分子生物学知识拓展21 (3).pdf

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1、Choteau et al.Gut Pathog (2017)9:9 DOI 10.1186/s13099-017-0158-0RESEARCHRole ofTLR1,TLR2 andTLR6 inthe modulation ofintestinal inflammation andCandida albicans eliminationLaura Choteau1,2,3,Hlne Vancraeyneste1,2,3,Didier Le Roy4,Laurent Dubuquoy1,2,Luiginia Romani5,Thierry Jouault1,2,Daniel Poulain1

2、,2,3,Boualem Sendid1,2,3,Thierry Calandra4,Thierry Roger4 and Samir Jawhara1,2,3*Abstract Background:Toll-like receptors(TLRs)are the major pattern recognition receptors that mediate sensing of a wide range of microorganisms.TLR2 forms heterodimers with either TLR1 or TLR6,broadening its ligand dive

3、rsity against pathogens.TLR1,TLR2 and TLR6 have been implicated in the recognition of Candida albicans,an opportunistic fungal pathogen that colonizes the gastrointestinal tract.In this study,we explored whether the deficiency in TLR1,TLR2 or TLR6 impacts C.albicans colonization and inflammation-ass

4、ociated colonic injury in the dextran sulfate sodium(DSS)-induced colitis in mice.Results:DSS treatment and C.albicans challenge induced greater weight loss,worse clinical signs of inflammation,higher histopathologic scores,and increased mortality rates in TLR1/and TLR2/mice when compared to TLR6/an

5、d wild-type mice.The number of C.albicans colonies in the stomach,colon and feces was decreased in TLR6/mice as compared to TLR2/,TLR1/and wild-type mice.Interestingly,the population of E.coli in colonic luminal contents,intestinal permeability to FITC-dextran and cytokine expression were significan

6、tly increased in TLR1/and TLR2/mice,while they were decreased in TLR6/mice.Conclusion:In contrast to TLR6,both TLR1 and TLR2 deficiencies increased intestinal inflammation,and the over-growth of C.albicans and E.coli populations in the colitis model,suggesting the involvement of TLR1 and TLR2 in epi

7、thelial homeostasis,and a role of TLR6 in increasing intestinal inflammation in response to pathogen-sensing.Keywords:TLR1,TLR2,TLR6,Candida albicans,E.coli,Colitis The Author(s)2017.This article is distributed under the terms of the Creative Commons Attribution 4.0 International License(http:/creat

8、ivecommons.org/licenses/by/4.0/),which permits unrestricted use,distribution,and reproduction in any medium,provided you give appropriate credit to the original author(s)and the source,provide a link to the Creative Commons license,and indicate if changes were made.The Creative Commons Public Domain

9、 Dedication waiver(http:/creativecommons.org/publicdomain/zero/1.0/)applies to the data made available in this article,unless otherwise stated.BackgroundToll-like receptors(TLRs)are the main family of pattern recognition receptors(PRRs)through which immune and non-immune cells sense pathogen-associa

10、ted molecu-lar patterns(PAMPs)1,2.Several TLRs are implicated in the recognition of fungal pathogens such as Candida albicans 3,4.The interaction between TLRs and yeasts during candidiasis stimulates immune cells to generate inflammatory and immunomodulatory mediators that shape the host immune resp

11、onse.Unlike TLR4,TLR2 recognizes both blastoconidia and hyphal forms of C.albicans 5.TLR2 forms heterodimers with either TLR1 or TLR6 which have been implicated in ligand discrimi-nation 6.TLR2 senses phospholipomannans,which are expressed in the cell wall of C.albicans 7.In addi-tion,TLR2 in combin

12、ation with galectin-3 also senses-mannosides 8.TLRs are expressed not only in myeloid cells and leu-kocytes,but also in the intestinal epithelium,which contributes to mucosal homeostasis by preventing the penetration of commensal microbiota into the intestine 9,10.In an animal model of colitis,TLR2/

13、mice developed more severe colonic inflammation than Open AccessGut Pathogens*Correspondence:samir.jawhara-3univ-lille2.fr 1 INSERM U995/2,Universit Lille Nord de France,1 Place Verdun,59000 Lille,FranceFull list of author information is available at the end of the articlePage 2 of 13Choteau et al.G

14、ut Pathog (2017)9:9 wild-type mice 11.Moreover,mutations in TLRs,including the TLR2 gene,have been associated with predisposition to and maintenance of inflammatory bowel disease(IBD)1214.Interestingly,in patients with ulcerative colitis,Pierik et al.15 observed an association between TLR1 and TLR2

15、gene polymor-phisms and pancolitis,and a negative relationship between TLR6 polymorphisms and pancolitis,suggest-ing that TLR2 and its co-receptors TLR1 and TLR6 are involved in the initial immune response to pathogens in the development of IBD.The aim of this study was to determine the impact of TL

16、R1,TLR2,and TLR6 deficiency on inflammatory parameters associated with C.albicans colonization and acute colitis induced by DSS by comparing wild-type,TLR1/,TLR2/,and TLR6/mice.We also assessed intestinal permeability,serological response,and colonic expression levels of pro-inflammatory and anti-in

17、flammatory cytokines in control and TLR-deficient mice.Finally,we explored the effects of TLR deficiency on neutrophil-mediated C.albicans phagocytosis/death.ResultsCandida albicans CFU instools andmouse body weightTLR1/,TLR2/,TLR6/,and wild-type mice were challenged with a single oral inoculum of C

18、.albicans(107 CFU)and the amount of yeast in stool samples was analyzed daily for 2weeks to assess the colonization rate(Fig.1a).C.albicans colonization was not observed in any of these mice a few days later.In the absence of DSS,no significant differences in body weight were observed between TLR de

19、ficient mice and wild-type mice that received C.albicans(Fig.1b).Additionally,there were no differences between wild-type and TLR deficient mice that challenged with C.albicans in terms of clinical and histological scores(data not shown).Mouse weight andsurvival analysis inDSSinduced murine colitisT

20、o assess the association between TLR1,TLR2 or TLR6 deficiency and C.albicans colonization in DSS-induced murine colitis,mice were monitored daily for 2weeks for body weight loss and survival after a sin-gle oral challenge with C.albicans and DSS treatment(Fig.2a).All mice treated with DSS showed sig

21、nifi-cant body weight loss,and no mortality was observed.Interestingly,C.albicans colonization caused a greater body weight loss in TLR1/and TLR2/DSS mice when compared to TLR6/and wild-type DSS-treated mice(Fig.2bd).From day 9,when compared to wild-type mice treated with C.albicans and DSS,there wa

22、s a significant decrease in body weight of TLR1/and TLR2/mice treated with C.albicans and DSS.Fur-thermore,the C.albicans and DSS-treated mouse sur-vival rate was lower in TLR1/and TLR2/mice(86%survival)than in TLR6/and wild-type mice(93%survival)(Fig.2e).Clinical andhistologic inflammation scoresTo

23、 evaluate clinical inflammation scores,mice were monitored daily using the parameters of stool con-sistency and the presence or absence of faecal blood(Fig.2f).In the absence of C.albicans challenge,no significant difference in clinical scores for inflamma-tion was observed between DSS-treated TLR6/

24、and DSS-treated wild-type mice.In contrast,clinical scores for inflammation were significantly higher in TLR1/and TLR2/mice than in wild-type DSS-treated mice.Importantly,the clinical symptoms of colitis,such as diarrhea and bloody stools,appeared more rapidly in TLR1/and TLR2/mice than in wild-type

25、 and TLR6/C.albicans and DSS-treated mice.Micro-scopic inflammatory changes were also assessed in mice Fig.1 Candida albicans colonization and body weight in mice chal-lenged with C.albicans.a Number of C.albicans colony forming units(CFU)recovered from stools.A single inoculum of 107 C.albicans was

26、 administered to mice on day 1.A total of 40 mice were divided into four groups composed of wild-type Candida(WT,n=10),TLR1/Candida(n=10),TLR2/Candida(n=10),and TLR6/Candida(n=10).Data are mean SE of two independent experiments.b Mouse body weight.There were no differences between wild-type and TLR

27、deficient mice in terms of body weight changes.Data are mean SE of two independent experimentsPage 3 of 13Choteau et al.Gut Pathog (2017)9:9 colons(Fig.2g).All surviving animals were sacrificed on day 14 and histologic injury scoring was performed on the colons.Histologic scores,based on the extent

28、of infiltration of inflammatory cells and the degree of tissue damage,ranged from 0 to 6,and reflected inflammation and crypt damage caused by DSS.No sig-nificant difference in the histologic score was detected between wild-type and TLR-deficient DSS-treated mice.In contrast,histologic scores were s

29、ubstantially higher in C.albicans and DSS-treated TLR1/and Fig.2 Increased morbidity and mortality of TLR1/and TLR2/mice due to C.albicans and DSS-induced colitis.a Schematic representation of the experimental procedure.A single inoculum of 107 C.albicans was administered to mice on day 1 and low do

30、ses of DSS(1.5%)were given in the drinking water for 2 weeks.A total of 120 mice were divided into eight groups composed of wild-type DSS(WT D,n=10),wild-type C.albicans and DSS(WT CaD,n=20),TLR1/D(n=10),TLR1/CaD(n=20),TLR2/D(n=10),TLR2/CaD(n=20),TLR6/D(n=10),and TLR6/CaD(n=20)mice.bd Mouse body wei

31、ght.Data are the mean SE of two independent experiments.+P0.05 for TLR-deficient CaD mice versus wild-type D mice.*P 0.05 for TLR-deficient CaD mice versus wild-type CaD mice.e Mouse survival.Results are expressed as percent survival from the time of C.albicans challenge and DSS treatment.The surviv

32、al data were significantly different by the log-rank test(P 0.05).f Clinical analysis of DSS-induced colitis in mice.Clinical score was determined by assessing weight loss,change in stool consistency and presence of gross bleeding.The clinical score ranged from 0 to 8(each value corresponds to the m

33、ean value of 14 days per group).+P0.05 for TLR1/DSS(d)and TLR2/D mice versus wild-type(WT)D mice;and*P 0.05 for TLR1/C.albicans and DSS(CaD)and TLR2/CaD mice versus wild-type CaD mice.g Histologic scores.Mice were exposed to 1.5%DSS in drinking water for 14 days.Scores range from 0(no changes)to 6(e

34、xtensive cell infiltration and tissue damage).*P 0.05 for TLR1/CaD and TLR2/CaD mice versus wild-type CaD micePage 4 of 13Choteau et al.Gut Pathog (2017)9:9 TLR2/as compared to TLR6/and wild-type mice(P0.05).Epithelial damage occurred throughout the colonic mucosa,and the infiltrated cells were most

35、ly polynuclear(Fig.3).In addition,cryptic abscesses and mucosal edema was more frequently observed in the colons of TLR1/and TLR2/mice than in those of TLR6/and wild-type C.albicans and DSS-treated mice(Fig.3).Effects ofDSSinduced colitis onC.albicans colonizationMice were challenged with a single o

36、ral inoculum of C.albicans(107 CFU)and given 1.5%DSS in drink-ing water for 14days(Fig.4a).Despite a trend towards higher number of C.albicans CFUs in the stools of TLR1/mice,there was no significant difference between wild-type,TLR1/,and TLR2/mice during the entire observation period.In contrast,th

37、e number of C.albicans CFUs strongly decreased in the stools of TLR6/mice on day 7 after challenge,and remained significantly lower than that of wild-type mice up to day 13(P0.05).To evaluate C.albicans colonization in the gut,the number of yeasts adhering to the stomach and colon was determined(Fig

38、.4b,c).Significantly higher numbers of CFUs were observed in the stomach of TLR1/mice when compared to TLR2/,TLR6/,Fig.3 Histologic analysis of the colon in C.albicans and DSS-induced colitis.a,c correspond to colon sections from wild-type mice receiving water as controls(CTL),and C.albicans(Ca)resp

39、ectively.e,g,i,and k correspond to colon sections from wild-type C.albicans and DSS(CaD),TLR1/CaD,TLR2/CaD,and TLR6/CaD mice,respectively.In the absence of DSS,no significant differences in the colon sections were observed between control animals(not inoculated)and those that received C.albicans(a,c

40、).The colon sections from wild-type CaD and TLR6/CaD mice show an inflammatory cell infiltrate in colonic wall structures(b,h).The colon sections from TLR1/CaD and TLR2/CaD mice display a high inflammatory cell infiltrate in colonic wall structures and massive tissue destruction(asterisk,h,j).The sc

41、ale bars represent 50 m(a,c,e,g,i,k)and 10 m (b,d,f,h,j l)Page 5 of 13Choteau et al.Gut Pathog (2017)9:9 and wild-type mice(P0.001).C.albicans did not dis-seminate to the lungs,heart,liver,spleen or kidneys of wild-type and TLR deficient mice(data not shown).Serologic analysis andintestinal permeabi

42、lityTo evaluate the association between anti-mannan antibody titers,C.albicans colonization,and TLR deficiency in DSS-treated mice,we assessed man-nanemia,which correlates to mannan levels in the blood(Fig.4d).Mannanemia was higher in TLR1/and TLR2/mice as compared to wild-type C.albicans and DSS-tr

43、eated mice(P0.05),which cor-related with the higher clinical and histologic scores for inflammation in TLR1/and TLR2/mice.Additionally,mannanemia was significantly lower in TLR6/than that of wild-type mice(P 0.05).Next,we assessed the in vivo intestinal permeabil-ity using fluorescein isothiocyanate

44、(FITC)-dextran(Fig.4e).Intestinal permeability of FITC-dextran was significantly increased in TLR1/and TLR2/as compared to TLR6/and wild-type C.albicans and DSS-treated mice(P 0.05).A damaged intestinal barrier in TLR1/and TLR2/mice following C.albicans colonization and DSS treatment is also con-sis

45、tent with the clinical symptoms and severe epithe-lial damage in the colons of these mice.Effects ofDSSinduced colitis andC.albicans colonization onthe growth ofthe E.coli populationTo assess the impact of DSS-induced colitis and C.albi-cans colonization on the growth of the E.coli popula-tion,the c

46、olonic luminal contents were analyzed at day 14 in all groups.We found that C.albicans alone did not induce any changes in the E.coli population.How-ever,DSS-induced colitis promoted an increased E.coli population in TLR1/,TLR2/and wild-type mice,but a decrease in TLR6/mice,regardless of C.albi-cans

47、 colonization(Fig.5).Colonic expression levels ofcytokinesIL-1 and TNF are major pro-inflammatory cytokines that are rapidly released after tissue injury and fun-gal infection,so we determined their expression levels in the colons of mice challenged with C.albicans and treated with DSS(Fig.6ad).Both

48、 the mRNA expres-sion levels and protein levels of IL-1 and TNF were significantly increased in the colons of TLR1/and TLR2/mice,whereas their expression levels were sig-nificantly reduced in TLR6/mice when compared to wild-type mice(P0.05).Interestingly,both TLR1/and TLR2/mice had higher expression

49、 levels of IL-10 and IL-17A when compared to TLR6/and wild-type mice(Figs.5f,6e,P0.05).Fig.4 Candida albicans colonization in mouse DSS-induced colitis.a Number of C.albicans colony forming units(CFU)recovered from stools.Data are the mean SD of 20 mice per group.*P 0.05 for TLR6/C.albicans and DSS(

50、CaD)mice versus wild-type(WT)CaD mice.b,c Number of C.albicans CFU recovered from the stomach and colon.Data are the mean SD of 20 mice per group(P 0.001).d Mannan levels in the serum of wild-type,TLR1/,TLR2/,and TLR6/CaD mice.*P 0.05 for TLR1/CaD,TLR2/CaDSS and TLR6/CaD mice versus wild-type CaDSS