Bifidobacterium infantis attenuates colitis by regulating T

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1 Sumit Mnuscript: Help Desk: DOI:.78/wjg.v.i8.86 World J Gstroenterol Decemer 8; (8): ISSN 7-97 (print) ISSN 9-8 (online) Bishideng Pulishing Group Inc. All rights reserved. ORIGINAL ARTICLE Bifidocterium infntis ttenutes colitis y regulting T cell suset responses Li Zuo, Ki-To Yun, Li Yu, Qing-Hong Meng, Peter Chee-Keung Chung, Ding-Hu Yng Li Zuo, Qing-Hong Meng, Peter Chee-Keung Chung, Deprtment of Immunology, Guiyng Medicl College, Guiyng 55, Guizhou Province, Chin Ki-To Yun, Deprtment of Surgery, the First Affilited Hospitl of Sun Yt-sen University, Gungzhou 58, Gungdong Province, Chin Li Yu, Deprtment of Peditrics, Gungzhou First Municipl People s Hospitl, Gungzhou 58, Gungdong Province, Chin Ding-Hu Yng, Deprtment of Heptoiliry Surgery, Nn Fng Hospitl, Southern Medicl University, Gungzhou 555, Gungdong Province, Chin Author contriutions: Zuo L nd Yun KT contriuted eqully to this work; Zuo L nd Yng DH designed the reserch; Zuo L, Yun KT, Yu L nd Meng QH performed the reserch; Chung PCK nd Yng DH contriuted to the new regents nd nlytic tools; Zuo L, Yun KT nd Yng DH nlyzed the dt; Zuo L nd Yun KT wrote the pper. Supported y Gungzhou Boxing Biotechnology Compny Correspondence to: Ding-Hu Yng, MD, PhD, Professor, Deprtment of Heptoiliry Surgery, Nn Fng Hospitl, Southern Medicl University, 88 North Gungzhou Ddo, Gungzhou 555, Gungdong Province, Chin. dhyng58@yhoo.com Telephone: Fx: Received: Octoer, Revised: My, Accepted: July, Pulished online: Decemer 8, Astrct AIM: to investigte the effect of Bifidocterium infntis (B. infntis ) on the T cell susets nd in ttenuting the severity of experimentl colitis in mice. METHODS: Norml BALB/c mice were fed different doses of B. infntis for wk, nd T cell susets nd relted cytokine profiles in mesenteric lymph nodes (MLNs) were detected y flow cytometry nd reltime RT-PCR. Colitis ws induced y dministrtion of trinitroenzene sulfonic cid (TNBS) in mice. Before colitis induction, mice were fed high dose B. infntis for wk. Cytokine profiles in MLNs nd histologicl chnges of colonic tissue were exmined 6 d fter colitis induction. RESULTS: No significnt chnge in cytokine profiles ws oserved in norml mice fed low dose B. infntis. However, Th-relted cytokines (IL-, IFN-γ, IL- p), Th7-relted trnscription fctor nd cytokines (RORγt, IL-, IL-), regultory T cell (Treg)-relted trnscription fctor nd cytokines (Foxp, IL-) were incresed in norml mice fed high dose B. infntis. Furthermore, flow cytometry ssy showed B. infntis incresed the numers of CD + Foxp + Tregs nd Th7 cells in MLNs. Colitis ws successfully induced y TNBS in mice, chrcterized y colonic inflmmtion nd errnt Th nd Th7 responses. Feeding high dose B. infntis for wk efore colitis induction decresed the inflmmtory cell infiltrtion nd golet cell depletion nd restored the intestinl epithelium. In ddition, B. infntis feeding reduced Th-relted cytokines (T-et, IL- nd IFN-γ) nd Th7-relted cytokines (IL-p, RORγt, IL-7A, IL- nd IL-), nd incresed Tregrelted molecules (Foxp, IL- nd TGF-β) in colitis mice. CONCLUSION: B. infntis effectively ttenutes TNBSinduced colitis y decresing Th nd Th7 responses nd incresing Foxp + Treg response in the colonic mucos. Bishideng Pulishing Group Inc. All rights reserved. Key words: Bifidocterium; Colitis; Cytokines; Th7; Regultory T cells Core tip: Inflmmtory owel disese is common utoimmune disorder chrcterized y chronic inflmmtion of the gstrointestinl trct. Anorml immune cell responses contriute to the pthogenesis of the colitis. Proiotics re found to regulte the intestinl immune system nd ply eneficil role in treting 86 Decemer 8, Volume Issue 8

2 colitis. In our study, we showed tht Bifidocterium infntis (B. infntis) reduced the intestinl inflmmtion in TNBS-induced colitis mice though decresing the Th nd Th7 responses nd promoting the Foxp + Treg response in mesenteric lymph nodes. This mechnism underlying the regultory effect of B. infntis on the immune system my hve significnt clinicl implictions in treting inflmmtory owel disese nd preventing colorectl cncer. Zuo L, Yun KT, Yu L, Meng QH, Chung PCK, Yng DH. Bifidocterium infntis ttenutes colitis y regulting T cell suset responses. World J Gstroenterol ; (8): Aville from: URL: com/7-97/full/v/i8/86.htm DOI: org/.78/wjg.v.i8.86 INTRODUCTION Inflmmtory owel disese (IBD) is n utoimmune disorder tht is chrcterized y chronic inflmmtion of the gstrointestinl trct. IBD comprises minly Crohn s disese (CD) nd ulcertive colitis. The pthogenesis of IBD involves genetic susceptiility, gut microiot nd host immune system []. It is widely ccepted tht the norml gut microiot plys key role in the pthogenesis of IBD. Severl studies showed striking reduction of commensl microiot including Firmicutes nd Bcteroides nd n overgrowth of Proteocteri in the intestinl tissue from IBD ptients []. Therefore, extensive reserch is focused on modifying the intestinl flor using proiotic cteri. Proiotics re live microorgnisms tht enefit helth when supplied in dequte mounts. Among them, Bifidocterium infntis (B. infntis) is commensl microe isolted from the humn gstrointestinl mucos nd hs een studied for its ility to tret irritle owel syndrome [], nd to protect ginst inflmmtory conditions including Helicocter pylori infection [] nd IBD [5-7]. The mechnisms ehind the meliortion of IBD y B. infntis re still lrgely unknown. IBD results from n indequte Foxp+ regultory T cell (Treg) response in the fce of overly exuernt Th nd Th7 responses in CD nd n norml Th response in ulcertive colitis [8]. B. infntis feeding reduced the numers of Th nd Th7 cells nd incresed the proportion of Foxp + Tregs within the lmin propri (LP) in DSS-induced colitis which mimics CD [5]. Moreover, B. infntis hd some influence on the expression of some cytokines. For instnce, B. infntis inhiited the expression of Th7 relted IL-7A nd induced the expression of IL- in DSS-induced colitis mice [7], nd decresed Th proinflmmtory cytokines (TNF-α, IL- nd IFN-γ) nd mintined the level of TGF-β in IL- knockout (KO) mice tht re spontneous genetic model of CD [6]. However, the effects of single B. infntis feeding on trinitroenzene sulfonic cid (TNBS)-induced colitis, which is n niml model of CD, nd the underlying immune mechnism were still poorly understood. We hypothesized tht B. infntis my meliorte the intestinl inflmmtion in TNBS-induced colitis y ffecting the differentition of CD + T susets nd cytokine expression. MATERIALS AND METHODS Animls Femle BALB/c mice (6-8-wk old) were purchsed from Chongqing Medicl University nd mintined in stndrd conditions. All the experimentl procedures were performed ccording to the guidelines of the Institutionl Animl Ethics Committee. Experiment design In the first experiment, norml mice were rndomly divided into groups of five nimls ech: control group, low dose B. infntis (6 7 cfu/d) group (Low-B. infntis) nd high dose B. infntis (6 8 cfu/d) group (High-B. infntis). B. infntis (6 9 cfu/g) ws provided y Gungzhou Boxing Biotechnology Compny (Gungzhou, Chin). Mice received orl dministrtion of low or high dose B. infntis once dily for wk. The control group received PBS. For induction of colitis, 5 μl TNBS [5 mg/kg ody weight (BW)] dissolved in.9% (w/v) NCl/ethnol (5:5 v/v) ws intrrectlly dministered to nesthetized mice cm proximl to the nl verge through.5f ctheter s descried previously [9]. TNBS ws purchsed from Sigm (St. Louis, MO, United Sttes). In the TNBS colitis model, three groups of five nimls ech were utilized. Group ws negtive control group, which ws dministered with PBS, insted of TNBS. Group ws designted s the TBNB- group s these mice were scrificed y cervicl disloction d fter dministrtion of TNBS. Group ws designted s the TNBS-6 group in which mice were scrificed 6 d fter dministrtion of TNBS. In the B. infntis treted model, two groups of five nimls ech were used. Group received dily orl dministrtion of high dose B. infntis (6 8 cfu/d) for wk efore colitis induction (B. infntis-tnbs group). The colitis control nimls were dministered with PBS using the sme technique efore colitis induction (PBS-TNBS group). Animls were scrificed y cervicl disloction 6 d fter TNBS induction, nd MLNs were selected crefully from the inflmed re of the owel in ll nimls. RNA extrction nd rel-time PCR Totl RNA ws extrcted from MLNs using TRIzol regent (Invitrogen) ccording to the mnufcturer s instructions. Totl RNA ( μg) ws trnscried to cdna using reverse-trnscription kit (TKR). Primer sequences were s follows: T-et (forwrd) 5 -CAACAA CCCCTTTGCCAAAG- nd (reverse) 5 -TCCCCCAAGCAGTTGACAGT-, IL- (forwrd) 87 Decemer 8, Volume Issue 8

3 5 -CCTGAGCAGGATGGAGAATTACA- nd (reverse) 5 -TCCAGA ACATGCCGCAGAG-, IFN-γ (forwrd) 5 -TCAAGTGGCATAGATGTGGAA, GAA- nd (reverse) 5 -TGGCTTGCAGGATTTTCATG-, IL-p (forwrd) 5 -GGAAGCACGGCAGCAGAATA- nd (reverse) 5 -AACTTGAGGGAGAAG TAGGAATGG-, GATA (forwrd) 5 -CCTACCGGGTTCGGATGTAA- nd (reverse) 5 -CACACACTCCCTGCCTTCTGT-, IL- (forwrd) 5 -ACAGGAGAA GGGACGCCAT- nd (reverse) 5 -GAAGCCCTACAGACGAGCTCA-, RORγt (forwrd) 5 -GCTGTCAAAGTGATCTGGAG- nd (reverse) 5 -GGTGGAACT TATGGGAAATC-, IL- (forwrd) 5 -GAGGACCCTTGTCTGTCTGG- nd (reverse) 5 -TCATCTTTTGAAGGAGCCATTT-, IL- (forwrd) 5 -GAGGTG GACTGGACTACCGA- nd (reverse) 5 -GGAACTGCTACTGCTCTTGA-, Foxp (forwrd) 5 -CCCATCCCCAGGAGTCTTG- nd (reverse) 5 -CCATGACTA GGGGCACTGTA-, IL- (forwrd) 5 -ATAACTGCACCCACTTCCCA- nd (reverse) 5 -TCATTTCCGATAAGGCTTGG-, TGF-β (forwrd) 5 -GAAGGCAGA GTTCAGGGTCTT- nd (reverse) 5 -GGTTCCTGTCTTTGTGGTGAA-, GAPDH (forwrd) 5 -CATCACTGCCACCCAGAAGA- nd (reverse) 5 -TGAAGT CGCAGGAGACAACC-. Rel-time reverse trnscription polymerse chin rection (RT-PCR) ws performed with the ABI 7 system. The rection mixture consisted of 5. μl of cdna,.5 μl of SYBR Green qpcr SuperMix-UDG/ROX (Invitrogen) nd 6.5 μl of ddh. All the PCR experiments were performed under the sme condition s follows: 95 for min, 95 for 5 s nd 6 for min, up to cycles. GAPDH ws used s n internl control. Gene expression ws clculted reltive to GAPDH. Flow cytometry nlysis Single cell suspensions were prepred from MLNs of ech group. In order to identify Tregs in MLNs, cells were first surfce leled with FITC leled nti-mouse CD (ebioscience) nd PE leled nti-mouse CD5 ntiodies (ebioscience). After tht, cells were fixed, permeted nd intrcellulrly stined with APC leled nti-foxp ntiody (ebioscience). To mesure Th7 cells, cells were pre-stimulted for h with PMA (5 ng/ml, Sigm) nd ionomycin (5 ng/ml, Sigm) in the presence of Brefeldin A ( mg/ml, ebioscience) t 7 nd 5% CO. Then, cells were wshed in PBS nd surfce leled with CD-FITC nd CD-PE-Cy5. For intrcellulr leling of IL-7A, these cells were permeilized with IL-7A fixtion/permeiliztion uffer (ebioscience) nd stined with nti-il-7a-pe (ebioscience). Cells were incuted with ffinity purified nti-mouse CD6/ to lock non-specific stining. IgG isotypes (BD phrmingen) were used s control in ll FACS experiments. Dt were cquired on FACS Cliur flow cytometer with CELLQuest softwre. Histologicl nlysis For histologicl exmintion, smples of colonic tissue locted precisely cm ove the nl cnl ws otined from the mice of ll groups. The colonic tissues were fixed in % neutrl uffered formlin nd emedded in prffin for histologicl nlysis. Four-micrometersections were deprffinized with xylene nd stined with hemtoxylin nd eosin using routine techniques. Sttisticl nlysis Sttisticl nlyses were crried out using GrphPd Prism for Windows (Version 5.). Significnt differences etween different doses of B. infntis groups were ssessed y one-wy nlysis of vrince (ANOVA) followed y Dunnett s multiple comprison test. Differences mong experimentl colitis groups induced y TNBS for d or 6 d were lso evluted using ANOVA followed y Dunnett s multiple comprison test. The difference etween PBS-TNBS group nd B. infntis- TNBS group ws mesured y pired t test. Dt re given s men ± SE. Differences were considered significnt t P <.5. RESULTS B. infntis ugments the expression of Th-, Th7- nd Treg-relted cytokines in MLNs in norml mice To investigte the effect of B. infntis on T cell cytokine expression, we studied the reltive expression of different cytokines in MLNs from norml mice y rel-time RT- PCR. Low dose B. infntis feeding (6 7 cfu/d) cused no significnt chnges in the expression of ll cytokines tested, ut high dose B. infntis feeding (6 8 cfu/d) ugmented the expression of Th-, Th7- nd Tregrelted cytokines in MLNs (Figure ). After high dose B. infntis feeding, the mrna expression of Th cytokines IL- nd IL-p incresed significntly in the MLNs when compred with PBS control (IL-, P <.5; IL- p, P <.5 ) (Figure A nd C). The mrna level of proinflmmtory cytokine IFN-γ lso seemed to elevte ut did not rech the significnce in comprison with the control group (Figure B). Th7-relted cytokine IL- nd trnscription fctor RORγ incresed more thn -fold in MLNs fter B. infntis feeding compred with the control group (P <.5) (Figure E nd G). IL- lso tended to rise. Moreover, mice receiving B. infntis hd significnt increse in Foxp nd IL- mrna expression in MLNs in comprison with PBS control (Foxp, P <.5; IL-, P <.5) (Figure H nd I). However, no difference in the mrna expression of Th-relted cytokine IL- ws detected fter B. infntis feeding (Figure D). B. infntis increses the levels of CD + T cells, Th7 cells nd CD + Foxp + Tregs in the MNLs in norml mice We mesured the numers of CD + T cells nd Th7 cells in the MLNs in norml mice y flow cytometry to determine if they were ltered fter B. infntis feeding. In order to identify CD + T nd Th7 cells, we utilized ntiodies ginst CD, CD, nd IL-7A to detect 88 Decemer 8, Volume Issue 8

4 A B C Low High IL- IFN-g IL-p D E F IL- RORgt IL- G H I IL- Foxp IL- Figure mrna expression of T cell-relted cytokines from mesenteric lymph nodes of norml BALB/c mice fed different doses of Bifidocterium infntis. A: IL-; B: IFN-γ; C: IL-p; D: IL-; E: RORγt; F: IL-; G: IL-; H: Foxp; I: IL-. : Mice from control group; Low: Mice treted with low dose Bifidocterium infntis (B. infntis); High: Mice treted with high dose B. infntis. Rel-time RT-PCR ws employed to exmine the expression of Th, Th7 nd Foxp + Treg relted cytokines. Dt re from 5 mice per group. Dt re expressed s reltive expression nd the control group ws chosen s the clirtor. Dt represent men ± SE. P <.5 vs the control group; P <. vs the control group. IL: Interleukin; IFN: Interferon. them. Flow cytometric nlyses reveled tht high dose B. infntis slightly incresed the percentge of CD + T cells in MLNs compred with PBS control (Figure A nd C). B. infntis lso up-regulted the level of Th7 cells (Figure B nd C). However, oth the increse of CD + T nd Th7 cells did not rech significnce (P >.5). For deciding the chnge of the popultion of Tregs fter B. infntis feeding, CD, CD5 nd Foxp were mesured y flow cytometry. B. infntis feeding slightly incresed the percentge of CD + Foxp + Tregs compred with control (9.99% ±.6% vs 9.% ±.8%) (Figure D). The incresed CD + Foxp + Tregs y B. infntis feeding were lso mostly derived from CD + CD5 + T cells, not from CD + CD5 - T (Figure B nd C). Colonic dmge in TNBS-induced colitis mice nd upregultion of Th- nd Th7-responses TNBS-induced colitis is well-chrcterized niml model of Th- nd Th7-medited colitis, nd it mimics humn CD. However, the pproprite time when the 89 Decemer 8, Volume Issue 8

5 A B. infntis C 6.%.77% CD %/CD + CD + T cells CD B. infntis B B. infntis D 6.%.7% IL-7A %/CD + CD7A + T cells CD B. infntis Figure Representtive flow plots nd levels of CD + T nd CD + IL-7A + T cells in mesenteric lymph nodes from norml mice fed high dose Bifidocterium infntis. A: Levels of mesenteric lymph node (MLN) CD + T cells in PBS-control nd Bifidocterium infntis (B. infntis) treted groups; B: Levels of CD + IL-7A + T cells in PBS-control nd B. infntis treted groups; C: The percentge of MLN CD + T cells; D: The percentge of CD + IL-7A + T cells. Results re either shown s men ± SE from three independent experiments, n = 5 (r grphs) or s representtive flow cytometry grph. pthologicl chnge of the colon ws drmtic ws not known. Therefore we decided to perform the histologicl nlysis in colitis control mice on dys nd 6 fter intrrectl instilltion of TNBS. Hemtoxylin nd eosin stining ws used to evlute the severity of TNBSinduced colitis. Integrl colonic epithelium nd no inflmmtory cell infiltrtion were oserved in the slices from the negtive control mice (Figure A). Sections of colonic tissues tken from mice on dy fter instilltion of TNBS showed extensive inflmmtory cell infiltrtion (Figure B). In contrst, mice on dy 6 fter the instilltion of TNBS exhiited golet cell depletion, colonic epithelil loss nd more inflmmtory cells infiltrted in the colon (Figure C). These microscopic evlutions of colons from mice with colitis induced y TNBS reveled pthologic correltions with CD nd longer durtion fter colitis induction ws linked to more severe inflmmtion in the colon. TNBS-induced colitis ws ssocited with excessive Th- nd Th7-responses. Therefore we mesured the reltive mrna expression of different T cell cytokines in MLNs y rel-time RT-PCR on dys nd 6 fter TNBS induction (Figure 5). Compred with the negtive control group, the mrna expression of Th cytokines (IL-, IFN-γ nd IL-p) displyed no chnge in colonic tissues from mice on dy fter TNBS induction (TNBS-) (Figure 5A-C). However, IL- nd IL-p incresed significntly in mice on dy 6 fter TNBS induction (TNBS-6) compred with PBS control group (IL-, P <.5; IL-p, P <.5) (Figure 5A nd C), nd the IFN-γ lso incresed little ut insignificntly. Mice in the TNBS-6 group hd significntly higher expression of Th7 relted molecules (RORγt nd IL- 7A) in the colonic tissue thn control mice (RORγt, P <.; IL-7A, P <.5) (Figure 5E nd H). The expression of other Th7-relted cytokines (IL- nd IL-) incresed drmticlly in the TNBS- group nd in the TNBS-6 group compred to the control group, though mice in the TNBS- group hd higher expression of oth cytokines thn mice in the TNBS-6 group (Figure 5F nd G). However, TNBS did not induce the chnge of IL- mrna representing Th (Figure 5D). Thus, we estlished the TNBS colitis model successfully nd the colon dmge ws ssocited with incresed ctivity of Th nd Th7 cells. Besides, we determined the pproprite time, on dy 6 fter colitis induction, to evlute the histologicl chnge nd Th- nd Th7- responses. Attenution of experimentl colitis y B. infntis is ssocited with downregultion of Th- nd Th7- responses We then investigted whether B. infntis reduced the severity of experimentl colitis. Mice were dily 8 Decemer 8, Volume Issue 8

6 A B. infntis 6 9.% 6.58% M 5 M Foxp B 9.6% 9.5% CD CD5.% Foxp C B. infntis.88%.86% CD CD5.% Foxp D %/CD + Foxp + Tregs 8 B. infntis Figure Representtive flow plots nd levels of Foxp + cells in mesenteric lymph nodes from norml mice fed high dose Bifidocterium infntis. A: Levels of Foxp + cells in mesenteric lymph nodes from PBS-control group nd Bifidocterium infntis (B. infntis) group; B: Levels of CD + Foxp + Tregs from PBScontrol group; C: Levels of CD + Foxp + Tregs from B. infntis group; D: The percentge of the CD + Foxp + Tregs. Dt re men ± SE with 5 mice per group from three independent experiments (r grphs) or s representtive flow cytometry grph. 8 Decemer 8, Volume Issue 8

7 A B C Figure Representtive histologicl imges of colon smples from trinitroenzene sulfonic cid-induced colitis mice. A: Colon imge from negtive control mouse; B: Colon imge from mouse on dy fter trinitroenzene sulfonic cid (TNBS) induction; C: Colon imge from mouse on dy 6 fter TNBS induction. All imges re t the sme originl mgnifiction ( ). dministrted with high dose B. infntis (6 8 cfu/d) for wk efore induction of colitis y the instilltion of TNBS. On dy 6 fter TNBS induction, sections of colonic tissues from mice in the PBS-TNBS group showed extensive golet cell depletion, epithelil loss s well s dense chronic inflmmtory infiltrtes in the LP (Figure 6A). In contrst, dministrtion of B. infntis drsticlly diminished golet cell depletion nd the intensity of inflmmtory infiltrtes nd restored the intestinl epithelium (Figure 6B). These results reveled tht B. infntis cn ttenute the intestinl inflmmtion in the experimentl colitis model. Next we tested whether Th/Th7 responses were downregulted fter B. infntis feeding in experimentl colitis mice. As shown in Figure 7, nlyses of cytokine mrna expression showed tht Th cytokines (IFN-γ nd IL-p) nd Th7 cytokines (IL-7A nd IL- ) were significntly decresed in the colons of mice receiving B. infntis efore induction of colitis in comprison with mice receiving PBS efore induction of colitis (IFN-γ, P <.5; IL-p, P <.5; IL-7A <.5; IL-, P <.) (Figure 7C, D, I nd J). Th cytokine IL- seemed to fll down ut not significntly (Figure 7B nd J). In ddition, B. infntis feeding led to lower levels of Th trnscription fctor T-et nd Th7 trnscription fctor RORγt compred with PBS control (T-et, P <.5) (Figure 7A nd G). No significnt difference in mrna expression of Th-relted GATA nd IL- ws mesured etween the PBS-TNBS group nd B. infntis- TNBS group. However, surprisingly, B. infntis feeding incresed the mrna expression of Foxp, IL- nd TGF-β in colitis mice significntly when compred with the PBS-TNBS group (Foxp, P <.; IL-, P <.5; TGF-β, P <.5) (Figure 7K-M). Tken together, these results show tht B. infntis feeding ttenutes the development of colonic inflmmtion in TNBS-induced colitis model in ssocition with downregultion of oth Th- nd Th7-responses. DISCUSSION This study showed tht B. infntis feeding to mice reduced the severity of TNBS-induced colitis, which ws ssocited with suppression of Th nd Th7 responses nd promotion of Foxp + Treg response within MLNs. This reveled tht the ttenution of TNBS-induced colitis y B. infntis feeding is medited y regulting the differentition of CD + T susets nd cytokine expression. We found tht B. infntis feeding reduced the severity of TNBS-induced colitis. TNBS instilltion resulted in extensive golet cell depletion, epithelil loss nd dense inflmmtory cell infiltrtions in the LP. However, colitis mice with B. infntis feeding showed noticele resolution of inflmmtory infiltrtes nd golet cell depletion nd restored intestinl epithelium. Next, the study explored the reltion etween the ttenution of colitis y B. infntis nd the chnge of T cell suset responses, given tht immune cell responses ply crucil role in the pthogenesis of colitis. Mucosl dendritic cells present gut ntigens to the dptive immune system nd then direct the polriztion of nïve CD + T cells towrds different effector T-helper cells (Th, Th nd Th7) nd Tregs []. These effector Th cells re importnt for comting pthogen infections; when errnt they relese enormous proinflmmtory cytokines nd initite excessive inflmmtion, resulting in chronic inflmmtory diseses. Nonetheless, the gut immune system hs mechnisms of tolernce tht void uncontrolled Th, Th or Th7 responses. Foxp + Tregs displyed immunosuppressive function y suppressing Th cells nd secreting nti-inflmmtory cytokines IL- nd TGF-β []. It is well known tht errnt Th nd Th7 responses nd deficient Treg response contriute to IBD. Clssiclly, CD hs een ssocited with Th response. In our study, levels of Th cytokines (IL-, IL-p nd IFN-γ) were up-regulted in the MLNs from TNBS-induced colitis mice. Th cells secrete high level of IFN-γ, proinflmmtory cytokine responsile for mcrophge ctivtion. IFN-γ is essentil for IBD pthogenesis s the dministrtion of nti-ifn-γ ntiody to mice cn prevent the development of colitis [] nd the tretment with fontolizum, n nti-ifn-γ ntiody, improved clinicl response nd remission in ptients with ctive CD compred with plceo []. Consistent with these results, our colitis mice were detected to hve 8 Decemer 8, Volume Issue 8

8 A 6 B C 8 TNBS- TNBS-6 6 IL- IFN-g IL-p D. E 8 F IL- RORgt IL- G 5 H IL- IL-7A Figure 5 mrna expression of T cell-relted cytokines from mesenteric lymph nodes of trinitroenzene sulfonic cid-induced colitis mice. A: IL-; B: IFN-γ; C: IL-p; D: IL-; E: RORγt; F: IL-; G: IL-; H: IL-7A. Rel-time RT-PCR ws employed to exmine the expression of Th, Th7 nd Foxp + Treg relted cytokines. : In the control group, the nimls were dministered with PBS; Trinitroenzene sulfonic cid (TNBS)-: In the TNBS- group, the nimls were scrificed d fter TNBS induction; TNBS-6: In the TNBS-6 group, the nimls were scrificed 6 d fter TNBS induction. Dt re from 5 mice per group. Dt re expressed s reltive expression nd the control group ws chosen s the clirtor. Dt represent men ± SE. P <.5 vs the control group; P <. vs the control group. IL: Interleukin; IFN: Interferon. reduction of IFN-γ mrna in MLNs fter B. infntis feeding. IL- hs two suunits, p nd p5, nd it is minly produced y mcrophges/monocytes nd cn e efficiently induced y intrcellulr cteri. IL- plys pivotl role in the Th differentition nd induces nïve T cells to produce IFN-γ. Administrtion of ntiody ginst its suunit IL-p ttenuted TNBS colitis with decresed levels of Th cells nd IFN-γ in the LP []. In ptients with ctive CD, nti-il-p tretment reduced Th-medited inflmmtory cytokines nd promoted clinicl remission [5]. In this experiment, colitis mice showed down-regulted mrna of IL-p following B. infntis feeding. Further, the study reveled tht Th trnscription fctor T-et ws up-regulted in colitis mice ut ws down-regulted y B. infntis feeding. T-et is memer of the T-ox trnscription fctor fmily tht is essentil for Th development s Th cells cnnot e generted either in vitro or in vivo without it [6]. These 8 Decemer 8, Volume Issue 8

9 A B Figure 6 Representtive histologicl imges of colon smples from trinitroenzene sulfonic cid-induced colitis mice fed high dose Bifidocterium infntis. A: Colon imge from trinitroenzene sulfonic cid (TNBS)-induced colitis mouse fed PBS; B: Colon imge from TNBS-induced mouse fed high dose Bifidocterium infntis. All imges re t the sme originl mgnifiction ( ). outcomes showed B. infntis seemed to weken the Th cell response vi regulting its differentition-relted fctors nd cytokine expression in TNBS-induced colitis mice. In the recent decde, the discovery of the Th7 suset hs rpidly expnded the understnding of IBD. High levels of Th7 nd its cytokine IL-7 were detected in inflmed colons of mice nd ptients with IBD [7]. IL- 7A is potent meditor of inflmmtory responses s it induces proinflmmtory cytokines such s IL-6, IL-8, nd monocyte chemotctic protein- (MCP-) [8]. Incresed IL-7A mrna ws mesured in our colitis model, though B. infntis reduced it. Likely, in DSSinduced colitis, B. infntis decresed Th7 cells nd IL- 7A [5]. Interestingly, IL-7A ws lso verified to induce production of IL- nd promote the Th response [9], implying tht IL-7A downregultion y B. infntis my fcilitte the inhiition of Th response in IBD. IL-, CD + T cell-derived cytokine produced in excess in IBD ws found to promote the differentition of Th7 cells from nïve CD + T cells nd prevent the TGF-βdependent expression of Foxp y nïve CD + T cells []. IL--deficient mice hve less Th7 cells with reduced severity of colitis fter induction y either DSS or TNBS []. However, we found tht B. infntis feeding led to the reduction of IL- in colitis mice. Orphn nucler receptor RORgmmt (RORγt) is constitutively expressed y Th7 cells nd is the key trnscription fctor to Th7 differentition []. In ddition, RORγt cn regulte the production of IL-7A nd IL-7F to induce chronic colitis []. The niml experiment showed reduction in RORγt mrna in colitis mice fed B. infntis. Another key cytokine, IL secreted y DCs potently promoted Th7 mplifiction, sustined nd strengthened the Th7 phenotype. Severl groups concurrently showed tht IL- plyed crucil role in development of IBD in different colitis models prtly ecuse IL- induced proinflmmtory cytokines such s TNF-α, IFN-γ nd IL-6 in the colon []. Moreover, other work hs shown tht IL- inhiited Foxp + Tregs to induce colitis [5]. It is, therefore, of gret importnce to find tht B. infntis feeding reduced the IL- mrna in the colitis model. Tken together, B. infntis suppressed Th7 responses in TNBS-induced colitis mice. Foxp + Tregs re specilized clss of lymphocytes chrcterized y the expression of the forkhed trnscription fctor Foxp. Foxp + Tregs re essentil for norml immunohomeostsis y suppressing Th effector cells []. The importnce of Tregs in the prevention of colitis hs een confirmed y previous studies showing tht trnsfer of nïve CD + T cells without Tregs into immunodeficient mice resulted in inflmmtion nd colitis, while co-trnsfer of Tregs meliorted or reversed pthology [6]. In IBD ptients, reduced numers of Foxp + Tregs were oserved in the peripherl lood nd greter numers of Foxp + Tregs in the intestinl mucos, lthough the increse in the intestine of IBD ptients ws lower compred with non-ibd inflmmtory conditions []. Given the lower Tregs, the expnsion of Tregs seems to e therpeutic tool for IBD. There is some evidence tht proiotic dministrtion is le to meliorte the colitis y incresing Foxp + Tregs in the colonic tissue, such s proiotic mixtures (lctocillus, ifidocterium nd streptococcus; lctocillus nd ifidocterium; ifidocterium, lctocillus nd enterococcus) [7-9]. In our norml mice, single B. infntis feeding incresed the Foxp + Tregs in the MLNs. Similrly, B. infntis feeding incresed Foxp mrna in colitis mice. Consistent with our results, humn volunteers tking B. infntis hd elevted levels of Foxp expression in PBMCs []. The expression of Foxp is criticl ecuse it controls the development of Tregs nd the immunosuppressive function of Tregs requires high level of Foxp expression []. Decresed Foxp expression could led to impired Treg function nd e cusl for immune disorders including humn immunodysregultion, polyendocrinopthy, enteropthy, X-linked (IPEX) syndrome []. Hence, the incresed Foxp mrna induced y B. infntis feeding my enhnce the immunosuppressive ctivities in colitis mice. Besides, B. infntis incresed Treg-relted IL- nd TGF-β in the colonic tissue in our colitis model. IL- is potent ntiinflmmtory cytokine, produced y oth nonimmune nd immune cells including Tregs. IL- hs prticulr significnce in IBD ecuse IL--deficient mice develop colitis [] nd Treg-specific deletion of IL- resulted in 8 Decemer 8, Volume Issue 8

10 A.5 B.5 C.5 PBS-TNBS B. infntis -TNBS T-et. IL-. IFN-g D.5 E.5 F IL-p. GATA. IL- G.5 H.5 I RORgt. IL-7A. IL- J.5 K.5 L IL-. Foxp. IL- 85 Decemer 8, Volume Issue 8

11 M.... TGF- Figure 7 mrna expression of T cell-relted cytokines from mesenteric lymph nodes of trinitroenzene sulfonic cid-induced colitis mice fed high dose Bifidocterium infntis. A: T-et; B: IL-; C: IFN-γ; D: IL-p; E: GATA; F: IL-; G: RORγt; H: IL-7A; I: IL-; J: IL-; K: Foxp; L: IL-; M: TGF-β. Rel-time RT-PCR ws employed to exmine the expression of Th, Th7 nd Foxp+ Treg relted cytokines. PBS-TNBS: In the PBS-TNBS group, the nimls were fed PBS efore TNBS induction; Bifidocterium infntis (B. infntis)-tnbs: In the B. infntis-tnbs group, the nimls were fed high dose B. infntis efore TNBS induction. Dt re expressed s reltive expression nd the control group ws chosen s the clirtor. Dt re men ± SE (n = 5, per group). P <.5 vs PBS-TNBS, P <. vs the control group. IL: Interleukin; IFN: Interferon; TNBS: Trinitroenzene sulfonic cid. spontneous colitis []. Aprt from IL-, Tregs produce lrge mounts of TGF-β. TGF-β is potent regultory cytokine tht inhiits Th cell prolifertion, differentition nd ctivtion, nd decreses secretion of hrmful cytokines [5]. Appliction of TGF-β locking ntiody eliminted the ility of Tregs to prevent colitis nd TGFβ-deficient Tregs filed to suppress colitis [6]. Wht s more, TGF-β is involved in the induction of Foxp nd drives the differentition of nïve T cells to Treg phenotype [7]. Overll, B. infntis promoted Foxp + Treg responses in ssocition with incresed levels of Foxp, L- nd TGF-β in the MLNs in TNBS-induced colitis mice. However, B. infntis feeding hd some different effects on the immune cells in different gut environments. In norml gut environment high dose elicited some Th, Th7 nd Treg responses with slight increses of Th7 cells nd Tregs nd their-relted cytokines, though this did not cuse pthologicl inflmmtion. One reson why B. infntis feeding resulted in little high immune response is tht B. infntis, which is derived from the humn gut, is regrded s foreign ntigen, therey triggering wek responses. But this immune response did not led to pthologicl inflmmtion, which indicted tht there ws proper lnce mong Th, Th7 nd Tregs in mintining gut homeostsis. This ws confirmed y evidence tht good lnce etween Tregs nd Th7 is essentil for oth eliminting potentil pthogens nd suppressing intestinl inflmmtion [8]. In some wy, proper increses in Th nd Th7 responses my enhnce the mucosl defense ginst pthogens nd thus e eneficil to the promotion of the host intestinl immunity. It is of gret importnce for the potentil of B. infntis to suppress IBD ecuse the risk of colorectl cncer is incresed in ptients with ulcertive colitis nd CD [9]. Animl models reveled tht the setting of intestinl inflmmtion contriuted to the tumorigenesis through T cell nd TLR-medited inflmmtion []. IL-7 immunorective cells were significntly expressed in the norml mucos of colon cncer ptient []. However, high density of Foxp + Tregs in tumor tissue ws ssocited with improved prognosis in colorectl cncer ptients in erly T stge []. Thus reducing chronic intestinl inflmmtion ppers to e good strtegy in preventing the incidence of colorectl cncer. The microil dysiosis ws found in colon cncer ptients [], indicting tht improvement of intestinl flor environment my enefit these ptients. Studies showed tht supplementtion of vile Bifidocteri efore surgery cn reduce infection compliction of surgery in ptients with colorectl cncer []. More importntly, proiotics cn dely or lock the development of tumor from inflmmtion disorders y reducing inflmmtion. Pretretment with the proiotic VSL#, mixture of lctocillus, ifidocterium nd streptococcus, cn ttenute the inflmmtion in TNBS-induced colitis, delying trnsition to dysplsi nd cncer []. Bifidocterium lctis prevents DSS-induced cute colitis nd DSS colitisssocited cncer in mice [5]. B. infntis hs the potentil to ttenute intestinl inflmmtion through suppressing Th nd Th7 cell responses s well s promoting Foxp + Tregs. Therefore, B. infntis my reduce risk of the trnsition from IBD to colorectl cncer, even slow down the existing cncer progression nd improve the prognosis. However, the mjor drwck of this study is tht we filed to mesure the frequencies of Th7 cells nd Foxp + Tregs nd identify their origin in colitis mice nd B. infntis-fed colitis mice. The expression of Foxp cn e induced in nturlly thymic-drived CD + CD5 + Tregs or in mture CD + conventionl T cells in vitro nd in vivo []. Nevertheless, Foxp + Tregs from CD + CD5 + Tregs or from CD + CD5 - conventionl T cells oth hve similr immunosuppressive functions. In norml mice, we found tht incresed Foxp + Tregs y B. infntis were mostly derived from the CD + CD5 + Tregs in the 86 Decemer 8, Volume Issue 8

12 MLNs. But whether the origin of the incresed Foxp mrna in MLNs in our colitis mice nd B. infntistreted colitis mice ws lso derived from CD + CD5 + Tregs or from peripherl CD + CD5 - T ws not known. Previous studies showed tht feeding mixed proiotics (ifidocterium, lctocillus nd enterococcus) or (lctocillus nd ifidocterium) led to elevted levels of CD + CD5 + Foxp + cells in MLNs or intrepithelil in TNBS colitis mice [8,9]. However, in the sme model, proiotic IRT5 dministrtion incresed the CD + CD5 Foxp + cells in MLNs [7]. For this discrepncy, one explntion is tht the individul component of proiotic mixtures hd different function on the popultion of Tregs in the intestine. In order to elucidte the origin of elevted levels of Foxp mrna in our B. infntis-treted colitis mice, further studies will e done in the future. In conclusion, we showed tht B. infntis ttenuted intestinl inflmmtion in TNBS-induced colitis mice y suppressing the Th nd Th7 responses nd incresing the Foxp + Treg response in MLNs. This mechnism underlying the regultory effect of B. infntis on the immune system my hve significnt clinicl implictions in treting IBD nd preventing colorectl cncer. ACKNOWLEDGMENTS The uthors thnk Gungzhou Boxing Biotechnology Compny for providing the Bifidocterium infntis. COMMENTS Bckground Crohn s disese (CD), one type of inflmmtory owel disese, is chrcterized y chronic inflmmtion of intestinl trct. Anorml microiot nd mucosl immune cell responses contriute to the pthogenesis of CD. Reserch frontiers Altertions of gut microiot cuse immune dysregultion, leding to utoimmune ttcks, s result of serious dmge of the intestinl mucosl rrier. CD involves errnt Th nd Th7 responses nd deficient T regultory cell response. This suggested tht regulting immune cell responses could potentilly ttenute intestinl inflmmtion. Innovtions nd rekthroughs In this study, the uthors found tht Bifidocterium infntis (B. infntis) hd the potentil to effectively ttenute the colitis induced y,,6-trinitroenzene sulfonic cid. These eneficil effects were ssocited with the role of B. infntis in the suppression of Th nd Th7 cell responses, the promotion of Foxp + T regultory cell response, nd restoring the lnce of T cell susets responses. Applictions Understnding the mechnism underlying the effects of B. infntis in treting experimentl colitis hs significnt clinicl implictions in effectively nd sfely treting inflmmtory owel disese nd further preventing colorectl cncer ssocited with intestinl inflmmtion. 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14 Zhng JW, Du P, Go J, Yng BR, Fng WJ, Ying CM. Preopertive proiotics decrese postopertive infectious complictions of colorectl cncer. Am J Med Sci ; : 99-5 [PMID: 9798 DOI:.97/MAJ.e8ce6] Appleyrd CB, Cruz ML, Isidro AA, Arthur JC, Join C, De Simone C. Pretretment with the proiotic VSL# delys trnsition from inflmmtion to dysplsi in rt model of colitis-ssocited cncer. Am J Physiol Gstrointest Liver Physiol ; : G-G [PMID: 976 DOI:.5/jpgi.67.] 5 Kim SW, Kim HM, Yng KM, Kim SA, Kim SK, An MJ, Prk JJ, Lee SK, Kim TI, Kim WH, Cheon JH. Bifidocterium lctis inhiits NF-kppB in intestinl epithelil cells nd prevents cute colitis nd colitis-ssocited colon cncer in mice. Inflmm Bowel Dis ; 6: 5-55 [PMID: DOI:./id.6] P- Reviewer: Ciccone MM, Foligne B, Lee I S- Editor: M YJ L- Editor: Wng TQ E- Editor: Wng CH 89 Decemer 8, Volume Issue 8

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