Immunological effects of dietary proteins

Conference presented at theAnnual Congress of SBI

Sociedade Brasileira de ImunologiaCampos do Jordão, SP

2001

Ana Maria Caetano de Faria

Mucosa:

margin of the immune system ?

Mucosal & circulating Immunoglobulins

blood & lymph

IgM IgG IgA

secretions

Mucosa:

physiology of the immune system

300 m2

Ig producing cells in physiological conditions

mice

0

2

4

6

8

10

12

14

16

P P

humans

0

1.000

2.000

3.000

4.000

5.000

6.000

mLN

10

10

pla

sm

cells

BM

Sp

leen

LN,

SPL,

B

M

gu

t

10

6 p

lasm

cells

gu

t

Lamina Propria e IELs* *

*

*

*

***

*

LP

CG

Peyer’s Patches

GALT

Peyer’s patch

lamina propria

M cell

GALT – Gut Associated Lymphoid Tissue

IEL

IL-7 SCF c-kit RAG

E-cadherin

E7

epithelial

Villus

GC

LTLT

YY

Y

Y

Y

Y

Y

YIgA

Y

Y

Y

Y

HEV

IEL

LTLB

CD8+

CD4+ Efferent

lymphatics

M cell

Y

TGF/IL10/IL-4

IgA

IL-6

MAdCAM-1

Epithelial cells

Lymphoid cells in the GALT

70% are activated (as opposed to 20% in spleen and peripheral lymph nodes)

IL-1, IFN-, TNF-, IL-6, chemokines, prostaglandins

IL-4, IL-10, TGF-, PGE-4

Presence of macrophages, eosinophils, mast cells, activated lymphocytes

Most activated cells in blood bear

microbiota

diet

Natural sources of antigen in the GALT

Immunological alterations in germ-free mice

Reduction in the size of Peyer’s patches

Reduction in the size of peripheral lymph nodes (small germinal centers)

IgG & IgA levels reduced, normal IgM levels

Reduction in the number of intraepithelial lymphocytes (IELs), B and T cells

Immunological alterations in antigen-free mice

Mucosal associated lymphoid tissue absent

Involution of peripheral lymph nodes (no germinal centers)

IgG and IgA absent, normal levels of IgM

Reduction in the cytotoxic activity of intraepithelial lymphocytes (IELs)

Immunological effects of stimulation by food

proteins

PhD thesis of Juscilene Menezes

Components for 1 kg of diet

Amid 680.0 gColin 4.0 gMetionin 10.0 gVitamins 10.0 gVitamin E 1 gotaCellulose 10.0 gMinerals 50.0 gSoy Oil 80.0 gNitrogen source * 150.0 gBHT (preservative) 0.1 g

Casein amino acids

Essencials % for 1 kgArginina 4.1 6.15 gHistidina 3.1 4.65 gIsoleucina 6.1 9.15 gLeucina 9.2 13.8 gLisina 8.2 12.30 gMetionina 3.2 4.80 gFenialanina 5 7.50 gTreonina 4.9 7.35 gTriptofano 1.2 1.80 gValina 7.2 10.80 gNon essencials % para 1 kgAlanina 3.0 4.50 gÁcido aspártico 7.1 10.65 gCistina 0.3 0.45 gÁcido glutâmico 22.4 33.60 gGlicina 2.7 4.05 gProlina 11.3 16.95g Hidroxiprolina 0 0Serina 6.3 9.45 gTirosina 6.3 9.45 g

Type of diet

Control dietTest diet

*Nitrogen source

CaseinCasein amino acids

Diet composition

.

Weaning(3-4 sem)

Control DietCasein

Experimental Diet aminoacids

MiceC57BL/6

Cas

Aa

Adults (8-10 sem)

Experimental Model

.

.

..

IntestineMucus

Immunoglobulins Histology Phenotype

Effects in the gut

Casein Aa

Intestinal MorphologyCas

Aa0

100

200

300

400

500

600

700

Duodenum P JejunumD Jejunum Ileum

Mic

rom

ete

rs (

µm

)

Cripta CasVillus Cas

Cripta AAVillus AA

Casein Aa

**

*

**

*

*

**

*

**

**

*

*****

LP

**

*

*

**

*

*

LP

*

IEL

02468

10121416

Du

od

en

um

Jeju

nu

m P

Jeju

nu

m D

Ileu

mIEL/

10

0 e

pit

helia

l ce

lls Lamina propria

020406080

100120

Du

od

en

um

Jeju

nu

m P

Jeju

nu

m D

Ileu

mNu

mb

er

of

cells

/vill

us

Casein

AA

Number of cells in the GALT

IEL Casein (%) AA (%)

TCR 41.3 ± 3.5 22.8 ± 3.2 p<0.001

TCR Thy1- 12.9 ± 2.4 11.4 ± 2.8 p< 0.1TCR Thy1+ 26.3 ± 1.5 9.9 ± 3.3 p<0.001

TCR CD5- 16.3 ± 2.8 11.0 ± 2.9 p<0.025TCR CD5+ 23.7 ± 1.2 10.0 ± 2.4 p<0.001

TCR 35.7 ± 2.9 45.2 ± 5.1 p< 0.001

TCR Thy1- 25.9 ± 1.8 26.8 ± 6.1 =TCR Thy1+ 10.6 ± 1.8 18.4 ± 1.7 p< 0.001

Sub-subsets of IELs

Globet Cells

Cas

Aa

Intestinal sIgA

0,00

0,01

0,02

0,03

0,04

0,05

0,06

0,07

Cas Aa

mg/m

l*

Withdrawl of either the microbiota or of food proteins has similar

immunological and morphological effects in the gut

Systemic Effects

..

Lymphoid organsSerum

Immunoglobulins Phenotype Cyotokine, AFC

Casein Aa

B220 TCR TCR CD3+

CD4+CD3+

CD8+CD3+

CD25+CD3+

CD45RBlo

45.8±3.0 28.8±6.8 1.6±0.3 15.3±3.8 9.7±2.3 1.9±06 36.4±7.841.6±3.0 22.9±2.4 2.2±1.0 12.7±1.0 9.51±2.2 1.3±0.3 46.8±6.1

3.1±0.9 6.5±2.5 0.2±0.1 3.6±1.6 2.9±1.4 0.4±0.1 3.1±1.50.6±0.4 2.0±0.8 0.1±0.1 1.1±0.4 0.8±0.5 0.1±0.1 0.9±0.5

9.0±3.2 15.0±3.6 0.7±0.3 10.0±2.6 6.4±1.7 1.1±0.3 7.7±1.44.0±1.4 6.8±1.4 0.3±0.05 4.2±0.8 2.9±0.9 0.8±0.4 3.5±0.9

6.0±2.0 11.5±3.9 0.3±0.1 7.3±3.1 4.7±0.8 1.0±0.4 5.1±2.35.2±2.0 13.8±6.5 0.3±0.2 8.6±4.4 5.97±2.5 0.8±0.2 6.0±2.6

pLN

Spl

mLN

cLN

CasAA

CasAA

CasAA

CasAA

Lymphocytes from different organs

Phenotype Casein (%) Aa (%)

CD3-CD4+ 35.81 ± 5.7 13.43 ± 3.0 p<0.05

CD3-CD8+ 76.47 ± 12.9 28.47 ± 3.1 p< 0.05

CD4+CD8+ 77.06 ± 13.5 29.59 ± 3.0 p<0.05

CD3+TCR 14.29 ± 5.3 8.11 ± 2.8 =

CD3+CD4+ 3.96 ± 1.5 2.00 ± 0.8 =

CD3+CD8+ 3.69 ± 1.3 2.17 ± 0.9 =

T cell precursors in the thymus

Serum Imunoglobulin

s0,0

0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

Ig IgM IgG IgAm

g/m

lCas

Aa

**

**

Spleen

0

1000

2000

3000

4000

5000

6000

7000

8000

Ig IgM IgG IgA

CFA

/10

6 C

els

*

*

* *

C57BL6/J BALB/c

In vitro production of IL-2 & IFN-

0

20000

40000

60000

Spl pLN cLN0

20000

40000

60000

Spl pLN mLN PP

pg

/ml

mLN

0

40000

80000

120000

Spl pLN mLN PP0

20000

40000

60000

Spleen pLN cLN

pg

/ml

CasAa

C57BL/6 BALB/c

CasAa

In vitro production of IL-4 & IL-10

0

3000

6000

9000

Spl pLN cLN0

4000

8000

12000

Spl pLN PP

pg

/ml

mLN

0

10000

20000

30000

Spl pLN cLN0

15000

30000

45000

Spleen pLN

pg

/ml

mLN

Neonate profile

Predominance of Th2 type cytokines

High IgM production, low IgG and IgA

Intestinal Morphology at Weaning

Immunological Activities

..

Infection with Leishmania major

Oral tolerance Systemic immunization

Casein Aa

Infection with Leishmania major

Footpad increment

0,000

0,500

1,000

1,500

2,000

2,500

4 Weeks 8 Weeks

mm

C57BL/6 CasC57BL/6 AaBALB/c

*

*

Oral Tolerance Induction and Specific Antibody Production

0,0

2,0

4,0

6,0

8,0

10,0

12,0

water fed OVA fed

ELI

SA

*

CasAa

c

a

b

a

El-Diet

• elemental diet

• available for human use

• therapeutic for Crohn’s disease post-surgery procedures, etc

GALTChow El Diet

0

2

4

6

8

10

12

14

Chow ED

IEL/

100

célu

las

epite

liais

*

Intraepithelial Lymphocytes

Chow El Diet

TCR 72 ± 10 47 ± 7*

TCR Thy1- 16 ± 6 9 ± 4TCR Thy1+ 56 ± 8 38 ± 4*

TCR CD5- 24 ± 7 17 ± 7TCR CD5+ 47 ± 10 30 ± 3*

TCR 17 ± 7 27 ± 8*

TCR Thy1- 3 ± 1 8 ± 1*TCR Thy1+ 14 ± 6 19 ± 7

IELs

Immunoglobulins

Serum

0,00

0,50

1,00

1,50

2,00

2,50

Ig Total IgM IgG IgA

ChowEl diet

*

**

Intestinal IgA

00,010,020,030,040,050,060,07

ChowEl diet

mg

/ml

*

Spleen

01000

2000

3000

4000

5000

6000

Ig Total IgM IgG IgA

**

*

Bone Marrow IgA

0200400600800

1000120014001600

ChowEl diet

*

CFA

/ 1

06

cells

Lymphoid cells

B220 CD3+ CD3+CD4+ CD3+CD8+ CD3+

CD45RBloCD3+

CD62Llo

SpleenChow 42.8±8.8 37.1±6.1 21.2±3.3 9.01±2.2 26.71±4.9 11.14±2.5

ED 40.2±7.1 37.19±7.0 22.1±3.0 9.53±3.3 36.51±4.8 17.46±2.7

PPChow 9.21±1.5 4.83±0.9 3.48±0.6 0.89±0.2 NF NF

ED 2.54±1.9* 1.28±0.6* 1.14±0.4* 0.30±0.1* NF NF

PLNChow 4.74±1.1 10.27±2.9 5.28±1.4 4.27±1.1 3.49±1.0 2.15±0.4

ED 1.79±0.7* 3.64±1.8* 1.92±0.9* 1.45±0.8* 1.65±0.8* 0.59±0.2*

Therapeutic Applications

Immunological disorders where dowmodulation is desirable (Crohn’s disease, autoimmune diseases, etc)

Collaborators

Nelson Vaz – UFMGJacqueline Alvarez-Leite - UFMGLeda Quércia Vieira - UFMGDenise Cara – UFMGRodrigo Corrêa Oliveira – CPqRRMomtchilo Russo – USPCláudia Rocha Carvalho - UFMG

Daniel Mucida – USPMariléia Andrade – UFMGElaine Speziali – UFMGMichele Barsante – UFMGAlice Kamphorst - UFMG

Frankcinéia Assis – UFMGVanessa Pinho – UFMGVirlânia Rodrigues - UFMGBeatriz Senra – UFMG