The 8th india probiotics symposium (2016) slide revised2

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


0 121 3 6

** *

a, b6

5

4

3


0 121 3 6

6

5

4

3

2


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

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

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

*


(mo)

1 3 6

Dia

rrhe

a (N

o./w

eek)

0

0.1

0.2

0.3

0.4

*


(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

The 8th india probiotics symposium (2016) slide revised2

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