Probiotics and systemic immunity

Raphaëlle Bourdet-Sicard, PhD Danone Research & BIOASTER

Commensal Flora, Fondation Mérieux Meeting

June 12th 2013

Definition of probiotics

FAO & WHO definition :

“ Probiotics are live microorganisms that, when administered in adequate amount, confer a

health benefit to the host ”

Probiotic

Gastrointestinal tract infections

Respiratory tract infections

Parenteral vaccination

Local effect

Beneficial health effects Defense against pathogens

Antibiotic-Associated Diarrhea

Travellers Diarrhea

Systemic and distal effect

Urogenital infections

Etc… Etc…

Immune modulation at systemic and distal mucosa

Probiotics and responses to vaccine

Vaccine. 2009 ;27:5677-84.

86 elderly in nursing home Vaccination 2005-2006

Probiotics & vaccine responses in seniors

Non fermented dairy control

L. casei CNCM I-1518 (Actimel®) 1010 cfu/2xday

4 weeks 3 weeks

Influenza Vaccination

Blood Blood

N=44

N=42

Antibody titers measured by Haemagglutination inhibition test (HI test)

Antibody levels for B strain (GMT)

p=0.020 0

40

80

120

An

tib

od

y le

vel

Actimel

Control

Seroconversion rate for B strain

0

5

10

15

20

25

30

0 20 40 60 80 100 120 140 160

Days after

vaccination

p=0.050

p=0.016

p=0.006

p= 0.002

p=0.017

Product consumption Follow-up

0

5

10

15

20

25

30

0 20 40 60 80 100 120 140 160

0

5

10

15

20

25

30

0 20 40 60 80 100 120 140 160

Days after

vaccination

p=0.050

p=0.016

p=0.006

p= 0.002

p=0.017

Product consumption Follow-up

Actimel

Control

Actimel

Control

Sero

co

nvers

ion R

ate

(%

)

Time post-vaccination (days)

Actimel

Control

Boge et al. Vaccine. 2009 ;27:5677-84.

Probiotics & vaccine responses in seniors

222 elderly in nursing home Vaccination 2006-2007, 13 weeks product consumption

Significant higher antibody levels and higher seroconversion rate against the B strain over the time (3, 6 and 9 weeks post vaccination) in subjects consuming Actimel® as compared to control group.

H1N1 and H3N2 were not significantly different between the 2 groups

Probiotics and response to vaccine Selection of clinical trials

Study criteria:

Probiotic product with no concomitant treatment (unless identical in test and control group)

Oral intake, concomitant with vaccination

Parenteral vaccine

Adults population

Randomised, double-blind, placebo-controlled trial

Publication in peer-reviewed journal, in english.

Young adults: 4 trials on influenza vaccine

3 trials on influenza vaccine

Seniors 6 trials on influenza vaccine

Probiotics vaccine N Method Intervention effect Trial reference

L. paracasei ssp paracasei CNCM I-1518 fermented milk (Actimel) 1010 cfu twice daily

Parenteral influenza vaccine - campaign 2005-2006 and 2006-2007

86 222

HI Pilot: Trend for higher H3N2 GMT Second study: Significantly greater GMT and seroconversion rate for B strain

Boge et al. Vaccine 2009

L. paracasei ssp paracasei Shirota fermented milk 6. 109 cfu twice daily

Parenteral influenza vaccine - campaign 2007-2008

565 HI No significant difference on non-seroprotected sub-group (737-172 = 565) for H3N2.

van Puyenbroeck et al., Am J Clin Nutr 2012

Probiotics and response to vaccine clinical trials in young adults

Young adults: 4 trials on influenza vaccine

Probiotics vaccine N Method Intervention effect Trial reference

L. fermentum CECT5716 1010 cfu/day

Parenteral influenza vaccine - campaign 2004/2005

50 ELISA Probiotic increased vaccine-specific IgA (no effect on IgG nor IgM) P< 0,05

Olivares et al., Nutrition 2007

L.fermentum VRI003 109 cfu/ day

Parenteral influenza vaccine - campaign 2006

47 HI Significant difference of H1N1 titers in Probiotic vs Placebo group. Non Seroconverted subject for H1N1 was lower in probiotic group (5,5%) vs 28% in placebo group.No difference for H3N2 nor B but very low response to vaccine for these 2 strains;

French & Penny, Int J Probiotics and Prebiotics 2009

B. animalis ssp lactis BB-12 or L. paracasei ssp paracasei 431 1 x 109 cfu/day

Parenteral influenza vaccine - campaign 2008-2009

211 ELISA Significantly greater increase in vaccine -specific plasma IgG titer in probiotic groups vs placebo, IgG1 and IgG3 (p< 0,001)

Rizzardini et al. Br J Nut 2012

L. rhamnosus GG 1010 cfu twice daily

Nasal Live attenuated trivalent influenza vaccine - campaign 2007/2008

39 HI Significant increase in seroprotection for H3N2 in probiotic group vs placebo (84% vs 55% p= 0,048). No effect on H1N1 nor on B strain, but very low vaccine response

Davidson et al., Eur j Clin Nutr 2011

Probiotics and resistance against respiratory infections

Lower average and cumulated duration of URTI and rhinopharyngitis in probiotic group versus control

Guillemard et al., Brit. J. Nutr. 2010, 103, 58-68.

Actimel® Control P-values1

All CIDs N=104 N=111

Mean (SD), days 7.4 (5.6) 9.8 (7.5) 0.008

Median. days 6.5 8.0

URTIs N=61 N=66

Mean (SD), days 7.7 (7.2) 11.0 (7.7) 0.0002

Median. days 7.0 8.0

Rhinopharyngitis N=61 N=58

Mean (SD), days 7.7 (7.2) 11.0 (8.1) 0.0007

Median. days 7.0 8.0

1 Mann-Whitney

Non fermented dairy control

L. casei CNCM I-1518 (Actimel®) 1010 cfu/2xday

3 months during winter

RCT

in 1072 free-living elderly 76·0 years (median)

Probiotics & respiratory infections in senior

Evidence for an potential effect on RTI : lower number and occurrence of CID (87% RTI) in Actimel group versus control

RCT in 1000 healthy shift workers, 32.1 ± 8.9 years

(mean ± SD)

0

10

20

30

40

50

60

70

0 1-2 > 2

All CIDs by class

Vo

lun

teers

wit

hin

fecti

on

, %

ITT population-3 class levels of CID

Actimel (N=500) Control (N=500)

0

10

20

30

40

50

60

70

0 1-2 > 2

All CIDs by class

Vo

lun

teers

wit

hin

fecti

on

, %

ITT population-3 class levels of CID

Actimel (N=500) Control (N=500)

p=0.017

Proportion of CID categories (%)

URTI 59%

LRTI 29%

GITI 13%

Proportion of CID during whole study

Subjects presenting CID

(Number and %)

Actimel Control P-value

213 (43%) 256 (51%) 0.005

Number of all CIDs-ITT

Probiotics & respiratory infections in young adults

Guillemard et al., J. Am. Coll. Nutr, 2010, 29(5):455-68.

Non fermented dairy control

L. casei CNCM I-1518 (Actimel®) 1010 cfu/2xday

3 months during winter

12

Probiotics & respiratory infections Selection of clinical trials

Study criteria:

Probiotic product with no concomitant treatment (unless identical in test and control group)

Oral intake

Randomised, double-blind, placebo-controlled trials (parallel groups or cross-over)

Results available on probiotics effect specifically on respiratory infection.

Publication in peer-reviewed journal, in english.

2001 Hatakka K et al., BMJ 322:1327, 2001. 2005 de Vrese M et al., Clin Nutr 24:481-491, 2005. Weizman et al. Pediatrics.;115(1):5-9, 2005. 2007 Hatakka K et al., Clin NutrJun;26(3):314-21, 2007. Marseglia GL et al. Ther Clin Risk Manag. 3(1):13-7, 2007. Tiollier et al., Mil Med.;172(9):1006-11, 2007. 2008 Cox AJ et al., Br J Sports Med, 2008. 2009 Leyer GJ et al., Pediatrics 124:e172-e179, 2009. Rautava S et al., Br J Nutr 101:1722-1726, 2009. 2010 Hojsak I. et al., Clin Nutr. Jun;29(3):312-316, 2010. Hojsak I et al. Pediatrics.;125(5):e1171-7, 2010. Namba K et al., Biosci Biotechnol Biochem.;74(5):939-45, 2010. Makino S et al. Br J Nutr. May 21:1-9, 2010. Merenstein et al., Eur J Clin Nutr.;64(7):669-77, 2010 Guillemard et al., Br J Nutr.;103(1):58-68, 2010.

2011 Taipale T. et al. Br J Nutr. 105(3):409-16.2011. Berggren A. et al. Eur J Nutr.;50(3):203-10, 2011. West NP et al. Nutr J.;10:30. 2011 Gleeson M et al. Int J Sport Nutr Exerc Metab.;21(1):55-64, 2011. Merenstein et al. Eur. J. Clin. Nutr.; 65, 447–453, 2011. 2012 Smith TJ et al. Br J Nutr. 1:1-9. 2012 Kumpu M et al. Eur J Clin Nutr. 66(9):1020-3, 2012. Shinkai S et al. Br J Nutr. 5:1-10, 2012. Rojas MA et al. Pediatrics;130(5):e1113-20, 2012. Di Pierro F et al. Int J Gen Med.; 5:991-7, 2012. Gleeson et al. Int J Sport Nutr Exerc Metab.;22(4):235-42, 2012. Rerksuppaphol S et al. Pediatr Int.;54(5):682-7, 2012. Van Puyenbroeck et al., Am J Clin Nutr.;95(5):1165-71, 2012. 2013 Di Pierro F et al. Expert Opin Biol Ther.;13(3):339-43, 2013.

Nagalingam et al., Trends in Micro 2013

Young, middle aged Adults: De Vrese et al., 2005

RCT in 479 healthy adults, 38+/-13 yrs (mean ± SD)

Probiotics for preventing acute upper respiratory tract infections (Hao et al., Cochrane Datab. Syst Rev. 2011 Sep 7;9).

Heterogeneity: Tau2 = 0.23; Chi2 = 16.15, df = 5 (P = 0.01); I2 =69%

Meta-analyses on probiotics and RTIs

Probiotics & respiratory infections Clinical outcomes

Probiotics were better than placebo on acute URTI for the following criteria: number of participants experiencing at least one or three episode, rate ratio of URTI, antibiotic prescription. Probiotics and placebo were similar for the mean duration (MD) of an episode of acute URTI.

Mechanisms of action

Probiotic

Respiratory tract infections

Parenteral vaccination

Impact on systemic immunity

Systemic & distal effect Gut Immune system (IS)

Systemic IS

Distant mucosal IS

?

?

Immunological markers

?

Probiotic

Clinical endpoints

blood

In case of infections, Leukocytes and Neutrophils blood counts were higher in Actimel group versus control. No difference in non-infected subjects

In 200 shift workers

Actimel

Control

0

1

2

3

4

5

6

Neu

tro

ph

ils c

ou

nt

(x1000/µ

l)Leukocytes

Neutrophils

Baseline

All subjects

0

1

2

3

4

5

6

7

8

9

10

Leu

ko

cyte

s c

ou

nt

(x1000/µ

l) P=0,43

P=0,829

-2

-1

0

1

2

3

4

Neu

tro

ph

ils c

han

ge f

rom

baseli

ne (

x1000/µ

l)

-2

-1

0

1

2

3

4

Neu

tro

ph

ils c

han

ge f

rom

baseli

ne (

x1000/µ

l)

All CIDs Rhino.

Change from baseline (product consumption phase)

Healthy subjects Infected subjects

-3

-2

-1

0

1

2

3

4

Leu

ko

cyte

s c

han

ge f

rom

baseli

ne (

x1000/µ

l)

-3

-2

-1

0

1

2

3

4

Leu

ko

cyte

s c

han

ge f

rom

baseli

ne (

x1000/µ

l)

All CIDs Rhino.

P=0,715 P=0,034 P=0,002

P=0,749 P=0,066 P=0,002

Guillemard et al., J. Am. Coll. Nutr, 2010, 29:455-68.

17

In case of infection, NK cells blood counts were higher in Actimel group versus control. No difference in non-infected subjects

-100

-50

0

50

100

150

NK

ce

lls

CD

16

+C

D5

6+

CD

3-

ch

an

ge

fro

m b

as

eli

ne

(/µ

l)

NK cells CD16+CD56+CD3-

Change from baseline (product consumption phase) Baseline

Healthy subjects Infected subjects All subjects

0

50

100

150

200

250

300

NK

cell

s C

D16+

CD

56+

CD

3-

co

un

t (/

µl)

-100

-50

0

50

100

150

NK

ce

lls

CD

16

+C

D5

6+

CD

3-

ch

an

ge

fro

m b

as

eli

ne

(/µ

l)P=0,503 P=0,243

Actimel

Control

Guillemard et al., J. Am. Coll. Nutr, 2010, 29:455-68.

Effect of probiotics on Dendritic cells in absence of infectious signal

In absence of inflammatory signal, induction of hyporesponsive suppressor T cells by probiotics-treated DCs, that will reinforce the gut tolerance

Baba et al., J Leukocyte Biol 2008

Probiotic + (200:1)

T cell proliferation IL-10 secretion

IFN- secretion IL-17 secretion

L. casei CNCM I-1518

Naive

CD4+ T Mature

DC

iDC

+

Effect of L. casei CNCM I-1518 on Dendritic cells in presence of infectious signal

19

Baba et al. (2009) Immunology 128, e523

Interleukin (IL)-12p70 profiles of DCs stimulated with Lactobacillus casei CNCM I-1518 +

various TLR agonists

P < 0.001

P < 0.05

DCs stimulated with L. casei plus poly(I:C) induces a higher amounts of IFN- in CD4+ T cells co-culture

In presence of an infectious signal (poly (I:C)), conversion of L. casei -treated DCs into potent promoters of Th1 cells.

Note : IL-12 stimulated production = unique profil of L. casei among eight different bacterial strains tested

Naive T

Mature

DC

Immature DC

Maturation

TH1

TReg

TH2

IFN-

IL-12

TH17

+ poly(I:C)

probiotic

In vitro

serum

Lung (BAL)

Villena et al., BMC Immunol 2012, 13:53

Oral administration of probiotic (L rhamnosus Lr1505 or Lr1506) 5 days Then, mice intranasaly infected with poly(I:C)

In vivo

Effect of probiotic on immunity in presence of infectious signal

Conclusion

Probiotics have been shown in clinical trials to confer health benefit beyond the gut area:

- Increase antibody response to parenteral vaccine - Increase resistance to respiratory tract infections (incidence, duration and /

or severity ) Mechanisms by which orally administered probiotics impact systemic and

distal mucosal immunity need to be further dissected both at • At the host level • At the probiotic level

Thanks to

Vaccination studies Thierry Boge Michel Remigy Sylvie van der Werf Sarah Vaudaine Sandrine Samson Jérome Tanguy Francoise Tondu

RTI studies F Lacoin S de La Motte J. Schrezenmeir Eric Guillemard A.L Flavigny Jérome Tanguy Francoise Tondu

DCs in vitro studies Nobu Baba Manuel Rubio Marika Sarfati Sandrine Samson