malaria is a serious disease caused by the plasmodium parasite and transmitted through the bite of the anopheles mosquito .
it is estimated that there were about 214 million cases of malaria in 2015 , resulting in approximately 440,000 deaths , most of which originating from sub - saharan africa and in children under 5 years of age .
malaria is characterized by signs and symptoms such as severe anemia , fever , vomiting , and fatigue . during the symptomatic phase of malaria several clinical complications
these complications are anemia , cerebral malaria , placental malaria , and acute lung injury / acute respiratory distress syndrome ( ali / ards ) . the ali / ards has been diagnosed in patients suffering from malaria caused by all the species that cause disease in humans , including p. knowlesi ; however it is more common in p. falciparum and p. vivax malaria .
ali / ards is characterized by high morbidity and , although more common in adults , also affects children and pregnant woman .
the most common manifestations of this syndrome are noncardiogenic pulmonary edema , increased phagocytic activity , dyspnea , reduction in the capacity of gas exchange , and increased levels of inflammatory mediators .
ali / ards is most commonly caused by bacteria , sepsis , viral pneumonia , gastric aspiration , severe trauma , adverse drug reactions , and fungal or parasitic infections of the lung .
the mechanisms that are critical in the initial and later stages of ali / ards are not well defined .
however , it is known that the presence of intravascular fluid in the lungs , due to increased permeability of the alveolar capillary membrane , is the key pathophysiological mechanism of ali / ards .
multiple factors are possibly involved in the increased vascular permeability , such as endothelium injury , increased levels of proinflammatory cytokines such as tnf- ( tumor necrosis factor alpha ) , interleukin 1 ( il-1 ) , or il-6 and il-8 , and endovascular occlusion associated with the accumulation of erythrocytes with reduced deformability , leukocytes , and platelets [ 6 , 9 ] .
different mouse models have been developed for the study of ma - ali / ards , showing similar aspects to human ali / ards [ 1013 ] .
dba/2 strain mice develop ali / ards when infected with the parasite plasmodium berghei anka ( pba ) . in this model , an average of 50% of the mice that die between days 7 and 12 after infection have dyspnea , hypoxemia , and reduced respiratory rate .
postmortem studies revealed that these mice had pleural effusion , containing cells such as neutrophils , lymphocytes , monocytes , and macrophages [ 10 , 14 ] .
furthermore , the vascular endothelial growth factor ( vegf ) has been identified as critical in increased pulmonary vascular permeability , a hallmark of ali / ards [ 10 , 15 ] .
the protective role of ho-1 enzyme and carbon monoxide ( co ) in experimental severe malaria episodes has been demonstrated in animal models [ 10 , 15 , 16 ] .
additionally , ho-1 was found to be increased in peripheral blood leukocytes , plasma , tissue macrophages , and monocytes of humans with severe malaria [ 17 , 18 ] .
ho-1 is an enzyme encoded by hmox-1 gene and is considered  protective  due to its anti - inflammatory , antiapoptotic , and antiproliferative actions in different cell types , including endothelial cells .
this enzyme participates in the free heme degradation , generating equimolar amounts of co , iron , and biliverdin and plays a protective role in modulating tissue response to injury in various organs , including the lung [ 2022 ] .
the malaria infection leads to the release of reactive oxygen species and free heme , harmful to the endothelial cells of the host .
ho-1 catabolizes free heme into iron , biliverdin , and co , which are less toxic to the cells .
therefore , some studies have shown that inducers of ho-1 such as hemin and cobalt protoporphyrin ix ( coppix ) protected mice infected with malaria , or suffering from other diseases such as polymicrobial sepsis , from developing ali / ards [ 16 , 24 , 25 ] .
additionally , it was observed in previous publications that ho-1 inducers are protective against experimental cerebral malaria   and that treatment of dba/2 mice with a co - releasing molecule is protective against ma - ali / ards .
meanwhile , in this study , it was observed , for the first time that ho-1 expression is increased in pba infected dba/2 mice lungs which develop ali / ards and that the induction of ho-1 protects these mice from developing ali / ards .
six- to ten - week - old male dba/2 mice were bred under pathogen - free conditions in isogenic mouse facilities , at the biomedical sciences institute of the university of so paulo . in all experiments ,
mice had ad libitum access to water and food ( nuvilab cr-1 , quintia s / a , so paulo , brazil ) .
mice were intraperitoneally infected with 1  10 plasmodium berghei anka ( clone 1.49l ) infected red blood cells ( irbcs ) , as previously described .
all experiments were performed in accordance with the ethical guidelines for experiments with mice , and the protocols were approved by the animal health committee of the biomedical sciences institute of the university of so paulo ( ceua number 146 , page 136 , book 2 ) .
the guidelines for animal use and care were based on the standards established by the brazilian college of animal experimentation ( cobea ) .
the mice were euthanized with an anesthetic overdose of ketamine ( 150  mg / kg ) ( vetbrands , brazil ) and xylazine ( 15  mg / kg ) ( syntec , brazil ) , and consciousness was checked by testing the pedal reflex , heartbeats , and breathing movements .
necropsy was performed in mice dying naturally from malaria or mice euthanized on the 20th day after infection ( dai ) to complete the experiment and to avoid animal suffering .
the lungs were collected and fixed in buffered 10% formalin for 24 hours and 70% alcohol for 2448 hours and then embedded in paraffin , sectioned at 5  m onto slides , and stained with hematoxylin - eosin ( he ) according to standard protocol .
histopathological analyses were performed under a axio imager m2 ( zeiss ) microscope using the axio cam hrc ( zeiss ) and the software axio vision , version 4.9.1.0 . in order to determine the alveolar area percentage ,
20 pictures per lung he section were taken and the software gimp , version 2.8.16 ( https://www.gimp.org/ ) was used to determine the alveolar area in pixels of each picture .
the percentage of alveolar area relatively to total area of the pictures were determined in ali / ards versus hp and in hemin treated versus nontreated ( saline ) mice .
respiratory patterns ( respiratory frequency ( rf ) and enhanced pause ( penh ) ) were monitored on the 7th dai by placing the mice in the plethysmography chamber ( wbp , buxco electronics , wilmington , north carolina , usa ) for 10 minutes ( basal level ) as described before .
the data were collected using biosystems xa software and included the rf ( breaths / minute ) and variables to calculate the penh , a theoretical variable that correlates with both pulmonary resistance and intrapleural pressure .
the penh is calculated by the following equation : ( 1)penh = peak  expiratory  heightpeak  inspiratory  heightexpiratory  timerelaxation  time1 .
identification of ali / ards in mice before death was done as described before . in short , we used two groups of pba infected mice ( 1012 mice per group ) : the survival group and the euthanized group in which the mice were euthanized on the 7th dai . in the survival group , in any mouse that died between the 7th and 12th dai showing pleural effusion or red and congested lungs at necropsy
, the cause of death was attributed to ali / ards . in contrast , in mice without pleural effusion that died after 13th dai with pale lungs and high levels of parasitemia , the cause of death was attributed to hyperparasitemia ( hp ) and , consequently , anemia . in the euthanized group , mice were classified as having been likely to die of ali / ards or hp , by comparing their respiratory patterns and parasitemia levels with the survival group , in which the causa mortis was known , as previously published . to perform the immunohistochemistry of ho-1 , slides containing the tissue sections were placed in an incubator at 60c for 20 minutes .
then they were incubated in xylene twice for 15  min at 60c and afterwards passed in absolute ethanol , in 95% alcohol , in 70% alcohol , in distilled water , and finally in 1x pbs ph 7.2 to 7.4 .
for the antigen retrieval , the slides were incubated in sodium citrate buffer ph 6 for 45 minutes at 95c .
endogenous peroxidase was blocked with 3% hydrogen peroxide for 15 minutes , twice , at room temperature and protected from light .
the tissue was probed with rabbit polyclonal to ho-1 ( 1  :  1000 ) antibody ( abcam , ab13243 ) overnight at 4c and then the kit reveal mouse / rabbit ( spring - code spd-015 ) was used in accordance with the manufacturer 's instructions .
the quantification of ho-1 in lung tissue was done by calculating the marked area in the entire tissue section .
the calculation was done in imagej ( version 1.50b ) software ( https://imagej.nih.gov/ij/ ) , using the plugin ihc toolbox ( http://rsb.info.nih.gov/ij/plugins/ihc-toolbox/index.html ) .
fresh frozen mice lung tissues were sonicated and homogenized in ice using the radio - immunoprecipitation assay ( ripa ) buffer composed of 50  mm tris - hcl ph 8.0 , 150  mm nacl , 0.5% sodium deoxycholate , 0.1% sodium dodecyl sulphate ( sds ) , 1  mm sodium orthovanadate , 1  mm naf , and a protease inhibitor tablet .
the samples were analyzed for protein content using a bradford protein assay ( biorad ) according to manufacturer 's instructions .
each sample was quantified , and then 9  g of protein was loaded onto a 12% tris - glycine sds - polyacrylamide gel , according to the manufacturer 's protocol ( biorad ) .
the gel was transferred to a pvdf membrane by electrophoresis at 30  v for 16  h. the membrane was blocked in pbs with 0.1% tween 20 ( pbs - t ) and 10% nonfat milk at room temperature for 2  h. all antibodies were diluted in the same buffer ( pbs - t ) with 1% nonfat milk .
the membrane was then probed with rabbit polyclonal ho-1 antibody ( abcam , ab13243 , 1  :  20,000 ) and then incubated for 1  h at room temperature .
after incubation , the membrane was washed five times with pbs - t and incubated with horseradish peroxidase  conjugated goat anti - rabbit igg ( millipore , ap307p , 1  :  20,000 ) for 1  h at room temperature . after washing five times with pbs - t , an ecl system ( clarity western ecl blotting substrate , biorad ) was used for detection of the proteins in a chemidoc xrs+ system ( biorad ) .
the ho-1 expression was calculated by densitometry , in the software imagej ( version 1.50b ) of the ho-1 bands in the immunoblot relative to the housekeeping protein -actin ( rabbit monoclonal to -actin , novus biologicals , nb600 - 501 , 1  :  500,000 ) . on the 7th dai
elisas kits were used to quantify ho-1 levels in serum and macerated lung tissue ( enzo lifesciences , adi-960 - 071 ) and bilirubin levels ( an indirect measure of ho-1 activity ) ( biomatik , eku08395 ) according to the manufacturer 's instructions .
the ho-1 level of the ali / ards and hp mice was expressed as fold increase in relation to noninfected mice .
extraction of total rna from lungs of mice was performed using rneasy mini kit ( qiagen ) , according to the manufacturer 's instructions .
one microgram of total rna was reverse - transcribed to single - strand cdna using the first - strand cdna synthesis kit ( roche ) amv reverse transcriptase protocol ( roche applied science ) .
ho-1 transcripts in the cdna obtained from the reverse transcriptase reaction were quantified by real - time quantitative fluorogenic pcr performed in the 7500 fast instrument ( applied biosystems ) .
sybr green pcr master mix ( applied biosystems ) was used to quantify gene expression according to the manufacturer 's instructions .
the gene expression was normalized by the housekeeping gene hypoxanthine guanine phosphoribosyltransferase ( hprt ) and using the relative quantification method 2 as described before .
the primers used were as follows : ho-1 : 5-tctcagggggtcaggtc-3 ( forward ) and 5-ggagcggtgtctgggatg-3 ( reverse ) ; ifn- : 5-cacactgcatcttggctttg-3 ( forward ) and 5-tctggctctgcaggattttc-3 ; hprt : 5-tgctcgagatgtgatgaagg-3 ( forward ) and 5-tcccctgttgactggtcatt-3 ( reverse ) .
hemin ( sigma - aldrich ) was diluted in 0.2  m naoh to a final concentration of 5  mm and ph 7.4 .
hemin was administered in two doses : the first was two days before the infection and another at 4th dai in single doses of 17  mg / kg or with saline solution ( control group ) , intraperitoneally . to measure parasitemias and survival rate of hemin treated and
ho-1 induction by hemin was also used to measure lung vascular permeability and vegf and cytokine serum levels . to investigate lung vascular permeability on the 7th dai ,
the hemin treated or saline treated infected mice and noninfected mice were injected intravenously with 0.2  ml of 1% evans blue ( sigma ) .
the mice were euthanized 45 minutes later , and the lungs were weighed and placed in 2  ml of formamide ( merck ) for 48 hours at 37c .
the amount of evans blue staining per gram of lung tissue was calculated from a standard curve .
the lung permeability was expressed as fold increase in relation to the noninfected mice . on the 7th dai ,
hemin treated or saline treated infected mice and noninfected mice were anesthetized , and their serum was collected by cardiac puncture . an elisa kit ( r&d systems ) was used to quantify vegf levels in the serum according to the manufacturer 's instructions .
the vegf level was expressed as fold increase in relation to that of the noninfected mice .
the cytokine levels were measured in serum from the hemin treated and saline treated dba/2 mice .
mice inflammation cba cytokine kit ( cytometric bead array , becton - dickinson ) was used to measure interferon , ifn- , tumor necrosis factor , tnf- , and il- and il-10 levels .
this kit was used for dosing cytokines from lung tissue lysates using flow cytometer ( bd facs calibur system , becton - dickinson ) and using the software cell quest pro , version 5.2 .
the individual standard curve range for a given cytokine was determined according to manufacturer instructions .
the primary microvascular lung endothelial cells ( pmlec ) were obtained from dba/2 mice , according to what was described before .
after euthanasia , the animal 's body was disinfected with iodine alcohol . then all blood was taken from the mice by cutting the carotid artery . in a laminar flow chamber ,
the lung tissue was cut into fragments of approximately 1  mm which were distributed among 6-well polystyrene plates . after being supplemented ,
dmem culture medium ( 20% serum fetal bovine ( fbs ) and antibiotics ) was added to each well .
the plate was then incubated at 37c and 5% co2 for 72  h. after this period , the tissue fragments were removed and 50% of the medium was replaced .
after 7 days of incubation , the cells were removed with trypsin 0.25% ( edta , gibco ) for 15  min and replaced in a culture flask of 75  cm .
the trypsinization procedure was repeated every 5 to 7 days . finally , the cells were cultured for 15 to 20 days ( 3rd and 4th passage ) until being used in the following trials .
the isolated pmlec were characterized by immunofluorescence with the antibodies anti - vwf , anti - vcam , anti - ace , anti - cd62e , anti - enos , anti - cd31 , and anti - ve - cadherin . to analyze the area of opening of interendothelial junctions ( oij ) ,
that , the lung endothelial cells were plated in 24 well plates ( 7  10 cells / well ) , adhered to gelatin on glass coverslips , and maintained at 37c and 5% co2 .
the cells were stimulated with pba lysate for 3  h , after incubation with hemin ( 5 , 10 , and 20  m during 24  h ) , or solely with dmem culture medium , supplemented with 20% fbs , in triplicate .
subsequently , the cells were fixed with 3.7% formaldehyde , permeabilized with acetone at 20c , and blocked with bovine serum albumin solution ( 1% bsa ) .
each slide , with fully confluent cells , was chosen randomly and ten to twenty pictures were taken and scanned in a  zig - zag  way , from top to bottom .
the images were acquired in the fluorescence axio imager m2 ( zeiss ) microscope using the axio cam hrc ( zeiss ) and the software axio vision , version 4.9.1.0 .
the total area of oij was measured in each picture using the software gimp , version 2.8.16 ( https://www.gimp.org/ ) .
the increased lung vascular permeability was analyzed in pmlec plated on inserts of permeable membranes with pores of 0.4  m ( transwell corning ) , pretreated with gelatin and coupled in 24-well polystyrene plates at a concentration of 2.2  10 cells per insert and maintained in dmem culture at 37c .
after 96 hours , until the cells reach confluency , the extract was applied for 3  h after incubation with hemin ( 20  m during 24  h ) or solely with 20% fbs supplemented dmem culture medium .
subsequently , the culture medium was replaced by hank 's balanced salt solution and in the upper compartment of each insert ( in contact with the cells ) ; 200  l of evans blue was incubated at a concentration of 2  mg / ml at 37c . after 30  min ,
the liquid from the lower compartment was collected and analyzed in a spectrophotometer at a wavelength of 650  nm ( nanodrop 2000 , thermo scientific ) .
finally , the concentration of evans blue in each sample was determined from a standard curve with previously known concentrations of evans blue ( 0.2  mg / ml to 0.0031  mg / ml ) . the data were analyzed by d'agostino - pearson normality test .
whitney test and kruskal  wallis test with dunn 's multiple comparisons test . the unpaired t - test and one - way anova with bonferroni multiple comparison test were used for parametric variables .
statistical analyses were performed in graphpad prism version 5.0 ( http://www.graphpad.com/scientific-software/prism/ ) , including assessments of sensitivity and specificity . to establish cut - off from data ,
roc curves were generated using the results of the control group in medcalc version 8.2.1.0 ( https://www.medcalc.org/ ) .
the development of ali / ards in the dba/2 mouse model occurred between the 7th and the 12th dai ( figures s1a and s1b in supplementary material , available online at http://dx.doi.org/10.1155/2016/4158698 ) , during which the mice have died presenting at necropsy reddish lungs , pleural effusion , and histologic changes such as congestion , alveolar edema and hemorrhage , inflammatory infiltrate , and damage to the alveolar wall ( figure s1c ) .
mice that survived after the 12th dai died on the 20th dai or were euthanized at the same day , showing grayish tone lungs , splenomegaly , and no pleural effusion , interstitial chronic pneumonia , and malarial pigment in the lung tissue ( figure s1c ) .
these two phenotypes were described in detail previously by our group [ 10 , 14 ] .
noninfected mice showed light pink lungs , and no liquid inside of the thoracic cavity was detected ( figure s1c ) .
these changes in lung histology were consistent with the percentage of alveolar area ; they were significantly lower in ali / ards than in hp mice ( figure s1d ) . by using the predictive model to identify ali / ards or hp in mice before death ,
the expression of ho-1 in the development of ali / ards was determined in the serum and lungs of infected dba/2 mice and compared to noninfected mice . in order to observe the protein expression of ho-1 , immunohistochemistry sections of lung tissue of dba/2 mice
the expression of ho-1 was higher in lungs of ali / ards - developing mice than in hp - developing mice and noninfected mice .
quantification of immunohistochemical stain of ho-1 in the lung was performed by image analysis ( figure 1(a ) ) .
ho-1 protein levels ( figure 1(b ) ) and ho-1 mrna expression ( figure 1(c ) ) , measured by western blot and qrt - pcr , respectively , in the mice lungs , were higher in ali / ards - developing mice compared to hp - developing mice on the 7th dai . also , ho-1 levels in the lung tissue cell lysate and in the sera were higher in ali / ards mice than in hp mice , as determined by elisa ( figures 1(d ) and 1(e ) ) .
in addition , we checked the activity of ho-1 measuring bilirubin in pba infected and noninfected mice by elisa , on the 7th dai .
the levels of bilirubin were significantly higher in ali / ards - developing mice than in noninfected mice ( figure 1(f ) ) .
however , the levels of bilirubin were not significantly different between ali / ards and hp mice .
mice were treated with 17  mg / kg of hemin , an inductor of ho-1 , on the second day before pba infection and on 4th dai .
the efficiency of induction of ho-1 protocol was checked and confirmed , showing that hemin treatment increased mrna and protein levels of ho-1 in the lungs and in the serum of noninfected animals ( figures s2a and s2b , resp . ) , which confirms it as an inducer of ho-1 .
the hemin treatment increased the survival with most of the mice alive after the 12th dai without developing ali / ards ( figure 2(a ) ) . additionally , the parasitemia was significantly different between the 5th and the 7th dai ( figure 2(b ) ) .
mice treated with saline ( control ) and sacrificed on 7th dai presented necropsy reddish lungs , pleural effusion , congestion , alveolar edema and hemorrhage , inflammatory infiltrate , and damage to the alveolar wall ( figure 2(c ) ) , similarly to what was observed in mice that developed ali / ards ( figure s1 ) [ 10 , 14 ] . on the other hand , hemin treated mice showed a phenotype similar to noninfected mice : light pink lungs , and no liquid inside of the thoracic cavity was detected ( figure 2(c ) ) . as it was observed in ali / ards versus hp mice , saline treated mice
also had significantly less percentage of alveolar area than hemin treated mice ( figure 2(d ) ) .
regarding the respiratory parameters , it was found that hemin treatment led to a significant amelioration , characterized by a decrease in enhanced pause ( penh ) ( figure 3(a ) ) and an increase in the respiratory frequency at 7th dai ( figure 3(b ) ) . also , treatment with hemin led to a significant reduction of ifn- mrna in lung and serum protein levels , il-10 in serum , mcp-1 protein in lung tissue lysates , and serum and tnf in serum ( figures 3(c)3(h ) ) .
the treatment of pba infected mice with hemin led to a decrease of vegf serum levels ( figure 4(a ) ) , a potent inducer of ali / ards in pba infected dba/2 . also , hemin treated mice showed protection of the alveolar capillary barrier , which can be seen by a reduction in lung vascular permeability after evans blue administration and quantification ( figure 4(b ) ) .
in addition , we observed the same effect in pmlec when they were hemin treated after being stimulated with pba lysate for 3 hours , in a transwell plate ( figure 4(c ) ) . to understand a possible mechanism of ho-1 action in reducing pulmonary vascular permeability , pmlec were treated with hemin in 5 , 10 , and 20  m , before pba stimulus .
the hemin action showed a significant reduction in the oij that are formed between the endothelial cells , when compared to nontreated cells ( figure 4(d ) ) .
the dba/2 developed ali / ards between the 7th and 12th dai , with an incidence of 50% ( average ) when infected with pba [ 10 , 14 ] .
our results reinforce this finding , showing that the animals that began to die after the 7th dai until the 12th dai showed typical signs of ali / ards , such as pleural effusion , alveolar edema and hemorrhage , alveolar wall damage , inflammatory infiltrates , and lower percentage of alveolar area .
the parasitemia reaches about 20% when mice start to develop ali / ards ( 7th dai ) and this value has a slight decrease from the 12th dai onwards .
interestingly , human malaria - associated ali / ards often occurs in patients who have already begun the antimalarial treatment , which also leads to a decrease in parasitemia .
ho-1 showed a protective role in experimental episodes of severe malaria , including cerebral malaria [ 15 , 16 , 31 ] .
moreover , treatment with ho-1 inhibitors such as zinc protoporphyrin ix ( znppix ) or tin protoporphyrin ix ( snppix ) led to an enhancement of ali / ards signals in the case of sepsis but had no effect in the cases of hyperoxia and experimental cerebral malaria [ 16 , 24 , 32 ] .
however , our data showed that ali / ards - developing mice had increased levels of mrna and ho-1 protein , when compared with hp - developing mice and noninfected mice .
additionally , the levels of bilirubin , which represent an indirect measure of ho-1 activity , were higher in ali / ards mice than in noninfected mice .
although it is not known whether this increase in ho-1 levels and ho-1 activity constitutes an effort to revert the ali / ards phenotype in the infected mice , it was showed in previous publications that the treatment of p. berghei anka infected dba/2 mice with co ( a product of ho-1 )   and with a co - releasing molecule   protected them against ali / ards .
therefore , in order to clarify whether ho-1 is protective in this model , its induction was performed by hemin .
the treatment with the inducer of ho-1 ( hemin ) had beneficial effects in pba infected dba/2 mice and in pmlec in contact with pba lysate .
in vivo , this treatment led to an improvement in the survival rate and in lung histology , with the absence of lung edema , higher alveolar area percentage , and the absence of pleural effusion at necropsy .
it is an interesting effect , since hemin was observed to lyse malaria parasites in culture .
additionally hemin treated mice infected with plasmodium chabaudi adami also have a reduction in parasitemia .
additionally , hemin induces ho-1 , which reduces the oxidative stress in the host , which may increase its capacity of parasite clearance .
hemin also led to an improvement of respiratory parameters , with a decrease of enhanced pause ( penh ) and increase of respiratory frequency reinforcing the mitigation of clinical signals of ali / ards , alongside an increase in survival rate .
these respiratory parameters were previously used in a predictive model of malaria - associated ali / ards in dba/2 mice . additionally , there is evidence of penh and respiratory frequency being altered in a model of lung injury by so2 exposure in rats .
in addition to improvements in survival , lung histology , penh , and respiratory frequency , the treatment with hemin led to a significant decrease in the levels of the proinflammatory cytokines ifn- , mcp-1 , and tnf- , which is in agreement with the survival increase and with improvement in lung parameters , as it was shown that tnf- induced pulmonary vascular endothelial injury in an animal model .
additionally , lung neutrophil accumulation and lung leak were abrogated in tnf- knockout mice that were subjected to hemorrhagic shock . ifn-
was also considered to be a key contributor to ali / ards in a hyperoxia mice model , where it was shown that this cytokine induced increases in lung alveolar permeability and neutrophil migration into lung air spaces .
increased levels of mcp-1 , chemokine involved in recruiting of monocytes , neutrophils , and lymphocytes , were found in pulmonary alveolar macrophages isolated from a rat model of immune complex - mediated acute inflammatory lung injury .
this shows that hemin reduced the levels of inflammatory cytokines that are important in ali / ards , which corroborates with previous results showing that ho-1 induction by hemin has anti - inflammatory effects in models of sepsis and lps induced ali / ards in mice [ 25 , 42 ] .
as an anti - inflammatory cytokine , which was increased in hemin treated animals , which were protected against endotoxic shock   and whose effect in ali / ards was shown to be protective , il-10 was not expected to be decreased in hemin treated animals in our data . however , this cytokine was observed to be increased in mice that developed ali / ards in our model before ( unpublished data ) . the treatment with hemin resulted in a reduction in serum levels of vegf , a factor that promoted the development of ali / ards in this model , in a previous study .
furthermore , siner and colleagues demonstrated that vegf is a potent inducer of ho-1 enzyme and that the induction of this enzyme reduced the acute lung injury in mice caused by hyperoxia .
the same could be happening in our model of ma - ali / ards , because the vegf levels are increased on the 7th dai , coinciding with an increase of ho-1 levels in serum and lungs .
on the other hand , it was shown that treatment of pba infected mice with vegf prevented them from developing experimental cerebral malaria .
however , the contribution of vegf to the increase of ho-1 might not occur soon enough to revert the ma - ali / ards phenotype in our model . in this model , vegf caused a deleterious effect , increasing the lung vascular permeability .
additionally , it was shown that alf492 , a co - releasing molecule , protected pba infected dba/2 from ali and reduced vegf levels of treated mice .
this corroborates our results that show a reduction in vegf levels on hemin treated mice .
however hemin treatment was also shown to have deleterious effects in a rat model of neuroinflammation , where it was responsible for increased levels of reactive oxygen species , brain tissue loss , microglial activation , and neuronal death .
in addition , improved pulmonary vascular permeability in hemin treated infected mice on the 7th dai was observed .
this corroborates previous studies that showed a reduction in the breakdown of alveolar capillary barriers in models of ali / ards induced by lps .
additionally , in experimental cerebral malaria , the induction of ho-1 reduced the permeability of the blood brain barrier in pba infected mice . also , in vitro , there was a reduction of the permeability of pmlec stimulated with pba and treated with hemin reinforcing the importance of ho-1 in this model .
finally , pmlec stimulated with pba lysate and treated with different hemin concentrations led to a decrease in the opening of interendothelial junctions , indicating that hemin acts at the cellular level , protecting from the deleterious effect of the parasite in endothelial cells and consequently reducing the lung vascular permeability .
this is in accordance with a previous publication where the presence of oij after acute lung injury initiation in an animal model was responsible for an increase in lung vascular permeability .
this data supports the hypothesis that the increase in ho-1 levels after the 7th dai is a late effort to reverse the ali / ards phenotype .
inducing ho-1 early in pba infection has a protective effect against ali / ards , making this enzyme a target for the prevention of ma - ali / ards .
 malaria is a serious disease , caused by the parasite of the genus plasmodium , which was responsible for 440,000 deaths in 2015 . 
 acute lung injury / acute respiratory distress syndrome ( ali / ards ) is one of the main clinical complications in severe malaria . 
 the murine model dba/2 reproduces the clinical signs of ali / ards in humans , when infected with plasmodium berghei anka . 
 high levels of ho-1 were reported in cases of severe malaria . 
 our data indicated that the ho-1 mrna and protein expression are increased in mice that develop malaria - associated ali / ards ( ma - ali / ards ) . 
 additionally , the hemin , a ho-1 inducing drug , prevented mice from developing ma - ali / ards when administered prior to the development of ma - ali / ards in this model . also , hemin treatment showed an amelioration of respiratory parameters in mice , high vegf levels in the sera , and a decrease in vascular permeability in the lung , which are signs of ali / ards . 
 therefore , the induction of ho-1 before the development of ma - ali / ards could be protective 
 . however , the increased expression of ho-1 on the onset of ma - ali / ards development may represent an effort to revert the phenotype of this syndrome by the host . 
 we therefore confirm that ho-1 inducing drugs could be used for prevention of ma - ali / ards in humans . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion