
 trypanosoma cruzi is an intracellular protozoan parasite causing chagas disease , which affects millions of people in latin america . during the acute inflammatory phase of the t. cruzi infection , high - level expression of inducible nitric oxide synthase
( inos ) , proinflammatory cytokines , and arachidonic acid- ( aa- ) derived lipids such as leukotrienes , lipoxins ( lxs ) , h ( p ) etes , prostaglandins , and thromboxane is prevalent [ 3 , 4 ] . in the early t. cruzi infection , nitric oxide ( no ) and
arachidonic acid metabolites could be attributed to resistance , but later on to tissue damage .
prostaglandins ( pgs ) are oxygenated lipid mediators formed from the 6 essential fatty acid , arachidonic acid ( aa ) .
the committed step in pg biosynthesis is the conversion of aa to pg h2 ( pgh2 ) , catalyzed by either pg endoperoxide h synthase-1 or -2 , commonly known as cyclooxygenase-1 ( cox-1 ) and cyclooxygenase-2 ( cox-2 ) , respectively [ 5 , 6 ] .
both cox-1 and cox-2 are nonselectively inhibited by nonsteroidal anti - inflammatory drugs ( nsaids ) such as aspirin and ibuprofen , whereas cox-2 activity is selectively blocked by cox-2 inhibitors called coxibs ( e.g. , celecoxib ) [ 7 , 8 ] .
the relevance of these enzymes and the bioactive lipids that they produce are not well understood in parasitic disease , although the role of eicosanoids in the pathogenesis of chagas disease is becoming more defined .
pharmacological antagonists of cox-1 ( aspirin , asa ) , cox-2 ( celecoxib ) , or both ( indomethacin ) have been found to increase mortality and parasitemia ( parasite load in peripheral blood and cardiac tissue ) regardless of which mouse or t. cruzi strains were used [ 913 ] .
conversely , others have found that inhibition of pg synthesis / release reduces parasitemia and extends survival of mice infected with t. cruzi [ 1417 ] .
this was often associated with a decrease in the levels of circulating inflammatory cytokines ( such as tnf- , ifn- , and il-10 ) .
more recently , treatment with asa during chronic infection was found to be beneficial with no increase in mortality and substantial improvement in cardiac function .
additionally , the protective effect of asa could be mediated by the synthesis of 15-epi - lipoxin a4 ( 15-epi - lxa4 ) .
given the increasing interest in the role of eicosanoids in t. cruzi infection , we decided to investigate the effect of prostaglandin synthesis inhibition with asa on inflammatory response and macrophage invasion by t. cruzi .
six- to eight - week - old balb / c female and male mice were supplied by the multi - institutional center for biological investigation , state university of campinas , brazil .
mice were maintained under standard conditions in the animal house of the department of pathological sciences , center for biological sciences , state university of londrina .
commercial rodent diet ( nuvilab - cr1 , quimtia - nuvital , colombo , brazil ) and sterilized water were available ad libitum .
all animal procedures were performed in accordance with the guidelines of the brazilian code for the use of laboratory animals .
the protocols were approved by the internal scientific commission and the ethics in animal experimentation committee of londrina state university ( approval number : ceea 5492.2012.22 ) . 

t. cruzi y   was maintained by weekly intraperitoneal inoculation of swiss mice with 2  10 trypomastigotes . to conduct our experiments , blood from previously infected mice was obtained by cardiac puncture without anticoagulant .
the blood was centrifuged at 1,500  g for 1  min and allowed to stand at 37c for 60  min . the supernatant serum containing most of the t. cruzi was centrifuged at 1,200  g for 15  min .
the sediment was resuspended in 1  ml of rpmi 1640 medium ( gibco , gran island , ny ) containing 10% inactivated fetal bovine serum ( fbs ) , 100 units of penicillin , and 100  g streptomycin ( gibco , gran island , ny ) .
trypomastigotes were derived from the supernatant of t. cruzi - infected llc - mk2 culture cells ( atcc ccl-7 ; american type culture collection , rockville , md ) grown in rpmi 1640 medium containing 10% inactivated fetal bovine serum ( fbs ) , 40  g  ml gentamicin , 100 units of penicillin , and 100  g streptomycin ( gibco , gran island , y ) .
free parasites were removed after 24  h and cultures were maintained in 10% fbs - rpmi 1640 .
mice were inoculated intraperitoneally with 2  ml of 5% thioglycollate and , 4 days later , the elicited cells from the peritoneal exudates were harvested in cold pbs .
mouse peritoneum was washed with 5  ml ice - cold , serum - free rpmi .
peritoneal cells from 36 mice were pooled and left to adhere in complete medium ( rpmi , 2  mm glutamine , 1  mm sodium pyruvate , 40  g ml gentamicin , and 10  mm hepes ) for 24  h in 24-well plates at 2  10 cells / well .
the macrophages were plated onto 13  mm round glass coverslips and washed in warm phosphate - buffered saline ( pbs ) before the interaction assays .
one set of plates was used to quantify il-1 and the other set for no detection . before the experiments , peritoneal macrophages previously washed were incubated for 30  min at 37c in a 5% co2 atmosphere in the presence of different concentrations of asa ( 2.5  mm , 1.25  mm , and 0.625  mm ) to test its effect on internalization of the parasite into the host cell .
after incubation , the medium containing asa was removed , and macrophages were allowed to interact with trypomastigote forms added in a ratio of 5 parasites per cell .
the interaction was allowed to proceed for 2  h , at 37c in a 5% co2 atmosphere .
the cells were then washed three times , fixed with bouin 's fixative , stained with giemsa ( merck ) stain , and observed with a light microscope at 1000x magnification .
other treatments included incubation with aminoguanidine ( 1  mm ) or l - name ( 1.0  mm ) for 60  min at 37c with or without asa .
the internalization index was calculated by multiplying the percentage of infected cells by the mean number of parasites per infected cell .
experiments were performed in triplicate , and six independent experiments were completed . all experiments included untreated , infected peritoneal macrophages as controls .
the quantification was carried out using light microscopy where a total of 500 cells were randomly counted .
the viability of the cells obtained from the cultures before and after incubation experiments was determined using mtt ( sigma - aldrich ) assay , showing the mitochondrial activity of living cells .
the culture medium was aspirated , and mtt ( 0.5  mg  ml ) was added to the cells prior to incubation at 37c for 4  h. the supernatant was aspirated and dimethyl sulfoxide ( sigma - aldrich ) was added to the wells .
insoluble crystals were dissolved by mixing and the plates were read using a biorad multiplate reader ( hercules , ca ) , at a test wavelength of 570  nm and a reference wavelength of 630  nm .
production of nitric oxide ( no ) was determined by measuring the level of accumulated nitrite , a metabolite of no in the culture supernatant using griess reagent ( sigma - aldrich ) .
after 24  h of treatment with asa ( 0.625  mm ) , the culture supernatants were collected and mixed with an equal volume of griess reagent in 96-well culture plates and incubated at room temperature for 10  min .
the absorbance was measured at 540  nm and nitrite concentrations were calculated by reference to a standard curve generated by known concentrations of sodium nitrite .
immunocytochemistry for inos was performed on coverslip - adherent cells using the labeled streptavidin biotin method with a lsab kit ( dako japan , kyoto , japan ) without microwave accentuation .
the coverslips were incubated with 10% triton x-100 solution for 1  h , washed 3 times in pbs , and treated for 40  min at room temperature with 10% bsa .
the coverslips were then incubated overnight at 4c with the primary antibody ( anti - inos rabbit monoclonal antibody diluted 1  :  200 , bd biosciences , catalog number 610599 ) , followed by secondary antibody treatment for 2  h at room temperature .
horseradish peroxidase activity was visualized by treatment with h2o2 and 3,3-diaminobenzidine ( dab ) for 5  min . at the last step
, the sections were weakly counterstained with harry 's hematoxylin ( merck ) . for each case , negative controls were prepared by omitting the primary antibody .
intensity and localization of immune reaction against primary antibody used were examined on all coverslips using a photomicroscope ( olympus bx41 , olympus optical co. , ltd . ,
after conversion of the images into grey scale ( adobe photoshop ) inos - positive pixels and total pixels became thresholds and were processed by image j program .
positive immunostained area was calculated as the proportion of positive pixels to total pixels ( % ) .
( 0.625  mm ) or untreated , either infected or not infected with t. cruzi , were incubated for 24  h. levels of il-1 in 100  l medium were measured by commercial elisa kits ( ready - set - go !
the statistical analysis was conducted using one way anova with bonferroni 's multiple comparison test .
statistical analysis was performed using the graphpad prism 5.0 computer software application ( graphpad software , san diego , ca , usa ) .
to determine whether cox - derived mediators are involved in t. cruzi entry into host cells , cells were treated with increasing amounts of asa for 30  min and after treatment , the medium containing asa was removed before macrophage invasion assay in order to guarantee that asa affected only the host cell and not the parasites .
after 2  h of incubation with parasites , which provides sufficient time for them to enter into macrophages , the free parasites were removed .
aspirin irreversibly inhibits cox-1 by acetylation of a single serine residue on the enzyme   and this inactivation persists , that is , 24 hours . in some cases , the medium with increasing asa concentrations was added every 24 hours until the end of the t. cruzi infection period ( 7 days , figure 4(d ) ) .   figure 1(a ) shows that asa markedly inhibited the internalization of trypomastigote by macrophages at all concentrations tested ( p < 0.0001 ) . thus , pge2 synthesis inhibition using asa improves macrophage response against t. cruzi infection .
the cytotoxicity of asa in macrophages was evaluated by mtt assay ( figure 1(a ) , insert ) .
asa did not induce cell death , as the concentrations of asa used in all experiments reported were too low to cause cytotoxicity .
light microscopy observations confirm t. cruzi inhibition invasion of asa treated cells ( figure 1(c ) ) .
we examined the combined effect of celecoxib ( cox-2 selective inhibitor ) and asa on the entry of t. cruzi into phagocytic cells .
peritoneal macrophages were incubated separately , with or without drugs ( asa and celecoxib ) either alone or in combination .  
figure 1(b ) shows that the drugs in combination significantly ( p < 0.01 ) restored the invasive capacity of trypomastigotes .
none of the drugs ( asa , celecoxib , or their combination ) showed cytotoxicity against uninfected peritoneal macrophages ( data not shown ) .
these results indicate that the enzyme activities of cox-1 and cox-2 favor invasion , but simultaneous inhibition of both activities also favors parasite invasion , probably by allowing a new panorama of eicosanoids production .
four days postinfection , macrophages began releasing trypomastigotes into the supernatant ( figure 4(d ) ) .
trypomastigotes release to culture supernatants from t. cruzi - infected macrophages was diminished by asa ( 0.625  mm ) .
this result is in agreement with previous reports , showing that aspirin and other cox inhibitors decrease t. cruzi infection in vitro [ 1416 , 23 ] .
lipoxins ( lxs ) are endogenous lipid mediators with potent anti - inflammatory and proresolving actions [ 24 , 25 ] .
native lxs and their stable analogues exert their biological effects by binding to a g - protein - coupled receptor , denoted as alx [ 26 , 27 ] . the effect of asa has been associated , in part , to a switch to the aa pathway linked to the acetylation of the cox-2 isoenzyme .
this reaction enables cox-2 to synthesize lxs as 15-epi - lxs ( 15-epi - lxa4 ) . to evaluate the effect of asa - triggered lxs on the entry of t. cruzi into phagocytic cells , peritoneal macrophages
were incubated separately either with or without drugs ( asa or boc-2 , a specific antagonist of the 15-epi - lxs ) either alone or in combination .
the inhibitory effect of asa on the entry of t. cruzi into macrophages was prevented by boc-2 demonstrating that the effect of asa on the entry of t. cruzi into macrophages could be mediated by the synthesis of 15-epi - lxa4 .
furthermore , boc-2 did not have any effect on the entry of t. cruzi into macrophages when used alone ( figure 2(a ) ) . 

figure 2(b ) ( insert ) shows that treatment of macrophages with high concentrations of pge2 causes inhibition of t. cruzi entry into macrophages . when pge2 ( 3.52  ng  ml ) was added alone or in combination with asa , the effect of asa was inhibited , indicating that pge2 is involved in the internalization of t. cruzi trypomastigotes into peritoneal macrophages .
the internalization of t. cruzi into macrophages stimulated the release of il-1 while asa increased il-1 production by infected macrophages ( figure 3(a ) ) .
the effect of asa on no production was evaluated by detection of nitrite in t. cruzi - infected macrophage supernatants using griess reaction ( figure 3(b ) ) . no production in macrophages
was stimulated by t. cruzi and was also increased by prior treatment of macrophages with asa . in addition , we observed that asa treatment stimulated inos expression in t. cruzi - infected macrophages ( figure 4(a ) ) . to confirm that the effect of asa on macrophage activity depends on no levels , we assessed the entry of trypomastigotes into macrophages incubated with aminoguanidine ( ag , inos inhibitor ) or with l - name ( c - nos inhibitor ) .
we found that both inhibitors reversed the effects of asa ( figures 4(b ) and 4(c ) ) .
previous studies have shown that the release of eicosanoids during infection with t. cruzi regulates host responses and controls disease progression [ 3 , 10 , 11 , 13 , 14 , 2831 ] .
pgs , together with no and tnf- , participate in a complex circuit that controls lymphoproliferative and cytokine responses in t. cruzi infection .
however , the involvement of cox - mediated pg production in the entry of t. cruzi into macrophages is largely unexplored .
the data shown herein demonstrates that the treatment of macrophages with asa significantly inhibits internalization of t. cruzi trypomastigotes and strongly supports the idea that the cox pathway plays a fundamental role in the process of parasite invasion .
in fact , pge2 production increases significantly in t. cruzi - infected macrophages as compared to uninfected macrophages   and synergistically enhances the activity of nifurtimox and benznidazole on infected raw 264.7 cells .
this is demonstrated by the observation that celecoxib reversed the aspirin - induced inhibition of entry of t. cruzi into macrophages .
the problem of nsaids coadministration is actively discussed in literature in the context of uncertainty of the resulting therapeutic and side effects arising from such combinations .
the mechanism of such a suppression of aspirin inhibitory effect on cox-1 by other nsaids has been difficult to satisfactorily explain .
this could be due to celecoxib binding strongly to a monomer of cox-1 without affecting aa oxygenation .
in fact , a related study has shown that celecoxib prevented asa inhibition in a dog model of thrombosis . however , the application of our approach to investigate the combined effect of aspirin and celecoxib on cox-1 during coadministration confirmed the ability of celecoxib to suppress the aspirin - mediated inhibition of cox-1 in vitro conditions .
in addition , the effects of asa on t. cruzi infection have been associated in part , to a switch to the aa pathway linked to the acetylation of the cox-2 isoenzyme .
such modification promotes the synthesis of the 15-r - hete intermediate , which can be transformed by 5-lipooxygenase to 15-epi - lxa4 , a lipid involved in the resolution of inflammation .
accordingly , we assessed the effect of boc-2 ( a specific antagonist of the 15-epi - lxs ) on low doses of asa , indicating that 15-epi - lxa4 is probably involved in the inhibitory effect exerted by asa on the internalization of t. cruzi by macrophages .
interestingly , when macrophages were treated with pge2 concentration as high as 3.52  ng  ml , we observed a reduction in the entry of parasites , but when we used low concentration of pge2 ( 0.35  ng  ml ) , the entry of t. cruzi was similar to that observed in untreated macrophages .
in addition , we found reversal of asa effect , even when pge2 concentration as low as 3.52  ng  ml was used , indicating that pge2 has an important role in asa effect .
inhibition of cox activity may increase no levels , thus restoring the antiparasitic activity of macrophages .
our results are in agreement with this hypothesis . to confirm that the effect of asa on macrophage activity depends upon restoring no levels , we assessed the invasive capacity of t. cruzi when cells were incubated with aminoguanidine ( ag ) , an inos inhibitor .
in addition , we showed that inos expression in macrophages was increased with asa treatment , suggesting that inos - dependent no production is responsible for asa effects .
we did find reversal of asa effect with l - name ( 1.0  mm ) , indicating the role of cnos in asa activity .
this can be explained as high concentrations of l - name may interfere in the selectivity for cnos and can inhibit other isoforms of nos , such as nnos and inos .
additionally , polyamines seem to be crucial for trypomastigote internalization process in , at least , some cellular types and infection progression . in t. cruzi - infected macrophages
, cox is related to the increase of ornithine decarboxylase ( odc ) activity , which might increase the polyamine content in macrophages . since t.
cruzi uses these polyamines to synthesize trypanothione ( an enzyme that participates in the hydroperoxide detoxification of t. cruzi ) , the inhibition of cox by asa probably resulted in a reduction in polyamine levels caused by inhibition of odc , indirectly contributing to decrease trypanothione synthesis in t. cruzi , as suggested by lpez - muoz and collaborators .
finally , in t. cruzi - infected macrophages , cox inhibition by asa was related to the increase of il-1 , which also might explain the increase of antiparasitic activity of macrophages treated with asa .
in fact , il-1 is critical for the restriction of leishmania amazonensis infection   and it recently was demonstrated that macrophages treated with il-1 released fewer trypomastigotes than untreated macrophages and il-1 triggered no release by t. cruzi - infected macrophages in a dose dependent manner .
in conclusion , this is the first report , to our knowledge , showing the in vitro effect of aspirin on t. cruzi entry into peritoneal macrophages and the influence of cox pathway on innate inflammatory response to t. cruzi infection , adding a new perspective to immune interventions against chagas disease .
 the intracellular protozoan parasite trypanosoma cruzi causes chagas disease , a serious disorder that affects millions of people in latin america . 
 cell invasion by t. cruzi and its intracellular replication are essential to the parasite 's life cycle and for the development of chagas disease . here 
 , we present evidence suggesting the involvement of the host 's cyclooxygenase ( cox ) enzyme during t. cruzi invasion . pharmacological antagonist for cox-1 , aspirin ( asa ) , 
 caused marked inhibition of t. cruzi infection when peritoneal macrophages were pretreated with asa for 30  min at 37c before inoculation . 
 this inhibition was associated with increased production of il-1 and nitric oxide ( no ) by macrophages . 
 the treatment of macrophages with either nos inhibitors or prostaglandin e2 ( pge2 ) restored the invasive action of t. cruzi in macrophages previously treated with asa . 
 lipoxin alx - receptor antagonist boc2 reversed the inhibitory effect of asa on trypomastigote invasion . 
 our results indicate that pge2 , no , and lipoxins are involved in the regulation of anti - t . 
 cruzi activity by macrophages , providing a better understanding of the role of prostaglandins in innate inflammatory response to t. cruzi infection as well as adding a new perspective to specific immune interventions . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Conclusion