ABSTRACT
http://dx.doi.org/10.1590/1678-775720150145
L a c t o b a c i l l u s r h a m n o s u s c o u l d i n h i b i t
Po r p h y r o m o n a s g i n g i v a l i s d e r i v e d CX CL8
at t enuat ion
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1- Gazi University, Faculty of Dentistry, Department of Medical Microbiology, Ankara, Turkey.
2- Hacettepe University, PEDI-STEM Center for Stem Cell Research and Development, Ankara, Turkey. 3- Gazi University, Faculty of Dentistry, Department of Oral and Maxilofacial Surgery, Ankara, Turkey. 4- Gazi University, Faculty of Dentistry, Department of Oral Pathology, Ankara, Turkey.
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6- Technopolis of Hacettepe University, Hemosoft IT & Training Services, Department of Life Sciences, Ankara, Turkey.
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312 203 4365 - Fax: +90 312 221 3202 - e-mail: aysegulmendi@gmail.com
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n incr easing body of evidence suggest s t hat t he use of pr obiot ic bact er ia is a pr om ising LQWHUYHQWLRQDSSURDFKIRUWKHWUHDWPHQWRILQÀDPPDWRU\GLVHDVHVZLWKDSRO\PLFURELDO et iology. P. gingiv alis has been not ed t o have a differ ent way of int eract ing w it h t he innat e im m une r esponse of t he host com par ed t o ot her pat hogenic bact er ia, w hich is a r ecognized feat ur e t hat inhibit s CXCL8 expr ession. Obj ect ive: The aim of t he st udy wast o det er m ine if P. gingivalisLQIHFWLRQPRGXODWHVWKHLQÀDPPDWRU\UHVSRQVHRIJLQJLYDO
st r om al st em cells ( G- MSSCs) , including t he r elease of CXCL8, and t he expr ession of TLRs and if im m unom odulat or y L. r ham nosus ATCC9595 could pr event CXCL8 inhibit ion LQH[SHULPHQWDOLQÀDPPDWLRQ0DWHULDODQG0HWKRGV*066&VZHUHSUHWUHDWHGZLWKL. r ham nosus ATCC9595 and t hen st im ulat ed w it h P. gingivalis ATCC33277. CXCL8 and I L- 10
OHYHOVZHUHLQYHVWLJDWHGZLWK(/,6$DQGWKH7/5DQGZHUHGHWHUPLQHGWKURXJKÀRZ cyt om et er analysis. Result s: CXCL8 was suppr essed by P. gingivalis and L. r ham nosus ATCC9595, w her eas incubat ion w it h bot h st rains did not abolish CXCL8. L. r ham nosus ATCC9595 scaled down t he expression of TLR4 and induced TLR2 expression when exposed
t o P. gingivalisVWLPXODWLRQS&RQFOXVLRQV7KHVH¿QGLQJVSURYLGHHYLGHQFHWKDW
L. r ham nosus$7&&FDQPRGXODWHWKHLQÀDPPDWRU\VLJQDOVDQGFRXOGLQWURGXFHP. gingivalis t o im m une syst em s by inducing CXCL8 secr et ion.
Ke y w or ds: CXCL8 chem okine. St r om a. Per iodont it is. Pr obiot ics. Recept or.
I N TROD UCTI ON
Th e g r a m - n e g a t i v e , a n a e r o b i c b a ct e r i u m
Porphyrom onas gingivalis is considered t o be one of
t he key pat hogens in per iodont it is20,24. P. gingivalis
possesses a num ber of pat hogenic pr oper t ies t hat
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lipopoylsaccharides, and gingipains24. Accum ulat ing
dat a sh ow s t h at gin gipain s ar e inv olv ed in t h e r eg u lat ion of h ost in f lam m at or y r esp on ses. P.
gingivalis st im ulat es an innat e im m une r esponse
a n d i n d u ce s t h e e x p r e ssi o n o f i n f l a m m a t o r y m ed i at o r s, b u t i t can d o w n r eg u l at e t h e h o st
im m une response at t he sam e t im e. I n ot her words,
P. gingivalis has evolved var ious m echanism s t o
escape host im m une syst em s by invading host cells and disrupt ing signaling pat hways t hrough cyt okine and r ecept or degrading18.
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disease r esult ing fr om a com plex poly m icr obial infect ion in w hich t he disrupt ion of t he hom eost asis b e t w e e n t h e su b g i n g i v a l m i cr o b i o t a a n d t h e h o st d ef en se l ead s t o t h e d est r u ct i o n o f t h e t oot h- suppor t ing t issue25. As a r esult of bact er ial
encount er s, t he host cells synt hesize and r elease
cells t o t h e sit e of in f ect ion1 9 , 2 4. CXCL8 is an
im p or t an t ch em ok in e t h at at t r act s n eu t r op h ils t o t he sit e of infect ion. The CXCL8 chem okine is ex pr essed and pr oduced by differ ent cell t y pes
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kerat inocyt es, epit helial cells, and lym phocyt es10.
P. gingivalis has a differ ent way of int eract ing w it h
t he host ’s innat e im m une r esponse, com par ed t o ot her pat hogenic, gram - negat ive bact er ia, w hich is r ecog n ized as in h ib it in g CXCL8 ex p r ession . The at t enuat ion of CXCL8 m ay delay t he defense m echanism s of t he host and allow P. gingivalis t o escape t he im m une sy st em , t hus cr eat ing m or e dam age t o t he sur r ounding t issue17.
The abilit y of t he im m une syst em of t he host t o sense, r ecognize, and r espond t o per iodont al associat ed pat hogens is an im por t ant det er m inant in t he pat hogenesis of per iodont it is. This abilit y is lar gely m ediat ed by t he innat e im m une sy st em via t he expr ession of t oll- like r ecept or s ( TLRs)16.
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3, 4, and 5, and ligands binding t o t hese r ecept or s leads t o t he secr et ion of CXCL821. Mor eover, TLR2,
w hich r ecognizes gram - posit ive bact er ial cell walls,
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gingivalis12. St udies have show n t hat P. gingivalis
could signal via TLR2, TLR4, or bot h29.
Conv en t ion al per iodon t al t r eat m en t is of t en n o t su f f i ci en t b y i t sel f t o co n t r o l d est r u ct i v e
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diseases1. This r equir es t he developm ent of novel
a n d ef f ect i v e t h er a p eu t i c st r a t eg i es t h a t a r e adj unct ive t o clinical per iodont al t r eat m ent . The use of pr obiot ics is one of t he several appr oaches being considered for t he t reat m ent of periodont it is3.
Pr ob iot ic t h er ap y h as r ecen t ly g ain ed m assiv e
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effect s on general and oral healt h as w ell as for b ei n g an i m p o r t an t co m p l em en t t o an t i b i o t i c t r eat m en t . Fu r t h er m or e, t h e ad m in ist r at ion is sim ple, inexpensive, and safe23. Anim al and hum an
st udies have show n t hat t he use of pr obiot ics is em er gin g as a pot en t ial adj u n ct iv e t h er apy for per iodont it is, alt hough t he under lying m echanism s
UHPDLQSRRUO\GH¿QHG22. Our t eam has suppor t ed
conduct ing in vit r o st udies befor e using pr obiot ics in clinical t rials. I n t his st udy, t he hypot hesis t est ed was t hat CXCL8 suppr ession by P. gingivalis could be pr event ed by t he pr obiot ic st rain L. r ham nosus ATCC 9595 t hr ough co- aggr egat ion, com pet it ive adhesion, and t he expr ession of TLRs.
M ATERI AL AN D M ETH OD S
I so l a t i o n a n d i d e n t i f i ca t i o n o f g i n g i v a l m e se n ch y m a l st r om a l st e m ce lls
Gingiva sam ples were obt ained from four healt hy subj ect s aged 21 t o 24 year s old dur ing w isdom
-t oo-t h ex -t rac-t ion. The ex per im en-t al pr o-t ocol was r eview ed and appr oved by t he I nst it ut ional Et hical Boar d, and infor m ed consent for m was obt ained fr om each subj ect . Gingival m esenchym al st r om al
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accor ding t o Zhang, et al.30 ( 2009) . Differ ent iat ion
pr ot ocol was conduct ed accor ding t o t he pr ot ocol descr ibed as Pit t enger, et al.26 ( 1999) .
Ba ct e r ia a n d gr ow t h con dit ion s
L. r ham nosus ATCC9595, t he pr obiot ic st rain,
and P. gingivalis ATCC33277 w er e obt ained fr om Am er ican Ty pe Cult ur e Collect ion. The pr obiot ic st rain was cult ur ed in MRS br ot h ( de Man, Rogosa, Sh a r p e, MERCK) u n d er a er o b i c co n d i t i o n s a t 37°C for 18 h. P. gingiv alis was cult ur ed under anaer obic condit ions ( 80% N2, and 10% H2) at 37°C in an anaerobic cham ber ( Elect ot ek Anaerobic w or k st at ion , UK) an d m ain t ain ed on Sch aedlar
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m l–1) . The num ber of bact er ia at t he beginning
of t he ex per im ent was adj ust ed t o McFar land 2 ( ~ 1× 108) and expr essed as cfu m L–1 by t he ser ial
dilut ion t echnique.
Au t o- a n d co- a ggr e ga t ion a ssa y s
Th e au t o- ag g r eg at ion assay w as p er f or m ed accor ding t o t he m et hods developed by Del Re, et al.9 ZLWK FHUWDLQ PRGL¿FDWLRQV %ULHÀ\
bact er ia w er e gr ow n for 18 h at 37°C w it h t he ap p r op r iat e g r ow t h m ed iu m . Th e cu lt u r e w as har vest ed by cent r ifugat ion at 5000 g for 15 m in, w ash ed t w ice, an d r esu spen ded in ph osph at e-b u f f er ed salin e ( PBS) t o g iv e v iae-b le cou n t s of ap p r ox im at ely 1 08 cf u m L- 1 . Cell su sp en sion s
( 4 m L) w er e m ix ed by v or t ex ing for 1 0 s, and aut o- aggr egat ion was det er m ined dur ing 4 h of incubat ion at r oom t em perat ur e. At t he end of t he incubat ion per iod, 0.1 m L of t he upper suspension was t ransfer r ed t o anot her t ube w it h 0.9 m L of PBS, and t he absor bance ( A) w as m easur ed at 6 0 0 nm . The aut o- aggr egat ion per cent age w as expressed as ( 1–( At/ A0) ) × 100, where At represent s t he absor bance at t he end of incubat ion t im e and A0 t he absor bance at t = 0.
by Handley, et al.15 ( 1987) :
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( ( Ax + Ay ) / 2 ) , w her e x and y r epr esent each of t he t w o st rains in t he cont r ol t ubes and ( x + y ) r epr esent s t he m ixt ur e.
Com pe t it iv e a dh e sion of ba ct e r ia l st r a in s t o G- M SSCs
For t he com pet it ive adherence assay, a t ot al cell num ber of 200 000 G- MSSCs per w ell w er e seeded on glass cover slips and incubat ed for 24 h in 6 w ell plat es. Befor e t he exper im ent , t he cult ur e m edium was changed wit h ant ibiot ic- free m edium . G- MSSCs w er e challenged w it h bact er ia at a m ult iplicit y of infect ion ( MOI ) of 1: 100 ( 108 bact eria well- 1) at 37°C
in 5% CO2 for 1 h. L. r ham nosus and P. gingivalis w er e pr ovided equal chances t o bind at t he sam e rat io and at t he sam e t im e8. At t he end of 1 h, t he
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light m icr oscopy, as descr ibed by Ber net , et al.2
( 1993) . For each m onolayer on a glass cover slip, t he num ber s of adher ent bact er ia on 100 differ ent , random ly select ed cells w er e evaluat ed.
Effe ct of L. r h a m n osu s on G- M SSCs u pon ,)1DŽRUP. gin giv a lis st im u la t ion
G- MSSCs w ere allow ed t o at t ach t o and grow on 24- w ell t issue cult ur e plat es ( 50 000 cells w ell- 1)
cont aining cult ur e m edium ( Cost ar, Cor ning, USA) . Aft er 24 h of incubat ion, t he m edium was discarded, washed w it h PBS, and r eplaced w it h new m edium w it hout Pen/ St r ep. cells t hat w er e pr et r eat ed w it h
L. rham nosus ( MOI 1: 100) for 12 h at 37°C and 5%
CO2. Subsequent ly, t he cult ur e w ells w er e washed
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( I nv it r ogen, USA) or P. gingiv alis ( MOI : 1: 100) for anot her 12 h. At t he end of t he exper im ent , cult ur e super nat ant s w er e har vest ed t o det er m ine t heir cyt okine levels, and t he cells w er e pr epar ed
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D e t e r m in a t ion of cy t ok in e pr odu ct ion
The co- cult ur e super nat ant s w er e collect ed and cent rifuged at 4000 g for 3 m in at 4°C. The am ount s of CXCL8 and I L- 10 secret ed int o t he m edium during t he co- cult ur ing w it h t he bact er ia w er e m easur ed b y u si n g t h e Hu m an Ul t r asen si t i v e ELI SA Ki t ( I nvit r ogen, USA) . The cult ur e super nat ant s w er e dilut ed according t o t he m anufact urer ’s inst ruct ions. The ELI SA lim it s w er e 0–25 pg m L- 1 for CXCL8 and
0–50 pg m L- 1 for I L- 10.
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G- MSSCs w er e p r ep ar ed b y t r eat m en t w it h Tr y psin / EDTA ( I nv it r ogen , USA) , t r an sf er r ed t o
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cent r ifuged ( 200 g × 5 m in) , and w ashed w it h PBS. Th e cells w er e f ix ed b y ad d in g 2 m L of
FACS lysing solut ion ( BD Bioscience, Heidelber g,
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for 10 m in at r oom t em perat ur e in dar k. G- MSSCs w er e cent r ifuged ( 200 g × 5 m in) and w ashed w it h 2 m L of wash buffer ( 1% BSA, 0.1 % NaN3 PBS) . Then, t he cells w er e st ained for expr ession of TLR- 2 and TLR- 4 on G- MSSCs w it h ant i-TLR- 2 FI TC and ant i-TLR- 4 PE ant ibodies ( eBioscience, USA) . The harvest ed cells were incubat ed wit hin t he
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isot ype I gG, aft er which t hey were used as cont rols. Appr opr iat e isot y pe cont r ols ( I gG- 2a) w er e also used in each case. Posit ively st ained cells w er e
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sy st em ( CEllQuest Pr oSoft war e, BD Biosciences, Heid elb er g , Ger m an y ) . Th e ex p er im en t s w er e done in t r iplicat e; r epr esent at ive hist ogram s ar e pr esent ed in t he Result s sect ion.
D a t a a n a ly sis
A g g r e g a t i o n , a d h e s i o n , ELI SA , a n d f l o w cyt om et er assays w ere carried out in t w o individual ex p er im en t s in v olv in g 3 r ep licat es. Au t o- an d co- aggr egat ion was analyzed by Spear m an’s r ho t est , w her eas Mann- Whit ney U t est was applied for com par ing adhesion and com pet it ive adhesion assays. CXCL8 and I L- 10 det erm inat ion from cult ure su per n at an t s w er e an aly zed by com par in g t h e G- MSSCs wit h bact eria t reat ed cells and I FN induced gr oups using Mann- Whit ney U and Kr uskal- Wallis t est . TLR expression on cell surfaces by I FN-J and/ or
P. gingivalis ATCC33277 and abilit y of t he pr obiot ic
st rain t o reduce TLR- 4 expression were invest igat ed by com par ing t w o pr opor t ions by Z- t est . A value o f p < 0 . 0 1 an d p < 0 . 0 5 w as co n si d er ed t o b e
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RESULTS
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Th e ex p an si o n o f G- MSSCs i n cu l t u r e w as su ccessf u l f or at least eigh t passages in all of t he sam ples. The com m on MSC m ar ker s CD106, CD1 0 5 , CD7 3 , CD2 9 , CD9 0 , CD1 4 6 , an d CD4 4 a n d t h e h em a t o p o i et i c m a r k er s, CD 3 , CD 4 5 , CD14, HLADR, HLA- ABC, and CD34, w er e t est ed, su ggest in g a m esen chy m al or igin f or t h e cells ( Figur e 1- i) .
G- M SSCs sh ow a dipoge n ic a n d ost e oge n ic diffe r e n t ia t ion ca pa cit y , bu t pot e n cy is low
Th e m or ph ology an d st ain in g ch ar act er ist ics of t h e dif f er en t iat in g cells w er e con sist en t w it h a p r ead ip ocy t e p h en ot y p e. Sm all, d ar k d ep osit s w er e visible on t he cult ur e plat es of G- MSSCs aft er 7–10 day s, and t hey incr eased in t he follow ing d ay s w it h in ost eog en ic d if f er en t iat ion cu lt u r e. Am or phous deposit s w er e pr esent on t he plat es by
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posit ive Alizarin Red S st aining at day 30 of cult ure, indicat ing t he in vit r o ost eogenic pot ent ial of t he cells ( Figur e 1- ii.b) .
L. r h a m n osu s cou ld co- a ggr e ga t e w it h P. gin giv a lis
The r esult s show ed t hat t he st rains exhibit ed a st r ong aut o- aggr egat ion phenot ype ( Table 1) . The
Figure 1- Gingival stromal stem cells (G-MSSCs) showed mesenchymal stem cell properties. i) CD44, CD29, CD106,
CD105, CD146, and CD90 are mesenchymal stem cell surface receptors that were detected on G-MSSCs, except CD106. CD106 is a subpopulation of mesenchymal stem cells that addresses immunomodulatory properties. Hematopoietic stem cell surface receptors CD14, CD34, CD45; CD3, HLA DR were found to be negative. Expression of CD73 as a cluster
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low adipogenic differentiation potential, whereas osteogenic differentiation was strong (a) Adipogenic differentiation: The black arrows show positively stained G-MSSCs for lipid vesicules with Oil Red O stain. (b) Osteogenic differentiation: The calcium granules were stained black with Alizarin Red stain in the osteogenic differentiation medium on the G-MSSCs (40x, Olympus CKX41, Tokyo, Japan)
Auto-aggregation (%) &RDJJUHJDWLRQ
Strains L. rhamnosus P. gingivalis L. rhamnosus and P. gingivalis
98.8±0.0001** 95.25±0.002 23.7±0.002
$ QHJDWLYH VLJQL¿FDQW FRUUHODWLRQ ZDV IRXQG EHWZHHQL. rhamnosus and P. gingivalis co-aggregation according to
Spearman's rho test
**± refers to the standard deviation
co- aggregat ion of L. rham nosus wit h P. gingivalis is expr essed as t he per cent age r educt ion aft er 4 h in t he absor bance of a m ixed suspension, com par ed w it h t he individual suspension.
L. r h a m n osu s in h ib it s t h e a d h e sion of P. gin giv a lis
The m orphology of G- MSSCs following t reat m ent w it h a v iable pr obiot ic st r ain an d P. gin giv alis
w as ex am ined by light m icr oscopy. No obv ious m or phological changes induced by t he bact er ia w er e obser ved. We dist inguished t he at t ached P.
gingiv alis fr om t he pr obiot ic st rain accor ding t o
t heir Gram st ain propert ies. We also det erm ined t he adhesion of t he st rains separat ely; L. r ham nosus showed higher adhesive propert ies t han P. gingivalis on G- MSSCs ( p< 0.01; Table 2) .
Figure 2- i. L. rhamnosus ATCC9595 modulated CXCL8 (a) and IL-10 (b) secretion. (a) The gingival stromal stem cells
(G-MSSCs) secreted an amount of CXCL8 without any stimulation. L. rhamnosus and P. gingivalis reduced CXCL8, as
NQRZQ7KHUHGXFWLRQZDVIRXQGWREHVLJQL¿FDQWDFFRUGLQJWR0DQQ:KLWQH\8WHVWS$L. rhamnosus and P. gingivalisFRFXOWXUHH[KLELWHGDVWLPXODQWHIIHFWIRU&;&/DVDQLQÀDPPDWRU\LQGXFHUDFFRUGLQJWRD.UXVNDO:DOOLVWHVW
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was found to be reduced on L. rhamnosus pretreated G-MSSCs when induced with IFN (p<0.05). (b) In contrast to CXCL8, the G-MSSCs did not secrete IL-10. L. rhamnosus and P. gingivalis increased IL-10 secretion, compared with G-MSSCs (p<0.05). The L. rhamnosus and P. gingivalis coculture reduced IL-10, since CXCL8 was increased (p<0.05). IL-10 was reduced on IFN-induced G-MSSCs, while CXCL8 was increased. L. rhamnosus -pretreated G-MSSCs induced IL-10 in IFN stimulation (p<0.05). ii . TLR expression was found to be synchronized with CXCL8 and IL-10 secretion. (a) G-MSSCs did not express TLR2 or 4 (<99.3%) b) G-MSSCs expressed TLR4 (42.1%) when stimulated with IFN (p<0.01). c) G-MSSCs pretreated with L. rhamnosusGHFUHDVHGWKHH[SUHVVLRQRI7/5LQ,)1LQGXFHGLQÀDPPDWRU\FRQGLWLRQVS d) P. gingivalis -induced TLR4 expression (11.1%; p<0.01). On the other hand, both TLR4 and TLR2 were expressed (1.1%). Decreased CXCL8 represents a gingipain effect, since we expected increased CXCL8 due to expressed TLR4 e) The L. rhamnosus and P. gingivalis coculture was able to reduce TLR4 expression to 0.5% (p<0.01). On the other hand, TLR2 was found to be 1.7% (p<0.01), which indicates the TLR2- dependent CXCL8 secretion. f) L. rhamnosus, when used
L. r h a m n o su s st r a i n s co u l d m a n a g e t o
in du ce CX CL8
Bot h CXCL8 ( Figur e 2- i. a) and I L- 10 ( Figur e 2 - i. b ) w er e f ou n d in t h e G- MSSC cell cu lt u r e
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alon e, L. r h am n osu s an d P. gin giv alis r edu ced CXCL8 levels and incr eased I L- 10 in cell cult ur e
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pg m L- 1) and decr eased I L- 10 ( 4.4 pg m L- 1) levels
in G- MSSCs ( p< 0.05) . The probiot ic st rain was able
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( r educed t o 14 pg m L- 1) and induced I L- 10 levels
( 9.26 pg m L- 1; p< 0.05) . On t he ot her hand, w hen
G- MSSCs were pret reat ed wit h L. rham nosus before
P. gingiv alis st im ulat ion, CXCL8 secr et ions w er e
found t o incr ease t o 22 pg m L- 1 ( p< 0.05) .
L. r h a m n osu s r e du ce s TLR- 4 e x pr e ssion on
H[SRVXUHWR,)1DŽDQGP. gin giv a lis
We t h e n e x a m i n e d t h e TLR- 2 a n d TLR- 4
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( Fi g u r e 2 - i i ) . Th e G- MSSCs u p r e g u l a t e d t h e
H[SUHVVLRQ RI 7/5 RQ ,)1DŽ DQGP. gin giv alis
st im u lat ion ( Fig u r e 2 - ii. d ; 4 2 . 1 % an d 1 1 . 1 % , r espect iv ely ) , com par ed w it h t h e u n st im u lat ed g r ou p ( Fig u r e 4 a; p < 0 . 0 1 ) . P. g in g iv alis also induced as m uch TLR- 2 expr ession as t he pr obiot ic st rain ( 0.8% ; p< 0.01) . L. r ham nosus r educed P.
gingivalis- induced TLR- 4 expression from 11.1% t o
0.8% ( p< 0.01; Figur e 2- ii.e) . Also, t he G- MSSCs pr et r eat ed w it h L. r ham nosus dow nr egulat ed
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( p < 0 . 0 1 ) ( Fig u r e 2 - ii. c) . P. g in g iv alis in d u ced bot h TLR- 4 and TLR- 2 expr ession in 1.1% of t he G- MSSCs ( Figur e 2- ii.d) . This value was r educed t o 0.5% w hen t he pr obiot ic st rain was pr et r eat ed in t his gr oup. An analogous r esult w as seen in
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pr obiot ic st rain w as able t o r educe t he num ber of cells expr essing bot h TLR- 4 and TLR- 2 ( 0.9% ; Figur e 2- ii.f ) . On t he ot her hand, w hen used alone, t he pr obiot ic st rain caused 4.5% of t he GMSSCs t o ex pr ess TLR- 4 and 0.8% of t he cells t o ex pr ess TLR- 2.
D I SCUSSI ON
This in vit r o st udy aim ed t o invest igat e w het her t he pr obiot ic st rain L. r ham nosus ATCC 9595 could pr event t he P. gingivalis w elded CXCL8 supr ession t h r ou gh co- aggr egat ion , com pet it iv e adh esion , and t he expr ession of TLRs. We chose t o use t he pr obiot ic L. r h am n osu s becau se t h e st r ain w as r epor t ed t o induce I L- 10 secr et ion and pr oduce high levels of exopolysaccharides ( EPSs) in different w or ks4,6. EPSs ar e one of t he pr im ar y m et abolic
pr oduct s of lact ic acid bact er ia, and have r eceived an incr easing am ount of at t ent ion because of t heir
KHDOWK EHQH¿WV22. The abilit y of a m icr oor ganism
t o sur r ound it self w it h a highly hydrat ed EPS layer m ay pr ovide it w it h pr ot ect ion against desiccat ion and pr edat ion. We suggest t hat in t er m s of oral en v i r o n m en t s i n cl i n i ca l st u d i es, co n si d er i n g t h e st r essf u l con d it ion s cr eat ed b y saliv a an d t oot h sur faces, high EPS- pr oduct ive st rains of L.
r ham nosus m ay sur vive bet t er.
Alt hough t he m echanism s of act ion ar e not fully under st ood, it is generally accept ed t hat t he abilit y of pr obiot ics t o co- aggr egat e w it h pat hogens is a desir ed pr oper t y. Co- aggr egat ion has an im por t ant eco l o g i ca l r o l e a s a n i n t eg r a l p r o cess i n t h e developm ent and m aint enance of m ixed- species b iof ilm com m u n it ies, esp ecially in or al cav it y.
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b a ct e r i a n e e d t o a ch i e v e a n a d e q u a t e m a ss t hr ough aggr egat ion. We used a sim ple and r obust spect rophot om et ric m et hod t hat has been shown t o cor r espond w ell t o m or e sophist icat ed radioact ive
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r ham nosus could co- aggr egat e w it h P. gingivalis
aft er 4 h. The co- aggregat ion abilit y of t he probiot ic st rain could enable t he for m at ion of a bar r ier t hat pr event s colonizat ion by pat hogenic bact er ia9.
I n addit ion t o aggr egat ion, at t achm ent is an
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and in m ost cases, aggr egat ion abilit y is r elat ed t o cell adher ence pr oper t ies9. We ex am ined t he
com pet it ive adhesion of t he pr obiot ic st rain and P.
gingivalis on G- MSSC m onolayer s t hat w er e gr ow n
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st udy invest igat ing L. rham nosus and P. gingivalis’s com pet it ive adhesion on G- MSSCs. L. r ham nosus
Adhesion ability (bacteria/cell)* &RPSHWLWLYHDGKHVLRQDELOLW\EDFWHULDFHOO
L. rhamnosus* 34.02±0.25*** 22±2
P. gingivalis 6.85±0.1 2±0.2
*Difference between L. rhamnosus and P. gingivalisDGKHVLRQRQ*066&VZDVIRXQGWREHKLJKO\VLJQL¿FDQWDFFRUGLQJWR Mann-Whitney U test (p<0.01 and p<0.05)
&RPSHWLWLYHDGKHVLRQZDVQRWIRXQGWREHVWDWLVWLFDOO\VLJQL¿FDQWDFFRUGLQJWR0DQQ:KLWQH\8WHVWS!DQGS!
***± refers to the standard deviation
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w hich is in good agr eem ent w it h st udies conduct ed wit h Caco- 2 cells11,27. I t is feasible t o t ake advant age
of L. r ham nosus, w hich could confer advant age t o t his st rain a com pet it ive in vivo. I t is w ell know n t hat P. gingivalis adher es t o and invades epit helial
FHOOVRU¿EUREODVWVin vit ro. The invasion of epit helial
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a m ech an ism applied by bact er ia t o evade t h e im m une syst em of t he host1. I t was r epor t ed t hat
invasion could occur aft er 6 h of incubat ion25. Based
on t hese r epor t s, w e incubat ed t he pr obiot ic st rain and P. gingivalis for 1 h t o prevent t he invasion int o t he GMSSCs. We show ed t hat t he pr obiot ic st rain inhibit ed t he adhesion of P. gingiv alis, alt hough t he m echanism r em ains unclear. Our r esult s do not explain w het her t he exclusion of P. gingivalis
ZDVGXHWRDFRPSHWLWLRQIRUVSHFL¿FVLWHVRQWKH VXUIDFHRI*066&VRUWKHFRQVWLWXWLRQRIDELR¿OP
of bact er ia pr event ed access t o t he cell sur faces of or ganism s. These r esult s show a pot ent ial for adhesive and co- aggregat ive L. rham nosus t o inhibit t he cell associat ion and cell ent r y of P. gingivalis. Many st udies have t r ied t o induce a m icr obiological sh ift or a clin ical pr obiot ic effect in an alr eady m at ured oral m icrobiological environm ent . However,
LWVHHPVORJLFDOWKDWDSURELRWLFZLOOKDYHGLI¿FXOW\LQ FRORQL]LQJWKHPRXWKDQGH[HUWLQJEHQH¿FLDOFOLQLFDO
effect s. We suggest t hat L. r ham nosus w it h it s co-aggregat ion abilit y and st rong adhesive propert ies, m ay sur vive bet t er.
As a consequence of adhesion, t he pr efer ence w ould or ient t he cell r esponse accor ding t o t he pr obiot ic st rain, since pr obiot ics can also act ivat e and m odulat e t he im m une syst em7. I n t he pr esent
st udy, w e dem onst rat ed t hat pr obiot ic P. gingivalis i n t e r a ct i o n s ca n i n h i b i t CXCL8 a t t e n u a t i o n . I t is k n ow n t h at secr et ed CXCL8 p r ot ein s ar e dow nr egulat ed w hen cells ar e challenged w it h P.
gingivalis14. The suggest ed m echanism com pr ises
t h e d o w n r e g u l a t i o n o f CXCL8 - m RN A a n d / o r t h e degr adat ion of CXCL8 by pr ot eases ( called g i n g i p ai n s) . St r o n g ev i d en ce h as sh o w n t h at using ant ibiot ics or delet ing one of t he pr ot ease gen es ( r gpA, r gpB, or k gp) in P. gin giv alis did not dram at ically affect CXCL8 at t enuat ion14. We
show ed t hat 12 h of exposur e t o viable P. gingivalis suppr essed CXCL8 pr oduct ion in t he cells, w hich is inconsist ent w it h lit erat ur e17,25. On t he ot her hand,
w hen t he cells w er e pr et r eat ed w it h t he pr obiot ic st rain befor e P. gingivalis st im ulat ion, t he CXCL8 lev els w er e found t o hav e incr eased, indicat ing t hat P. gingiv alis pr ot eases m ight be degraded by L. r ham nosus or P. gingivalis- pr obiot ic st rain co- aggr egat ion, w hich m ay act ivat e or deact ivat e any st r uct ur es on t he bact er ia cell wall r esponsible for t he degradat ion of CXCL8. I ndeed, w e need t o dem onst rat e t he m olecular m echanism in t his
int eract ion. We scr eened t he effect s of bact er ia on cy t ok in e secr et ion s w h en u sed alon e. Low levels of CXCL8 and I L- 10 w er e det er m ined in t he G- MSSCs cult ur e super nat ant , as cor r oborat ed by Tonet t i, et al.28 ( 1994) , w ho r epor t ed t hat low- level
expr ession of CXCL8 in healt hy t issue m ost likely cont r ibut es t o t he r em ar kable abilit y of t he host t o lim it per iodont al bact er ial gr ow t h. To fur t her in v est ig at e t h e ef f ect of p r ob iot ics on CXCL8
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levels w er e dow nr egulat ed and I L- 10 levels w er e found t o have incr eased. Such im m unom odulat ion induced by pr obiot ics is im por t ant for m aint aining t he host - m icr obe hom eost asis w it hout t r igger ing
H[DJJHUDWHGGHWULPHQWDOLQÀDPPDWRU\UHVSRQVHV
Becau se TLR act iv at ion play s a v it al r ole in cyt okine pr oduct ion, w e m easur ed t he expr ession of TLRs aft er t r eat m ent w it h t he pr obiot ic st rain a n d P. g i n g i v a l i s. I t m u st b e n o t e d t h a t P.
g i n g i v a l i s l i p o p o l y sa cch a r i d e ( LPS) , a n o t h e r
put at ive v ir ulence fact or, is suggest ed t o evade r ecognit ion by t he host v ia TLR- 429. The lack of
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t o P. gingivalis is count er int uit ive, since t hat t his is a gram negat ive bact er ium t hat expr esses a LPS. How ever, P gingivalisXWLOL]HVDVSHFL¿FOLSLGDQG 4- phosphat ases and a deacylase, which, in concert , generat e a t et racylat ed and dephosphorylat ed lipid, a st r uct ur e t hat is biologically iner t7. I n cont rast
t o t his r epor t , in our st udy w e obser v ed TLR- 4 ex pr ession ( 1 1 . 1 % ) , bu t r edu ced CXCL8 lev els and TLR- 2. This r esult could be explained by t he d eg r ad at ion of secr et ed CXCL8 b y g in g ip ain s. Mor eover, P. gingivalis’s effect was clear ly alt er ed w h en L. r h am n osu s w as added t o t h e cu lt u r e. CXCL8 and TLR- 2 were found t o be enhanced, while TLR- 4 ex pr ession w as r edu ced. Sev er al gr ou ps have found t hat TLR- 2 is r equired for a full cyt okine r esponse t o infect ion w it h P. gingivalis, raising t he quest ion of w het her P. gingivalis evades clearance b y r ed u cin g r ecog n it ion t h r ou g h TLR- 2 , r at h er t han TLR- 45,13. Our r esult s clear ly show ed t hat L.
rham nosus t oget her wit h P. gingivalis boost s CXCL8
pr oduct ion w hile enhancing TLR 2 and inhibit ing 4 expr ession. Pr evious r epor t s show ed TLR-2, rat her t han TLR- 4, t o be cr it ical for t he host response t o infect ion wit h P. gingivalis5,13. Gingipain
CON CLUSI ON
Th e i m m u n o m o d u l at o r y p r o b i o t i c st r ai n L.
r ham nosus ATCC 9595 is pr oposed t o be essent ial
for m aint aining healt hy t issue, w it h m ult iple r oles including co- aggr egat ion, adhesion, and pr im ing
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defen se again st P. gin giv alis ATCC 3 3 2 7 7 . Th e default st at e of oral t issues, such as in t he gut , is
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p r ob iot ics t h at m od u lat e TLR sig n alin g . Th er e
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evaluat ed t he effect s of probiot ics, possibly because t hey used differ ent doses, t r eat m ent durat ions, bact er ial species, an d applicat ion for m s. Th ese
FRQÀLFWLQJUHVXOWVSRLQWRXWWKDWQRWDOOSURELRWLFV KDYH EHQH¿FLDO HIIHFWV RQ SHULRGRQWDO GLVHDVHV 7KHUHIRUHLWVHHPVQHFHVVDU\WRSHUIRUPVSHFL¿F
scr eenings t o select appr opr iat e pr obiot ic st rains for pr event ing gingivit is or per iodont it is and ot her oral healt h diseases. Thus, new in v it r o st udies
ZLWK JUHDWHU VFLHQWL¿F ULJRU VKRXOG EH GHYHORSHG WR HYDOXDWH WKH VDIHW\ DQG HI¿FDF\ RI SURELRWLFV
befor e t hey ar e int r oduced int o clinical pract ice for per iodont al t herapy. The r esult s of t he pr esent
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L. r ham nosus ATCC 9595 t o pr event P. gingivalis
in d u ced in f lam m at ion an d CXCL8 at t en u at ion . How ev er, our r esult s should be pr ov en in v iv o, t hr ough bot h hum an and anim al t r ials.
ACKN OW LED GEM EN TS
Th i s st u d y w a s su p p o r t e d b y t h e Tu r k i sh Sci en t i f i c an d Tech n o l o g i cal Resear ch Co u n ci l ( TUBI TAK, Pr oj ect no: SBAG 11S520 and SBAG-1002, 108S107) . We specially t hank t o I r em Akar
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CON FLI CT OF I N TERESTS
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