
 human parainfluenza virus type 1 ( hpiv-1 ) , which is a member of the family paramyxoviridae , is an enveloped virus with a single - stranded rna genome [ 1 , 2 ] .
hpiv-1 infects the upper and lower respiratory tract and causes acute respiratory infections ( aris ) , ranging from mild infections , such as the common cold and laryngitis , to severe infections , such as croup , pneumonia , and bronchiolitis .
hpiv-1 is responsible for almost half of all hospitalizations due to aris both in patients younger than 5 years old and in the elderly ; additionally , hpiv-1 is the most common cause of infectious laryngotracheitis ( croup ) in children [ 36 ] .
the therapy used to treat symptoms of inflammation is based on glucocorticoid and ephedrine , also humidifying the airway ; however , this is not always effective [ 79 ] .
there is no evidence indicating that mitogenic signal activation is required in the early stages of infection [ 10 , 11 ] .
il-8 is a mediator responsible for the recruitment of neutrophils that participate in the local inflammatory infiltrate , contributing to airway closure [ 12 , 13 ] .
it has been reported that epithelial cells , alveolar macrophages , and neutrophils secrete il-8 [ 1417 ] .
other authors have reported that infection with respiratory syncytial virus ( rsv ) , varicella - zoster virus , and smallpox virus activates il-8 secretion without viral replication [ 18 , 19 ] .
these observations indicate that the interaction between the virus and its receptor is sufficient to promote the signalling pathways that activate the il-8 gene ; however , replication is necessary in other viruses , such as vaccinia virus and rhinovirus [ 2123 ] .
it has been shown that viruses have different effects on the regulation of il-8 expression and secretion .
the most prominent examples include the filoviruses marburg and ebola and arenaviruses , such as lassa and junin .
other viruses such as rsv , adenovirus , vaccine virus , and herpes virus secrete il-8 immediately [ 24 , 25 ] .
the primary signalling pathways that elicit a response by chemokines are the mapks and transcription factor nf kappa b pathways .
mapks are a family of proteins that activate kinases through a cascade of intracellular phosphorylation events and signal transduction from the cell surface to the nucleus .
they are composed of three well - characterized subfamilies : extracellular signal - regulated kinases ( erk1/2 ) , c - jun n - terminal kinases ( jnks ) , and p38 mitogen - activated proteinkinases ( p38 ) .
each subfamily includes a kinase that sequentially acts on three proteins : mekk , mek , and mapk .
the mapk family substrates in the cytoplasm and nucleus include additional kinases , transcription factors , phospholipases , and cytoskeletal proteins .
erk1/2 is associated with anabolic processes , such as cell division , growth , and differentiation , while jnk and p38 are associated with cellular responses to stress conditions , death , and inflammation [ 2628 ] .
the molecular mechanisms in which larynx epithelial cells play a role and their active involvement in the inflammatory infiltration response to infection by hpiv-1 through production of small chemoattractants that recruit neutrophils have not been investigated sufficiently to generate a strategy that counteracts pathogenesis and to determine whether viral replication is necessary . in this study ,
an in vitro model of hpiv-1 infection of hep-2 and a549 cells was used to simulate the upper and lower airways .
the aim of this study was to determine whether viral replication induces the il-8 secretion and mapk kinase signalization .
the hep-2 human laryngeal epithelioma type 2 cells ( atcc ccl-23 , usa , which reported a contamination with hela cells ) and a549 human alveolar type ii - like epithelial cells ( atcc , ccl-185 , usa ) were acquired from american type culture collection ( manassas , va , usa ) .
cell lines were propagated in 25  cm flasks ( nunc thermo scientific inc . , turnberry , usa ) and petri dishes with eagle 's minimal essential medium ( mem ) , supplemented with 10% foetal bovine serum and antibiotics ( penicillin 100  u / ml , streptomycin 100  g / ml , amphotericin - b 1  g / ml ) from invitrogen life technologies ( gaithersburg , md ) .
cells were maintained at 37c in 5% co2 and were stored and grown in accordance with the manufacturer 's instructions .
virus strain was obtained from american type culture collection , manassas , va , usa .
confluent monolayers of hep-2 and a549 cells in 25  cm flasks were inoculated with hpiv-1 and incubated at 37c in 5% co2 until a cytopathic effect was observed 48 hours after inoculation ( formation of syncytia ) .
one aliquot of the virus was transferred to 1.8  ml cryotubes after titration and stored at 70c , and another aliquot was used to infect cell monolayers at a 0.01 multiplicity of infection ( moi ) .
uninfected hep-2 and a549 cell monolayers were used as controls . a portion of hpiv-1 ( 5  ml of virus at a titre of 6  10  pfu ) in serum - free medium ( rpmi ) was placed in a sealed box and irradiated at a distance of 10  cm for 10 minutes with optimal radiation levels ( 1,200  units  100  j / cm ) using an fb - uvxl 1000  uv cross - linker fisher biotech ( thermo fisher scientific inc . , ky , usa ) as previously described .
haemagglutination assays were performed in 96-well plates to verify the haemagglutinin activity of hpiv-1 using serial dilutions of o - positive human blood ( 1  :  2 , 1  :  4 , etc . ) .
hep-2 and a549 cells were infected with live and inactive hpiv-1 at an moi of 0.01 , supernatant was collected at different times ( 30 , 45 , 60 , 90 , and 120 minutes and 12 and 24 hours ) , and uninfected hep-2 and a549 cells were used as controls .
il-8 concentrations were quantitated by elisa according to the procedure described by kittigul et al .
( 1998 )   using anti - human cxcl8/il-8 monoclonal antibodies from r&d systems , inc .
. optical density at 450  nm was measured on an elisa reader spectrophotometer from thermo labsystems ( santa rosa , ca , usa , and helsinki , finland ) . the expression of mrna for il-8 was determined by reverse transcription - polymerase chain reaction ( rt - pcr ) .
total rna was isolated by using sv total rna isolation system ( promega , madison , usa ) following the manufacturer 's protocol .
a 0.5  g portion of total rna was reverse - transcribed for subsequent pcr amplification for each pair of primers in a volume of 20  l , including 200  u m - mlv rt reverse transcriptase ( invitrogen , ca , usa ) , 50  u of rnase inhibitor ( sigma ) , 0.2  g of random primer , 10  mm of dntp mix ( invitrogen , ca , usa ) , and 5x first - strand buffer ( 250  mm tris - hcl , ph 8.3 ; 375  mm  kcl ; 15  mm  mgcl2 ) provided by life technologies .
. a 20  l portion of the rt products was then brought to a volume of 20  l containing 10  mm of each dntp , 1  u of taq polymerase ( promega , madison , usa ) , 10  pmol of both the upstream and downstream pcr primers , and 1  x  pcr buffer ( proprietary formulation supplied at ph 8.5 containing blue dye and yellow dye ) provided by promega .
the primers for il-8 are as follows : forward , 5-atg act tcc aag ctg gcc gtg gct-3 and reverse , 5-tct cag ccc tct tca aaa act tct-3 with a product of 289  bps . for glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) ; the forward primer was 5-ccagccgagccacatcgctc-3 ; and the reverse primer was 5-atgagccccagccttctccat-3 , giving a 390 bps pcr product .
the gapdh gene was amplified in the same reaction to serve as the reference gene .
amplification was carried out in a biometra personal thermal cycler after an initial denaturation at 94c for 3  min .
this was followed by 35 cycles of pcr using the following temperature and time profile : denaturation at 94c for 40  s , primer annealing at 58c for 40  s , primer extension at 72c for 1  min , and a final extension of 72c for 6  min .
the pcr products were visualised by electrophoresis on a 2% agarose gel and visualized by uv transillumination . hep-2 and a549 cells were infected with live or inactive hpiv-1 at an moi of 0.01 with the same kinetics as that described above in the previous assays .
cells were harvested , and total protein was extracted and quantified by the bradford method .
cells were boiled at 100c for 10 minutes in a buffer solution supplemented with -mercaptoethanol .
the obtained proteins were separated on 15% sds - page gels and transferred to a 0.45 m nitrocellulose membrane from bio - rad laboratories ( hercules , ca ) for 3.5 hours , and western blot assays were performed .
transfer efficiency was determined by staining with 0.1% ponceau red in 5% glacial acetic acid from sigma - aldrich ( st .
louis , mo ) , and measurements were performed using laemmli 's sds - page system .
membranes were washed , blocked , and probed overnight with mapk phospho - erk1/2 ( sc-81492 ) , mapk erk1/2 ( sc-292838 ) , phospho - p38 mapk ( sc-7973 ) , p38 mapk ( sc-535 ) , phospho - jnk ( sc-135642 ) , and jnk ( sc-571 ) antibodies ( 1  :  3  000 dilution ) purchased from santa cruz biotechnology ( santa cruz , ca ) .
the bound antibody was coated with a peroxidase - conjugated goat anti - mouse igg secondary antibody ( sigma - aldrich ) ( 1  :  3  000 ) for 2 hours .
membranes were washed and incubated with a luminal chemiluminescent substrate from santa cruz biotechnology , before exposure to x - ray film .
the band intensity was quantified using the quantity one ( bio - rad ) image analysis software . for the best reproduction of larynx conditions
, assays were conducted using hep-2 cells that were pretreated with the kinase inhibitors pd980559 for erk1/2 , sb203580 * for p38 mapk , and sp600125 * for jnk from calbiochem millipore * ( san diego , ca ) .
the concentrations used were from 5 to 20  m ( 1.3 to 5.2  l ) in dmso ; the same dmso concentration was added to control cells and was incubated at 37c .
one ml of hpiv-1 at an moi of 0.01 was added , and infected cells were incubated under the same conditions .
supernatants from hep-2 cells without inhibitors collected at the same infection time were used as controls .
when the mapk inhibitor was used , cells were pretreated with the inhibitor 1 hour before infection . because mapk inhibitors were diluted in dmso , equal concentrations of dmso were added to the untreated infected cells as control . after treatment with the inhibitor ,
the analysis done in the western blot bands was performed in the quantity one software from bio - rad .
individual quantification of the bands was made from the intensity of light generated in them , due to the fluorescence emitted in the radiographic plate .
data were tested for normality ( shapiro - wilk ) and since data did not show normal distribution , kruskal - wallis test was used to compare the different treatments .
the assays were performed in triplicate , and the results were statistically analysed using statistica software 5.0 ( stat soft , tulsa , oklahoma ) . a p value < 0.05 was considered to indicate a statistically significant difference .
the inactivated viruses were created to demonstrate whether viral replication was involved in the activation of the secretion of il-8 or viral structure alone was sufficient to generate the response to this chemokine .
the virus was inactive with uv radiation ; plaque assays were performed to verify that the virus was found inactive and will not be replicated .
exponential virus dilutions are presented in figure 1 to determine the number of infectious particles .
( a ) the first two rows correspond to the viral activity with the inactive virus , where there was no replication due to the absence of pfu . in the remaining rows of live virus activity in both cell types was evaluated .
panel ( b ) corresponds to viral dilutions and panel ( c ) refers to the number of infective particles in a dilution type cellular .
because the inactive virus did not replicate , haemagglutination tests were made showing that inactive virus particles had the same ability to agglutinate erythrocytes and the active virus ( l ) . in both lytic and haemagglutination assays exponential dilutions
these tests are often used to demonstrate the viral replication , for the presence of hemagglutinin glycoprotein or viable structures .
il-8 secretion was quantified during hpiv-1 infection suggesting that infection of cell lines with live hpiv-1 induced a gradual increase in il-8 secretion .
the highest il-8 secretion in hep-2 and a549 was observed at 12 hours after infection , whereas the cells with inactive virus showed a marginal response at 24 hours ( 2.05 and 2.0 ) .
the higher secretion of il-8 was found using infected hep-2 cells followed by infected a549 cells ( table 1 ) , compared with the cells with inactive virus where a minimal response was achieved . in the case of hep-2 cells and a549 cells treated with inactive virus , no il-8 secretion
was observed neither in the uninfected cells used as controls . the mrna expression of il-8 in a549 and hep-2 cells was determined by rt - pcr analysis . in the analysis , uninfected cells , cells infected with hpiv - infected cells , and hpiv-1 inactive were used .
the increased expression of il-8 originated from infected cells , followed by uninfected cells and the inactivated virus , and the expression in the latter two was almost equal .
infection was performed at different times and behavior for each was similar in both cell lines ( figure 3 ) .
gapdh was constitutively expressed in both cell lines and expression did not vary by infection . to establish whether the inactive virus stimulated the phosphorylation of mapks ( erk , jnk , and p38 ) western blot tests were made ( figure 4 ) .
the two different cell lines hep-2 cell line for the upper portion of the tract and the a549 for the bottom line of the same tract were used to simulate the respiratory tract .
western blot assays were performed to determine the phosphorylation status of the mapks erk1/2 , jnk , and p38 . in hep-2 cells , infection with live hpiv-1 promoted early erk1/2 phosphorylation , beginning at 60 minutes of interaction ( figures 5(a ) and 5(b ) ; band intensities were quantified through densitometric analysis ) .
however , erk phosphorylation was higher at 12 hours than at any other selected time and decreased at 24 hours . in the case of inactivated virus , the 12-hour band was only in lane 3 because no evidence of phosphorylation was observed in the kinetics selected times .
the same criteria were applied to the western blot figures ; only a band corresponding to a 12-hour interaction was observed with no increase in phosphorylation before and after this time point .
controls consisted of uninfected cells , which showed no significant changes . in a549 cells infected with live hpiv-1 ,
the detection of erk1/2 phosphorylation began at 60 minutes of interaction ; a small phosphorylation signal was observed at 90 minutes of interaction ( figures 5(c ) and 5(d ) ) , increasing slightly at 120 minutes and remaining constant until the 24-hour time point .
phosphorylation was slightly detected for the inactivated virus and control samples . in controls ( uninfected cells ) , inactive virus samples , and samples
that were incubated for 30 and 45 minutes phosphorylation was only slightly detected . with inactive viruses ,
the result was similar to the previous western blot ( figures 5(c ) , lane 4 , and 5(d ) , lane 3 ) .
jnk phosphorylation in hep-2 cells also began at 60 minutes and gradually increased until the 90-minute time point but decreased at 120 minutes .
by 12 and 24 hours , phosphorylation ceased ( figures 6(a ) and 6(b ) ) .
as in hep-2 cells , jnk phosphorylation in a549 cells began at 60 minutes , increased at 120 minutes , and decreased at 12 and 24 hours , with no further increase ( figures 6(c ) and 6(d ) , lanes 9 and 10 ) .
likewise , p38 phosphorylation in the hep-2 cell line began at 30 minutes ( figures 7(a ) and 7(b ) ) , increased again at 120 minutes , and subsequently remained stable until the 24-hour time point .
in a549 cells , p38 phosphorylation levels were similar , beginning at 30 minutes ( figures 7(c ) and 7(d ) ) but with a gradual increase from the 90-minute time point and achieving a maximum at 12 and 24 hours .
the kinase inhibition assay was performed on the cell line hep-2 , derived from human laryngeal epithelioma , given that is a suitable target for the in vitro virus assay .
our results suggest that p38 , erk1/2 , and jnk are activated following hpiv-1 infection . therefore , to determine whether these kinases are required for il-8 gene expression , elisas were performed using the supernatant from hep-2 cells pretreated with erk1/2 , jnk , and p38 kinase inhibitors and infected with hpiv-1 at the following time points : 45 , 60 , and 90 minutes and 12 and 24 hours ( figure 9 ) .
results demonstrate that the p38 inhibitor repressed il-8 production upon viral infection , whereas erk1/2 and jnk mapk inhibitors did not inhibit il-8 production ( figures 9(a ) and 9(b ) ) , suggesting that p38 mapk signalling is involved in il-8 production .  
figure 9(c ) depicts the average of three experiments with hep-2 cells infected with hpiv-1 and inhibitors .
controls without inhibitors for each kinase were included for all time points . treating the cells with jnk and
erk1/2 inhibitors did not impair il-8 production , but treating cells with an inhibitor of p38 mapk significantly impaired il-8 secretion in most of the incubation times ( before 24  h ) .
erk1/2 and jnk inhibitors were not different from the control group ( table 2 ) .
total inhibitory dose mapks ( erk1/2 , jnk , and p38 ) were evaluated , showing that the optimal concentration to suppress phosphorylation was 20  m ( figure 8) .
in the present study , an in vitro model with two cell lines that were infected with hpiv-1 at different time points was used to determine whether an increase in il-8 secretion occurred .
these results are consistent with reports that other respiratory viruses , including influenza a virus ( h3n2 ) and rsv , induce il-8 secretion from bronchial epithelial cells [ 36 , 37 ] .
it is known that il-8 is responsible for the recruitment of neutrophils and macrophages and that it performs other functions , such as adhesion and degranulation .
likewise , the presence of il-8 in inflammatory infiltrates in lung tissues has been reported .
studies performed with different viruses have revealed that epithelial cells infected with specific viruses release il-8 without requiring viral replication , as what occurs in rsv infections [ 18 , 19 ] .
contact with the cellular receptor is sufficient to trigger il-8 secretion , whereas other viruses only promote il-8 synthesis during replication [ 21 , 23 ] . in this study
, assays using both live and inactivated hpiv-1 were performed to determine whether replication - independent il-8 release occurs with hpiv-1 , a virus from the same family as rsv .
the results indicate that contact between the virus and target cell is insufficient , even when haemagglutination activity is intact in the inactivated virus , and viral replication is required to stimulate il-8 secretion .
previous reports have shown that il-8 secretion is involved in mapk pathway activation [ 12 , 16 ] .
these kinases respond to different ligands and stimuli and are involved in signalling pathways and cellular processes , such as proliferation , differentiation , inflammation , immune response , and apoptosis .
hpiv-1 does not use the jnk pathway for il-8 activation , whereas p38 and erk are phosphorylated when il-8 is present in the supernatant after infection .
the results of this study showed that hpvi-1 does not use the jnk pathway for activation of il-8 , while the mapks p38 and erk are phosphorylated by the production of il-8 after the infection , similar results have been described after rsv infection ( virus belonging to the paramyxoviridae family ) in human lung endothelial cells where erk phosphorylation enabled il-8se creation . therefore , to define which kinases are required for il-8 secretion , the same assays were performed using specific inhibitors for each kinase .
the results showed that , even in the presence of specific inhibitors of erk and jnk , il-8 was still secreted , but there was no secretion in the presence of p38 inhibitor . in this case , a complete suppression of il-8 secretion was observed , which suggests that p38 mapk is the pathway through which hpiv-1 induces il-8 production .
it is known that the inhibitor sb203580 is a potent , cell - permeable , selective , and reversible inhibitor of p38 and  isoforms .
inhibition is competitive with atp and requires substrates to accommodate the fluorophenyl ring structure of the pyridinyl imidazole in their atp - binding pocket .
structures of other p38 isoforms , jnks , and erk1/2 do not allow inhibitor binding at equivalent positions [ 38 , 39 ]
. many members of the paramyxovirus family have been shown to be potent inducers of chemokines of the cxc family such as interleukin-8 ( il-8 ) , including respiratory syncytial virus ( rsv ) , sendai virus , human parainfluenza virus type 2 ( hpiv-2 ) , hpiv-3 , and newcastle disease virus .
the receptor - binding glycoprotein is hn in parainfluenza virus and mumps virus , h in measles virus , and g in pneumoviruses ( rsv and metapneumovirus ) .
the second glycoprotein is the fusion protein ( f ) , which catalyses membrane fusion ( viral and cellular ) .
it has been suggested that hn glycoproteins form a complex that is initiated by the binding hn , activating the fusion protein that acts immediately in such a way that the virus can enter the cell .
however , it must be noted that glycoprotein hn acts as both a haemagglutinin ( ha ) and a neuraminidase ( na ) , binding to sialic acid molecules through its ha activity , mediating the enzymatic cleavage of sialic acid and promoting fusion through its neuraminidase activity .
glycoprotein h of morbillivirus only has haemagglutinin activity , and glycoprotein g has neither of these two activities [ 4144 ] .
such differences are likely related to signalling pathways that are activated when viruses interact with the cell .
although hpiv-1 and rsv belong to the same family , the two viruses have different glycoproteins , which may explain why rsv uses erk kinase and hpiv-1 uses p38 kinase .
following virus binding to its cellular receptor , the extracellular stimulus is transmitted to the nucleus to activate the expression of various genes involved in a given biological activity .
the virus activates these pathways according to the coevolution of the parasite - host complex , and it is likely that the virus also can take advantage of the activation of the pathway to provide a better environment and thus freely replicate .
viruses , such as herpesviruses , hepatitis b virus , hiv , influenza virus , coxsackie virus , and vaccinia virus , activate the mapk pathway , particularly erk , in the early stages of infection . in this study
, we found that mapks are activated during the early stages of infection ( 60 minutes ) .
it is possible that the activation of the p38 kinase pathway following hpiv-1 infection plays a similarly essential role during other stages of infection .
importantly , the stimulus by hpiv-1 increased and was maintained up to 24 hours ; therefore , it was detectable at the first stages of infection , even when cytopathic effect was clearly visible in at least 90% of the culture .
several authors have asserted that kinase activation could be a requirement for signalling regulation associated with different biological functions during infection with other viruses .
for example , the first events during hiv-1 infection depend on the mapk / erk pathway , which improves viral infectivity . the mechanism by which hpiv-1 stimulates p38 activation needs to be further investigated .
however , a product of any of the virus genes may be involved in the process , considering that no kinase activation was observed when viruses were uv - irradiated .
additionally , it is possible that mapks also influence virus replication , promoting the synthesis of some proteins necessary for the virus .
children with croup are regularly admitted to the emergency department with their lives at risk due to airway closure .
currently , the treatment for patients with moderate to severe croup is oral dexamethasone at 0.6  mg / kg ( maximum 1012  mg ) doses , which acts on these and additional cell types involved in the immune response .
however , data show that p38 mapk inhibitors may reverse glucocorticoid insensitivity and have beneficial effects on glucocorticoid concentrations in patients with asthma as well as airway inflammation , in addition to its role as a treatment substitute
. a better understanding of hpiv-1 pathogenesis will guide the design of strategies and therapies allowing the inhibition of excessive cell recruitment and il-8 synthesis to reduce the uncontrolled inflammatory process .
for now , the exact mechanism in inflammatory and immune processes must be defined , and further research is required to understand whether these pathways are significant for hpiv-1 pathogenesis .
an in vitro model with two cell lines infected with hpiv-1 at different time points was used to determine whether an increase in il-8 production occurred .
the results showed that il-8 production gradually increased during the time points assessed and that viral replication is required for il-8 secretion .
no kinase activation was observed when viruses were inactivated , suggesting that a product of any of the virus genes may be involved in the process
. when a p38 inhibitor was used , il-8 concentrations decreased , suggesting that hpiv-1 induces il-8 secretion through p38 .
additionally , it is possible that mapks also affect virus replication , promoting the synthesis of some proteins necessary for the virus .
however further studies are needed to understand whether these pathways are significant for hpiv-1 pathogenesis , in order to develop strategies and therapies allowing the inhibition of excessive cell recruitment and il-8 synthesis to reduce the uncontrolled inflammatory process .
 human parainfluenza virus type 1 ( hpiv-1 ) is the most common cause of croup in infants . 
 the aim of this study was to describe molecular mechanisms associated with il-8 production during hpiv-1 infection and the role of viral replication in mapk synthesis and activation . 
 an in vitro model of hpiv-1 infection in the hep-2 and a549 cell lines was used ; a kinetic - based elisa for il-8 detection was also used , phosphorylation of the mitogen - activated protein kinases ( mapks ) was identified by western blot analysis , and specific inhibitors for each kinase were used to identify which mapk was involved . 
 inactivated viruses were used to assess whether viral replication is required for il-8 production . 
 results revealed a gradual increase in il-8 production at different selected times , when phosphorylation of mapk was detected . 
 the secretion of il-8 in the two cell lines infected with the hpiv-1 is related to the phosphorylation of the mapk as well as viral replication . 
 inhibition of p38 suppressed the secretion of il-8 in the hep-2 cells . 
 no kinase activation was observed when viruses were inactivated . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions