a number of studies have documented the potential benefits of probiotic bacteria which prevent and ameliorate human t - helper-2-cell- ( th2- ) related diseases , such as atopic dermatitis and asthma . at the helper t - cell level , a number of probiotic lactobacilli promote the production of t - helper-1-cel - l ( th1- ) skewing cytokines , such as interleukin ( il- ) 12 and interferon ( ifn- )  , and suppress the production of ige and th2 cytokines .
other studies have demonstrated that bifidobacteria suppress th2 cytokine and ige production , without significant induction of th1 cytokines .
the th2-suppressive effect of probiotic bacteria is mediated by regulatory t ( treg ) cells linked to the induction of regulatory cytokines , including il-10 and transforming growth factor ( tgf- )  [ 4 , 5 ] .
it has been reported that macrophages stimulated with lactococcus   lactis w58   produce il-10 and that tgf- is induced by lactobacillus paracasei ncc246 in mixed lymphocyte reactions .
in addition , recent clinical studies have proven the prevention and curative features of probiotics in some intestinal disorders , such as inflammatory bowel diseases ( ibd ) including crohn 's disease and ulcerative colitis .
it was demonstrated that administration of bifidobacterium longum bb536 was effective in inducing remission of patients with ulcerative colitis .
multiple mechanisms of action have been suggested to explain the protective effects of probiotics in intestinal inflammation .
these can be broadly classified as follows : ( 1 ) suppression of growth or epithelial binding / invasion by pathogenic bacteria , ( 2 ) improved epithelial barrier function , and ( 3 ) immunoregulatory activities . as for immunoregulation , several probiotics can induce protective cytokines , including il-10 and tgf- , and suppress pro - inflammatory cytokines , such as tnf- , in the mucosa of patients with paucities and crohn 's disease . besides th1 ,
th2 , and treg , t helper 17 cells ( th17 ) have received considerable recent attention as they exhibit effector functions distinct from th1 and th2 cells [ 9 , 10 ] .
differentiation factors ( tgf- plus il-6 or il-21 ) , growth and stabilization factor ( il-23 ) , and transcription factors ( signal transducer and activator transcription 3 ( stat3 ) , retinoic acid receptor - related orphan receptor ( ror- ) t and ror- ) involved in the development of th17 cells have been identified .
notably , th17 cells produce the pleiotropic cytokine il-17 , which potently induces tissue inflammation and is associated with the pathogenesis of many human diseases , such as ibd   and rheumatoid arthritis .
thus , modulation of th17 cells is currently viewed as a potentially positive pharmacological outcome .
although some studies have revealed that lactic acid bacteria and bifidobacteria may have potential use as anti - inflammatory agents in certain chronic inflammatory diseases , such as ibd , it is unclear whether these bacteria are able to suppress excess th17 activity .
we recently reported that bifidobacterium longum infantis jcm 1222 ( type strain ) suppresses il-17 production in murine splenocytes in vitro and in inflamed intestinal cultures ex vivo . to identify other bacterial strains with potential th17 modulating activity
, we previously performed an in vitro assay with 200 strains of streptococci and found that streptococcus thermophilus st28 exhibited potent inhibitory activity towards il-17 production .
the main objective of this study was to evaluate the effects of oral administration of st28 on il-17 production by murine lamina propria lymphocytes ( lpls ) and on the expression of th17-related surface markers in inflamed intestines .
we first evaluated and compared in vitro cytokine production patterns in th17-skewed conditions following the exposure of splenocytes to st28 and atcc 19258 ( type strain ) .
the in vivo effects of st28 were also evaluated using flow cytometry and real - time pcr in dextran - sodium - sulphate- ( dss- ) induced colitis mice , a human ibd model in which il-17 plays a pivotal role in pathogenesis .
two strains of s. thermophilus , atcc 19258 ( type strain ) and st28 , were obtained from the american type culture collection and glico dairy products ( tokyo , japan ) , respectively .
each strain was cultured in m17 broth ( merck , whitehouse station , nj , usa ) at 37c for 1765 hours . the cultured cells were washed with sterile distilled water and incubated at 100c for 50  min . following the incubation , the heat - killed bacteria were lyophilized and used for subsequent experiments .
six - week - old female balb / c mice were obtained from charles river japan ( kanagawa , japan ) , and all experimental protocols involving animals were approved by the animal care committee , graduate school of biosphere science , hiroshima university . to harvest splenocytes ,
mice were sacrificed by cervical dislocation , spleens were removed , and suspensions of splenocytes ( 1.2  10 cells ) were incubated with 2  ng / ml tgf- ( r&d systems , minneapolis , minn , usa ) plus 20  ng / ml il-6 ( r&d systems ) at 37c for 72 hours in 120  l rpmi 1640 medium ( life technologies , foster city , california , usa ) supplemented with 10% fetal bovine serum ( fbs , icn biomedicals , osaka , japan ) , 10  m 2-mercaptoethanol , 10  mm hepes , 5 units / ml penicillin ( life technologies ) , and 5  g / ml streptomycin ( life technologies ) in a 96-well cell culture plate ( thermo fisher scientific , waltham , ma , usa ) .
heat - killed bacteria ( 1.2  10 cells ) were then added to the cell cultures . a culture to which no tgf- plus il-6 or heat - killed bacterial cells were added was included as a control . following the incubation ,
cells were applied to flow cytometry ( figure 1 ) , and culture supernatants were assayed for cytokine concentrations ( table 1 ) as described below .
the splenocytes ( 1  10 cells / ml ) were incubated at 37c for 4 hours in rpmi 1640 medium containing 0.5  g / ml phorbol-12-myristate-13-acetate ( mp biomedicals , aurora , ohio , usa ) , 1  g / ml ionomycin ( wako pure chemical industries , osaka , japan ) , and 3  g / ml brefeldin - a ( wako pure chemical industries ) . then , anti - mouse cd32/16 antibody was added and incubated 4c for 30  min .
for analysis of surface markers , fluorescein isothiocyanate ( fitc ) anti - mouse cd11c antibody ( biolegend , san diego , calif , usa ) , allophycocyanin ( apc ) anti - mouse cd11b antibody ( ebioscience , san diego , calif , usa ) , or apc anti - mouse cd4 antibody ( ebioscience ) was added to the cell suspension , which was then incubated in the dark at 4c for 30 min . for isotype controls , fitc - armenian hamster igg , apc rat igg2a ( biolegend ) , or apc rat igg2b ( ebioscience )
, the cells were permeabilized by intraprep ( beckman coulter , marseille codex , france ) for intracellular cytokine staining .
phycoerythrin ( pe ) anti - mouse il-17 antibody ( ebioscience ) or pe rat igg2a ( ebioscience ) was added to the cell suspension and incubated in the dark at 4c for 30  min . following the incubation ,
the cells were washed twice with 5% fbs - hanks ' balanced salt solution ( hbss ) and suspended in 300  l of 5% fbs - hbss .
fluorescence intensity was measured on a guava easycyte flow cytometry system ( millipore , billerica , mass , usa ) .
cytokine ( ifn- , il-2 , il-4 , il-5 , il-10 , il-12(p70 ) , tumor - necrosis - factor- ( tnf- )  , and granulocyte macrophage colony - stimulating factor ( gm - csf ) ) concentrations of the culture supernatants were determined by the microbead method using the bio - plex suspension array system ( biorad laboratories , hercules , calif , usa ) , according to the manufacturer 's instructions .
briefly , culture supernatants were incubated with beads conjugated with anti - ifn- , -il-2 , -il-4 , -il-5 , -il-10 , -il-12(p70 ) , -tnf- , and -gm - csf antibodies ( biorad laboratories ) followed by sandwich immunoassay using biotinylated secondary antibodies .
the beads were washed three times after each incubation , and pe - streptavidin was used as a reporter .
the relative fluorescence units were determined by counting 100 beads using the bio - plex system .
data were evaluated with the bio - plex manager software 6.0 ( biorad laboratories ) .
separately , the il-17 concentration of culture supernatants was measured using a duoset sandwich elisa kit ( r&d systems ) following the manufacturer 's instructions .
six - week - old female balb / c mice were housed in an air - conditioned room under a 12 hours light - dark cycle and were allowed ad libitum access to tap water and a standard diet ( mf ; oriental yeast , tokyo , japan ) .
acute colitis was induced in mice by adding 3.5% ( w / v ) dss ( molecular weight , 36,00050,000 ; mp biomedicals ) to their drinking water for 5 days .
the experimental protocols ( figure 1(a ) ) were approved by the animal care committee , graduate school of biosphere science , hiroshima university .
group 1 ( control group ) mice were treated orally with phosphate - buffered saline ( pbs ) from days 1 to 3 , while group 2 ( control group ) received drinking water containing 3.5% ( w / v ) dss and were orally administered pbs .
groups 3 and 4 also received drinking water containing 3.5% ( w / v ) dss , and , 24 hours after the start of dss administration , the mice were orally administered 10  cfu / day of heat - killed st28 ( group 3 ) or atcc 19258 ( group 4 ) in pbs for 3 consecutive days ( figure 2(a ) ) .
the severity of intestinal inflammation was assessed on day 5 by assigning a score for each of the 2 stool scores , consistency , and bleeding ( figure 2(b ) ) .
the scale used for consistency was 0 ( normal stool ) , 1 ( loose stool ) , 2 ( diarrheal stool ) , and 3 ( watery stool ) , and that used for bleeding was 0 ( normally colored stool ) , 1 ( blood visibly present in the stool ) , 2 ( adherent of blood on anus ) , and 3 ( gross bleeding on anus ) .
then , the total score was expressed as the sum of the scores of the 2 items ( total score , 6 ) .
after the experimental period , the entire colon was removed from mice ( figure 2(c ) ) , and lamina propria from the excised colons was removed .
pooled ( n = 4 ) colon tissue was suspended in hbss containing 10  mm dithiothreitol ( wako pure chemical industries ) and incubated at 20c for 10  min with shaking .
the tissue was further treated with 2.1  mg / ml collagenase d ( wako pure chemical industries ) at 37c for 20  min in a shaking water bath [ 19 , 20 ] .
only live cells were collected from single - cell suspensions from the lpls by dead cell removal kit ( miltenyi biotec , auburn , calif , usa ) , and used for subsequent experiments .
portion of lpls ( 1  10 cells ) were suspended in the same rpmi 1640 medium as described above and incubated at 37c for 48 hours in the presence of 1  g / ml anti - cd3 ( ebioscience ) and 1  g / ml anti - cd28 ( ebioscience ) . following the incubation ,
cells were applied to flow cytometry , and culture supernatants were assayed for il-17 and ifn- concentrations ( figure 3 ) . for analysis of intracellular il-17 ( figure 4(a ) ) ,
lpls ( 1  10 cells / ml ) were incubated at 37c for 4 hours in rpmi 1640 medium containing 0.5  g / ml phorbol-12-myristate-13-acetate , 1  g / ml ionomycin , and 3  g / ml brefeldin a. then , apc anti - mouse cd4 antibody or apc rat igg2a was added to the cell suspension .
after the incubation , the cells were permeabilized by intraprep , and pe anti - mouse il-17 antibody or pe rat igg2a was added to the cell suspension . following the incubation ,
the cells were applied to flow cytometry .       for analysis of surface markers ( figure 4(b ) ) , freshly prepared lpls were incubated with fitc anti - mouse cd11c antibody and apc anti - mouse cd86 antibody ( ebioscience ) , or with fitc armenian hamster igg and apc rat igg2a , were added to the cell suspension and applied to flow cytometry . pooled ( n = 4 ) colon tissue from other dss colitis mice was suspended in hbss containing 5  mm edta and incubated at 37c for 20  min in a shaking water bath .
the tissue was further treated with 1  mg / ml collagenase d ( roche applied science , nonnenwald , germany ) and 3  mg / ml dispase ii ( life technologies ) at 37c for 30  min in a shaking water bath .
briefly , tissue was resuspended in 40% percoll ( ge healthcare biosciences ab , uppsala , sweden ) and overlaid on 80% percoll , after which density gradient was performed by centrifugation at 1,000  g for 20  min at 25c .
lpls were subsequently collected from the interface fraction between the 40% and 80% percoll 's layers [ 19 , 20 ] .
rna was extracted from the lpls using trizol ( life technologies ) following the manufacturer 's instructions .
reverse transcription reactions were performed with high - capacity cdna reverse transcription kit ( life technologies ) at 25c for 10  min and 37c for 120  min . the reaction was terminated by heating at 85c for 5  sec , followed by cooling at 4c .
real - time pcr was performed using kapa sybr fast abi prism qpcr kit ( kapa biosystems , woburn , mass , usa ) and the primer sequences listed ( /see/ in supplementary material available on line at doi : 10.1155/2011/378417 ) .
reactions were performed at 95c for 2  min , followed by 40 cycles of 95c for 5  sec and 60c for 30  sec .
the dissociation stage was analyzed at 95c for 15  sec , followed by 1 cycle of 60c for 15  sec and 95c for 15  sec .
the fluorescence of the sybr green dye was determined as a function of the pcr cycle number , giving the threshold cycle number at which amplification reached a significant threshold .
data were analysed by the ct method and presented as fold changes in gene expression after normalization to the internal control -actin gene expression level ( supplementary figure  1 ) .  
briefly , bone marrow cells were collected from femurs of six - week - old female balb / c mice .
bone marrow mononuclear cells ( 2  10 cells / ml ) were cultured at 37c for 7 days in rpmi1640 medium containing mouse 200 units
/ ml granulocyte macrophage colony - stimulating factor ( miltenyi biotec ) , 10% fbs , 10  m 2-mercaptoethanol , 10  mm hepes , 5 units / ml penicillin , and 5  g / ml streptomycin in a tissue culture flask . on day 7 , bmdc ( 1  10 cells / ml ) were seeded in 24-well cell culture plate and matured by 1  g / ml lipopolysaccharide ( lps , sigma - aldrich , st .
louis , mo , usa ) for 24 hours . on day 8 , heat - killed bacteria ( 1  10 cells / well ) were added to the cell cultures . after 24-hour incubation ,
il-6 and il-10 concentrations of culture supernatants were measured using a duoset sandwich elisa kit ( r&d systems ) following the manufacturer 's instructions .
statistical analysis was performed using one - way anova followed by tukey 's post hoc test .
      when live st28 was added to murine splenocytes and coincubated at 37c for 72 hours , st28 grew in rpmi 1640 medium , and it became impossible to properly examine the effect of st28 on splenocytes .
thus , in subsequent experiments , st28 and atcc 19258 were used after heat killing .
prior to the experiment , we first purified t cells from splenocytes by magnetic bead - based cell separation ( macs ) , and stimulated the macs - purified t cells with tgf- plus il-6 . however , il-17 production was not detectable ( data not shown ) , which suggested that il-17 production from th17 cells in splenocytes required the coexistence of non - t cells .
therefore , we alternatively stimulated whole splenocytes with tgf- plus il-6 for further analyses , after which the stimulated splenocytes were applied to flow cytometry in order to analyze cd4 , cd11b , and cd11c cells separately .
as a result , most of the il-17 cells were cd4 t cells , which indicated that the major source of il-17 was th17 cells .
more importantly , it was confirmed that st28 decreased the percentage of cd4 il-17 cells ( figure 1 ) .
as shown in table 1 , st28 significantly ( p < 0.01 , (  ) versus st28 ) repressed il-17 production ( 78% reduction ) .
on the other hand , atcc 19258 failed to repress its production . based on these results ,
st28 was judged to be a suitable candidate strain for the treatment of th17-mediated diseases , and its suppressive effects were further examined in vivo using dss - induced colitis mice .       in regard to other cytokines than il-17 , the productions of all cytokines tested were changed by the stimulation with tgf- plus il-6 . among them , ifn- and tnf- productions in the st28 group were significantly higher than those in the atcc 19258 group , and il-2 and il-4 productions were significantly lower . on the other hand , there were not significantly differences in il-10 ( anti - inflammatory cytokine ) and il-12 ( proinflammatory cytokines ) productions between the two groups .
acute colitis was induced in mice by exposure to 3.5% dss for 5 days , which resulted in an increase in colon inflammation score compared to the control group ( score , 2.5  1.5 versus 0.5  0.3 ) ( figure 2 ) . however , oral treatment with st28 ameliorated the inflammation ( score , 1.0  0.4 ) .
atcc 19258 exerted the same ameliorative effect on the intestinal inflammation , but the effect was weaker ( figures 2(b ) and 2(c ) ) .
next , lpls from the mice was applied to ex vivo culture experiment ( figure 3 ) .
although differences in ifn- concentrations in lpls culture were not prominent among four groups , lpls from dss colitis mice produced substantial level of il-17 . however , st28 markedly suppressed il-17 production from lpls culture ( 86% suppression ) , while atcc 19258 failed to suppress il-17 production . from the analyses of mrna expression in lpls , it was revealed that the expression of both il-17 and ror-t , the master regulator of th17 , in lpls from the dss - administered group was drastically upregulated , compared to control mice ( supplementary figure  1 ) .
notably , st28 markedly inhibited both expressions , which indicated that st28 suppressed the differentiation of nave t cells towards th17 in lpls .
cd11c and cd86 were also found to be downregulated by the oral administration of st28 .
it was also confirmed by flow cytometry that cd4il17 cells ( th17 cells ) disappeared in lpls by the oral administration of both st28 and atcc 19258 ( figure 4(a ) ) . on the other hand
, the effects of dss and those of the administration of st28 and atcc 19258 on cd11c cd86 cells , inflammatory dendritic cells ( dc ) , were unclear , although atcc 19258 seemed to decrease the percentage of cd11c cd86 cells . to clarify this point
the effect of st28 on lps - matured bmdc was evaluated ( figure 5 ) .
il-10 production was not significantly decreased by the addition of st28 and atcc 19258 . however , both st28 and atcc 19258 significantly suppressed il-6 production .
accordingly , the percentage of cd86cells , which is a hallmark of maturation , was also decreased by st28 and atcc 19258 .
therefore , it is highly probable that st28 suppressed th17 cells via a dc - dependent mechanism .
in medical therapy for ibd , the modulation of immune responses and the regulation of intestinal inflammation is essential .
although the pathogenesis of ibd is complex , involving environmental , microbial , genetic , and immunological factors , several recent studies have shown that th17 cells play a significant role in ibd pathology [ 23 , 24 ] . here , we demonstrated that s. thermophilus st28 repressed il-17 production in mouse splenocytes under th17-skewed conditions in vitro ( table  1 ) .
moreover , oral treatment of dss - induced colitis mice with st28 suppressed inflammatory th17 cells in lpls .
although s. thermophilus is not commonly considered a probiotic bacterium , the findings of the present study imply that this species would be useful in the treatment of ibd by suppressing exaggerated th17 activity in inflamed intestines .
th1/th2 balance can influence the direction and outcomes of immune responses , due to the mutually antagonistic nature of th1 and th2 responses . in this regard ,
the modulating activity of probiotic and commensal bacteria on th1/th2 balance is commonly evaluated [ 3 , 4 ] . here ,
as ifn-/il-4 production ratio in st28 group was high even in th17-skewed conditions ( table 1 ) , it is expected that st28 would suppress not only th17 , but also th2 cells .
this response would also affect ige production in allergic conditions , because th2 cells drive ige class switching by allergen - stimulated b cells .
suppressing both th2 and th17 cells is desirable for the repression of ige - mediated reaction , because these cell types are closely related with each other in allergic patients . to confirm that st28 also suppressed th17 cells in vivo , acute colitis was induced by dss treatment of mice .
dss induces acute inflammation and recruitment of immune cells , whose subsequent activation directly causes epithelial cell . in dss - induced colitis mice , which serve as a useful model of human ibd , st28 administration improved inflammation score ( figure 1 ) , repressed il-17 production ( figure 3 ) , and decreased th17 cells ( figure 4 ) in lpls isolated from inflamed intestines . moreover , st28 decreased the mrna expressions of both rort and il-17 in lpls ( supplementary figure  1 ) . considering all of the data ,
although several lines of evidence indicate probiotic bacteria have beneficial effects on dss colitis [ 7 , 14 , 26 ] , the mechanisms by which bacteria mitigate intestinal inflammation have not been fully clarified .
we propose that st28 ameliorates dss - induced colitis in mice by suppression of inflammatory th17 cells .
it should be noted that even  heat - killed  bacteria could suppress il-17 production in this study . therefore , some heat - resistant components of st28 might exert th17 supprressive activity .
it is generally accepted that bacterial components are recognized by members of the pattern recognition receptor family , such as toll - like receptors ( tlrs ) . in regard to the epithelial barrier , some studies have shown that the tlr2 ligand pcsk ameliorated tnf--induced intestinal barrier impairment in the human epithelial caco-2 cells [ 28 , 29 ] and dss colitis mice .
it is likely that the recognition of heat - resistant bacterial components by these kinds of receptors is also involved in the suppression of th17 cells .
it has been recently suggested that cd86 plays a critical role in the initiation of t - cell responses including th17 cells .
for example , gene silencing of cd40 , cd80 , and cd86 protected collagen - induced arthritis , one of the th17-related inflammation . in the effector cd4 t - cell responses inducing antigen - induced arthritis , blockade of cd86 significantly suppressed il-17 production in splenocytes , and cd86 enhanced disease severity by upregulating il-17 production .
also , high cell surface expression of cd86 was observed in patients with ibd   and in mice with cd4 t - cell - induced colitis .
these findings lead us to examine the effect of st28 on the expression of cd86 .
the mrna expressions of cd86 and cd11c were drastically upregulated in lpls from dss colitis mice , but they were downregulated by the oral administration of st28 ( supplementary figure  1 ) .
st28 also significantly decreased the percentage of cd86cells in matured bmdc ( figure 5 ) .
collectively , it is highly probable that st28 ameliorated dss induced colitis by suppressing cd86 dc , which needs to be clarified in detail .
it has been described that il-17 is involved in the pathogenesis of dss - induced colitis in mice , and il-17 receptor signaling plays a critical role in the development of trinitrobenzenesulfonic acid - induced acute colitis in mice , yet another report has suggested that il-17 might offer an inhibitory role in the development of dss - induced colitis . o'connor
et al .   also demonstrated the data on a protective function for il-17 in t - cell - mediated intestinal inflammation .
for example , buonocore et al .   have recently suggested that a novel innate lymphoid cell population accumulates in the inflamed colon and induces il-17 and ifn-. further investigations on th17/th1 in the intestinal inflammation are required to clarify the beneficial functions of st28 on th17-mediated diseases . in conclusion , we have demonstrated that st28 ameliorates intestinal inflammation in dss - induced colitis mice at least partially through suppression of inflammatory th17 cells .
the use of probiotic bacteria in ibd therapy has been investigated in a number of clinical studies .
although the efficacy of this treatment approach was demonstrated for ulcerative colitis , the results for crohn 's disease are not yet clear owing to conflicting results and a paucity of trials . in any case , the majority of the demonstrated curative and palliative effects of probiotic bacteria appear to be mediated by modulation of the intestinal immune system .
clinical approaches using probiotics are appealing due to a lack of toxicity and patient desire for the use of natural physiological approaches to treating disease . since s. thermophilus has a long history of being safely consumed in yogurt , its further application to the treatment and/or prevention of th17-mediated diseases such as ibd
 the effects of streptococcus thermophilus st28 on cytokine production by murine splenocytes stimulated with transforming growth factor- plus interleukin- ( il- ) 6 were evaluated . 
 the addition of st28 significantly repressed il-17 production compared to atcc 19258 ( type strain ) . 
 st28 also decreased the number of th17 cells in the stimulated splenocytes . 
 the anti - inflammatory effects of st28 administration were evaluated in mice with colitis induced by dextran sodium sulphate ( dss ) . 
 oral treatment of mice with st28 ameliorated the intestinal lesions by dss . upon dss treatment , il-17 production in lamina propria lymphocytes ( lpls ) was induced , but st28 significantly decreased its production . 
 st28 also decreased the percentage of th17 cells in lpl from dss - induced colitis . 
 the present results imply that st28 suppresses the th17 response in inflamed intestines and would be useful in the treatment of th17-mediated diseases , such as inflammatory bowel disease . 