Probiotics in Health – Emerging OpportunitiesDec 3-4, 2016, Chennai
Probiotics and the Elderly Microbiome- Is it ever too late to change? -
Masanobu NannoYakult Central Institute, Tokyo, Japan
By J.O.
Today’s topics
1. Health problems in the elderly – Is microbiome related? ー
2. Probiotics are promising agent to improve the altered gut microbiome.
3. Probiotics are beneficial for health management in the elderly .
Demographics in India and Japan
1950 2010 2025 2050 21000
20
40
60
80
100
India
<20 20 ~ 6465 ~ 74 >75
%
1950 2010 2025 2050 21000
20
40
60
80
100
Japan
<20 20 ~ 6465 ~ 74 >75
%
Data from Statistical Handbook of Japan 2016
The proportion of elderly is gradually increasing in India and Japan!Age Age
Data from Ministry of Health, Labor and Welfare (1996)
Inci
denc
e (N
o./1
05 p
opul
atio
n)
Mor
talit
y
Incidence
Mortality
Age
Effect of age on incidence and mortality
of influenza infection in Japan
Data from National Cancer Centre Japan (2015)
Effect of age on cancer incidence in Japan
40-44
45-49
50-54
55-59
60-64
65-69
70-74
75-79
80-84
>850
500
1000
1500
2000
2500
3000
3500
4000
4500
Male Female
Inci
denc
e(N
o. o
f pat
ient
s/10
5 po
pu-
lati
on)
Age
Immune functions in the elderly
Innate immunity: Neutrophils: reduction of capabilities to migrate and uptake opsonized particles and pathogens Monocytes: reduction of ROS production and phagocytosis, and dysregulation in the release of cytokines Natural killer cells: defective capacity to secrete chemokines and reduction of cytotoxic potential Acquired immunity: Dendritic cells: reduction of mobilization and impairment of cytokine release T lymphocytes: reduction in the frequency of naïve T cells and lower capacity to induce antigen-specific T cell responses accumulation of memory T cells B lymphocytes: decrease in the number and change in the relative frequencies of different B cell subsets
Susceptibility to infection and cancer development in elderly
Pinti M et al. Eur J Immunol. 46:2286-2301 (2016)
Health problems in the elderly Pathogenic infection Cancer Allergies – food allergy, pollen allergy Autoimmune diseases – rheumatoid arthritis Metabolic syndrome – obesity, diabetes, cardiovascular
diseases Mental diseases – depression, dementia
Gut microbiome
Food
Livingenvironment
Stress
Drugs
Aging and gut microbiome of healthy subjects in Japan
2
4
6
8
10
D1-D3 D7-M1 M3-M6 Y3-19 Y20-59 Y60-79 >Y80
No.
of b
acte
ria
(Lo
g 10 c
ells
/g o
f fec
es)
Age
Total bacteriaC. coccoides groupC. leptum subgroupB. fragilis groupPrevotellaBifidobacteriumEnterobacteriaceaeLactobacillusStaphylococcus
Nomoto K et al. Intestinal Microbiota. 29:9-18 (2015)
Gut microbiome in the elderly
Claesson MJ et al. Nature. 488:178-184 (2012)
Gut microbiome in the elderly: different from the core microbiome in younger
adults higher Firmicutes in subgroup (1) and higher
Bacteroidetes in subgroup (4) most diverse in low fat/high fiber group to
which the majority of subgroup (1) belongs higher inflammation markers (TNF-a, IL-6, IL-
8, CRP) in subgroup (4)
Subjects: (1) the community-dwelling elderly (n=83) (2) the elderly attending an out-patient day hospital (n=20) (3) the elderly in short-term rehabilitation hospital care (n=15) (4) the elderly in long-term residential care (n=60) (5) younger adults (n=13)
Summary (1)
• Population aging is continuing in many countries of the world.
• The elderly is highly susceptible to infection and carcinogenesis due to decline of immune functions.
• Gut microbiome is dysregulated in the elderly, the extent of which is dependent on their lifestyle.
By J.O.
Definition of probioticsLive microorganisms which, when administered in adequate amounts, confer a health benefit on the host(FAO/WHO, 2001)
Is Lactobacillus casei Shirota (LcS) regarded as probiotics ?
*, P<0.05: vs. before ingestion**, P<0.01 : vs. before ingestiona, P<0.01 : vs. 3 months after ingestion b, P<0.01 : vs. 6 months after ingestionWang C et al. Annal Nutr Metabol. 67:257-266 (2015)
Improvement of gut microbiome by L. casei Shirota in the children
Bact
eria
l cou
nt
(Log
10 c
ells
/g o
f fec
es)
Bifidobacterium Enterobacteriaceae S. aureus C. perfringens
* ** b
Ingestionperiod (mo)
0 121 3 6
11
10
9
8
76
5
* **
b
10
9
8
76
5
4
Ingestionperiod (mo)
0 121 3 6
** *
a, b6
5
4
3
Ingestionperiod (mo)
0 121 3 6
6
5
4
3
2
Ingestionperiod (mo)
0 121 3 6
*44(%)
b
35 35 7 63
Improvement of gut microbiome by L. casei Shirota in the elderly
Bact
eria
l cou
nt (
Log 1
0 cel
ls/g
of f
eces
) Bifidobacterium L. casei ShirotaLactobacillus
3456789
10
3456789
1011
ND3456789
10 * * *** ***
beforeafter ingestion (mo)
1 3 6 beforeafter ingestion (mo)
1 3 6 beforeafter ingestion (mo)
1 3 6
C. perfringens
345678
*
71(%) 48 60 61
Staphylococcus10
4
6
89
7
5 62(%) 70 51 65
**
Pseudomonas8
2
4
67
5
3 33 29 16
*
45(%)
beforeafter ingestion (mo)
1 3 6 beforeafter ingestion (mo)
1 3 6 beforeafter ingestion (mo)
1 3 6
Bian L et al. Int J Prob Preb. 6:123-132 (2011) *, P<0.05; **,P<0.01; ND, Not detected.
Improvement of clinical symptomsby L. casei Shirota in the elderly
DiarrheaConstipationAttack of fever (>37℃)
Feve
rish
dur
atio
n (d
ays/
wee
k)
0
2.0
0.5
1.0
1.5
** * *
beforeafter ingestion
(mo)
1 3 6
Cons
tipa
tion
(N
o./w
eek)
0
0.2
0.8
0.6
0.4
*
beforeafter ingestion
(mo)
1 3 6
Dia
rrhe
a (N
o./w
eek)
0
0.1
0.2
0.3
0.4
*
beforeafter ingestion
(mo)
1 3 6
Bian L et al. Int J Prob Preb. 6:123-132 (2011) *, P<0.05; **,P<0.01, vs. before ingestion
Improvement by L. casei Shirota of gut discomfort in the elderly
Japan study (Mean age 85.9, Intervention 1 year; Sekita Y et al. 2015)
Before
inges
tion
After i
ngestio
n0 5
10 15 20 25 30 35
Evacuation
No.
per
mon
th
Before
inges
tion
After i
ngestio
n0
5
10
15
20
25
Suppository
No.
per
yea
rBef
ore in
gestio
n
After i
ngestio
n0 2 4 6 8
10 12 14
Physical condition
Day
s pe
r ye
arx1.21x0.64 x0.63
Netherlands study (Mean age 83.8, Intervention 6 weeks; van den Nieuwhoer M et al. 2015)
Before
inges
tion
After i
ngestio
n0 1 2 3 4 5 6
Evacuation
No.
per
wee
k
Before
inges
tion
After i
ngestio
n0 2 4 6 8
10 12 14
Constipation
%
0 5
10 15 20 25 30 35 40 45
Diarrhea
%
x1.04
x0.41
x0.85
Lactobacillus casei Shirota (LcS)・ Survives in the gut lumen and is recovered
alive from the feces.・ Normalizes the altered gut microbiota and
improves the bowel movement.・ Decreases the production of noxious
substances in the gut lumen and allows their clearance.
Lactobacillus casei Shirota is probiotics !
Van Puyenbroeck K et al.(2012)
Fujita R et al.(2013)
Belgium studyAge: >65 yearsIntervention: 6 months
Japan studyAge: Mean 83 yearsIntervention: 7 months
Improvement by L. casei Shirota of infectious diseases in the elderly
0 1 2 3 4 50.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Risk of RTI
Odd
s Ra
tio
Placebo LcS-drink0
1
2
3
4
5
6
Duration of URTI
Day
s
Japan studyAge: Mean 85 yearsIntervention: 6 months
Before
Afte
r 1 m
onth
Afte
r 3 m
onth
s
Afte
r 6 m
onth
s0
1
2
3
Duration with fever
Placebo LcS-drink
Day
sNagata S et al.
(2016)
Time after ingestion (month)
Asahara T et al. J Appl Microbiol. 110:163-173 (2010)
Survival rate (%)
Days after infection with Salmonella
0
20
40
60
80
100
0 3 15 216 9 12 18
InfectedInfected + killed LcS
Infected + live LcS
P<0.05
pH
6.4
6.6
6.8
7.0
7.4
7.2
P<0.01
Not infected Infected
Infected + live LcS Infected + killed LcS
Organic acid
mm
ol/g
of c
ecal
con
tent
s
0
20
40
60
80
100P<0.01
Anti-infectious mechanism of L. casei Shirota
- Production of short chain fatty acids -
Anti-infectious action of L. casei Shirota- Enhancement of natural killer activity -
Hori T et al. Clin Diag Lab Immunol. 9:105-108 (2002)
Protocol: BALB/c mice at 15 months old ⇒ LcS feeding for 4 months ⇒ IFV infection NK activity assay
0
2
4
6
8
10
Control LcS
**
Spec
ific
lysi
s (%
)
NK activity of splenocytes
0
1
2
Viru
s ti
ter
(10n )
*3
Control LcS
IFV titer in nasal washing
*, P<0.05; **,P<0.01
Placebo LcS0
10
20
30
40
Administration
Inci
denc
e (%
)
< 4 times > 4 times0.0
0.2
0.4
0.6
0.8
1.0
1.2
Frequency
Odd
s ra
tio
None LcS0
10
20
30
40
50
Administration
Inci
denc
e (%
)
Toi M et al. (2013)Ishikawa H et al. (2005)Aso Y et al. (1995)
Superficial bladder cancer
(recurrence rate 1 year after operation)
High grade colon cancer(crisis rate 4 years
after operation)Breast cancer(relative risk)
Prevention by L. casei Shirota of cancer development
Takagi A et al. Carcinogenesis. 22:599-605 (2001)
Canc
er d
evel
opm
ent
rate
(%
)
0
10
20
30
40
50
1 2 3 4 5 6 7 8 9 10
Development of cancer
contLcS
Time after 3-MC administration (wks)
Anti-cancer mechanism of L. casei Shirota
- Enhancement of natural killer activity -
25 50 1000
4
8
12
16
NK cell activity
C57BL/6 cont
C57BL/6 LcS
Beige cont
Beige LcS
E/T ratio
Lys
is o
f ta
rget
cel
ls (
%)
Protocol: C57BL/6 mice ⇒ 3-MC injection ⇒LcS feeding for 10 weeks ⇒ cancer development NK activity assay
Subjects: 55 ~ 74 years old; male n=12, female n=18Protocol: cross-over experiment probiotics-drink (1.3 x 1010 L. casei Shirota/130 ml) or skimmed milk, each 4 weeks intake
Dong H et al, Eur J Nutr, 52:1853-1863 (2013)
Enhancement of natural killer activity
by L. casei Shirota in the elderly
0 50 100 1500
10
20
30
Before
Placebo LcS-drink
E/T ratio
% o
f sp
ecifi
c ly
sis
0 50 100 1500
10
20
30
After
Placebo LcS-drink
E/T ratio%
of
spec
ific
lysi
s
4 weeks
* *, P<0.05
Anti-cancer mechanism of L. casei Shirota
- Amelioration of inflammatory response -
Matsumoto S et al. Immunology. 128:e170-e180 (2009)
LcS
LcSDPSPG-I
Control
0 20 40 60 80IL-6/G3PDH mRNA
Expression of IL-6
P <0.05
P <0.05
0 1 2 3Tumor count/mice
Tumor count
P <0.01
P <0.05
DSS-induced colitis-associated colon cancer
Protocol: BALB/c mice ⇒ DSS treatment ⇒LcS feeding for 20 weeks ⇒ cancer development
Modified from nature INSIGHT 16 June 2011
Increased gene expression(cryptidin, PPARg)
IL-12
LcS
Epithelial cell layer
Treg
Mf
Th17Th1NK
IL-6
Intestinal lumen
Intestinal mucosa
Immunostimulation
Mucous layer
Normal flora
Anti-inflammation
Mf
Maintenance of homeostasis Immune balance
Short-chain fatty acid
Immuno-modulating mechanism of L. casei Shirota
Summary (2)
• Probiotics improves the gut microbiota of healthy persons irrespective of age.
• Probiotics helps the elderly to maintain the healthy state.
• Probiotics in cooperation with gut microbiota improves the immune functions through various pathway.
By J.O.
Inflammatory bowel diseases
YesUlcerative
colitis
Infection
YesDiarrhea
ColdYes
Constipation
Intestinal discomfort
Predicted effects
Cancer
YesSuperficial
bladder cancer
Colon cancer
Autoimmune diseases
MaybeArthritis
Intriguing effects
Psychological stress
YesMedical students
Desk workers
Amazing effects
Effectiveness of L. casei Shirota confirmed in clinical trials
Individual difference of gut microbiomeHow can the healthy gut microbiome be
defined?Regional difference of gut microbiome Is the role of gut microbiome common
in all the populations of the world? Importance of infant gut microbiome to
keep health for all one’s lifeWhen do we start to ingest probiotics
for our health?Responder vs non-responder to
probioticsWhat determines the efficacy of
probiotics? Is it necessary to find the individual-
specific probiotics?
Issues to be examined in the future
Acknowledgments Juntendo University: Satoru Nagata, Yuichiro Yamashiro, Kazuyoshi Takeda, Ko Okumura The University of Tokyo: Retsu Fujita, Yasuo Ohashi Yakult Central Institute: Hirokazu Tsuji, Takuya Takahashi, Takashi Asahara, Koji Nomoto, Akira Kushiro, Akimitsu Takagi, Takeshi Matsuzaki Kan Shida, Tetsuji Hori, Satoshi Matsumoto
Thank you for your attention
!
Cell Wall Polysaccharide I
Cell Wall Polysaccharide II
Cell Wall
Cell Wall Polysaccharide II
L. casei Shirota D-PSPGI
L. casei Shirota
Cell Wall
Cytoplasm