the most common inflammatory bowel diseases ( ibds ) are crohn 's diseases ( cd ) and ulcerative colitis ( uc ) . it is recognized that aberrant immune response in the mucosa of the gastrointestinal tract plays an important role in pathogenesis of ibds [ 1 , 2 ] . a large body of evidence from experimental models of ibd has shown that cd4 + t cells ( t helper cells th1 and th2 ) play a major role in initiating and regulating the immunopathologic process , largely based on the production of proinflammatory cytokines , such as tumor necrosis factor ( tnf ) [ 3 , 4 ] . more recently , another subset of t helper cells that produces the cytokine interleukin ( il-17 , also called il-17a ) was identified as th17 cells and has gained particular attention due to its proinflammatory role in the mucosal immune response [ 5 , 6 ] . the number of th17 cells and il-17 expression were found to be significantly enhanced in the inflamed gut of cd and uc patients [ 7 , 8 ] . in addition to the il-17 isoforms ( il-17a and il-17f ) , th17 cells also secrete il-6 , il-21 , il-22 , and il-26 . il-17 drives microbial defense , contributes to neutrophil migration and function , and promotes t - cell priming and cellular production of inflammatory mediators , such as il-1 , il-6 , tnf , granulocyte macrophage colony - stimulating factor ( gm - csf ) , nitric oxide synthase ( nos)-2 , prostaglandin e2 , and metalloproteases . therefore , control of the differentiation and function of th17 cells is potentially significant for ibd treatment . it has been recently demonstrated that differentiation of th17 cells is initiated by transforming growth factor 1 ( tgf1 ) and il-6 [ 9 , 14 ] . both tgf1 and il-6 regulate th17 differentiation by activating the transcription factor signal transducer and activator of transcription 3 ( stat3 ) [ 15 , 16 ] . a line of evidence has also indicated a significant role of il-23 in th17 differentiation . il-23 was initially thought to be critical for th17 differentiation ; however , it was later found to be rather important for th17 cell expansion , stabilization , and/or conditioning for a fully inflammatory cell phenotype [ 17 , 18 ] . the il-23/th17 immune axis has thus been shown to play a crucial role in a number of chronic inflammatory diseases including ibd . the lactobacillus species can rebalance homeostasis in gastrointestinal inflammatory diseases and thus have a protective role against ibd . we have previously shown that lactobacillus ( l. ) acidophilus treatment can efficiently ameliorate dextran sodium sulfate- ( dss- ) induced experimental colitis in mice . in the present study , we examined whether the therapeutic effect of l. acidophilus is achieved through suppression of the il-23/th17 pathway , including th17 cell differentiation and the expansion factors il-23 , tgf1 , and stat3 , as well as th17 cell - secreted cytokines il-17 and tnf. we found that oral administration of l. acidophilus at all doses induced a significant downregulation of colitis - enhanced expression of all the examined factors . l. acidophilus was isolated from a normal human intestinal tract smc - s095 sample and sequence - verified by our laboratory . after culturing under anaerobic conditions with de man , rogosa , sharpe ( mrs ) medium for 24  h , the bacteria were collected , quantified by a spectrophotometer , and diluted with normal saline to 10  cfu / ml . seventy - two female balb / c mice ( 68 weeks old , 20.0  2.0  g mean body weight ) were purchased from hunan agricultural university and housed under standard conditions ( 50%  10% humidity , 12  h light / dark cycle , and ad libitum access to standard mouse chow ) . colitis was established in 64 mice by adding 5% dss ( mw 50000 ; sigma chemical co. , st . louis , mo ) to the drinking water and allowing ad libitum access for 7 days . the mice were randomly divided into the following control and experimental model groups ( n = 8 each ; day 0 ) : the nontreated model group ; the vehicle treated model group ( oral gavage of 1  ml/10  g normal saline ) ; the l. acidophilus - treated groups c4c8 ( oral gavage of 10 , 10 , 10 , 10 , or 10  cfu/10  g body weight , resp . ) ; the prednisone acetate ( an anti - inflammatory agent ) treated positive control ( administered at 45  g/10  g body weight ) . eight mice that were given regular water and received no subsequent treatments served as the normal control group . different doses were applied in order to identify an appropriate condition that can efficiently modulate the expression of factors related to th17 cell function . on posttreatment day 7 , all mice were sacrificed with ether and the colon was collected . a 0.5  cm segment of the distal colon that is 1  cm proximal to the anus was excised and fixed with 10% formalin for later paraffin embedding and sectioning . the rest of the distal colon was stored in liquid nitrogen for rna and protein extractions . the colonic tissue was homogenized , and rna was extracted by using trizol ( invitrogen , grand island , ny ) according to the manufacturer 's instructions . first - strand cdna was synthesized using a reverse transcription kit ( mbi , ottawa , ca ) . 5-taaaacgcagctcagtaacagtccg-3 ( product length of 349  bp , tm 60c , 30 cycles ) , il-17 forward primer 5-tcagactacctcaaccgttcc-3 and reverse primer 5-cagtttccctccgcatt-3 ( product length of 129  bp , tm 54.5c , 30 cycles ) , il-23 forward primer 5-aataatgtgccccgtatcca-3 and reverse primer 5-aggctcccctttgaagatgt-3 ( product length of 144  bp , tm 58c , 28 cycles ) , tnf forward primer 5-ggttgtctttgagatccatgc-3 and reverse primer 5-acgtggaactggcagaagag-3 ( product length of 411  bp , tm 54.5c , 30 cycles ) , tgf forward primer 5-gaagtggatccacgagcccaag-3 and reverse primer 5-gctgcacttgcaggagcgcac-3 ( product length of 247  bp , tm 60c , 30 cycles ) , and stat3 forward primer:5-acccaacagccgccgtag-3 and reverse primer 5-cagactggttgtttccattcagat-3 ( product length of 192  bp , tm 62c , 30 cycles ) . multiplex reactions were run in duplicate , and pcr products were run on agarose gels . the intensity of dna bands was measured with quantity one software ( bio - rad , hercules , ca ) , and the results were normalized to the internal control -actin . the total homogenate was then centrifuged at 12,000  rpm for 30 minutes at 4c . the supernatant was collected , and protein concentration was measured using the dc protein assay method ( bio - rad , hercules , ca ) . twenty g proteins were separated on sds - page gels and transferred to pvdf membranes . the membrane was then blotted with primary antibodies for 1  h at room temperature . antibodies to il-17 ( h-132 ) , tnf ( n-19 ) , tgf1 ( v ) , stat3 ( h-190 ) , and -actin were purchased from santa cruz biotechnology ( santa cruz , ca ) , and anti - il-23 ( p19 ) and anti - p - stat3 ( y705 ) antibodies were obtained from abcam ( cambridge , uk ) . antibody dilution ratios : ( 1 ) il-17 and il-23 , 1  :  400 ; ( 2 ) stat3 , p - stat3 and tgf1 , 1  :  500 ; ( 3 ) tnf , 1  :  2000 ; and ( 4 ) -actin , 1  :  5000 . after three 10  min washes , membranes were incubated with anti - mouse , -rabbit , or -goat horseradish peroxidase ( hrp ) conjugated secondary antibodies ( zsgb - bio , beijing , china ) and washed , and detection was achieved using a dab kit ( zsgb - bio ) . densitometric analysis was performed using quantity one software , and the results are expressed as the relative intensity ( od ) of target protein to that of -actin control . nonspecific blocking was obtained by incubation in phosphate - buffered saline ( pbs ) containing 5% normal goat serum for 30 minutes at room temperature . tissue sections were then incubated with anti - p - stat3 ( 1  :  200 ) antibody for 1  h at room temperature . sections were washed with pbs , incubated in the abc reagent for 1 hour at room temperature , washed again , and incubated in a peroxidase solution ( zsgb - bio ) . the signal of immunohistochemical staining was analyzed with image - pro plus 6.0 software ( media cybernetics , maryland , usa ) . all statistical analyses were carried out using the spss statistical software suite ( version 16.0 ; spss inc . , single - factor analysis of variance ( one - way anova ) was used to analyze the differences between groups , with p < 0.05 considered as statistical significance . we have recently shown that oral administration of l. acidophilus ameliorates the pathogenesis of colitis in mice . given the critical role of th17 cells in autoimmune diseases including ibd , we hypothesized that l. acidophilus might improve colitis at least partially through suppression of th17 cell function . we thus initially sought to examine whether colonic expression of il-17 , the hallmark cytokine of th17 cells , is modulated by l. acidophilus treatment . the expression level of il-17 was examined in all nine groups at mrna and protein levels . we found that both mrna and protein expression of il-17 were significantly ( p < 0.05 ) enhanced in dss - induced colitis ( figures 1(a ) and 1(b ) ) , which is consistent with the results of a previous report . l. acidophilus application at different doses in the c4c8 groups all significantly ( p < 0.05 ) attenuated colitis - increased il-17 expression ( figures 1(a ) and 1(b ) ) compared to levels in the nontreated or vehicle treated groups . of all the l. acidophilus - treated groups , c5 showed the greatest inhibition of il-17 expression at both the mrna and protein levels , and this inhibition was even superior to the therapeutic effect of prednisone treatment ( figure 1(c ) ) . the electrophoresis images showing the mrna and protein expression of internal control -actin are presented for il-17 expression in figures 1(a ) and 1(b ) and are not repeatedly shown for the other examined molecules . because tnf is another important proinflammatory cytokine secreted by th17 cells , we also examined the effect of l. acidophilus treatment on tnf expression . similarly to il-17 , tnf expression at both the mrna and protein levels was significantly ( p < 0.05 ) decreased by l. acidophilus treatment in groups c4c8 compared to the vehicle treated control group ( figures 2(a ) and 2(b ) ) . however , unlike il-17 , tnf expression was lowest in group c6 among all the dss - treated groups ( figure 2(c ) ) . il-23 and tgf1 have been shown to play essential roles in promoting th17 cell differentiation , expansion , and function [ 9 , 14 , 17 , 18 ] . we tested whether the inhibitory effect of l. acidophilus on th17-associated cytokine expression is achieved through downregulation of il-23 and/or tgf1 expression , thereby suppressing th17 cell differentiation and stabilization . interestingly , treatment of colitis with a lower dose of l. acidophilus ( c4c6 groups ) caused a dramatic decrease in il-23 mrna expression , which reached a level comparable to that of the normal control group ( figure 3(a ) ) . moreover , all l. acidophilus - treated groups showed a significant ( p < 0.05 ) decrease in il-23 protein expression ( figure 3(b ) ) . notably , both the mrna and protein levels of il-23 expression were lowest in group c5 among all the colitis groups ( figure 3(c ) ) , at a level similar to that observed for il-17 expression . likewise , l. acidophilus administration at all doses induced marked inhibition of tgf1 mrna expression ( figure 4(a ) ) . a dramatic attenuation of tgf1 protein expression was also observed in the low dose groups , c4 and c5 ( figure 4(b ) ) . again , mice treated with 10  cfu/10  g l. acidophilus , group c5 , showed the lowest expression of tgf1 ( figure 4(c ) ) . stat3 is an important transcription factor that mediates the establishment of th17 cells in response to il-23 and tgf1 [ 15 , 16 ] . we therefore examined whether stat3 expression is altered with diminished expression of il-23 and tgf1 upon l. acidophilus treatment . we observed that stat3 expression was upregulated in the colon of dss - mediated colitis , which is consistent with previous reports [ 22 , 23 ] . although the decrease in the stat3 mrna level was relatively moderate in the l. acidophilus - treated groups ( figure 5(a ) ) , an apparent decrease in stat3 protein level was observed in contrast to the vehicle treated control group ( figure 5(b ) ) . among all the l. acidophilus - treated groups , inhibition of stat3 protein expression was most obvious in c5 ( figure 5(c ) ) . because phospho- ( p- ) stat3 is the functional form , it is important to understand whether the phosphorylation level of stat3 is altered by l. acidophilus treatment . we observed that p - stat3 was significantly ( p < 0.05 ) lower in all groups ( c4c8 ) relative to the untreated or vehicle treated control groups by western blotting analysis ( figure 6(a ) ) . moreover , immunohistochemical staining showed that p - stat3 localized in the nuclei was predominantly expressed in the inflamed area in the dss - induced untreated control group ( figure 6(b ) ) . importantly , l. acidophilus treatment caused a significant ( p < 0.05 ) reduction in p - stat3 signal , with a stronger effect in the c5 and c6 groups ( figures 6(b ) and 6(c ) ) . these findings suggest that administration of l. acidophilus improves colitis by restoring the homeostasis of p - stat3-mediated cellular events , such as th17 differentiation and il-17 secretion . ibds , including uc and cd , are considered a consequence of aberrant immune response in the gastrointestinal tract . it is traditionally recognized that cd is associated with th1 cytokines and uc is modulated by th2 cytokines . although controversy remains , th17 cells are generally accepted as the mediators of intestinal inflammation via the secretion of proinflammatory cytokines , such as il-17 [ 22 , 23 ] . therefore , targeting th17 cells can be a potential approach for treating ibd . with the accumulation of evidence supporting the significant role of microbiota in gut homeostasis [ 24 , 25 ] , probiotics have nowadays been more and more frequently used in treating ibd , especially uc , through induction of remission and prevention of pouchitis . however , whether and how probiotics modulate th17 cell fate in the gut remains poorly understood . we found the expression of il-17 , a hallmark cytokine characterizing th17 cells , was dramatically increased in colitis , which is consistent with previous reports [ 7 , 8 , 21 ] . moreover , we herein provide the first evidence that oral administration of l. acidophilus , one of the most common natural inhabitants of the human gut , suppressed the upregulation of the proinflammatory cytokine il-17 in colitis . that oral administration of l. gasseri suppresses il-17 induction in allergen - induced airway inflammation in mice . more recently , amdekar et al . showed that administration of l. acidophilus downregulates the expression of il-17 and to a lesser extent that of tnf in rats with collagen - induced arthritis . similarly , we also observed an inhibitory effect of l. acidophilus on colitis - mediated tnf expression in mouse colon . the decrease in il-17 expression might be an important contributor to the ameliorative effect of l. acidophilus on the pathogenesis of colitis as observed in our previous report , because inhibition of il-17 function with a chemical inhibitor or anti - il-17 antibody has been extensively demonstrated to be effective in ibd treatment [ 2830 ] . the observed downregulation of il-17 further indicates that administration of l. acidophilus may have modulated the fate of th17 cells . in inflammation , th17 cell differentiation is induced by paracrine signaling and th17 cells are further expanded to achieve full involvement . accumulating evidence demonstrates that tgf1 is critically involved in th17 cell differentiation , whereas il-23 plays an essential role in maintaining proliferation and survival of th17 cells . moreover , recent studies have shown that the expression levels of tgf1 and il-23 are both enhanced in ibd [ 3133 ] , thereby promoting the progression of inflammation by activating th17 differentiation and function . in this study , we showed for the first time that l. acidophilus inhibits the colitis - mediated increase in tgf1 and il-23 expression in mouse colon . these results implicate a decrease in tgf1 and il-23 expression upon l. acidophilus treatment might alter th17 cell population . however , this suspection needs be testified by immunohistochemical staining of th17 cell - specific marker , such as il-17 , in colitis treated with and without l. acidophilus . although we did not attempt to elucidate the molecular mechanisms that mediate the inhibitory role of l. acidophilus in il-23 regulation in the present study , we suspect that reduced expression of tnf may be responsible . in fact , previous studies have clearly shown that nuclear factor- ( nf- ) b , a critical mediator of tnf signaling , regulates the transcription of the il-23p19 gene . a recent finding by the de vrese lab showed that probiotics bifidobacterium breve and lactobacillus rhamnosus gg ( lgg ) inhibit lipopolysaccharide- ( lps- ) activated expression of il-23 in cultured intestinal cells via inhibition of histone acetylation and enhancement of dna methylation , providing another potential mechanism for l. acidophilus - mediated downregulation of il-23 . the results of this study further indicate that the efficacy of different concentrations of commensal probiotics on the expression of proinflammatory cytokines and relevant effectors must be carefully determined . we found that the greatest inhibitory effect was achieved in the c5 or c6 group depending on the molecule examined , which is largely consistent with our earlier observation that the optimal therapeutic effect against disease activity was from the c6 group . probiotic overdose might break the homeostasis of the gut community and cause aberrant immune response of the gastrointestinal defense system and thus altered expression of cytokine profiles . in conclusion , we showed that oral administration of l. acidophilus suppressed colitis - associated hyper - response of the il-23/th17 axis . specifically , l. acidophilus treatment inhibited the secretion of proinflammatory cytokine il-17 by downregulating il-23 and tgf1 , which are required for th17 cell differentiation and stabilization . these findings indicate that the therapeutic role of l. acidophilus in ibd treatment , at least in part , involves modulating the il-23/th17 immune axis .
the aim of this paper is to determine the modulatory effects of lactobacillus acidophilus on the il-23/th17 immune axis in experimental colitis . 
 dss - induced mouse models of uc were to be saline , hormones , and different concentrations of lactobacillus acidophilus intervention . 
 the expression of interleukin- ( il- ) 17 , tumor necrosis factor  ( tnf ) , il-23 , transforming growth factor 1 ( tgf1 ) , signal transducer and activator of transcription 3 ( stat3 ) , and phosphorylated ( p)-stat3 was examined by rt - pcr , western blotting , and immunohistochemical analysis . 
 and the results showed that administration of l. acidophilus suppressed th17 cell - mediated secretion of proinflammatory cytokine il-17 through downregulation of il-23 and tgf1 expression and downstream phosphorylation of p - stat3 .