad spectrum of antiviral activity (Castilla et al., 1998; Wachsman et al., 1998; Alche et al., 2002). Several studies performed in our laboratory have shown that MA has a potent antiviral activity against several RNA and DNA viruses without cell cytotoxicity (Wachsman et al., 1987; Wachsman et al., 1998; Andrei et al., 1988). Of particular interest is that MA strongly inhibited the replication of HSV-1 in Vero cells (Andrei et al., 1994) and exhibited a synergistic antiviral activity when combined with acyclovir (Barquero et al., 1997). Electron microscopic studies performed by Alché on infected Vero cells incubated in the presence of meliacine suggested that MA exerted its antiviral action on both, the synthesis of viral DNA and maturation and egress of HSV-1 particles (Alche et al., 2002). Interestingly, Meliacine significantly reduced the incidence and the severity of blepharitis, neovascularization and stromal keratitis in a murine experimental model, due to its antiviral action and immunomodulating properties against HSV-1 infection (Alche et al., 2000; Alche et al., 2002). Additionally, when female mice were infected with HSV-2 and then treated topically with MA, an overall protective effect was observed: animal survival increased, the severity of the disease was reduced, life span was extended and virus shedding in vagina fluids diminished. Meliacine also reduced the amount of virus that migrated to the brain and vaginal fluids presented higher levels of IFN-γ and TNF-α than untreated infected mice (Petrera and Coto 2009).

Bioassay guided purification of MA led to the isolation of the limonoid 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM), a tetranortriterpenoid with high antiviral activity against vesicular stomatitis (VSV), HSV-1 and HSV-2 viruses (Alche et al., 2003; Petrera and Coto, 2006). Further investigations demonstrated that CDM affected the intracellular transport of virus glycoproteins gB, gC and gD of HSV-1, and gG of VSV which were confined to the Golgi apparatus (Barquero et al., 2004; Barquero et al., 2006).

During an inflammatory process, viruses are able to alter host metabolism and modulate cellular signaling pathways to support infection (Paludan, 2000). In genital infections, the pro-inflammatory mediator nitric oxide (NO) is generated by the inducible isoform of NO synthase (iNOS), and a large amount of pro-inflammatory cytokines are produced principally in macrophages to obtain complete clearance of the infecting virus (Moncada and Palmer, 1991; Yun et al., 1996; Ellermann-Eriksen 2005). To combat HSV-2 infections, the ideal microbicide should inhibit virus replication and increase the immune response to control the infection. Both properties were observed with Meliacine in vivo (Petrera and Coto, 2009) and in vitro (Petrera and Coto, 2003).

Taking into consideration these results, we decided to evaluate if the cytokine increase observed in animals was due to the combined effect between MA and HSV-2. For that purpose, we analyzed here the synthesis of TNF-α, IFN-γ, IL-6, nitrites and IL-10 in peritoneal macrophages treated with CDM, the active principle of Meliacine, alone or in combination with HSV-2. In addition, we also studied the antiviral action of CDM against HSV-2 replication in macrophages and Vero cells.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. Conflict of Interest
  8. REFERENCES

Plant material

CDM was purified in our laboratory from M. azedarach L leaves, as described by Alché et al., (Alche et al., 2003).

Cell culture and virus

Vero and L929 cells were obtained from ATCC. Peritoneal macrophages were harvested from female BALB/c mice (6–8 weeks old, I.N.T.A. Castelar, Buenos Aires) at day 5 following intraperitoneal injection with sterile thioglycolate broth (Difco Laboratories, Detroit, MI). All cells were cultured in Iscove's modified Dulbecco's medium (Sigma, USA) containing 50 µg/mL of gentamycin and 10% fetal bovine serum (Gibco) in a humidified incubator with 5% CO2 at 37 °C.

Triplicate macrophage monolayers seeded at a density of 5×105 cells per well (90% macrophages) were treated with 104 PFU/mL of HSV-2 MS or 100 UI/mL of IFN-γ (BD Pharmingen), alone or in combination with CDM. Cell-free supernatants were harvested at the designated time points and kept frozen until cytokine or nitrites determination.

Wild-type HSV-2 strain MS was obtained from ATCC and propagated in Vero cells.

Cell viability assay

Cell viability was determined by MTT assay. Vero cells or peritoneal macrophages, treated with two-fold serial dilution of CDM (from 7 to 900 μM) for 24 h, were incubated for 4 h with culture medium containing 0.5 mg/mL of MTT. Then, formazan salts produced were dissolved with ethanol, and the absorbance was measured at 570 nm. The 50% cytotoxic concentration (CC50) was defined as the compound concentration required to reduce cell viability by 50%.

Viral replication assays

For virus replication assays, Vero cells or macrophages were seeded in 24-well plates, and 24 h later, cultures were inoculated with HSV-2 MS. After adsorption, fresh culture medium was added with different concentrations of CDM. Twenty-four and 48 h p.i., titres of infectious virus in cell supernatants were determined by a standard plaque assay. Briefly, Vero cells monolayers grown in 24-well plates were incubated for viral adsorption with samples’ dilutions for 1 h at 37 °C. Infected monolayers were overlaid with MEM supplemented with 0.7% of methyl cellulose and maintained for 48 h at 37 °C, then fixed and stained with crystal violet. Viral plaques were counted, and the number of PFU per milliliter was calculated.

Acridine orange staining of living cells

Macrophages grown on coverslips were incubated with culture medium or CDM (75 μM) for 15 min. After that, cells were stained with acridine orange (1 µg/mL) for 15 min at 37 °C, washed twice with cold PBS, mounted in PBS and visualized on a Zeiss Axioplan fluorescence microscope (magnification ×1000).

Measurement of NO

NO2- accumulation was used as an indicator of NO production (Ding et al., 1988). Supernatants were mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride and 2% phosphoric acid) and incubated at room temperature for 15 min. NaNO2 was used to generate a standard curve, and optical density at 540 nm was measured.

TNF-α bioassay

Active TNF-α was determined using a bioassay based on TNF-α cytotoxic activity (Petrera and Coto 2003). Briefly, monolayers of L-929 cells were incubated at 38.5 °C for 20 h with serial two-fold dilutions of test supernatants containing 5 µg/mL of actinomycin D (Biosidus, Argentina). Plates were then fixed in 10% formaldehyde, stained with crystal violet, and the absorbance at 600 nm was measured. TNF-α titres were calculated by determination of the dilution resulting in 50% cytotoxicity in comparison with a standard calibration curve obtained with TNF-α mouse recombinant (Sigma, USA). The cytotoxic principle was identified as TNF-α by neutralization with specific polyclonal goat anti-mouse TNF-α antibodies (Sigma).

ELISA assay

Cytokines (IFN-γ, IL-6 and IL-10) were determined with a capture ELISA assay. Briefly, MaxiSorp 96 plates (Nunc, USA) were coated with a capture monoclonal anti-mouse IFN-γ, anti-IL-10 or IL-6 antibody (BD Pharmingen, USA) at 2 µg/mL and 4 °C. Plates were blocked with PBS plus 10% FBS for 4 h at room temperature and loaded with 100 μL of samples. Incubation was carried out at 4 °C overnight. Unbound material was washed off, and biotinylated monoclonal anti-IFN-γ, anti IL-10 and anti IL-6 antibodies (Pharmingen) were added at the concentration of 2 µg/mL for 1 h. Plates were developed with Streptavidin-HRP 1:2000 and 3,3’, 5,5’-tetramethylbenzidine (BD PharMingen). The reaction was stopped by the addition of 2 N H2SO4, and the absorbance at 450 nm was determined with a microplate reader.

Statistical analysis

All results are expressed as means ± SEM for three experiments. Statistical significance was determined by one-way analysis of variance followed by Tukey's testing for three or more groups. Significance was considered for p < 0.05.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. Conflict of Interest
  8. REFERENCES

The present study was performed to evaluate the possibility that the limonoid CDM, the active principle of Meliacine could act as a microbicide in vivo. We studied the antiviral action of CDM in epithelial cells, and its immunomodulatory properties on macrophages confronted with herpes infection.

First, we evaluated CDM effect on cell viability by MTT assay. The CC50 values obtained were >900 μM for both Vero and macrophages cells.

It was previously reported that CDM inhibited VSV uncoating by cytoplasmic alkalinization of Vero cells (Barquero et al., 2004) To test CDM action on macrophages vesicles, a vital fluorescence microscopic study with acridine orange was performed. The absence of orange fluorescence observed in 100% of the treated cells revealed that CDM affected the acidic pH of macrophages intracellular vesicles (Fig. 1B), like in Vero cells (Barquero et al., 2004).

image

Figure 1. Effect of CDM on macrophages. Macrophages were treated with culture medium (A) or CDM (B) during 15 min, then incubated for 15 min with acridine orange and visualized on a Zeiss Axioplan fluorescence microscope. Magnification × 1000. (C) For virus replication assays, Vero cells (▲) or macrophages (o) were inoculated with HSV-2 MS (103 PFU). After 1 h adsorption, the inoculum was removed, and fresh culture medium was added to each well. Twenty-four and 48 h p.i. titres of infectious virus in cell supernatants were determined by plaque assay. (D) Macrophages were incubated with different CDM concentrations, and, 24 h later, TNF-α presence in supernatants was determined by a bioassay. The data represents the mean ± SEM of three experiments. This figure is available in colour online at wileyonlinelibrary.com/journal/ptr.

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To study HSV-2 replication in the presence of CDM, macrophages and Vero cells were infected with 1 PFU/cell of HSV-2 strain MS. At various times post-infection, the amount of infectious virus was determined titrating by a plaque assay. HSV-2 replication in macrophages was dramatically lower than in Vero cells. At 48 h post-infection, the amount of infectious virus from macrophages cultures was >1000-fold less compared to Vero cells (Fig. 1C). These results agree with a previous work by Sit et al., which analyses the intrinsic resistance of peritoneal macrophages to HSV-2 infection. The work demonstrated that, despite thioglycollate-elicited inflammatory macrophages were slightly more permissive than resident peritoneal macrophages, the amount of infectious virus from macrophages cultures was >4000-fold less than in Vero cells. (Sit et al., 1988)

Then, CDM antiviral activity on HSV-2 infected cultures was tested. Although in Vero cells, the IC50 value of CDM after 24 h of treatment was 7 μM, the IC50 value in peritoneal macrophages could not be determined because HSV-2 did not replicate. For that reason, these peritoneal macrophages constituted a good model to study CDM immunomodulatory effect without the interference of its antiviral activity.

Since macrophages are the first line of defence in inflammatory responses against pathogens, the effect of CDM on TNF-α production was evaluated. When macrophages were treated with CDM for 24 h, a concentration-dependent increase in the level of TNF-α expression was found, indicating that immunomodulating properties displayed by CDM were specific (Fig. 1D). This result agrees with our previous report showing that Meliacine increased TNF-α production in peritoneal macrophages (Petrera and Coto, 2003) and it allowed us to select 75 μM as the optimal concentration of CDM which produced 0.85 ng/mL of TNF-α.

In an attempt to simulate an in vivo situation, we studied the effect of CDM addition on TNF-α production in peritoneal macrophages induced by HSV-2 (Shimeld et al., 1997; Paludan 2000). Active TNF-α concentration was determined in macrophages treated with 75 μM of CDM, 104 PFU/mL of HSV-2 MS or their combination during 8 or 24 h. Even though BALB/c peritoneal macrophages were not permissive to HSV-2 MS replication, some experiments were performed with UV-inactivated virus to avoid any antiviral intrinsic effect of CDM, which could influence the results. There were no significant differences between the induction of TNF-α with HSV-2 UV-irradiated or not, and the increased concentration of TNF-α observed at 8 h was maintained until 24 h post induction in both treatments (Fig. 2A). Interestingly, after 8 h of induction, CDM induced as much TNF-α as HSV-2. Following 24 h of treatment, CDM increased TNF-α production more than HSV-2 alone (and twice as high as 8 h values), indicating that CDM is an immunomodulator per se (Fig. 2A). When CDM was added in combination with HSV-2 UV-irradiated or not, the increase in the amount of TNF-α was approximately the sum of both individual activities (Fig. 2A).

image

Figure 2. CDM increases TNF-α production in macrophages. (A) Macrophages were treated with culture medium (●), 75 μM of CDM (o), 104 PFU of HSV-2 (■), 104 PFU of UV-inactivated HSV-2 (▲) or their combination: HSV-2 + CDM (□) and UV-inactivated HSV-2 + CDM (Δ). (B) Macrophages were treated with HSV-2 (104 PFU) (▲), IFN-γ (100 IU) (▼), HSV-2 + IFN-γ (■) or HSV-2 + IFN-γ + CDM (●). (C) Macrophages were treated with IFN-γ (100 IU) (▲), CDM (75 μM) (○) or CDM + IFN-γ (■). After different times, culture supernatants were harvested to determine the presence of active TNF-α by using a bioassay. The data represents the mean ± SEM of three experiments.

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Paludan et al., demonstrated that co-treatment of macrophages with HSV-2 and IFN-γ enhanced synergistically TNF-α secretion, which in turn is responsible for activation of the transcription factor NF-kB (Paludan 2000). To see the effect of CDM on this system, active TNF-α concentration was determined in cultures of macrophages treated with 104 PFU of HSV-2, 100 IU of IFN-γ, 75 μM of CDM or their combinations during 8, 12 and 24 h. As expected, HSV-2 and IFN-γ added together increased TNF-α production synergistically, reaching a maximum at 12 h (Fig. 2B). CDM presence did not alter the kinetics of TNF-α production, and, besides, it prolonged the response maintaining a high TNF-α concentration at least for 24 h of treatment. Since CDM did not synergize with HSV-2 for TNF-α production (Fig. 2A), its combination with IFN-γ alone was evaluated. Surprisingly, the kinetics and the amount of TNF-α produced by CDM treatment were similar to that induced by IFN-γ and the combination of both increased synergistically the amount of TNF-α, reaching a value of 258 ng/mL after 24 h of treatment (Fig. 2C). Paludan reported that HSV-2 infection of macrophages quickly induces TNF-α secretion, via a mechanism that is strongly amplified by IFN-γ, TNF-α then functions synergistically with IFN-γ to induce expression of many genes involved in generating inflammatory processes (Paludan, 2000). Taking that in consideration, we postulate that CDM would exert its synergistic effect on IFN-γ-induced TNF-α production by triggering autocrine TNF-α secretion, which in turn would stimulate gene transcription by activating NF-κB.

As CDM increased TNF-α production in macrophages, its effect on the induction of other cytokines was determined. Then, macrophages were treated with 75 μM of CDM, 104 PFU/mL of HSV-2 or their combination during 24 h. Table 1 shows that the induction with HSV-2 increased the synthesis of IFN-γ, nitrites, IL-10 and IL-6. CDM per se increased five-fold the production of IL-6 with respect to the control, but it behaved as a weaker inductor of IFN-γ, nitrites and IL-10 than HSV-2. The combination of CDM plus HSV-2 enhanced cytokines production in all cases in comparison with values obtained with individuals inductions (Table 1). Synthesis of NO through the iNOS pathway in macrophages is known to be induced by inflammatory stimuli such as HSV infection and IFN-γ among other cytokines (Moncada and Palmer 1991; Ellermann-Eriksen 2005). The augmented production of nitrites observed when CDM was added together with HSV-2 could be due to the stimulation of iNOS activity because its expression analyzed by western blot did not suffer modifications (data not shown). This result agreed with a previous report where it was necessary a viral infection to induce the expression of the enzyme (Baskin et al., 1997)

Table 1. Cytokine production in macrophages treated with HSV-2 and CDM
 IL-6 (pg/mL)Nitrites (μM)IFN-γ (pg/mL)IL-10 (pg/mL)
  1. Macrophages were treated with culture medium alone as a control, CDM, HSV-2 or HSV-2 + CDM; 24 h later supernatants were removed to determine cytokine concentration. IL-6, IFN-γ and IL-10 were determined by ELISA, and nitrites were determined by Griess reaction.

  2. To compare, data were derived by subtracting the basal level of cytokines (obtained with control treatment) to each of the values obtained in all experiments

  3. Presented values represent the mean ± SEM from three independent experiments.

CDM388.5 ± 6.43.13 ± 0.67365 ± 7.0711.25 ± 0.07
HSV-227.5 ± 3.510.92 ± 0.97496 ± 5.6626.45 ± 2.89
HSV-2 + CDM481.5 ± 4.914.8 ± 1.13902.5 ± 10.635.95 ± 0.77

It was previously reported that bafilomycin A1, a V-ATPase inhibitor, which also alkalinized intracellular vesicles, increased TNF-α, IFN-γ, IL-6 and NO production in mouse macrophages by activating both NF-kB and AP-1 (Hinoki et al., 2006; Hong et al., 2006). On the other hand, bafilomycin also synergized with IFN-γ to induce 20 times more TNF-α than did IFN-γ alone and had an effect on the duration of the response: TNF-α production normally declined to background levels by 24 h after stimulation, when V-ATPase activity was blocked, significant levels of TNF-α were still produced at 24 h. This prolonged response was similar to the effect obtained with CDM and IFN-γ.

How CDM increased cytokine production could not be explained so far, but taking into account all the information presented above, CDM could maybe act in an analogous way to V-ATPase inhibitors. Further studies are needed to investigate the possibility of CDM exerting its immunomodulatory effect via NF-κB activation.

Inflammatory responses are induced to combat invading pathogens and are mediated by cytokines. Modulation of cytokine production seems to be critical for clearance of microbes and has been studied in depth in macrophages, major regulatory cells of innate immune response (Unanue, 1981). Hence, an appropriate therapy to control the dissemination of microbes could be a way to imitate or enhance the host immune response.

The results obtained in this study showed that in a hypothetic genital herpes infection, CDM could not only directly inhibit its replication in epithelial cells but also reduce its propagation by increasing pro-inflammatory cytokines. The IFN-γ produced by CDM treatment could synergize with HSV-2, increasing TNF-α production with the consequent viral clearance.

Although further investigations are needed to clarify the mechanism of action, we postulate here that this natural product could be important in immunomodulatory treatment modalities imitating the early non-specific antiviral defense in herpes virus infections.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. Conflict of Interest
  8. REFERENCES

This work was supported by grants from the Universidad de Buenos Aires (UBA X-046) and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) (PIP 1007).

Conflict of Interest

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. Conflict of Interest
  8. REFERENCES

Authors declare no financial/commercial conflicts of interest.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS AND DISCUSSION
  6. Acknowledgements
  7. Conflict of Interest
  8. REFERENCES
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