T-Bet Is Dependent on Stat-4 Inhibiting Acute Colitis but Not Stat …downloads.hindawi.com/journals/tswj/2018/8571920.pdf · 2018-11-12 · ey were obtained from Pusvetma Surabaya

Research ArticleT-Bet Is Dependent on Stat-4 Inhibiting Acute Colitis but NotStat-1 Using L4 Somatic Antigen of Heligmosomoides polygyrus

Agustina Tri Endharti 12 and Sofy Permana3

1Department of Parasitology Faculty of Medicine Brawijaya University Indonesia2Biomedical Central Laboratory Faculty of Medicine Brawijaya University Indonesia3Department of Biology Faculty of Mathematics and Natural Sciences Brawijaya University Indonesia

Correspondence should be addressed to Agustina Tri Endharti tinapermanayahoocom

Received 5 January 2018 Revised 25 March 2018 Accepted 17 April 2018 Published 7 June 2018

Academic Editor Srinivas Mutalik

Copyright copy 2018 Agustina Tri Endharti and Sofy PermanaThis is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Helminths may alter the immunoinflammatory reactions of colitis Proteins derived from H polygyrus have prospective therapyfor colitis The goal of this study was to interpret the protective mechanisms of L4 somatic antigen (LSA) from Heligmosomoidespolygyrus against an inflammatory response to the pathogenesis of DNBS-induced colitis Colitis was actuated in mice by rectalinstillation of DNBS The mice were randomly divided into five groups containing control DNBS alone and three groups withdifferent doses of LSA (50 100 and 200 120583gmL) respectively Mice initiated colitis by rectal administration of DNBS and after thatwere immunized with LSA for 14 days Mice treated with LSA inhibited wasting disease compared with DNBS only group Thepercentages of cells producing IFN-120574 were reduced by LSA treatment The level of T lymphocytes CD4+IFN-120574+ cells in the LPLwas significantly diminished by LSA at both 100 and 200 120583gmL groups (plt005) The mRNA expression of T-bet was significantlydeclined in LSA immunizedmice but not ROR120574-TmRNA whereas GATA-3 expression tended to increaseThe activation of STAT-4 significantly reduced LSA-treatedmice but not STAT-1 It can be concluded thatT-bet is required for optimal production of IFN-120574in colitis

1 Introduction

Ulcerative colitis (UC) is an inflammatory bowel diseasethat causes long-lasting inflammation in the inner mostlining of the colon [1 2] UC is a chronic immune dis-order affecting the gastrointestinal tract It is a chronicgastrointestinal illness described by inflammatory changesand mucosal tissue damage [3 4] Obsessive signs of UCincorporate ulceration of the mucosa decreasing and lossof the crypts and infiltration of inflammatory cells [5 6]The signs of colitis stimulated by 24-dinitrobenzene sulfonicacid (DNBS) in a rodents models are similar to humanUC these incorporate looseness of the bowels loss of bodyweight shortening of the colon ulceration of themucosa andhistopathological lesions as in humanUC [5ndash9] UC is for themost part related to a T helper type 1 (Th1) immune response[8 9] Studies have proposed thatTh1 cytokinesmay assume apart in the enhanced inflammation perceived in the intestinal

epithelium of patients with UC [8 10] Besides the mucosaof patients diagnosed with Crohnrsquos disease is dominatedby Th1 which contains greater part of CD4+ lymphocytes[8 10] UC is associated with proinflammatory cytokinesresponses eg IFN-120574 increasing IFN-120574 production by Tcell lymphocytes [10] Many researchers described that atreatment including infection with live worms decreases theinflammation related to autoimmune diseases as Crohnrsquosdisease [8 10] In contrast the application of live parasitesis disadvantageous because as a side effect of a live wormtreatment the parasite might invade other tissues of thehuman host [8ndash12] H polygyrus is regulated by IFN-120574-producingCD4T cells directly correlatedwith the percentageof T-bet GATA-3 controls Th2 activity by inducing Th2cytokine gene expression Additionally T-bet and GATA-3are transcription factors required to promote Th1 and Th2differentiation [8 12] Generation of Th1 cells from naiveprecursors requires T-bet signaling that leads to activation

Hindawie Scientific World JournalVolume 2018 Article ID 8571920 9 pageshttpsdoiorg10115520188571920

2 The Scientific World Journal

of STAT-4 [2 13] STAT-4 induces expression of T-bet thatacts as a key regulator of Th1 cells [13ndash15] H polygyrus waseffectively used to maintain typical conditions in chroniccolitis [2 16] Allowing for the fact that helminthes maymodulate the inflammatory response of colitis we exploredthe effect of H polygyrus on mice in an experimental colitismodel In any case little is thought about the basic mech-anisms of the caring effect of somatic antigen derived fromL4H polygyrus larvae against an inflammatory response Weare concerned about proving the expression level of tran-scription factor reflecting inhibition of inflammation duringDNBS

T-bet performs a crucial function in immune responseby modulating the some genes involved in the inflammatoryresponses [16] Hence by monitoring the transcription ofinflammatory cytokine genes T-bet plays a central rolein regulating the immune responses and inflammationT-bet is a critical transcription factor that controls dif-ferentiation of Th1 cells it specifically binds to the pro-moter of the IFN-120574 gene and activates its transcription[17ndash19]

Worm therapy has been established recently The mecha-nism of human disease of the numerous worm species canbe studied using a mouse model of H polygyrus infection[11 16] As confirmed by many live nematode treatment isconsidered an alternative therapy [18ndash20] this way has alittle harm because patients are infected by live nematodes[17ndash23] Additionally the specific mechanisms sustainingthe therapeutic effect of gastrointestinal nematodes are notopenly understood Moreover the pathology related to worminfection requires for the therapy using live worms to beexchanged with nematodes-derived proteins [2 11 16] Theexcretory-secretory proteins derived from H polygyrus havea potential in the therapy of colitis [24 25] A protein basedtreatment could overcome the disadvantages of usage thatrelies on living parasitesThe goal of this study was to explorethe effect of L4 larvae extract (LSA) on clinical indicationsof UC including body weight and extent of occult bleedingin a mouse model Studies reported that mice with DNBS-induced colitis exhibit characteristics similar to chronic UCin human [17 24 25] In addition prolonged and chronic UCdevelop to a colorectal cancerTh1 andTh2 cells are associatedwith increased expression of Gata-3 and reduced expressionof T-bet inmemoryCD4T cells CD4T cells expressingGata-3 were significantly increased in H polygyrus induced miceThis study also verifies the assessment of the effect of LSA onthe expression of T-bet and GATA-3 in DNBS treated colonictissue To verify the role of STATs signaling in the regulationof T-bet in LSA-treatedmice we examined whether the T-betgene targets were generally expressed as STAT1 and STAT4Therefore we explored the possibility that STATs activitycould play a major role in modulating the LSA mediated T-bet expression

It is greatly likely that studies employing the modelsused in the current study might contribute key informationabout the pathogenesis of human inflammatory colonicdiseases in the future Certainly the exact mechanism bywhich LSA affects T-bet and GATA-3 should be investigatedfurther

2 Materials and Methods

21 H polygyrus Establishment Third-stage infective larvae(L3) of H polygyrus were obtained from Jikei University ofTokyo and maintained at Parasitology Laboratory MedicalFaculty Brawijaya University Indonesia Female Balbc micewere 8 weeks old at the start of the study Mice wereinoculated by oral gavage with 200 L3 of H polygyrususing a 20-gauge ball-tip feeding tube Infective unsheathedL3 of H polygyrus (Parasitology Laboratory Collection noH120) were obtained from fecal cultures of eggs according toChavarrıa et al [22] with modification L3 collections werepropagated and stored at 4∘C until used

22 Mice Female Balbc mice weighing 25-30g were usedThey were obtained from Pusvetma Surabaya The micewere keep under standard conditions of temperature of 25-27∘C relative humidity (55plusmn5) and 12h12h lightdark cycleMice were given normal drinking water ad libitum duringthe experimental periods All experiments were conductedaccording to the principles of Guide for the Care and Useof Laboratory Animals in Indonesia and were approvedby the Ethical Committee of Brawijaya University MalangIndonesia (No 171-KEP-UB)

23 Preparation of L4 Somatic Antigen (LSA) From Hpolygyrus Previously third-stage infective larvae (L3) wereadministered orally for each mouse with single dose of 200L3 Mice were sacrificed on day 15 after LSA treatmentand then L4 were collected from mouse intestine LSA wasprepared from L4 of H polygyrus according to Chavarrıaet al [22] Briefly LSA were collected from 100 L4 whichwere washed several times in Phosphate Buffer Saline (PBS)supplemented with 100mgmL penicillin and 100 mgmLstreptomycin Total L4 were homogenized in a cocktail ofextraction buffer (2mM EDTA 10mM PMSF and 30mMpotassium phosphate in PBS) followed by centrifugation at10000 rpm for 15 minutes The final protein concentrationof L4 homogenate was measured by the Bradford techniqueTotal protein of antigen containing lt20 endotoxin unitsmgwas collected and stored at -30∘C until used

24 Induction of Experimental Colitis and Injection of LSAMice were randomly divided into five groups includinggroup 1 including vehicle (control) groups 2-5 with rectaladministration of DNBS and groups 3-5 that were injected(intraperitoneally) with LSA (50 100 or 200 120583gmL respec-tively) once during 14 days (Figure 1) Colitis was inducedby rectal instillation of 5mgkgBW of DNBS (Sigma Chem-ical Co St Louis USA) according to procedure describedby Morampudi et al [23] with few modifications Brieflycolitis was induced in mice by rectal instillation of 5 mgof DNBS100 120583l of 50 ethanol into the rectum through acatheter inserted 4 cm proximally to the anus The volumeof DNBS enema was 100 120583L Thereafter the mice were keptfor 90 seconds in a head-down position to avoid loss of theDNBSThe injection of LSAwas started from second day andcontinued until 14 days Development of colitis was assesseddaily by using an occult blood detection kit (Hemoccult)

The Scientific World Journal 3

Figure 1 Experimental colitis was induced by administration ofDNBS followed by LSA injection Firstly colitis was induced inBalbc mice by rectal instillation of DNBS followed by regularinjection of LSA (L4 somatic antigen) for four times a week (1 3 5 79 11 and 13) For the experiment mice were randomly divided into5 groups I received 50 ethanol only (control) II was given DNBSonly IIIndashV groups were injected with different concentrations ofLSA (50 120583gmL 100 120583gmL and 200 120583gmL) respectively until 14daysThe injection of LSA was started from day 1 until day 13 At theend of 14 days mice in all groups were sacrificede 50 ethanol darrDNBS LSA

At the end of day 14 mice in all groups were sacrificed andcolon tissues were removed and cleaned and then subjectedto histological examination RNA isolation PCR and flowcytometry

25 Clinical Assessment of Colitis Body weight diarrheascores and bleeding scores were assessed daily [17 24 25]Body weight change was monitored The body weights andoccult blood test results were recorded

26 Histological Score of Colitis The microscopic cross sec-tions of the colons were histologically investigated For histo-logical examination the colonic tissue was fixed in 10 for-malin dehydrated paraffin-embedded processed and slicedinto 5120583m thick sections Furthermore the sections weredeparaffinized with xylene and stained with hematoxylin-eosin Histological changes were graded semiquantitativelyfrom 0 to 4 according to Erben et al [26] that describedcriteria as follows (0) no symptoms of inflammation at all(1) very low levels of leukocyte infiltration and loss of lt10crypts (2) low level of leukocyte infiltration and loss of cryptsbetween 10 and 20 (3) high level of leukocyte infiltrationhigh vascular density and thickening of the colon wall andloss of crypts between 20 and 30 (4) severe ulcerationtransmural infiltration loss of goblet cells high vasculardensity and thickening of the colon wall with loss of gt30crypts To have a quantitative estimation of colon damageall slides were evaluated using light microscope and scoredby two independent observers blinded to the experimentalgroups

27 Isolation and Culture of Mouse Colon Lamina PropriaLymphocyte (LPL)Cells TheculturemediumusedwasRPMI1640 (Gibco) supplemented with 10 Fetal Bovine Serum(FBS) 20 mM Hepes 4 mM glutamine 100 Uml penicillin

and 100 Uml streptomycin (Sigma) Briefly colon tissueswere cut off into 1 mm pieces This tissue fragments wereincubated in HBSS (Sigma) at 37∘C under shaking for 30min in the presence of 200 Uml type I collagenase (Sigma)and 100 Uml hyaluronidase (Sigma) 2 mM EDTA and 25mM Tris (Sigma) The tissue fragments were suspended andincubated The incubation procedure was repeated for twotimesThe supernatant containing the purified epithelial cellswas collected by centrifugation at 1200 rpm at 4∘C and thenseparated by Percoll (Sigma) density gradient

28 Flow Cytometry and Intracellular Staining All antibodiesused for cell labeling were purchased from eBioscienceFor intracellular cytokine measurement Lamina PropriaLymphocyte (LPL) cells (1x106) were stimulated for 5 h withPMA (1 120583gmL Sigma Aldrich) and ionomycin (50 120583gmLBD Biosciences) in the presence of monensin (01 mgmLSigma Aldrich) and placed in 37∘C and 5 CO

2 LPL cells

were washed with PBS (pH 72) and surface-labeled withanti-CD4-FITC (Biolegend Uithoorn Netherlands) LPLcells were fixed and permeabilized (CytofixCytoperm BDBiosciences) and stained intracellularly with anti-IFN-120574-PE(BDBiosciences)The stained cells were analyzed using FACSCalibur and the data were analyzed using Cell Quest Prosoftware

29 RNA Isolation and mRNA Gene Expression Colonwere removed from euthanized mice The colons werehomogenized for total RNA extractions and oligonucleotideprimer was purchased from Integrated DNA technologies(Coralville) Total RNA was isolated using Tri reagent (MPBiomedical Santa Ana CA USA) according to manufac-turerrsquos instructions The yield and purity of RNA werequantified by nanospectrophotometry at 260 and 280 nmTotal RNA (1 120583gml) was reverse-transcribed to cDNA byusing First Strand cDNA Synthesis (Gbioscience) accordingto the manufacturers A real time assay was performedaccording to Light Cycler-Fast Start DNAMasterPLUS SYBRGreen I (Roche Applied Science Light Cycler DiagnosticUSA) Primer sequences were as follows T-bet forward5rsquo-GGATTCTGGGGTTTACTTCTT-3rsquo T-bet reverse 5rsquo-TTCTCTGTTTGGCTGGCTGTT-3rsquo ROR120574-T forward 51015840-GACAGGGAGCCAAGTTCTCAG-31015840 ROR120574-T reverse 51015840-TCGGTCAATGGGGCAGTTC-31015840 GATA-3 forward 5rsquo-ATCAAGCCCAAGCGAAG-3rsquo GATA-3 reverse 5rsquo-GCT-CTGCCTCTCTAACC-3rsquo 120573-actin forward 5rsquo-TGGAAT-CCTGTGGCATCCATGAAAC-3rsquo 120573-actin reverse 5rsquo-TAA-AACGCAGCTCAGTAACAGTCCG-lsquo3 STAT-1 forward5rsquo-TCT GTG TCT GAA GTC CAC C-3 STAT-1 reverse5-CAAGAAAGCGAGCTTAGTGATAC-3 STAT-4 for-ward 51015840-CACCTGCCACATTGAGTCAACTA-31015840 STAT-4 reverse 51015840-TAAGACCACGACCAACGTACGA-31015840 Theamplification program consisted of the following steps aninitial denaturation at 95∘C for 4 min the 35 cycles of95∘C for 25 seconds of denaturation 56∘C for 10 seconds ofannealing and 72∘C for 20 seconds of extension followedby final extension for 3 minutes at 72∘C Each target geneexpression was normalized with 120573-actin mRNA contentThe relative induction of mRNA expression comparative Ct


Figure 2 LSA instillation effects on mice with DNBS-inducedcolitis Data were obtained from five independent experiments(n = 5) each of which contained five mice (total n = 25 errorbar = SD) Body weight loss was observed in mice without LSAinstillation (Group II) andGroups IIIndashVwere injectedwith differentconcentration of LSA Mice of Group II (DNBS only) showedsignificant body weight loss compared to LSA-injected mice (a plt 005) whereas Group V (LSA 200 120583gmL) injected mice weightswere not significantly decreased compared to control b p lt 0001Representative results obtained from five independent experimentsare shown (ns not significant)

was calculated using ΔΔCt using the following formula (Cttarget - Ct 120573-actin) treatment - (Ct target - Ct 120573-actin)nontreatment

210 Statistical Analysis All datawere presented as themeansplusmn standard deviation An ANOVA test (analysis of variance)was conducted to determine the statistical significance ofdifference between groups with p lt 005 being consideredsignificant

3 Results

31 Changes in Body Weight following Intrarectal Administra-tion ofDNBS Body growth ratewas slightly lower in themicethat received DNBS administration only (Figure 1)

32 LSA Treatment Attenuates DNBS-Induced Acute Colitis inMouseModel We examined whether LSA could enhance theDNBS-induced acute colitis Mice that had been given DNBSdeveloped severe bloody diarrhea followed by an extensivewasting disease Mice given DNBS alone revealed substantialloss of bodyweight ie almost 8between days 4 and 14Theprogression of wasting disease was hindered in mice treatedwith LSA compared with DNBS only group The injection ofLSA clearly enhanced body weight reduction in the recoveryperiod this effect was continuous during 14 days (Figure 2)

33 Histological Changes during DNBS-Induced Acute ColitisTo assess the effect of LSA on the degree of colitis micewere sacrificed 14 days after colonic administration of DNBSThe result was consistent with previous findings DNBS-induced colitis was characterized by the loss of body weightwith extensive ulceration with severe inflammatory cellsinfiltration in the DNBS only group The disease was more

pronounced in mice only given DNBS Histopathologicalanalysis revealed a diffuse leukocyte infiltrate in the mucosaof colon sections fromDNBS-inducedmice (Figure 3(a))TheLSA treatment significantly reduced the extent and severityof the colonic injury even though injection of 50 120583gmLLSA slightly decreased histological score These results havedemonstrated that LSA is able to reduce colon inflammationwhereas mice given 100 120583gmL LSA had essentially decreasedmononuclear cellrsquos infiltration (Figure 3(b)) Further inves-tigation proves that injection of 200 120583gmL LSA was ableto reduce inflammatory cellrsquos infiltration LSA treatment hasprotecting effects against acute colitis

34 Generation of Th1 Cells Requires IFN-120574 Production Toexplore whether LSA could regulate the improvement ofTh1the intracellular cytokines expression in CD4+T cell fromDNBS-induced mice was characterized by flow cytometryColonic injury due to DNBS induction was also consideredby an increase in IFN-120574 production The percentage of IFN-120574 from LPL cells was measured by intracellular staining Ourresults show that IFN-120574 production of LPL was significantlyhigher in DNBS only group while IFN-120574 production ofCD4+ was clearly lower in mice injected with LSA thanthose in DNBS-induced mice (Figure 4(A)) The percentageof IFN-120574+CD4+ cells in the LPL was significantly declinedby LSA treatment at both 100 and 200 120583gmL treatmentgroups (plt005) for each group compared to DNBS onlygroup (Figure 4(B)) Results shown are meanplusmnSD with n=4replicates in each group lowastplt005 lowastlowastplt0001

35 LSADirectly Suppresses Transcription Factor T-Bet DNBS-Induced Th1 Responses To determine whether T-bet genetargets were generally more highly expressed inTh1 cells thanTh2 cells and confirm a functional role of T-bet gene target inLSA-treatedmice we next measured the mRNA gene expres-sion of transcription factor T-bet from LPL cells Results ofreal time PCR analysis are shown as bar graphs (Figures5(a)ndash5(c))The result revealed that the mRNA expressionof T-bet was significantly diminished in LSA-treated mice(Figure 5(a)) whereas ROR120574-T was not significantly alteredafter treatment of LSA-treated mice (Figure 5(b)) Thesestudies suggest that LSA directly suppresses T-bet in DNBS-induced mice In contrast expression of the transcriptionfactor GATA-3 indicated no significant differences betweengroups (Figure 5(c))

36 LSA Treatment Suppressed T-Bet Related Pathway inSTATs-Mediated T Lymphocytes Activation Moreover LSApronouncedly inhibited the transcription expression of genesengaged in the IFN-120574 associated STATs signaling pathwayincluding STAT1 and STAT4 STAT-4 expression was sig-nificantly reduced in LSA-treated mice Likewise STAT-4expression was significantly higher in mice receiving LSAcompared with control counterparts However the expres-sion of STAT-1 was not significantly different between DNBSonly and LSA-treated groups (Figures 6(a)-6(b)) Thesefindings demonstrate that LSA treatment is able to reduceSTAT-4 activation It was noticed that STAT-4 activity mightplay a role in regulating LSA mediated T-bet expression


(a) (b) (c)

(d) (e)

(f)

Figure 3 Histological changes with DNBS-induced colitis in mice after 14 days (a) Mice having instillation of 50 ethanol were used asthe control (b) Mice had rectal instillation of DNBS only (cndashe) Mice had rectal instillation of DNBS and then were injected with differentconcentration of LSA (50 120583gmL 100 120583gmL and 200 120583gmL) respectively (f) Histological colitis scores of individual mice are shown ascircles and the averages of each group are shown as horizontal bars Each symbol represents an individual animal Representative pictures ofeach group are shown Groups B and C had significantly higher colitis scores than Group E ((a) p gt 005 and (b) p lt 001 respectively) L gutlumen M muscle U ulcer arrow inflammatory infiltrate Magnification 200times

4 Discussion

Helminthic therapy is used to maintain the balance viamodulation of the host immune system The ability of wormsto protect the host from various diseases such as colitisencephalitis rheumatoid arthritis asthma and diabetes mel-litus has been studied experimentally [23 24 26]

It is realized that the inflammatory response is caused bya parasite and the protective consequences are related to thenematode staying in the hostrsquos body during treatment Onthe other hand nematodes are allowed to be infected andstay alive which the patients still find difficult to accept Wethink that the somatic antigen derived from nematodes L4stage induces the immune response toTh1-mediated diseasesThe reciprocal cross-regulation between Th1 and Th2 cellssuggests that mobilization of a Th2 response by nematodescould reduce the effects of Th1-mediated diseases [24 26]

In the current study we also demonstrated that the somaticantigen got from L4 stage H polygyrus has been enabledafter DNBS treatment Further investigation revealed thatLSA injection diminished inflammatory cell infiltration intocolon DNBS-induced colitis is mediated by Th1 responsesresulting in enhanced production of T-bet in the inflamedcolonic tissue T-bet assumes a basic part in the pathogenesisof colitis inflammation and critical transcription factor forTh1 cell differentiation Our experiments demonstrated thatLSA injection significantly reduced the percentage of T-betin LPL cells in DNBS-induced mice These perceptions indi-cated that the anti-inflammatory effect of LSA was mediatedthrough a suppressive activity on T lymphocytesThe currentstudy revealed that the LSA has a capacity to suppress colitisinduced by DNBS

Increased DNA-binding activity of T-bet associated withthe secretion of high levels of IFN-120574 has been reported


Figure 4 Effect of LSA on suppressing the development of colitis via percentage of IFN-120574 A percentages of lymphocytes T CD4 +IFN-120574+were observed inmicewith or without LSA injection B the percentages of lymphocytes TCD4 +IFN-120574+ cells were shown Representative flowcytometry plots show cells gated as CD4+IFN-120574+ Data are representative of four independent experiments with similar results Percentagesof cells in each quadrant are shown inside the panels Representative results of five mice in each group are shown

(a)

(b) (c) (d)

Figure 5 Relative gene expression of T-bet ROR120574-T and Gata-3 from the colon of CAC models Amplification was performed by gene-specific primers with 2 120583L cDNA for 25 cycles (120573-actin) or 35 cycles (transcription factor) (b) The fold change in T-bet mRNA (normalizedto 120573-actin) expression in colon (c) The fold change in ROR120574minusT mRNA (normalized to 120573-actin) expression in colon (d) The fold changein Gata-3 mRNA (normalized to 120573-actin) expression in colon Results shown are meanplusmnSD with n=4 replicates in each group lowastplt005lowastlowastplt0001 (ns not significant) Relative gene expression of p53 from the colon of CACmodels RNAwas extracted from colons and p53 geneexpression was analyzed by real time PCR and normalized by 120573-actin mRNA Results are representative of four independent experimentsData with no statistically significant differences (ns not significant)

for UC patients [27] Accordingly the nuclear localizationarrangement of T-bet allowed its translocation into thenucleus where it binds to DNA and initiates transcriptionof T-bet dependent genes which were associated with theimmune and inflammatory responses T-bet is recognizedas an ideal target for molecular therapies for the treatmentof inflammatory diseases [24 25 27] T-bet expressionprotects against colitis by inducing Th1 expression on LPLand response of mucosal immune system to intraluminal byregulating IFN-120574production in the colon T-bet also regulatesIFN-120574 production in LPL [25 27 28] These observations

suggested that T-bet is induced by STATs signaling during Tcell activation

The aims of the current study were to prove the cureresponse of DNBS-induced colitis by the L4 somatic antigenSurprisingly LSA derived fromH polygyrus is able to inhibitTh1 cells LSA was able to cure colitis in the DNBS-inducedmice Our research has shown that LSA enhances GATA-3 expression This data indicates that GATA-3 controls Th1expression In addition somatic antigen treatment resultsin the modulation of immune activity and the reduction ofcolitis associated with Th1 cells responses [16 29ndash32] These


(a)

(b) (c)

Figure 6 The mRNA expression of STAT-1 and STAT-4 Agarose gel electrophoresis was used for semiquantitative determination of PCR-amplified transcription factor of mRNA in colons total RNA was extracted and was reverse-transcribed to cDNA Amplification wasperformed by gene-specific primers with 2 120583L cDNA for 25 cycles (120573-actin) or 35 cycles (transcription factor) (b)The fold change in STAT-1mRNA (normalized to 120573-actin) expression in colon (c) The fold change in STAT-4 mRNA (normalized to 120573-actin) expression in colon ThePCR band density is automatically generated by the ImageJ software Results shown aremeanplusmnSD with n=4 replicates in each group lowastplt005lowastlowastplt0001 (ns not significant)

findings indicate a novel function for T-bet as a target geneand provide new perspectives on the pathophysiology ofcolitis The anticolitis activity of the somatic antigen attenu-ates the histopathological changes associated with colitis anddeclines inflammatory cell infiltration in the mouse colitismodel [31ndash33]

IFN-120574 has been described as inducing T-bet expres-sion with a potential to include differentiation of the Th1cells Mouse colitis exhibits markedly increased T-bet DNA-binding activity [16 33] In addition LSA could suppress T-bet expression and considerably reduced the production ofproinflammatory cytokines such as IFN-120574 Consistently withour results of the current study LSA treatment suppressed thedifferentiation of naive CD4 T cells into Th1 subsets whichhave been considered in the mouse colitis model [16] Inthe current study we observed that the T-bet activity wassignificantly higher in the colonic tissues of LSA- treatedmicethan in the control group

Based on the above results we propose that LSA inhibitsT-bet activity which leads to the suppression of transcriptionof inflammatory mediator genes Our perceptions recom-mend the existence of an endogenous program for Th2cell differentiation in vivo which is usually repressed byT-bet during Th1 cell differentiation Taken together thesefindings suggest that LSA exerts an inhibitory effect on theinflammatory response in colitis through the regulation ofT-bet activation Previous study reported that specific DNAsequences are bound with the transcription factor T-betthat translocates in nucleus and activates gene transcription

[16 34] so by controlling the transcriptional arrangementsof inflammatory cytokine genes T-bet plays a critical rolefor the regulation of immune responses and inflammationin spite of the fact that molecular mechanisms underlyingantiproliferative activity of T-bet remain to be explained

Interestingly the activation of STAT-1 did not sig-nificantly differ between LSA-treated mice ImportantlyLSA dose-dependently inhibited IFN-120574 stimulated STAT-4nuclear translocation inTh1 cells however it did not influenceSTAT-1 Despite the suggestion that Stat-4 mainly plays arole in Th1 expansion or survival downstream of T-bet therole of T-bet in Th1 induction is still controversial [34 35]Consequently the functional correlation between Stat-4 andT-bet in developing Th1 cells may be more complex thanis presently appreciated These data suggest that STAT-4 isassociated with a proinflammatory profile promoting tissueinfiltration and proliferationTherefore the protein therapiesusing protein extract are more favorable compared withmaintaining nematodes alive in host We further proved theeffect of H polygyrus antigen on DNBS-induced colitis

5 Conclusions

In this study we found that LSA injection had a signifi-cant inhibitory effect on mRNA T-bet expression and IFN-120574 percentages Decreased percentage of intracellular IFN-120574producing CD4 T cells in LSA-treated mice caused T-betdownregulation in LSA-treated mice We demonstrated thatinjection of LSAmay be effective in protecting the mucosa of


mice in DNBS-induced colitis which consequently preventsproinflammatory mediators from acutely increasing Thesestudies suggested that LSA injection could inhibit T-betdifferentiation by STAT-4 Our results suggest that LSA mayhas therapeutic potential in colitis This study indicates thatLSA is promising therapeutic agent in ulcerative colitis

Abbreviations

DNBS 24-Dinitrobenzene sulfonic acidGATA-3 GATA-binding protein 3LPL Lamina propria T lymphocytesLSA L4 somatic antigenSTAT Signal transducer and activator of transcriptionROR120574-T RAR-related orphan receptor gammaT-bet T-box expressed in T cellsUC Ulcerative colitis

Data Availability

The data used to support the findings of this study areavailable from the corresponding author upon request

Conflicts of Interest

The authors declare that there are no conflicts of interestregarding the publication of this paper

Acknowledgments

The authors gratefully thank Professor Roger Price for hiskind assistance in reviewing the manuscript They would liketo acknowledge Directorate General of Higher EducationMinistry of National Education and Culture of RepublicIndonesia for the provided grant for this research

References

[1] T T MacDonald I Monteleone M C Fantini and G Mon-teleone ldquoRegulation of homeostasis and inflammation in theintestinerdquo Gastroenterology vol 140 no 6 pp 1768ndash1775 2011

[2] P K Randhawa K Singh N Singh and A S Jaggi ldquoA reviewon chemical-induced inflammatory bowel disease models inrodentsrdquo Korean Journal of Physiology amp Pharmacology vol 18no 4 pp 279ndash288 2014

[3] H J McSorley J P Hewitson and R M Maizels ldquoImmuno-modulation by helminth parasites defining mechanisms andmediatorsrdquo International Journal for Parasitology vol 43 no 3-4 pp 301ndash310 2013

[4] H JMcSorley andRMMaizels ldquoHelminth infections andhostimmune regulationrdquo Clinical Microbiology Reviews vol 25 no4 pp 585ndash608 2012

[5] D B Cury S J Mizsputen C Versolato et al ldquoSerum calpro-tectin levels correlate with biochemical and histological mark-ers of disease activity in TNBS colitisrdquoCellular Immunology vol282 no 1 pp 66ndash70 2013

[6] R C Dutra M Cola and D F P Leite ldquoInhibitor of PI3K120574ameliorates TNBS-induced colitis in mice by affecting thefunctional activity of CD4+CD25+FoxP3+ regulatory T cellsrdquo

British Journal of Pharmacology vol 163 no 2 pp 358ndash3742011

[7] D Ramanan R Bowcutt S C Lee et al ldquoHelminth infectionpromotes colonization resistance via type 2 immunityrdquo Sciencevol 352 no 6285 pp 608ndash612 2016

[8] W C Gause T AWynn and J E Allen ldquoType 2 immunity andwound healing evolutionary refinement of adaptive immunityby helminthsrdquo Nature Reviews Immunology vol 13 no 8 pp607ndash614 2013

[9] F Lopes J L Reyes A Wang G Leung and D M McKayldquoEnteric epithelial cells support growth of Hymenolepis dimin-uta in vitro and trigger TH2-promoting events in a species-specific mannerrdquo International Journal for Parasitology vol 45no 11 pp 691ndash696 2015

[10] J VWeinstock ldquoAutoimmunity the worm returnsrdquoNature vol491 no 7423 pp 183ndash185 2012

[11] R M Maizels J P Hewitson and K A Smith ldquoSusceptibilityand immunity to helminth parasitesrdquo Current Opinion inImmunology vol 24 no 4 pp 459ndash466 2012

[12] J E Allen and R M Maizels ldquoDiversity and dialogue inimmunity to helminthesrdquo Nature Reviews Immunology vol 11no 6 pp 375ndash388 2011

[13] C Ippolito C Segnani M Errede et al ldquoAn integrated assess-ment of histopathological changes of the enteric neuromuscularcompartment in experimental colitisrdquo Journal of Cellular andMolecular Medicine vol 19 no 2 pp 485ndash500 2015

[14] L Hang A M Blum T Setiawan J P Urban Jr K MStoyanoff and J V Weinstock ldquoHeligmosomoides polygyrusbakeri infection activates colonic Foxp3+ T cells enhancing theircapacity to prevent colitisrdquoThe Journal of Immunology vol 191no 4 pp 1927ndash1934 2013

[15] A M Blum L Hang T Setiawan et al ldquoHeligmosomoidespolygyrus bakeri induces tolerogenic dendritic cells that blockcolitis and prevent antigen-specific gut T cell responsesrdquo TheJournal of Immunology vol 189 no 5 pp 2512ndash2520 2012

[16] K Donskow-Łysoniewska J Bien K Brodaczewska KKrawczak and M Doligalska ldquoColitis Promotes Adaptation ofan Intestinal Nematode A Heligmosomoides Polygyrus MouseModel Systemrdquo PLoS ONE vol 8 no 10 Article ID e780342013

[17] A T Endharti A Wulandari A Listyana E Norahmawatiand S Permana ldquoDendrophthoe pentandra (L) Miq extracteffectively inhibits inflammation proliferation and induces p53expression on colitis-associated colon cancerrdquo BMC Comple-mentary and Alternative Medicine vol 16 no 1 article no 3742016

[18] D E Elliott and J V Weinstock ldquoHelminth-host immunolog-ical interactions prevention and control of immune-mediateddiseasesrdquo Annals of the New York Academy of Sciences vol 1247no 1 pp 83ndash96 2012

[19] W J Sandborn D E Elliott J Weinstock et al ldquoRandomisedclinical trial the safety and tolerability of Trichuris suis ovain patients with Crohnrsquos diseaserdquo Alimentary Pharmacology ampTherapeutics vol 38 no 3 pp 255ndash263 2013

[20] J V Weinstock and D E Elliott ldquoTranslatability of helminththerapy in inflammatory bowel diseasesrdquo International Journalfor Parasitology vol 43 no 3-4 pp 245ndash251 2013

[21] J L Reyes A Wang M R Fernando et al ldquoSplenic B cellsfrom hymenolepis diminuta-infected mice ameliorate colitisindependent of T cells and via cooperation with macrophagesrdquoThe Journal of Immunology vol 194 no 1 pp 364ndash378 2015


[22] F Hernandez-Chavarrıa and L Avendano ldquoA simple modifica-tion of the Baermannmethod for diagnosis of strongyloidiasisrdquoMemorias do Instituto Oswaldo Cruz vol 96 no 6 pp 805ndash8072001

[23] V Morampudi G Bhinder X Wu et al ldquoDNBSTNBS ColitisModels Providing Insights Into Inflammatory Bowel Diseaseand Effects of Dietary Fatrdquo Journal of Visualized Experimentsvol 27 no 84 Article ID e51297 2014

[24] A T Endharti A D Baskoro and E Norahmawati ldquoTherapeu-tic effect of soluble worm protein acting as immune regulatoryon colitisrdquo Asian Pacific Journal of Tropical Biomedicine vol 7no 1 pp 70ndash77 2017

[25] D E Elliott and J V Weinstock ldquoWhere are we on wormsrdquoCurrent Opinion in Gastroenterology vol 28 no 6 pp 551ndash5562012

[26] U Erben C Loddenkemper K Doerfel et al ldquoA guide tohistomorphological evaluation of intestinal inflammation inmouse modelsrdquo International Journal of Clinical and Experi-mental Pathology vol 7 no 8 pp 4557ndash4576 2014

[27] J E Allen and T A Wynn ldquoEvolution of Th2 immunity arapid repair response to tissue destructive pathogensrdquo PLoSPathogens vol 7 no 5 Article ID e1002003 2011

[28] S Keely E L Campbell A W Baird et al ldquoContributionof epithelial innate immunity to systemic protection affordedby prolyl hydroxylase inhibition in murine colitisrdquo MucosalImmunology vol 7 no 1 pp 114ndash123 2014

[29] E Antoniou G A Margonis A Angelou et al ldquoThe TNBS-induced colitis animal model an overviewrdquo Annals of Medicineand Surgery vol 11 pp 9ndash15 2016

[30] A B A De Almeida M Sanchez-Hidalgo A R Martın etal ldquoAnti-inflammatory intestinal activity of Arctium lappa L(Asteraceae) in TNBS colitis modelrdquo Journal of Ethnopharma-cology vol 146 no 1 pp 300ndash310 2013

[31] J L Reyes M R Fernando F Lopes et al ldquoIL-22 RestrainsTapeworm-Mediated Protection against Experimental Colitisvia Regulation of IL-25 Expressionrdquo PLoS Pathogens vol 12 no4 Article ID e1005481 2016

[32] F Isik T Tunali Akbay A Yarat et al ldquoProtective effects ofblack cumin (Nigella sativa) oil on TNBS-induced experimentalcolitis in ratsrdquo Digestive Diseases and Sciences vol 56 no 3 pp721ndash730 2011

[33] I Ferreira D Smyth S Gaze et al ldquoHookworm excre-torysecretory products induce interleukin-4 (IL-4)+ IL-10+CD4+ t cell responses and suppress pathology in amousemodelof colitisrdquo Infection and Immunity vol 81 no 6 pp 2104ndash21112013

[34] A T Endharti Y Okuno Z Shi et al ldquoCD8+CD122+ regulatoryT cells (Tregs) and CD4+ Tregs cooperatively prevent and cureCD4+ cell-induced colitisrdquoThe Journal of Immunology vol 186no 1 pp 41ndash52 2011

[35] A T Endharti and S Permana ldquoExtract frommangomistletoesDendrophthoe pentandra ameliorates TNBS-induced colitis byregulating CD4+ T cells in mesenteric lymph nodesrdquo BMCComplementary and Alternative Medicine vol 17 no 1 articleno 468 2017

Stem Cells International

Hindawiwwwhindawicom Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwwwhindawicom Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwwwhindawicom

Submit your manuscripts atwwwhindawicom


of STAT-4 [2 13] STAT-4 induces expression of T-bet thatacts as a key regulator of Th1 cells [13ndash15] H polygyrus waseffectively used to maintain typical conditions in chroniccolitis [2 16] Allowing for the fact that helminthes maymodulate the inflammatory response of colitis we exploredthe effect of H polygyrus on mice in an experimental colitismodel In any case little is thought about the basic mech-anisms of the caring effect of somatic antigen derived fromL4H polygyrus larvae against an inflammatory response Weare concerned about proving the expression level of tran-scription factor reflecting inhibition of inflammation duringDNBS

T-bet performs a crucial function in immune responseby modulating the some genes involved in the inflammatoryresponses [16] Hence by monitoring the transcription ofinflammatory cytokine genes T-bet plays a central rolein regulating the immune responses and inflammationT-bet is a critical transcription factor that controls dif-ferentiation of Th1 cells it specifically binds to the pro-moter of the IFN-120574 gene and activates its transcription[17ndash19]

Worm therapy has been established recently The mecha-nism of human disease of the numerous worm species canbe studied using a mouse model of H polygyrus infection[11 16] As confirmed by many live nematode treatment isconsidered an alternative therapy [18ndash20] this way has alittle harm because patients are infected by live nematodes[17ndash23] Additionally the specific mechanisms sustainingthe therapeutic effect of gastrointestinal nematodes are notopenly understood Moreover the pathology related to worminfection requires for the therapy using live worms to beexchanged with nematodes-derived proteins [2 11 16] Theexcretory-secretory proteins derived from H polygyrus havea potential in the therapy of colitis [24 25] A protein basedtreatment could overcome the disadvantages of usage thatrelies on living parasitesThe goal of this study was to explorethe effect of L4 larvae extract (LSA) on clinical indicationsof UC including body weight and extent of occult bleedingin a mouse model Studies reported that mice with DNBS-induced colitis exhibit characteristics similar to chronic UCin human [17 24 25] In addition prolonged and chronic UCdevelop to a colorectal cancerTh1 andTh2 cells are associatedwith increased expression of Gata-3 and reduced expressionof T-bet inmemoryCD4T cells CD4T cells expressingGata-3 were significantly increased in H polygyrus induced miceThis study also verifies the assessment of the effect of LSA onthe expression of T-bet and GATA-3 in DNBS treated colonictissue To verify the role of STATs signaling in the regulationof T-bet in LSA-treatedmice we examined whether the T-betgene targets were generally expressed as STAT1 and STAT4Therefore we explored the possibility that STATs activitycould play a major role in modulating the LSA mediated T-bet expression

It is greatly likely that studies employing the modelsused in the current study might contribute key informationabout the pathogenesis of human inflammatory colonicdiseases in the future Certainly the exact mechanism bywhich LSA affects T-bet and GATA-3 should be investigatedfurther

2 Materials and Methods

21 H polygyrus Establishment Third-stage infective larvae(L3) of H polygyrus were obtained from Jikei University ofTokyo and maintained at Parasitology Laboratory MedicalFaculty Brawijaya University Indonesia Female Balbc micewere 8 weeks old at the start of the study Mice wereinoculated by oral gavage with 200 L3 of H polygyrususing a 20-gauge ball-tip feeding tube Infective unsheathedL3 of H polygyrus (Parasitology Laboratory Collection noH120) were obtained from fecal cultures of eggs according toChavarrıa et al [22] with modification L3 collections werepropagated and stored at 4∘C until used

22 Mice Female Balbc mice weighing 25-30g were usedThey were obtained from Pusvetma Surabaya The micewere keep under standard conditions of temperature of 25-27∘C relative humidity (55plusmn5) and 12h12h lightdark cycleMice were given normal drinking water ad libitum duringthe experimental periods All experiments were conductedaccording to the principles of Guide for the Care and Useof Laboratory Animals in Indonesia and were approvedby the Ethical Committee of Brawijaya University MalangIndonesia (No 171-KEP-UB)

23 Preparation of L4 Somatic Antigen (LSA) From Hpolygyrus Previously third-stage infective larvae (L3) wereadministered orally for each mouse with single dose of 200L3 Mice were sacrificed on day 15 after LSA treatmentand then L4 were collected from mouse intestine LSA wasprepared from L4 of H polygyrus according to Chavarrıaet al [22] Briefly LSA were collected from 100 L4 whichwere washed several times in Phosphate Buffer Saline (PBS)supplemented with 100mgmL penicillin and 100 mgmLstreptomycin Total L4 were homogenized in a cocktail ofextraction buffer (2mM EDTA 10mM PMSF and 30mMpotassium phosphate in PBS) followed by centrifugation at10000 rpm for 15 minutes The final protein concentrationof L4 homogenate was measured by the Bradford techniqueTotal protein of antigen containing lt20 endotoxin unitsmgwas collected and stored at -30∘C until used

24 Induction of Experimental Colitis and Injection of LSAMice were randomly divided into five groups includinggroup 1 including vehicle (control) groups 2-5 with rectaladministration of DNBS and groups 3-5 that were injected(intraperitoneally) with LSA (50 100 or 200 120583gmL respec-tively) once during 14 days (Figure 1) Colitis was inducedby rectal instillation of 5mgkgBW of DNBS (Sigma Chem-ical Co St Louis USA) according to procedure describedby Morampudi et al [23] with few modifications Brieflycolitis was induced in mice by rectal instillation of 5 mgof DNBS100 120583l of 50 ethanol into the rectum through acatheter inserted 4 cm proximally to the anus The volumeof DNBS enema was 100 120583L Thereafter the mice were keptfor 90 seconds in a head-down position to avoid loss of theDNBSThe injection of LSAwas started from second day andcontinued until 14 days Development of colitis was assesseddaily by using an occult blood detection kit (Hemoccult)









2 LPL cells







3 Results









(a) (b) (c)

(d) (e)

(f)


4 Discussion







(a)

(b) (c) (d)






(a)

(b) (c)







5 Conclusions




Abbreviations


Data Availability




Acknowledgments


References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



























2 LPL cells







3 Results









(a) (b) (c)

(d) (e)

(f)


4 Discussion







(a)

(b) (c) (d)






(a)

(b) (c)







5 Conclusions




Abbreviations


Data Availability




Acknowledgments


References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























3 Results









(a) (b) (c)

(d) (e)

(f)


4 Discussion







(a)

(b) (c) (d)






(a)

(b) (c)







5 Conclusions




Abbreviations


Data Availability




Acknowledgments


References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a) (b) (c)

(d) (e)

(f)


4 Discussion







(a)

(b) (c) (d)






(a)

(b) (c)







5 Conclusions




Abbreviations


Data Availability




Acknowledgments


References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















(a)

(b) (c) (d)






(a)

(b) (c)







5 Conclusions




Abbreviations


Data Availability




Acknowledgments


References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of




















(a)

(b) (c)







5 Conclusions




Abbreviations


Data Availability




Acknowledgments


References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of





















Abbreviations


Data Availability




Acknowledgments


References










































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of






































of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of























of




Disease Markers



OncologyJournal of





PPAR Research



Volume 2018


Journal of

ObesityJournal of



















Documents

T-Bet Is Dependent on Stat-4 Inhibiting Acute Colitis but Not Stat …downloads.hindawi.com/journals/tswj/2018/8571920.pdf · 2018-11-12 · ey were obtained from Pusvetma Surabaya