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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
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Mucosa:
margin of the immune system ?
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Mucosal & circulating Immunoglobulins
blood & lymph
IgM IgG IgA
secretions
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Mucosa:
physiology of the immune system
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300 m2
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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
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Lamina Propria e IELs* *
*
*
*
***
*
LP
CG
Peyer’s Patches
GALT
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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
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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
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microbiota
diet
Natural sources of antigen in the GALT
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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
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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)
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Immunological effects of stimulation by food
proteins
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PhD thesis of Juscilene Menezes
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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
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.
Weaning(3-4 sem)
Control DietCasein
Experimental Diet aminoacids
MiceC57BL/6
Cas
Aa
Adults (8-10 sem)
Experimental Model
.
.
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..
IntestineMucus
Immunoglobulins Histology Phenotype
Effects in the gut
Casein Aa
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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
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Casein Aa
**
*
**
*
*
**
*
**
**
*
*****
LP
**
*
*
**
*
*
LP
*
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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
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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
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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*
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Withdrawl of either the microbiota or of food proteins has similar
immunological and morphological effects in the gut
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Systemic Effects
..
Lymphoid organsSerum
Immunoglobulins Phenotype Cyotokine, AFC
Casein Aa
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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
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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
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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
*
*
* *
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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
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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
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Neonate profile
Predominance of Th2 type cytokines
High IgM production, low IgG and IgA
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Intestinal Morphology at Weaning
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Immunological Activities
..
Infection with Leishmania major
Oral tolerance Systemic immunization
Casein Aa
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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
*
*
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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
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El-Diet
• elemental diet
• available for human use
• therapeutic for Crohn’s disease post-surgery procedures, etc
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GALTChow El Diet
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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
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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
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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*
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Therapeutic Applications
Immunological disorders where dowmodulation is desirable (Crohn’s disease, autoimmune diseases, etc)
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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