Protective Role of Acetylsalicylic Acid in Experimental Trypanosoma cruzi Infection: Evidence of a 15-epi-Lipoxin A₄-Mediated Effect

Ethic statement

All animal handling protocols were performed according to the “Guide for the Care and Use of Laboratory Animals”, from the National Institute of Health, USA [15], and approved by the Institutional Ethical Committee at the Faculty of Medicine, University of Chile (Protocol CBA# 0277 FMUCH), associated to FONDECYT-Chile grant number 1090078.

In vivo infection model

Adult male BALB/c mice (20–25 g) were obtained from the Animal Facility at the Faculty of Medicine, University of Chile. Animals were first infected intraperitoneally with 30,000 T. cruzi blood trypomastigotes (Dm28c strain). Afterwards, animals were randomized to receive the different treatments. T. cruzi infection was followed daily by parasitemia through direct microscopic visualization of circulating trypomastigotes from peripheral blood, as previously described [16].

Treatment administration

Acetylsalicylic acid (Sigma, USA) was given diluted in the drinking water at concentrations that ranged from 19.5 to 390 mg/L, to achieve final doses from 5 to 100 mg/Kg/day, based in the observation that mice drank 6.4 mL of water daily. The water was available ad libitum. The bottles were replaced every morning with fresh water or drug solution, and the residual volume of water in bottles was measured to assure that the mice drank the intended water volume [17], [18]. The treatment was initiated 48 hours after parasite inoculation, for 20 days at the doses indicated in each figure. As a measure of the pharmacological effect of orally administered aspirin, we determined the bleeding time in mice. To do this, we made a cut in the tail tip and bleeding was evaluated with filter paper every 15 seconds. These determinations were made at the day 10 post-infection (p.i.). (Table 1). 5(S),6(R),15(R)-Lipoxin A₄ (Cayman Chemicals, USA), was diluted daily in fresh PBS and administered i.p. for 10 days, starting at the fourth day p.i., using the doses indicated in each figure. Controls received PBS exclusively. Mortality rate was recorded daily.

Cardiac histopathological analysis

Hearts were extracted at the moment of death from those mice dead before the end point. Surviving mice were euthanized at day 20 p.i. and their hearts were extracted. Hearts were longitudinally sectioned to further analysis by histopathology and qPCR. Samples were fixed in 10% formaldehyde 0.1 M phosphate buffer (pH 7.3) for 24 h, dehydrated in alcohol, clarified in xylene, and embedded in paraffin. 5 µm sections were obtained and stained with hematoxylin-eosin for routine histopathological analysis as well as to evaluate the presence of T. cruzi amastigote nests and inflammation of the myocardium [19]. The automated morphometric analysis was performed by methodology developed by us, using the public domain software ImageJ (ver 1.46). The photographs of at least four nonconsecutive slides of tissue from different mice were assessed. The contrast of each photograph was increased automatically. To facilitate visualization of cell nuclei, the color channels (red, blue and green) were split. To isolate nuclei marks, the red channel was transformed in a binary image, using the “threshold” tool. To separate particles corresponding to adjacent nuclei, the “watershed” filter was applied. To exclude amastigote nuclei, only particles over 50 pixels were included in the particle count. Results of this procedure were compared with those obtained from manual count to verify the accuracy of the automatic counting. There was less than 10% variation between manual and automated count. To eliminate bias in the application of the methodology, the procedure was kept as a “macro” tool to apply the count automatically without intervention of the researcher.

Real-time PCR

Hearts from infected animals treated with ASA or 15-epi-LXA₄ were homogenized, and DNA was isolated using the Wizard Genomic DNA Purification Kit (Promega, USA), following manufacturer's instructions. DNA was quantified through 280 nm absorbance measurements using a Varioskan spectrophotometer (Thermo Scientific, USA). Parasite DNA quantification was performed using the primers TCZ-F (5′-GCTCTTGCCCACAMGGGTGC-3′) and TCZ-R (5′-CCAAGCAGCGGATAGTTCAGG-3′), designed to amplify a 195 bp Satellite-DNA sequence of T. cruzi [20]. We used the TNFα-5241 (5′-TCCCTCTCATCAGTTCTATGGCCCA-3′) and TNFα-5411 (5′-CAGCAAGCATCTATGCACTTAGACCCC-3′) primers, which amplify a 170 bp sequence of the Mus musculus TNF-α gene as loading control [20], [21]. PCR amplifications were carried out in the 7300 Real Time PCR system (Applied Biosystems, USA). All reactions were performed using 10 ng of DNA and using the SensiMix SYBR Hi-Rox Kit (Bioline, UK) at a final volume of 20 µL. For both primer pairs, the thermal cycles consisted of one 10 min step of polymerase at 95°C, followed by 40 cycles of 15 s at 95°C, 15 s at 60°C, and 30 s at 72°C. Fluorescence was measured at the end of each amplification cycle. Finally, the melting curve was performed between 60 and 95°C.

Cell culture and in vitro infection model

RAW 264.7 cells (murine macrophages, ATCC number CRL-2922) were cultured at a density of 250,000 cells/cm², in RPMI 1640 medium, supplemented with 5% fetal bovine serum, in humidified air with 5% CO₂, at 37°C. RAW cells were infected with T. cruzi trypomastigotes (Dm28c strain) at a 3∶1 ratio (trypomastigote∶RAW cell). Trypomastigotes were allowed to infect cells for 24 hours, after which supernatant was extracted. Cells were then washed twice with sterile PBS (pH 7.4) to extract extracellular trypomastigotes.

Western blot determination of COX isoforms and 5-LOX

For protein isolation, RAW cells were washed with PBS, scraped, and lysed by sonication. Hearts from control or infected mice were homogenized at 4°C in a Potter-Elvejem homogenizer. All samples were homogenized in lysis buffer at pH 8, containing Tris 0.01 µM, SDS 1%, and protease inhibitor cocktail (Complete Mini EDTA-free, Roche, USA). Total protein was quantified using the bicinchoninic acid method, using the BCA Pierce kit (Pierce Biotechnology, USA), following manufacturer instructions. For electrophoresis, extracted proteins were mixed with loading buffer (10% SDS, 50% glycerol, 0.5 M Tris, 0.1% bromphenol blue, and 1 M dithiothreitol, pH 6.8), and 40 µg (from the in vitro samples) or 30 µg (from the in vivo samples) of total protein was loaded into 8% polyacrylamide gels. After, proteins were transferred to nitrocellulose membranes, and were blocked overnight with BSA (3% in PBST 0.05%). After three washes with PBST, membranes were incubated overnight at 4°C with primary polyclonal antibodies against COX-1 (ab59964, Abcam, UK), COX-2 (ab52237, Abcam, UK) or 5-LOX (ab59341, Abcam, UK), diluted in PBST. Membranes were washed with PBST, and incubated with a HRP-conjugated anti-rabbit-IgG secondary antibody (ab6721, Abcam, UK) for 1 hour. Afterwards, membranes were washed with PBST, and developed through chemiluminescence using the Pierce ECL Western Blotting Substrate (Pierce Biotechnology, USA). After developing, membranes were incubated on stripping solution (62.5 mM Tris-HCl (pH 6.8), 2% SDS and 50 mM of mercaptoethanol) at 50°C for 30 minutes. Membranes were blocked and incubated with a primary antibody against β-actin (ab3280, Abcam, UK), overnight at 4°C. We used a polyclonal anti-mouse-IgG Ab (ab6728, Abcam, UK) HRP-conjugated as secondary antibody. Developed films were scanned, and band densitometry analysis was performed using ImageJ software.

PGE₂, LTB₄ and 15-epi-LXA₄ determination

For PGE₂ and LTB₄ in vitro determinations, 10⁶ RAW cells were cultured in 24 well plates, and exposed to 3×10⁶ trypomastigotes (Dm28c strain). After 24 hours, supernatant was removed and infected cells were washed twice with PBS (pH 7.4), and treated with ASA at different concentrations. After 48 hours of treatment, PGE₂ and LTB₄ were assayed in the supernatant, using the Prostaglandin E₂ EIA Kit and Leukotriene B₄ EIA Kit (Cayman Chemicals, USA) immunoassays, following manufacturer instructions. LTB₄ from animal plasma was measured using the Parameter(r) Kit (R&D Systems, USA), following manufacturer's instructions. 15-epi-LXA₄ was assayed in the same model in vitro and in vivo, using the LXA₄-15epi BioAssay ELISA Kit (USBiological, USA). For in vivo determinations, plasma from infected mice was collected after 10 days p.i., and assayed directly following manufacturer instructions.

Statistical analysis

For all experiments, the statistical significance was established at p<0.05. Results represent mean ± SD of triplicates. All statistical analyses were performed using GraphPad Prism (5.0) software. Normal distribution of data was assessed using D'Agostino-Pearsons analysis. One- and two-way ANOVA analyses (with Tukey post-test) or non-parametric Kruskal-Wallis analyses (with Dunns post-test) were performed when required. For survival analysis, the log rank test was performed.

Outcome of infected mice treated with ASA does not correlate with administered dose

We evaluated the effect of ASA on BALB/c mice infected with trypomastigotes of T. cruzi (Dm28c strain), at doses ranging from 5 to 100 mg/Kg. Figure 1A shows that treatment with 25 and 50 mg/Kg ASA significantly increased the survival of infected mice (p<0.01 and p<0.05, respectively). However, when the highest doses were used, (75 and 100 mg/Kg), the mortality rate was similar to that observed in the infected control. Similarly, lower ASA doses (<25 mg/Kg) did not affect mortality (data not shown). Thus, ASA impact upon survival was only observed at intermediate doses. Parasite DNA content was quantified by qPCR (Figure 1B), no significant differences were observed between the control and the ASA-treated groups.

ASA treatment was able to decrease parasitemia peaks at 25 and 50 mg/Kg (Figure 1C). In mice treated with ASA 25 mg/Kg the parasitemia was decreased significantly on days 8 and 14 p.i. (p<0.001 and p<0.01 respectively), while in the 50 mg/Kg group, parasitemia decreased significantly only on day 14 (p<0.01) (Figure 1C). Similarly, at 75 mg/Kg, the parasitemia peak on day 8 was decreased (p<0.05), although at a lower magnitude than that observed at 25 mg/Kg. No differences were observed at 100 mg/Kg when comparing to control. Finally, 5 and 10 mg/Kg of ASA did not show any differences when compared to control group (data not shown).

When we evaluated the cardiac structure of mice (Fig. 1D), we found that infected mice exhibited severe inflammatory infiltration, associated edema, and amastigote nests. At 25 mg/Kg ASA, there was less inflammation and edema, and heart tissue histology seemed normal. These protective effects disappeared when the ASA dose was increased, as seen in 100 mg/Kg ASA-treated mice, which showed more edema and inflammation than controls. In addition, amastigote nests were more evident in 75 and 100 mg/Kg treated mice. A quantification of inflammatory infiltrate (Figure 1E) showed that ASA 25, 50 and 75 mg/Kg decreased significantly the number of infiltrate cells. Although ASA 100 mg/Kg seems to have less infiltrate than control, this difference did not have statistical significance with the infected mice without treatment.

Infected cells treated with ASA are able to synthetize 15-epi-LXA4

We have previously reported that the effect of ASA upon in vitro T. cruzi infection is not reversed by exogenous PGE₂ administration. Thus, we hypothesized that COX inhibitors, ASA in particular, have alternative mechanisms involved in this phenomenon. One possible mechanism could be ASA induction of a shift in the arachidonic acid metabolic pathway towards the production of 5-lipoxygenase derivatives [22]. Thus, we explored the variation in the metabolic pathway of AA, induced by ASA in an in vitro infection model. We assayed three concentrations of ASA, using 50% of our previously reported effect as a reference [23]. T. cruzi infection increased the PGE₂ and LTB₄ production in RAW 264.7 cells (Figure 2A and 2B). Correlated with the increase of PGE₂ production, the COX-2 levels in RAW cells infected with T. cruzi also increased significantly (Figure 2D and 2E). As expected, ASA inhibited PGE₂ synthesis at all tested concentrations (Figure 2A). In contrast, LTB₄ production in ASA treated cells did not increase. Unexpectedly, we observed a significant low level of LTB₄ in the 0.5 mM ASA treated cells, when compared with infected cells (Figure 2B). COX-2 acetylation by ASA modifies its activity, promoting the synthesis of 15-epi-LXA₄, a lipid involved in the resolution of inflammation [24], [25]. Accordingly, we assessed the production of 15-epi-LXA₄ in T. cruzi infected RAW cells treated with ASA. 15-epi-LXA₄ production was significantly increased by ASA in these cells (Figure 2C). Interestingly, the production of 15-epi-LXA₄ was inversely correlated to the concentration of ASA, reaching a level similar to the control at 0.5 mM ASA.

Infected Balb/c mice treated with ASA have circulating 15-epi-LXA₄

Based on the previous in vitro results, we evaluated the generation of 15-epi-LXA₄ and LTB₄ in T. cruzi-infected mice. In agreement with in vitro data, low doses of ASA increased the circulating levels of 15-epi-LXA₄, which decreased in a dose-depended manner (Figure 3A). Unpredictably, infection alone produced an increase in 15-epi-LXA₄ in mice. This discrepancy in the 15-epi-LXA₄ production observed between infected cells and mice may be due to the lack of cooperative systems in single mammalian cell model. In fact, coincubation of macrophages with polymorphonuclear neutrophils increases the production of 15-epi-LXA₄ [26]. Therefore, it is expected that 15-epi-LXA₄ production in mice to be more efficient than in RAW cells monoculture. ASA did not modify LTB₄ production in infected mice (Figure 3B). Furthermore, T. cruzi infection did not change the basal levels of LTB₄ (Figure 3B), contrary to what we observed in infected RAW cells (Figure 2B).

In the heart tissue of T. cruzi infected mice, COX-2 levels presented a two-fold increase compared with control (Figure 3C and 3D). These results are in agreement with previously reported data from immunohistochemical determinations [7]. Interestingly, the COX-2 levels appear to decrease with increasing ASA doses (Figure 3C and 3D), indeed the COX-2 levels in the 75 and 100 mg/Kg treated groups were statistically different with infected control. This data could explain why 15-epi-LXA₄ levels are decreased in infected mice treated with ASA 75 or 100 mg/Kg. In addition, COX-1 and 5-LOX levels in cardiac tissue did not change with infection or treatment (Figures 3C, 3E and 3F). Thus, the changes in 15-epi-LXA₄ production could be related with ASA effects on the COX-2 enzyme.

15-epi-LXA₄ decreases parasite burden and cardiac inflammation in mice infected with T. cruzi

Since ASA treatment can modify synthesis of 15-epi-LXA₄ both in vitro and in vivo, we evaluated the effect of exogenous administration of 15-epi-LXA₄ in the T. cruzi infection outcome in BALB/c mice. Infected mice were treated with 5 or 25 µg/Kg 15-epi-LXA₄ according to previously reported schemes for other murine models [27]. 15-epi-LXA₄ at 5 µg/Kg had no effect on the survival rate and cardiac parasite burden when quantified by relative DNA load (Figures 4A and 4B). In contrast, 15-epi-LXA₄ at 25 µg/Kg significantly increased survival and decreased cardiac parasite load (Figure 4A and 4B). On the other hand, treatment with 5 and 25 µg/Kg 15-epi-LXA₄ significantly decreased the parasitemia peaks observed on days 12 and 14 p.i. (Figure 4C). Cardiac histopathological analysis showed that 25 µg/Kg 15-epi-LXA₄ decreased number of amastigote nests and the inflammatory infiltration (Figures 4D and 4E). Nevertheless, at 5 µg/Kg, focal inflammatory infiltration and amastigote nests persisted, as compared with untreated infected controls.

15-epi-lipoxin A₄ restores the protective effect on T. cruzi infected-mice treated with high doses of ASA

Considering the above results, do high ASA doses lose its general protective effect on T. cruzi infected mice due to absence of 15-epi-LXA₄ production? To answer this question, we administered 25 µg/Kg of 15-epi-LXA₄ to T. cruzi-infected mice, treated with either 75 or 100 mg/Kg ASA. Although there were no significant effects of 15-epi-LXA₄ on survival or cardiac parasite burden (Figures 5A and 5B), parasitemia, inflammatory infiltrate, and amastigote nests decreased when 15-epi-LXA₄ was administered to the 75 mg/Kg ASA treated mice (Figures 5C, 5D and 5E). 15-epi-LXA₄ did not produce effect in the 100 mg/Kg ASA treated-mice (Figure 5).