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Hindawi Publishing CorporationGastroenterology Research and PracticeVolume 2011, Article ID 971938, 16 pagesdoi:10.1155/2011/971938
Review Article
Probiotics, Nuclear Receptor Signaling, andAnti-Inflammatory Pathways
Sonia S. Yoon1 and Jun Sun1, 2
1 Division of Gastroenterology and Hepatology, Department of Medicine, Strong Memorial Hospital,University of Rochester Mediacal Center, University of Rochester, Rochester, NY 14642, USA
2 Department of Microbiology and Immunology, University of Rochester, P.O. Box 646,601 Elmwood Avenue, Rochester, NY 14642, USA
Correspondence should be addressed to Jun Sun, jun [email protected]
Received 2 February 2011; Revised 28 March 2011; Accepted 19 May 2011
Academic Editor: Genevieve B. Melton-Meaux
Copyright © 2011 S. S. Yoon and J. Sun. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.
There is increased investigation of the human microbiome as it relates to health and disease. Dysbiosis is implicated in variousclinical conditions including inflammatory bowel disease (IBD). Probiotics have been explored as a potential treatment for IBDand other diseases. The mechanism of action for probiotics has yet to be fully elucidated. This paper discusses novel mechanismsof action for probiotics involving anti-inflammatory signaling pathways. We highlight recent progress in probiotics and nuclearreceptor signaling, such as peroxisome-proliferator-activated receptor gamma (PPARγ) and vitamin D receptor (VDR). We alsodiscuss future areas of investigation.
1. Introduction
Probiotics are ingestible microorganisms with health bene-fits. Increased interest in the intestinal microbiome and itseffect on health and disease is evidenced by the concomi-tant increase in peer-reviewed clinical trials investigatingprobiotics as therapy since 1999 [1]. Studies of the varioussignaling pathways involved in the response to bacteria andinflammation have led to a more detailed understanding ofmechanisms and actions of probiotics. This paper discussesprogress in understanding how probiotics contribute tointestinal mucosal function, particularly in relation to anti-inflammatory signaling pathways.
2. Intestinal Microflora
The intestinal microflora, as a whole, serves importantfunctions in metabolism, intestinal epithelial cell functionand health, immunity, and inflammatory signaling [2, 3].Recently, there has been increasing interest in the role ofthe intestinal microflora and its total genetic composition,together referred to as the microbiome in the development,
maintenance, and perpetuation of various clinical condi-tions, both intestinal and extraintestinal.
Dysbiosis has been implicated in various clinical condi-tions including atopy, irritable bowel syndrome (IBS), col-orectal cancer, alcoholic liver disease in animal and humanstudies, obesity and other metabolic disorders, and chronicinflammatory diseases such as IBD [4–11]. Decreaseddiversity of the intestinal microbiota was seen in fecalsamples obtained from children who subsequently developedallergic disease [6, 7]. Altered microbiota composition incolon cancer patients when compared to patients withnormal colonoscopies and in patients with IBS comparedto unaffected patients has also been demonstrated [5, 9].Alcohol feeding resulted in enteric bacterial overgrowth ina mouse model [8]. The role of the microbiota in obesityhas been extensively studied and carefully reviewed in theliterature [12, 13]. Microbial composition in IBD patientswith ulcerative colitis (UC) or Crohn’s disease (CD) as com-pared to unaffected individuals has been studied and showsdecreased diversity [4, 14–19]. This altered microflora mayhave significant implications for the intestinal milieu, with asyet incompletely understood effects. The pathogenesis of IBD
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2 Gastroenterology Research and Practice
likely involves a combination of factors including intestinaldysbiosis in conjunction with environmental factors in agenetically susceptible host [20].
Based on the concept of a dysregulated or dysfunctionalmicrobiota in disease, various methods to attenuate theeffects of an altered microbiome have been attempted.
3. Probiotics
“Probiotics” were first described in the literature by Lillyand Stillwell in 1965 as growth-promoting factors producedby certain microorganisms [21] although it may have beendescribed as early as 1908 [22]. Recently, probiotics weredefined as “live organisms which, when consumed in ade-quate amounts as part of food, confer a health benefit on thehost” (Report of a Joint FAO/WHO Expert Consultation onEvaluation of Health and Nutritional Properties of Probioticsin Food Including Powder Milk with Live Lactic AcidBacteria (October 2001), “Health and Nutritional Propertiesof Probiotics in Food Including Powder Milk with LiveLactic Acid Bacteria”, Food and Agriculture Organizationof the United Nations, World Health Organization). Themechanisms of action of probiotics include immune modu-lation, direct effect on commensal and pathogenic bacteria toinhibit infection and restore homeostasis, and modificationof pathogenic toxins and host products [23]. The efficacy ofprobiotics in various clinical conditions both in the pediatricand adult patient population has been extensively studiedand carefully reviewed [1, 19, 24–32].
Rectal infusion of normal stool via enemas to treatpseudomembranous colitis has been described as early as1958 [70]. Infusion of stool via nasogastric tube to thesmall intestine or via colonoscopy to the colon for CDADhas also been described and shows high response rates[71–74]. A recent study showed that fecal bacteriotherapywas effective in relief of clinical symptoms in a patientwith recurrent CDAD and that this was accompanied bythe repopulation of the diseased intestinal microbiota withbeneficial species that were diminished pretreatment [75].Other methods to supply live, nonpathogenic organisms tothe intestinal microbiota in AAD and CDAD include orallyadministered probiotics. The efficacy of various probioticformulations in AAD and CDAD has been extensivelystudied and carefully reviewed [1]. A recent study showedthat the probiotics Lactobacillus acidophilus and Lactobacilluscasei were well tolerated and effective in reducing therisk of the development of AAD and CDAD [76]. Theutility of the probiotic yeast Saccharomyces boulardii for avariety of conditions including traveler’s diarrhea, enteralnutrition-associated diarrhea, AAD, and CDAD has beeninvestigated, and according to a recent meta-analysis, strongevidence exists for advocating its use in traveler’s diarrheaand AAD [77]. Recent trials using Bifidobacterium bifidumand Saccharomyces boulardii demonstrated improvement inclinical IBS symptoms and quality of life [78, 79], and severalreviews of the evidence for the utility of probiotics in IBShave been published [80–82].
For IBD therapy, treatment with different strains ofprobiotics has shown varied results. Small trials have shown
promise for probiotic use in the induction and maintenanceof remission in UC. VSL#3 has been shown to be safe andeffective in the treatment of acute mild to moderately activeUC [83]. Patients with mild to moderate UC unresponsive toconventional therapy achieved a combined induction remis-sion/response rate of 77% with treatment with VSL#3 [84].E. coli Nissle 1917 was found to be effective and equivalent tomesalazine in maintaining remission in UC [85]. In anotherstudy, Lactobacillus rhamnosus GG (LGG) was equivalent tomesalazine in the maintenance of remission in UC, however,appeared to be more effective in prolonging the relapse-free time [86]. Evidence also exists for the role of probioticsin prophylaxis of pouchitis after surgery in UC patientsas well as induction of remission in chronic pouchitis[87, 88].
Studies of probiotic use in induction and maintenanceof remission and prevention of postoperative recurrencein CD have been less consistent than those for UC. Asmall study of LGG for the prevention of recurrence aftersurgery in CD did not show any improvement over placebo[89]; however, Saccharomyces boulardii appears useful inmaintaining remission in CD [90, 91]. The progress in theuse of probiotics for IBD has been carefully reviewed [92, 93];however, there remains a relative lack of well-designed, large,randomized, placebo-controlled trials.
Several barriers exist to advocating broad use of probi-otics in clinical practice, not least of which is the considerableheterogeneity in the experimental designs with respect tospecies and strains of probiotics and the various animalmodels utilized [94]. Although clinical trials examining therole of probiotics in the treatment and/or prevention of AAD,CDAD, IBD including UC, CD, and pouchitis, necrotizingenterocolitis, infectious gastroenteritis, radiation-inducedenteritis, and colitis, IBS and various atopic diseases havebeen reported [1, 24, 25, 28, 29, 31, 87, 95–97]; in manycases, results have been inconsistent, and large, well-designedtrials are lacking. An additional complicating factor pertainsto issues of quality control. Determining whether a commer-cially available probiotic actually contains the live organismsit purports to contain and determining if there is rationalselection of component probiotic strains in “cocktails” areissues that must be considered [22]. Future research to refinetechniques to accurately identify “normal” and “diseased”microbiota and to further elucidate the specific effects andmechanisms of actions of individual probiotic strains will aidin optimizing therapeutic efficacy.
4. Mechanisms for Probiotics inAnti-Inflammation
There has been and continues to be considerable research indelineating the underlying mechanisms by which probioticsexert their beneficial effects. The mechanisms regulating thefunction of probiotics are very diverse. It is well accepted thatprobiotics use distinct cellular and molecular mechanisms,including blocking pathogenic bacterial effects, regulat-ing immune responses, and altering intestinal epithelialhomeostasis by promoting cell survival, enhancing barrierfunction, and stimulating protective responses [32].
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Gastroenterology Research and Practice 3
Table 1 outlines representative publications on probioticmechanisms of actions. The probiotic-host interaction iscomplex and further complicated by the fact that certainprobiotic effects appear to be species and strain specific.Different probiotics have been shown to exert both pro-inflammatory [98] and anti-inflammatory effects on den-dritic cells [99]. A recent study demonstrated that the anti-inflammatory effect of certain lactobacilli is via NOD2-mediated signaling [100]. NOD2/CARD15 is a member ofa superfamily of genes involved in intracellular bacterialrecognition and has been identified as an important suscep-tibility gene for CD [101, 102]. The authors speculate thatthe inconsistent clinical results of lactobacilli use in patientswith CD may be related to a relative deficiency of NOD2.Probiotic effect on the innate immune responsive pathwaysincluding toll-like receptor (TLR), nuclear factor kappa B(NF-κB), mitogen-activated protein kinase (MAPK), c-JunNH2-terminal kinase (JNK) has been extensively investigated(Table 1). Activation of specific TLRs also appears to bespecies specific [47, 48]. The action of E. coli Nissle 1917 onCaco-2 cells was found to be mediated by flagellin possiblyvia a TLR pathway [103]. The probiotic-induced effect on theNF-κB signaling pathway is well represented in the literatureand is generally characterized by inhibition (Table 1).
Defective epithelial barrier function has been implicatedin IBD and can predict relapse during clinical remission[104–109]. One way by which probiotics have been shownexert their action is by stabilizing tight junctions (TJs)and enhancing barrier function of intestinal epithelial cells(Table 1).
Abnormal STAT/suppressor of cytokine signaling(SOCS) signaling has been demonstrated in CD patients[110], and probiotics are also shown to modulate the JAK-STAT signaling in human placental trophoblast cells [111].Increasing evidence further demonstrates that metabolism,xenobiotics, and nuclear receptor signaling are involved inthe action of probiotics [67, 68].
Induction of heat shock proteins (HSPs) and endogenousantimicrobial peptides (defensins) via activation of NF-κB,MAPK, and JNK has also been linked to probiotic action [35,41, 43]. Since defensins are implicated in the pathogenesis ofIBD, increased expression by probiotics provides a possiblemechanism for clinical efficacy seen in certain IBD patientsand deserves further study.
5. Defensins and Nuclear Receptor Signaling
Defensins are a class of endogenous antimicrobial peptidesinvolved in innate immunity which is highly evolutionarilyconserved and represents a primary line of defense againstvarious microbial pathogens [112–114]. Antimicrobial pep-tides are widely distributed throughout the animal and plantkingdom, and despite their evolutionary heritage, remaineffective antimicrobial agents [114]. This is due, in largepart, to their mechanism of action involving membranedisruption and pore formation, which is not easily exploitedby pathogens to confer resistance [112–115]. Importantantimicrobial peptides in humans include defensins, cathe-licidins, lysozymes, and other antimicrobial antiproteases
[116]. There are three known defensin subfamilies; α and βdefensins are expressed mainly in immune cells and epithelialcells while the θ defensin is found mainly in immune cellsof the Rhesus macaque [117, 118]. In the gastrointestinaltract, β defensin expression is seen in multiple sites, whereasα defensin expression is largely in the small intestine[119]. In the uninflamed colon, human β defensin 1 isthe predominant defensin and human β defensin 2 and 3are induced with inflammation or infection [120]. In micelacking functional cryptidins (murine α defensins), increasedsurvival and virulence of orally administered bacteria wereseen and intestinal peptide preparations had decreasedantimicrobial activity [121].
The possible role of a deficiency in defensins in thepathogenesis of IBD has been proposed [116, 122]. ThePaneth cells of the small intestine are the major source ofendogenous antimicrobials, including α defensins [102]. Inaddition, The Paneth cells have been shown to express NOD2[123]. In patients with ileal CD, human α defensin 5 and6 production is reduced, and this effect is magnified inthose patients with a concomitant NOD2 mutation [124].For β defensins, CD patients with colonic disease exhibitnormal levels of β defensin 2 and 3 whereas UC patientshave increased levels, suggesting a role of failure of β defensininduction in the pathogenesis of CD [125]. Constitutivehuman β defensin 1 expression is reduced in CD patientswith colonic involvement independent of inflammation,and recently, the maintenance of constitutive β defensinexpression was shown to be activated by the nuclear receptorperoxisome-proliferator-activated receptor gamma (PPARγ)[122].
Further contributing to the effect of a defensin defi-ciency in the pathogenesis of IBD may be the diminisheddiversity of the intestinal microbiota seen in IBD patients.The interaction of commensal bacteria with antimicrobialpeptide synthesis is not well understood; however, it hasbeen suggested that commensal bacteria provide chronicstimulation of epithelial cells to produce antimicrobialpeptides at levels sufficient to kill microbial pathogens [114,126].
Probiotics, but not fecal isolates, have been shown toinduce human β defensin 2 in intestinal epithelial cells [41,42]. Wehkamp et al. and Schlee et al. have reported thatNF-κB and activator protein-1 (AP-1) mediate inductionof human β defensin 2 in intestinal epithelial cells by theprobiotic E. coli Nissle 1917 and VSL#3 [41, 42].
Interestingly, nuclear receptors are known to regulate theexpressions of defensins [122, 127]. Nuclear receptors rep-resent a class of intracellular transcription factors activatedby ligands which can directly interact with DNA; as a result,nuclear receptors play significant roles in the regulation ofmetabolic, reproductive, developmental, and immune pro-cesses [128–131]. Nuclear receptors regulate transcriptionalactivity by several distinct mechanisms, including “ligand-dependent transactivation, ligand-independent repression,and ligand-dependent transrepression” although the rangeof transcriptional activities of each nuclear receptor variesand even the transcriptional effects of a single nuclearreceptor may be cell specific [132]. A detailed discussion
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4 Gastroenterology Research and Practice
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colit
isan
dpr
oin
flam
mat
ory
cyto
kin
ese
cret
ion
inW
Tbu
tn
otT
LR2-
orT
LR4-
knoc
kou
tm
ice
(ii)
EC
Nre
sult
edin
redu
ctio
nof
inte
rfer
on(I
FN)-γ
secr
etio
nin
TL
R2
knoc
kou
t(i
ii)
Cyt
okin
ese
cret
ion
was
alm
ost
un
dete
ctab
lean
dn
otm
odu
late
dby
EC
Nin
TLR
-4-k
noc
kou
tm
ice
(iv)
Incr
ease
dT
LR2
and
TLR
4pr
otei
nex
pres
sion
and
NF-κB
acti
vity
via
TL
R2
and
TL
R4
was
seen
wit
hE
CN
and
hum
anT
-cel
lcoc
ult
ure
[48]
-
6 Gastroenterology Research and Practice
Ta
ble
1:C
onti
nu
ed.
Invo
lved
path
way
sP
robi
otic
sIn
vitr
osy
stem
Invi
vosy
stem
Sum
mar
yR
ef.
MA
PK
(p38
/ER
K)
(i)
LP(i
i)L
c(i
)M
ice
per
iton
eal
mac
roph
ages
(i)
LPst
ron
gly
indu
ced
IL-1
0an
dw
eakl
yin
duce
dIL
-12;
Lcst
ron
gly
indu
ced
IL-1
2an
dw
eakl
yin
duce
dIL
-10
(ii)
LP,c
ompa
red
toLc
,dem
onst
rate
dm
ore
rapi
dan
dst
ron
gac
tiva
tion
ofM
AP
Ks,
espe
cial
lyof
ER
K(i
ii)
Blo
ckin
gL
P-in
duce
dE
RK
acti
vati
onre
sult
edin
decr
ease
dIL
-10
prod
uct
ion
and
incr
ease
dIL
-12
prod
uct
ion
(iv)
Com
bin
edst
imu
lati
onw
ith
LPan
dL
cre
sult
edin
syn
ergi
stic
indu
ctio
nof
IL-1
0pr
odu
ctio
n;t
his
was
trig
gere
dby
the
key
fact
ors:
cell
wal
ltei
choi
cac
idan
dlip
otei
choi
cac
ids
(v)
Teic
hoi
c-ac
id-i
ndu
ced
IL-1
0pr
odu
ctio
nw
asm
edia
ted
byT
LR2-
depe
nde
nt
ER
Kac
tiva
tion
[49]
MA
PK
(p38
/ER
K)
Tig
ht
jun
ctio
ns
(TJ)
(i)
LGG
(i)
CaC
o-2
(i)
LGG
-pro
duce
dso
lubl
epr
otei
ns
(p40
and
p75)
dim
inis
hed
the
hydr
ogen
-per
oxid
e-in
duce
dde
crea
sein
TE
Ran
din
crea
sein
inu
linpe
rmea
bilit
yan
din
duce
din
crea
sein
mem
bran
etr
ansl
ocat
ion
ofpr
otei
nki
nas
eC
(PK
C)
beta
I,P
KC
epsi
lon
,an
dle
velo
fph
osph
o-E
RK
1/2
inth
ede
terg
ent-
inso
lubl
efr
acti
ons
(ii)
LGG
-pro
duce
dso
lubl
epr
otei
ns
(p40
and
p75)
prev
ente
dhy
drog
en-p
erox
ide-
indu
ced
redi
stri
buti
onof
occl
udi
n,z
onu
laoc
clu
den
s(Z
O)-
1,E
-cad
her
in,a
nd
beta
-cat
enin
from
inte
rcel
lula
rju
nct
ion
san
dth
eir
diss
ocia
tion
from
the
dete
rgen
t-in
solu
ble
frac
tion
s(i
ii)
p40-
and
p75-
med
iate
dre
duct
ion
ofhy
drog
en-p
erox
ide-
indu
ced
tigh
tju
nct
ion
disr
upt
ion
and
inu
linp
erm
eabi
lity
was
atte
nu
ated
byU
0126
(aM
AP
kin
ase
inh
ibit
or)
[50]
MA
PK
(p38
/ER
K)
TJ
(i)
B.i
nfan
tis
(i)
T84
(i)
IL-1
0,IL
-1de
fici
ent
mic
e
(i)
B.i
nfan
tis-
con
diti
oned
med
ium
(BiC
M)
incr
ease
dT
ER
,ZO
-1,a
nd
occl
udi
nex
pres
sion
and
decr
ease
dcl
audi
n-2
expr
essi
on;t
his
was
asso
ciat
edw
ith
incr
ease
dph
osph
o-E
RK
and
decr
ease
dph
osph
o-p3
8(i
i)T
NF-α
-an
dIF
N-γ
-in
duce
ddr
ops
inT
ER
and
rear
ran
gem
ent
ofT
Jpr
otei
ns
wer
epr
even
ted
byB
iCM
(iii
)In
hib
itio
nof
ER
Kat
ten
uat
edth
epr
otec
tion
from
TN
F-α
and
IFN
-γan
dpr
even
ted
BiC
M-i
ndu
ced
incr
ease
inT
ER
(iv)
Invi
vo,o
ralB
iCM
redu
ced
colo
nic
perm
eabi
lity,
inIL
-10-
defi
cien
tm
ice,
lon
g-te
rmB
iCM
decr
ease
dco
lon
ican
dsp
len
icIF
N-γ
secr
etio
n,a
tten
uat
edin
flam
mat
ion
,an
dn
orm
aliz
edco
lon
icpe
rmea
bilit
y
[51]
TJ
(i)
LPM
B45
2(i
)C
aCo-
2
(i)
LPM
B45
2in
crea
sed
TE
Rac
ross
Cac
o-2
cell
mon
olay
ers
indo
se-d
epen
den
tm
ann
er(i
i)A
lter
edex
pres
sion
ofse
vera
ltig
ht-
jun
ctio
n-r
elat
edge
nes
(in
clu
din
goc
clu
din
and
asso
ciat
edpl
aqu
epr
otei
ns)
was
seen
inre
spon
seto
LPM
B45
2(i
ii)LP
MB
452
cau
sed
chan
ges
inge
ne
expr
essi
onle
vels
oftu
bulin
and
prot
easo
me
(iv)
LPM
B45
2-tr
eate
dce
llssh
owed
incr
ease
dfl
uor
esce
nce
inte
nsi
tyof
the
fou
rti
ght
jun
ctio
npr
otei
ns
wh
enco
mpa
red
tou
ntr
eate
dco
ntr
ols
[52]
-
Gastroenterology Research and Practice 7T
abl
e1:
Con
tin
ued
.
Invo
lved
path
way
sP
robi
otic
sIn
vitr
osy
stem
Invi
vosy
stem
Sum
mar
yR
ef.
TJ
PK
C(i
)LP
(i)
CaC
o-2
(i)
Un
con
juga
ted
bilir
ubi
n(U
CB
)ca
use
dde
crea
sed
PK
Cac
tivi
ty,s
erin
eph
osph
oryl
ated
occl
udi
ng,
and
ZO
-1le
vels
(ii)
Hig
hco
nce
ntr
atio
ns
ofU
CB
cau
sed
cyto
toxi
city
and
decr
ease
dT
ER
(iii
)Tr
eatm
ent
wit
hL
Pm
itig
ated
the
effec
tsof
UC
Bon
TE
Ran
dap
opto
sis,
prev
ente
dab
erra
nt
expr
essi
onan
dre
arra
nge
men
tof
TJ
prot
ein
s,an
dpa
rtia
llyre
stor
edP
KC
acti
vity
and
seri
ne
phos
phor
ylat
edT
Jpr
otei
nle
vels
[53]
TJ
(i)
LPD
SM26
48(i
)C
aCo-
2
(i)
LPD
SM26
48re
duce
dth
ede
lete
riou
seff
ect
ofE
sche
rich
iaco
li(e
nte
ropa
thog
enic
E.c
oli(
EP
EC
))O
127:
H6
(E23
48/6
9)on
TE
Ran
dad
her
ence
wit
hsi
mu
ltan
eou
sor
prio
rco
cult
ure
com
pare
dw
ith
EP
EC
incu
bati
onal
one
[54]
TJ
TL
R(i
)LP
(i)
CaC
o-2
(i)
Hu
man
(i)
LPin
duce
dtr
ansl
ocat
ion
ofZ
O-1
toth
eT
Jre
gion
inan
invi
tro
mod
el,b
ut
the
effec
tson
occl
udi
nw
ere
min
orco
mpa
red
wit
heff
ects
seen
invi
vo(i
i)L
Pac
tiva
ted
TL
R2
sign
alin
g,an
dtr
eatm
ent
wit
hT
LR
2ag
onis
tPa
m(3
)-C
ys-S
K4(
PC
SK),
incr
ease
dfl
uor
esce
nt
stai
nin
gof
occl
udi
nin
the
TJ
(iii
)P
hor
bol-
este
r-in
duce
ddi
sloc
atio
nof
ZO
-1an
doc
clu
din
and
asso
ciat
edin
crea
sein
epit
hel
ialp
erm
eabi
lity
wer
eat
ten
uat
edw
ith
pret
reat
men
tw
ith
LPor
PC
SK
[55]
TJ
(i)
B.b
ifidu
m(i
)R
ats
(i)
B.b
ifidu
mde
crea
sed
the
inci
den
ceof
nec
roti
zin
gen
tero
colit
is(N
EC
),n
orm
aliz
edIL
-6,m
uci
n-3
,an
dT
ff3
leve
lsin
the
ileu
mof
NE
Cra
ts,a
nd
nor
mal
ized
the
expr
essi
onan
dlo
caliz
atio
nof
TJ
and
adh
eren
sju
nct
ion
(AJ)
prot
ein
sin
the
ileu
mco
mpa
red
wit
han
imal
sw
ith
NE
C(i
i)B
.bifi
dum
did
not
affec
tre
duce
dm
uci
n-2
prod
uct
ion
inth
eN
EC
rats
[56]
TJ
(i)
VSL
#3(i
)B
AL
B/c
mic
e
(i)
VSL
#3tr
eatm
ent
prev
ente
dth
ein
crea
sein
epit
hel
ialp
erm
eabi
lity
inac
ute
colit
is,d
ecre
ase
inex
pres
sion
and
redi
stri
buti
onof
occl
udi
n,Z
O-1
,an
dcl
audi
n-1
,cla
udi
n-3
,cla
udi
n-4
,an
dcl
audi
n-5
,an
din
crea
seof
epit
hel
ial
apop
toti
cra
tio
[57]
TJ
(i)
Lr(i
i)L
a(i
)M
ice
(i)
Pre
trea
tmen
tw
ith
com
bin
atio
nof
Lran
dL
asi
gnifi
can
tly
prev
ente
dde
crea
sein
the
mem
bran
e-bo
un
dA
TPa
ses
and
redu
ced
expr
essi
onof
tigh
tju
nct
ion
prot
ein
sin
the
mem
bran
e[5
8]
TJ
(i)
B.l
acti
s42
0(i
i)B
.lac
tis
HN
019
(iii
)L
aN
CFM
(iv)
L.sa
livar
ius
Ls-3
3
(i)
CaC
o-2
(i)
B.l
acti
s42
0an
dE
sche
rich
iaco
liO
157:
H7
(EH
EC
)su
per
nat
ant
had
oppo
site
effec
tsin
tigh
tju
nct
ion
inte
grit
y;B
.lac
tis
420
sup
ern
atan
tpr
otec
ted
the
tigh
tju
nct
ion
sfr
omE
HE
C-i
ndu
ced
dam
age
wh
enad
min
iste
red
befo
reE
HE
Csu
pern
atan
t(i
i)E
HE
Can
dpr
obio
tics
had
reve
rse
effec
tsu
pon
cycl
o-ox
ygen
ase
expr
essi
on
[59]
TJ
(i)
EC
N(i
)B
AL
B/c
mic
e
(i)
EC
Nco
lon
izat
ion
ofgn
otob
ioti
cm
ice
resu
lted
inu
preg
ula
tion
ofZ
O-1
mR
NA
and
prot
ein
leve
lsin
IEC
s(i
i)E
CN
adm
inis
trat
ion
redu
ced
loss
ofbo
dyw
eigh
tan
dco
lon
shor
ten
ing
inD
SS-t
reat
edm
ice
(iii
)E
CN
inoc
ula
tion
amel
iora
tes
the
infi
ltra
tion
ofth
eco
lon
wit
hle
uko
cyte
s
[60]
TJ
PK
C(i
)E
CN
(i)
T84
(i)
EP
EC
wit
hE
CN
coin
cuba
tion
:add
itio
nof
EC
Naf
ter
EP
EC
infe
ctio
nre
stor
edba
rrie
rin
tegr
ity
and
abol
ish
edba
rrie
rdi
sru
ptio
n(i
i)E
CN
alte
red
the
expr
essi
on,d
istr
ibu
tion
ofZ
O-2
prot
ein
and
ofdi
stin
ctP
KC
isot
ypes
;ZO
-2ex
pres
sion
was
incr
ease
din
para
llelt
oit
sre
dist
ribu
tion
tow
ards
the
cell
bou
nda
ries
(iii
)E
CN
indu
ces
rest
orat
ion
ofa
disr
upt
edep
ith
elia
lbar
rier
;th
isis
tran
smit
ted
byP
KC
zeta
sile
nci
ng
and
ZO
-2re
dist
ribu
tion
[61]
-
8 Gastroenterology Research and Practice
Ta
ble
1:C
onti
nu
ed.
Invo
lved
path
way
sP
robi
otic
sIn
vitr
osy
stem
Invi
vosy
stem
Sum
mar
yR
ef.
TJ
(i)
LGG
(i)
MD
CK
-1,T
84
(i)
EH
EC
-in
duce
dde
crea
sein
elec
tric
alre
sist
ance
and
the
incr
ease
inba
rrie
rp
erm
eabi
lity
assa
ysw
ere
atte
nu
ated
bypr
obio
tic
pret
reat
men
t(i
i)LG
Gpr
otec
ted
epit
hel
ialm
onol
ayer
sag
ain
stE
HE
C-i
ndu
ced
redi
stri
buti
onof
clau
din
-1an
dZ
O-1
prot
ein
s(i
ii)
Hea
t-in
acti
vate
dLG
Gdi
dn
otaff
ect
EH
EC
bin
din
gor
disr
upt
ion
ofba
rrie
rfu
nct
ion
[62]
JAK
/STA
TM
AP
K(p
38/E
RK
)
(i)
Stre
ptoc
occu
sth
erm
ophi
lus
(ii)
La
(i)
HT
29/c
l.19A
(ii)
Cac
o-2,
(i)
Stre
ptoc
occu
sth
erm
ophi
lus
(ST
)/La
orth
eco
mm
ensa
lBac
tero
ides
thet
aiot
aom
icro
n(B
T)
prev
ente
dth
eT
NF-α
-an
dIF
N-γ
-in
duce
dre
duct
ion
inT
ER
and
incr
ease
inep
ith
elia
lper
mea
bilit
y(i
i)ST
/La
orB
Tpr
even
ted
IFN
-γin
hib
itio
nof
agon
ist-
stim
ula
ted
chlo
ride
secr
etio
n(i
ii)
ST/L
aor
BT
rest
orat
ion
ofC
l(-)
secr
etio
nw
asbl
ocke
dby
inh
ibit
ors
ofp3
8M
AP
K,E
RK
1,2,
and
PI3
K(i
v)ST
/La
pret
reat
men
tre
vers
edth
eIF
N-γ
-in
duce
ddo
wn
regu
lati
onof
the
cyst
icfi
bros
istr
ansm
embr
ane
con
duct
ance
regu
lato
r(C
FTR
)an
dth
eN
KC
C1
cotr
ansp
orte
r(v
)T
he
effec
tsof
ST/L
aor
BT
onT
ER
and
per
mea
bilit
yw
ere
pote
nti
ated
bya
Jan
us
kin
ase
(JA
K)
inh
ibit
orbu
tn
otby
p38,
ER
K1,
2,or
PI3
Kin
hib
itio
n(v
i)R
edu
ced
acti
vati
onof
supp
ress
orof
cyto
kin
esi
gnal
ing
(SO
CS)
3an
dST
AT
1,3
was
seen
only
inpr
obio
tic-
trea
ted
epit
hel
ialc
ells
expo
sed
tocy
toki
nes
[63]
JAK
/STA
TN
F-κB
(i)
LcS
(i)
RA
W26
4.7
(ii)
LI-
LP
MC
(iii
)U
C-P
BM
C
(i)
SAM
P1/
Yit
mic
e
(i)
LcS
impr
oved
chro
nic
ileit
isin
SAM
P1/
Yit
mic
e(i
i)L
csim
prov
edm
uri
ne
chro
nic
colit
is,a
nd
this
was
asso
ciat
edw
ith
decr
ease
dIL
-6pr
odu
ctio
nby
larg
ein
test
inal
lam
ina
prop
ria
mon
onu
clea
rce
lls(
LI-L
PM
Cs)
and
dow
nre
gula
tion
ofpr
oin
flam
mat
ory
cyto
kin
essu
chas
IL-6
and
IFN
-γpr
odu
ctio
nin
LPM
C(i
ii)
IL-6
rele
ase
inLP
S-ac
tiva
ted
LI-L
PM
C,R
AW
264.
7,an
du
lcer
ativ
eco
litis
-per
iph
eral
bloo
dm
onon
ucl
ear
cells
(UC
-PB
MC
s)w
asin
hib
ited
byLc
S-de
rive
dpo
lysa
cch
arid
e-pe
ptid
ogly
can
com
plex
(PSP
G)
(iv)
Inlip
opol
ysac
char
ide-
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ear
tran
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nof
NF-κB
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dow
nre
gula
ted
byL
cS
[64]
-
Gastroenterology Research and Practice 9
Ta
ble
1:C
onti
nu
ed.
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lved
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MA
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biot
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amm
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id,a
nd
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obio
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abol
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iin
the
CD
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fru
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ein
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glu
cose
accu
mu
lati
on
[68]
Rea
ctiv
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s(R
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nt
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SH)
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llin
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seen
inth
ein
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ines
ofm
ice
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owin
glo
calR
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erat
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and
the
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ltan
tU
bc12
inac
tiva
tion
(iii
)P
refe
edin
gLG
Gpr
even
ted
TN
F-α
-in
duce
dac
tiva
tion
ofin
test
inal
NF-κB
[69]
-
10 Gastroenterology Research and Practice
of nuclear receptors and their mechanisms of action isbeyond the scope of this article; however, further discussionof two nuclear receptors (peroxisome-proliferator-activatedreceptor gamma (PPARγ) and vitamin D receptor (VDR))with putative roles in inflammation is warranted.
PPARγ is a member of a class of nuclear receptors thatform obligate heterodimers with the retinoid X receptor(RXR) [129]. The PPAR family has been shown to affectvarious cellular functions including “adipocyte differentia-tion, fatty-acid oxidation, and glucose metabolism” [129].PPARγ is highly expressed in the large intestine [133], and itsactivation has been shown to be protective in animal modelsof colitis [134, 135]. Decreased PPARγ expression in UCpatients has been shown [136], and the anti-inflammatorycompound 5-aminosalicylic acid (5-ASA) commonly uti-lized in IBD therapy was shown to be a PPARγ agonist,thereby establishing a possible mechanism by which it exertsits anti-inflammatory effects [137]. PPARγ also plays a rolein the maintenance of “constitutive epithelial expression of asubset of β defensins in the colon” [122].
6. Vitamin D and Vitamin D Receptor (VDR)
Vitamin D receptor (VDR) is a nuclear receptor that medi-ates most known functions of 1,25-dihydroxyvitamin D(1,25(OH)2D3), the active form of vitamin D [138]. VDRheterodimerizes with RXR once VDR is activated by1,25(OH)2D3. VDR binds to the vitamin D response elementin the target gene promoter to regulate gene transcription[139]. VDR downstream target genes include antimicrobialpeptides such as cathelicidin and β defensin.
VDR is critical in regulating intestinal homeostasis bypreventing pathogenic bacterial invasion, inhibiting inflam-mation, and maintaining cell integrity [140–145]. VitaminD directly modulates the T-cell receptor (TCR) [146], andvitamin D has also been shown to downregulate the expres-sion of proinflammatory cytokines and have regulatoryeffects on autophagy and various immune cells includingT cells, B cells, macrophages, dendritic cells, and epithelialcells [147, 148]. It has been reported that 1,25(OH)2D3suppresses the development of IBD in animal models [149].Deficiency of 1,25(OH)2D3 has been reported in patientswith IBD [150, 151], and, recently, using a novel vitamin Dbioavailability test, vitamin D deficiency or insufficiency wasseen in more than 70% of patients with quiescent CD [152].Given the diverse immune functions of vitamin D, deficientlevels may have important implications for the developmentand maintenance of intestinal homeostasis. A possible roleof vitamin D status and VDR signaling in modulating theeffects of intestinal microflora in other conditions suchas asthma and obesity has been suggested [100]. Whilepresent literature has primarily focused on elucidating theimmunoregulatory effects of vitamin D, there is a paucity ofdata on the status and function of VDR [147]. In addition,probiotic-induced modulation of anti-inflammatory VDRsignaling in colitis remains virtually unexplored.
Recent studies indicate that VDR−/− mice haveincreased bacterial loading in the intestine [145, 153]. Ourmicroarray data found that VDR signaling responds to
pathogenic Salmonella in intestinal colitis in vivo [154]. Datafrom a recent study demonstrate that bacterial stimulation,both commensal and pathogenic, regulates VDR expressionand location and that VDR negatively regulates bacterial-induced intestinal NF-κB activation [153]. In general,probiotic-induced nuclear receptor signaling is not well char-acterized. The probiotic VSL3# was associated with nuclearreceptor signaling in the IL10−/− colitis model [67]. Nuclearreceptors have been shown to negatively regulate bacterial-stimulated NF-κB activity in intestinal epithelium [153, 155].Our recent data show probiotic treatment is able to enhanceVDR expression and activity in the host. An increase inVDR expression and a concomitant increase in cathelicidinmRNA in cultured intestinal epithelial cells when treatedwith Lactobacillus plantarum were seen [156]. We used aprobiotic monoassociated pig model to assess the probioticeffect on VDR expression in vivo and found intestinal VDRincreased significantly after probiotic colonization comparedto the ex-germ-free pig. Furthermore, our unpublished dataindicate that probiotics did not inhibit inflammation in micelacking VDR.
The presence of VDR in various tissues along with itsability to exert diverse actions in differentiation, growth, andanti-inflammation sets the stage for exploitation of VDRligands for the treatment of various inflammatory conditions[157, 158]. Although the potential importance of VDR as atherapeutic target has been appreciated [159], no approachto date has safely and effectively altered VDR’s activity.Hence, understanding VDR’s contribution to probiotic-induced anti-inflammation may provide significant insightin the pathogenesis of inflammatory conditions such as IBD,and thereby, guide the development of novel treatments.Further investigation of the complex interplay of nuclearreceptors, defensins, probiotics, and inflammatory pathwaysmay provide significant insight into the mechanisms ofaction of probiotics in anti-inflammation.
7. Current Problems and Future Directions
The individual diversity of the intestinal microflora under-scores the difficulty of identifying the entire human micro-biota and poses barriers to this field of research. In addition,it is apparent that the actions of probiotics are species andstrain specific [19]. It is also apparent that even a single strainof probiotic may exert its actions via multiple, concomitantpathways. Current investigation into the mechanism ofaction of specific probiotics has focused on probiotic-induced changes in the innate immune functions involvingTLRs and its downstream systems including NF-κB, JAK-STAT, MAPK, and SAPK/JNK pathways. Future research onnovel mechanisms of action for probiotics involving nuclearreceptor signaling, including PPARγ and VDR, is needed.With evolving knowledge, effective probiotic therapy will bepossible in the future.
AbbreviationsAAD: Antibiotic-associated diarrheaAP-1: Activator protein-1CD: Crohn’s disease
-
Gastroenterology Research and Practice 11
CDAD: Clostridium-difficile-associated diseaseCDM: Chemically defined mediaCM: Conditioned mediaCTFR: Cystic fibrosis transmembrane
conductance regulatorECN: E. coli Nissle 1917EGFR: Epidermal growth factor receptorEHEC: E. coli O157:H7EPEC: Enteropathogenic E. coliERK: Extracellular-signal-regulated kinaseGSH: GlutathionehBD: Human beta defensinHSP: Heat shock proteinsIBD: Inflammatory bowel diseaseIBS: Irritable bowel syndromeIEC: Intestinal epithelial cellIFN: InterferonIL: InterleukinIκB: Inhibitor of kappa BJAK/STAT: Janus kinase/signal transducers and
activators of transcriptionLa: Lactobacillus acidophilusLc: Lactobacillus caseiLcS: Lactobacillus casei ShirotaLGG: Lactobacillus rhamnosus GGLI-LPMC: Large intestinal lamina propria
mononuclear cellsLP: Lactobacillus plantarumLPS: LipopolysaccharideLr: Lactobacillus rhamnosusMAPK: Mitogen-activated protein kinaseMCP: Monocyte chemotactic proteinNEC: Necrotizing enterocolitisNF-κB: Nuclear factor kappa BPKC: Protein kinase CPPARγ: Peroxisome-proliferator-activated
receptor gammaROS: Reactive oxygen speciesRXR: Retinoid X receptorSAPK/JNK: Stress-activated protein kinase/c-Jun
NH2-terminal kinaseSb: Saccharomyces boulardiiSOCS: Suppressor of cytokine signalingTCR: T-cell receptorTER: Transepithelial electrical resistanceTJs: Tight junctionsTLR: Toll-like receptorTNF: Tumor necrosis factorUC: Ulcerative colitisUCB: Unconjugated bilirubinVDR: Vitamin D receptorZO: Zonula occludens.
Acknowledgments
This work was supported by the National Institutes of Health(DK075386-0251, R03DK089010-01) and the IDEAL awardfrom the New York State’s Empire State Stem Cell Board(N09G-279) to J. Sun.
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