regular exercise offers protection against and may be useful as a treatment for a wide variety of chronic diseases associated with low - grade inflammation [ 13 ] . indeed , exercise triggers important adaptations in the inflammatory system , which vary depending on the type and duration of the exercise intervention .
although the inflammatory response to acute exercise may not fully resemble disease associated inflammation , it represents a well - established and useful model to investigate the mechanisms controlling inflammation . in particular ,
studies employing eccentric exercise have highlighted the impact of this type of exercise on the inflammatory response .
thus , an acute bout of eccentric exercise induces a marked proinflammatory response , while an eccentric training program attenuates the activation of different signaling pathways involved in inflammatory processes [ 68 ] .
recently , the regulation of toll - like receptors ( tlrs ) as key factors involved in the inflammatory responses to exercise has received great attention [ 9 , 10 ] .
tlrs are type - i transmembrane glycoproteins expressed in different cells of the innate immune system and in other cell types , such as fibroblasts or epithelial cells .
tlrs mediate the recognition of pathogen - associated molecular patterns and play an important role in the immune and inflammatory responses .
among all the tlrs described , tlr4 has been shown to highly respond to different exercise protocols [ 6 , 1214 ] . in conjunction with cd14
, tlr4 orchestrates several processes in the inflammatory cascade   and can activate different signaling pathways that increase , or inhibit , the activity of several transcription factors .
three major transcription factors are controlled , at least to some extent , by tlr4 signaling pathways : nuclear transcription factor b ( nf-b ) , activator protein-1 ( ap-1 ) , and interferon regulatory transcription factor 3 ( irf3 ) .
a recent study showed that eccentric exercise plays an important role in tlr4-induced nf-b activation through both the myeloid differentiation factor 88- ( myd88- ) dependent and independent pathways in peripheral blood mononuclear cells ( pbmc ) of young men .
however , data explaining possible exercise - induced tlr4 regulation of ap-1 and irf3 are scarce .
most of the tlr4 signaling data have been collected with elderly populations [ 10 , 1618 ] , and although few reports include young men [ 6 , 19 ] , tlr4 response to exercise is practically unknown in young women .
this may seem surprising given that women are more susceptible to autoimmune and inflammatory diseases , probably due to their higher basal inflammatory status [ 20 , 21 ] .
in fact , the sex difference in subcutaneous adiposity and a differential regulation of cytokine production are probably important factors explaining the sex differences in proinflammatory markers concentrations [ 22 , 23 ] . given the great benefits of exercise as a potential tool to protect against the development of chronic inflammatory diseases later in life , research assessing exercise - induced tlr4 activity in young women is warranted .
first , we aimed to describe the regulation of nf-b , irf3 , and indirectly ap-1 through tlr4 signaling pathway activation in pbmc in response to an acute bout of eccentric exercise in young female subjects . considering that tlr4 signaling was upregulated in men after a similar bout of exercise
, we hypothesized that the acute eccentric bout would induce an upregulation of the tlr4-related pathways .
in addition , to further understand the effects of eccentric training on tlr4 regulation , we evaluated the influence of this training paradigm on the activity pattern of different tlr4-initiated pathways .
it was hypothesized that the activation of the different tlr4 signaling pathways , for example , nf-b , ap-1 , and irf3 , in response to an acute eccentric bout would be reduced after the eccentric training period .
all the activities involved in this study , including familiarization sessions , were performed during a 12-week period . initially , two groups of young women completed maximal strength tests , followed by an acute eccentric bout some days later .
after 2 weeks , one of the groups ( training group , tg ) followed a 6-week eccentric training program , while the rest of the participants ( control group , cg ) followed their daily routines .
one week after the last training session , all the subjects repeated the strength tests and the acute eccentric bout .
blood samples were obtained before and immediately after and 2  h after each acute eccentric bout . peripheral blood mononuclear cells ( pbmc )
all subjects were healthy sport science undergraduate students involved in recreational physical activities 35  h per week ( team and ball sports ) .
none of them reported previous or present resistance exercise training experience or muscle joint or bone injuries .
subjects reporting inflammatory or autoimmune conditions or taking any medication known to affect their inflammatory , hormonal , or metabolic response were excluded from the study .
none of the participants were smokers . to minimize the effects of different sex hormone concentrations , for example
, estrogens may modulate innate immunity both at rest and during exercise [ 20 , 23 ] , all the volunteers from the present investigation carried out the first and the second ( 1 week ) acute eccentric bouts during the follicular phase of the menstrual cycle ( self - reported ) .
this phase has been described as an appropriate moment to evaluate the effects of exercise in women [ 23 , 25 ] .
participants were randomly assigned to either a training group ( tg ; n = 12 ) or to a control group ( cg ; n = 8) .
age , height , body mass , body mass index , and fat % ( analyzed by skinfold measurements using yuhasz equation ) were 22.5  0.3  yrs , 163.8  1.4  cm , 60  2.1  kg , 22.4  3.2  kg / m , and 18.5  2.8% fat , respectively , in women from tg and 22.5  2.3  yrs , 160.7  1.8  cm , 59.7  3.1  kg , 23.0  2.5  kg / m , and 17.9  3.1% fat , respectively , in women form cg .
the purposes and possible risks associated with participation in the study were explained to the subjects before written consent for participation was obtained .
the study followed the principles of the declaration of helsinki , and all procedures were approved by the local ethics committee .
maximal strength tests took place after two familiarization sessions , where appropriate squat exercise technique was explained .
three to five days before the first and the second acute eccentric bouts all participants performed a maximal voluntary isometric contraction ( mvic ) and a one repetition maximum ( 1rm ) tests using a multipower device ( e.g. , guided barbell squat exercise , salter , barcelona , spain ) . after a standardized warmup , subjects carried out the mvic test in a squat position , with 110 knee flexion .
each participant completed two mvic tests lasting 5 seconds , with 1  min of rest in between .
force was registered using a strain gauge ( globus ergometer , codogn , italy ) .
if the values obtained after the two trials differed more than 5% , a third test was performed .
briefly , participants had to lift an estimated load from 90 knee flexion to full extension ( 180 ) .
the load was increased 10  kg if the participant succeeded or decreased 5  kg if they failed .
the protocol followed during the acute eccentric - damaging bouts was similar to fernndez - gonzalo et al . and garca - lpez et al .
the eccentric insult corresponded to the negative phase of the squat exercise performed using a guided barbell multipower device .
the bout comprised 12 sets of 10 repetitions with a load equivalent to 60% of the mvic .
a 3-min rest period was allowed between sets . once the subject completed the eccentric action ( lower the load from 180 , full extension , to 90 knee flexion ) , research assistants raised the load up using a pulley system .
subjects lowered the load with the most comfortable velocity for them , although they were requested to control the descent of the barbell during the full range of motion , allowing the subjects to stop the movement at ~90 knee flexion .
an encoder system ( globus real power ) was used to register distance , time , and velocity of the vertical displacements of the barbell .
the first acute eccentric bout was performed ~2 weeks before the first training session , whereas the second bout was carried out ~1 week after the last training session .
all participants completed both acute eccentric bouts . over 6 weeks , tg subjects completed 18 training sessions ( 3 sessions per week ) with at least 48  h between sessions .
the training program was initiated ~2 weeks after the first acute eccentric bout and it was completed ~1 week before the second acute eccentric bout . the eccentric action ( full extension to 90 knee flexion ) and
the multipower device described for the acute eccentric bouts were used during the training sessions .
the velocity of each eccentric action was monitored to offer real - time feedback to the subjects , allowing them to perform the movement with a similar velocity to the initial acute bout .
the load and volume of the training was progressively increased in a weekly manner : weeks 1 and 2 : 3  10 and 5  10 at 40% of mvic , respectively ; weeks 3 and 4 : 3  10 and 5  10 at 45% of mvic , respectively ; weeks 5 and 6 : 3  10 and 5  10 at 50% of mvic , respectively .
blood samples ( 30  ml ) were obtained from the brachiocephalic vein before , immediately after , and 2  h after each acute eccentric bout using vacutainer system ( bd , franklin lakes , nj ) with edta .
basal blood collection was performed under conditions of fasting and rest ( 8:00 a.m ) .
all participants remained in the exercise physiology laboratory at the university of len until finishing acute testing and sampling . during this time , the subjects ingested only water .
relative quantity of pbmc subpopulations was assessed using an automatic analyzer in 5  ml of total blood ( technicon h1 , bayer , tarrytwon , ny ) .
density gradient centrifugation on ficoll separating solution ( biochrom ag , berlin , germany ) was employed to separate pbmc from total blood .
total rna was isolated from pbmc using a ribopuretm - blood kit ( ambion , paisley , uk ) and quantified by spectrophotometry ( nanodrop 1000 , thermo scientific , waltham , ma , usa ) .
dnase i ( rnase - free ) ( ambion ) was used to removed residual genomic dna .
first - standard cdna was synthesized using high - capacity cdna archive kit ( applied biosystems , paisley , uk ) and then it was amplified using taqman universal pcr master mix ( applied biosystems ) on a steponeplustm real - time pcr systems ( applied biosystems ) . taqman primers and probes for cd14 ( genbank m86511.1 and hs00169122_g1 ) ,
tlr4 ( genbank u88880.1 and hs00370853_m1 ) , traf6 ( genbank bc031052.1 and hs00377558_m1 ) , and 18srrna as housekeeping gene ( genbank x03205.1 and hs99999901_s1 ) were derived from the commercially available taqman assays - on - demand gene ( applied biosystems ) .
relative changes in gene expression levels were determined using the 2-ct method as described previously .
the cycle number at which the transcripts were detectable ( ct ) was normalized to the cycle number of gapdh detection , referred to as ct .
lysate proteins were fractionated by sds - page and western blotting was performed using the corresponding primary antibodies .
antibodies against tlr4 ( 96  kda ) , myd88 ( 33  kda ) , p65 ( 65  kda ) , perk ( 4244  kda ) , tbk1 ( 80  kda ) , irf3 ( 50  kda ) , protein c - reactive ( pcr ) ( 30  kda ) , and tnf ( 27  kda ) were purchased from santa cruz biotechnology ( santa cruz , ca ) ; antibodies against cd14 ( 40  kda ) , traf6 ( 55  kda ) , trif ( 66  kda ) , and ikki / ikk ( 80  kda ) were purchased from abcam ( cambridge , uk ) ; and antibodies against pikk ( 8587  kda ) , pib ( 40  kda ) , p38 ( 43  kda ) , pp38 ( 43  kda ) , and pirf3 ( 4555  kda ) were purchased from cell signaling technology ( beverly , ma ) .
equal loading of protein was demonstrated by probing the membranes with a rabbit antilamin - b polyclonal antibody ( santa cruz biotechnology ) ( 67  kda ) or rabbit anti--actin polyclonal antibody ( sigma - aldrich ) ( 42  kda ) .
the density of the specific bands was quantified with an imaging densitometer ( image j , national institute of health , bethesda , md ) .
data are expressed as means of percentages from resting values  standard error of means ( sem ) .
shapiro - wilk test was used to verify normal data distribution . to compare differences within each group
, data were analyzed using an analysis of variance ( anova ) with repeated measures for training ( pre- and posttraining ) and time ( baseline , immediately , and 2  h after acute bout ) .
to compare results between tg and cg after the first and the second acute eccentric bout , a two - way anova with repeated measurements for group ( tg and cg ) and time ( baseline , immediately , and 2  h after acute bouts ) was used .
all analyses were performed with spss version 17.0 statistical software ( chicago , il ) .
the 6-week eccentric training program took place during the 9-week interval between the two acute eccentric bouts . at baseline
, there were no differences in 1rm or mvic between groups ( p > 0.05 ) .
one repetition maximum ( 126  4 versus 140  5  kg ) and mvic ( 140  4 versus 159  3  kg ) increased in tg after the training program , but not in cg ( 137  7 versus 139  7  kg and 149  8 versus 142  7  kg , for 1rm and mvic , resp ) .
no differences between exercise bouts or groups were found in the distribution of pbmc subpopulations ( table 1 ) .
muscle soreness ( mm in vas ) before and 48 hours after the first acute eccentric bout was 2.9  5.3 and 43.6  16.9 , respectively , for tg and 2.5  4.3 and 49.3  16.3 , respectively , for cg .
compared with the first , soreness was lower in both groups ( p < 0.05 ) 48 hours after the second acute eccentric bout ( 13.0  5.7 for tg ; 31.7  9.1 for cg ) , and the decrease was greater for the tg ( p < 0.001 ) .
cd14 and tlr4 mrna levels increased after both acute eccentric bouts for tg and cg ( p < 0.04 ) , with no differences between bouts or groups ( figures 1(a ) and 1(b ) ) .
the first acute bout of eccentric exercise induced an increase of cd14 and tlr4 protein content in cg and tg that was significant immediately after exercise ( p < 0.05 ) and after 2  h ( p < 0.05 ) .
similar results were observed after the second bout in cg group . however , after the training program no significant change was detected following the eccentric bout in tg ( figures 1(c ) and 1(d ) ) .
myd88 ( figure 2(a ) ) increased after the first acute bout in tg and cg ( p < 0.02 ) .
however , this protein was downregulated after the second bout in tg when compared with the first eccentric bout and with cg ( p < 0.03 ) .
similarly , trif ( figure 2(b ) ) also increased significantly ( p < 0.03 ) in response to the first acute eccentric bout in both cg and tg , but only in cg after the second bout .
trif protein expression was significantly reduced after the second bout in tg ( p < 0.01 ) when compared with the first bout or with cg .
traf6 mrna levels were also analyzed . in the same line with cd14 and tlr4 ,
traf6 mrna levels ( figure 3(a ) ) increased after both acute eccentric bouts for tg and cg ( p < 0.05 ) .
the first acute eccentric bout triggered a significant ( p < 0.04 immediately after and p < 0.03 2  h after the first acute eccentric bout ) increase in traf6 protein concentration ( figure 3(b ) ) in tg and cg .
after the second acute bout , cg still showed such upregulation , while tg values did not differ from basal .
phospho - ib protein levels ( figure 3(c ) ) increased significantly ( p < 0.02 ) in response to the first acute eccentric bout in both cg and tg .
similar results were observed after the second bout in cg group , but a reduction in phospho - ib was found after the second bout in tg ( p < 0.01 ) when compared with the first bout or with cg .  
figure 3(d ) shows p65 protein content in nuclear extracts , which increased progressively reaching maximal levels 2  h after the first acute bout ( p < 0.01 ) .
posttraining values were lower in tg group , but they remained elevated in cg after the second acute bout .
a marked increase of cytosolic phospho - erk-1/2 protein concentration was evident immediately after the first acute eccentric exercise and was maintained 2  h after ( p < 0.01 and p < 0.01 , resp . ) in cg and tg group ( figure 3(e ) ) . however , 6 weeks of eccentric training reduced the phosphorylation of erk-1/2 to basal values after the acute bout in tg .
tnf and crp protein levels were measured as an estimation of the proinflammatory status ( figures 4(a ) and 4(b ) , resp ) .
tnf increased after the first acute eccentric bout in both tg and cg ( p < 0.04 ) , but only in cg after the second bout ( p < 0.04 ) .
furthermore , tg showed lower tnf protein expression after the second acute eccentric bout when compared with the first bout immediately after and 2  h after , and with cg 2  h after the bout ( p < 0.05 ) .
the first acute eccentric bout also triggered a significant increase of crp ( p < 0.04 ) in tg and cg .
after the second acute bout , cg still showed upregulation of crp ( p < 0.04 ) , while tg values were markedly reduced after the second bout ( p < 0.05 ) .
the inducible ikk ( figure 5(a ) ) increased immediately after the first acute eccentric bout and was maintained 2  h after in both groups ( p < 0.04 ) .
after the second bout this upregulation persisted in cg but was blunted in tg ( p < 0.03 ) .
no changes were observed in the phosphorylation state of irf3 in response to acute exercise either before or after 6 weeks of training ( figure 5(b ) ) .
the current study investigated the effects of eccentric exercise on the tlr4-mediated inflammatory response in healthy young women .
the results indicate that 6 weeks of submaximal eccentric training attenuate several intermediates related to the nf-b and ap-1 activation and , consequently , the proinflammatory response detected after an acute bout of eccentric exercise .
this response was associated with a downregulation of the tlr4 signaling through both myd88-dependent and independent pathways .
our data further suggest that perhaps other tlr4 signaling pathways , such as irfs , could be implicated in the beneficial adaptations induced by training .
previous studies have shown that tlr4 may play a critical role as a link between inflammatory cytokine production and a physically active lifestyle [ 6 , 9 , 17 ] .
thus , resistance - trained older women had significantly lower inflammation and tlr4 mrna levels than sedentary counterparts . moreover , physical activity status , but not age , influenced tlr4 cell - surface expression .
our results confirm that a single bout of eccentric exercise increases tlr4 and cd14 mrna and protein contents in young women , as it had previously been reported in men .
interestingly , only the protein content overexpression of the receptor and coreceptor in pbmc was lower following the eccentric training program .
therefore , these data provide further support for a training - induced downregulation of tlr4 and its coreceptor cd14 at protein level . confirming previous results in young men
, there was a discrepancy between cd14 and tlr4 mrna levels and protein concentration , which may indicate posttranscriptional adaptations of cd14 and tlr4 in response to eccentric training .
different stimuli induce tlr4 activation via recruitment of different adaptor / signaling molecules to the cytoplasmic domain of the receptor , thus resulting in different cellular responses .
in addition , the cell surface or endosomal localization of tlr4 drives recruitment of myd88 or toll / interleukin-1 receptor ( tir ) domain containing adapter inducing interferon- ( trif ) , respectively . in the present study ,
an acute eccentric bout activated both the myd88-dependent pathway and the trif - dependent pathway , which mediate the early- and late - phase activation of nf-b , respectively .
this activation was blunted in young women after 6 weeks of training , which resembles previous reports in both human and animal models [ 6 , 31 ] .
early- and late - phase activation of nf-b through tlr4 signaling is mediated by tnf receptor - associated factor 6 ( traf6 ) .
traf6 recruits the protein kinase complex tak1 ( transforming growth factor beta - activated kinase-1 ) and tabs ( tak1 binding proteins ) [ 33 , 34 ] , which then activate two distinct pathways involving the ib kinases ( ikk ) complex ( nf-b activation ) and the mitogen - activated protein kinase ( mapk ) ( erk , jnk , p38 ) pathway , for example , ap-1 activation . the results from the present study indicate that an acute bout of eccentric exercise induced an increase of traf6 protein concentration , which was associated with ikk and mapk signaling pathway activation .
this resembles previous observations where eccentric contractions also induced a significant increase in nf-b activation [ 7 , 8 ] and mapkerk1/2 phosphorylation .
remarkably , the eccentric training reduced the protein concentration of traf6 , phospho - ib , p65 , and mapkerk1/2 in tg after the second acute eccentric bout compared with the first bout and with cg .
the proinflammatory cytokine tnf- , which is regulated by transcription factors such as nf-b and ap-1 , increased its content in pbmc after the first acute bout in both groups .
however , this upregulation was blunted by the training program after the second acute bout .
taken together , these findings support the role of the eccentric training as a valuable asset to decrease the inflammation associated with acute exercise .
thus , upon stimulation , tlr4 is internalized into endosomes where two transcriptional events occur : ( 1 ) delayed activation of nf-b and ( 2 ) initiation of the irf3 transcription program . in the latter case , trif activates two ikk related proteins through traf3 , inducible ikk ( ikki or ikk ) , and tank - binding kinase1 ( tbk1 ) .
ikk and tbk1 activate interferon regulatory factors , such as irf3 , which dimerise , translocate to the nucleus , and bind interferon response elements ( isre ) , resulting in the induction of type-1 interferons [ 30 , 33 ] . in the present study
trif protein levels increased after the acute eccentric exercise in parallel to an activation of ikk , and both effects were inhibited following 6 weeks of submaximal eccentric training .
however , our data indicate a nonsignificant increase immediately after and at 2  h after the acute bouts in the irf3 protein .
first , we can not exclude the existence of a methodological limitation since data in the literature show that other authors used a phospho - specific antibody against ser386 to demonstrate that overexpressed irf3 is phosphorylated on ser386 and that phosphorylation on ser386 is observed in the dimeric form of irf3 [ 37 , 38 ] .
however , phosphorylation on ser396 ( antibody used in this study ) alleviates autoinhibition to allow interaction with cbp ( creb - binding protein ) and facilitates phosphorylation at ser386 .
second , tbk1 and ikki can also phosphorylate and activate irf7 , which is the member of irf family most closely related to irf3 . whereas irf3 is ubiquitously
expressed and not inducible , irf7 is expressed at low levels in most types of cells but is strongly induced in response to various stimuli .
it has been shown that the expression of some downstream effectors of the tlr signaling pathways , such as irf7 , was reduced by addition of resveratrol to exercise training in rat heart .
furthermore , irf7 , which is mainly activated by tlr7 and 9 , seems to be the master regulator of type - i interferon - dependent immune responses and exerts transcriptional control over a large set of proinflammatory genes .
the activity profile of the tlr4 pathway may be affected by exercise - induced changes in the pbmc subpopulations . in this study , there were no differences in the distribution of pbmc subpopulations between the first and the second acute eccentric bouts within a group or between groups .
therefore , differences in tlr4 signaling activity between tg and cg and between acute eccentric bouts can only be explained by the eccentric - based training intervention followed by tg .
additionally , it should be noted that none of the proinflammatory markers analyzed were downregulated in the cg .
thus , although the adaptations induced by a single bout of eccentric exercise may decrease the muscle damage from a subsequent bout ( i.e. , repeated bout effect ) , it appears that such adaptations do not affect the inflammatory response associated with eccentric exercise in young women .
indeed , the repeated bout effect failed to protect against the proinflammatory response in young men [ 6 , 7 ] . in summary , this study provides evidences indicating that an acute eccentric exercise increases the tlr4-mediated proinflammatory response through both myd88- and trif - dependent pathways in young women , supporting previous reports in men .
more importantly , we show that regular eccentric training may be an effective method to prevent the upregulation of some of the signaling pathways controlling the proinflammatory response in young women .
this may be of great importance given the higher risks of women to suffer from autoimmune and inflammatory diseases .
further research assessing long - term effects of eccentric exercise on inflammatory profile in young women is therefore warranted .
the present study also supports the notion that tlr4 signaling pathway is a key regulator of the exercise - induced inflammatory response , highlighting the role of tlr4 as a target for future exercise - based anti - inflammatory interventions .
however , more research is needed to fully evaluate the potential associations between exercise , tlr4 , and activation of the irf transcription factors .
the current study provides novel understanding of the molecular mechanisms behind the inflammatory response controlled by the tlr4 signaling pathway after acute exercise in trained and untrained young women .
it is suggested that different signaling pathways involving ikk and the transcription factors irf are modulated by tlr4 in response to exercise .
additionally , the eccentric training is purported to be a valuable tool to decrease exercise - induced inflammation through a downregulation of the tlr4 downstream signaling in young women .
in contrast , the repeated bout effect does not seem to modify the eccentric exercise - induced inflammatory response in women .
these data may have implications in the prevention and rehabilitation programs currently employed for autoimmune and inflammatory diseases in this population .
 this study assessed the inflammatory response mediated by the toll - like receptor 4 ( tlr4 ) signaling pathway after acute eccentric exercise before and after an eccentric training program in women . 
 twenty women performed two acute eccentric bouts using a squat machine over a ~9 week interval . 
 the training group ( tg ) carried out an eccentric training program during 6 weeks , while the control group ( cg ) did not follow any training . 
 protein content of markers involved in the tlr4-mediated activation of several nuclear transcription factors , such as nuclear factor b ( nf-b ) , and interferon regulatory transcription factor 3 ( irf3 ) , was analyzed . 
 the inflammatory response after the first acute bout was similar between tg and cg , showing an upregulation of all the markers analyzed , with the exception of irf3 . 
 after the second bout , the upregulation of tlr4 signaling pathway was blunted in tg , but not in cg , through both the myeloid differentiation factor 88- and toll / interleukin-1 receptor domain containing adapter inducing interferon--dependent pathways . 
 these results highlight the role of the tlr4 in controlling the exercise - induced inflammatory response in young women . more importantly , 
 these data suggest eccentric training may help to prevent tlr4 activation principally through nf-b , and perhaps irf3 , downstream signaling in this population . 