Nutritional Immunomodulation:

1
Nutritional Immunomodulation Glutamine, Arginine
Antioxidants Philip C. Calder Institute of
Human Nutrition, University of Southampton
2
Will cover

• Introduction to the immune system
• The immune system in health and disease
• Glutamine, immunity infection
• Arginine, immunity infection
• Antioxidants, immunity infection
• Will include cell culture, animal and clinical
  studies (bench to bedside)

3
The immune system . is a cell and tissue
system that protects the host from invading
pathogens and from noxious environmental
agents distinguishes self from non-self
4
The immune response

• The response to infection by bacteria, viruses,
  fungi, and parasites
• Involved in protection against growth of cancer
  cells and in the response to injury and trauma
• Typified by cellular interactions and movement of
  cells to sites of infection
• Normally it is protective (and so beneficial)
• It includes both non-specific, innate (natural)
  responses (including inflammation) and specific,
  acquired responses
• Includes a component of memory
• Involves various cells including antigen
  presenting cells (especially dendritic cells),
  macrophages and T and B lymphocytes

5
Interaction amongst immune cells
NK
B
Th (CD4)
Activate
Activate
Activate
Tc
Antigen presentation
Activate
Antibodies
Antigen presentation
Lysis
Phagocyte
Bacteria
Virus infected cell
6
Th1 vs. Th2 paradigm
IL-12 IFN-g
B cells Cytotoxic T cells NK cells Macrophages
IL-2 IFN-g
Th1
-

-
CELL-MEDIATED IMMUNITY ANTI-BACTERIAL ANTI-VIRAL A
NTI-FUNGAL ANTI-TUMOUR
Undifferentiated T helper cell CD4
IL-4
-
Th2
-

IL-4
IL-5
B cells Eosinophils
IL-10
IFN-g
HUMORAL IMMUNITY ANTI-PARASITE
7
1000 800 600 400 200 0
Death
CD4 cells
CD4 T cells per ml
Opportunistic infections
HIV titer
0 2 4 6 8 10
12
YEARS
8
Th1 gone wrong Chronic inflammation
Self antigen
Antigen presenting cell
Y
Antigen-specific IgG
B
Y
Y
Y
MHCII
IFN-g IL-2
Peptide
T cell receptor
Th
Th 1
IL-12
IFN-g
IFN-g
Inflammation
Th 2
Macrophage
9
Th2 gone wrong Allergic inflammation
Allergen
Eosinophil
Antigen presenting cell
Inflammation
MHCII
IL-5
Peptide derived from allergen
B
T cell receptor
Th
IL-4
Th 2
IL-13
IL-4
Y
Allergen-specific IgE
IFN-g IL-12
Y
Y
Y
Allergen
Y
Y
Th 1
IgE receptor
Inflammatory mediators
Mast cell
10
Th1 vs. Th2 paradigm
IL-12 IFN-g
B cells Cytotoxic T cells NK cells Macrophages
IL-2 IFN-g
Th1
-

-
CELL-MEDIATED IMMUNITY ANTI-BACTERIAL ANTI-VIRAL A
NTI-FUNGAL ANTI-TUMOUR CHRONIC INFLAMMATION
Undifferentiated T helper cell
IL-4
-
Th2
-

IL-4
IL-5
B cells Eosinophils
IL-10
IFN-g
HUMORAL IMMUNITY ANTI-PARASITE ALLERGIC
INFLAMMATION
11
ACUTE CORONARY EVENT (HEART ATTACK)
ATHEROSCLEROSIS
12
Hypothetical time course of sepsis Expression
of pro- andanti-inflammatory cytokines
These cytokines (TNF-a, IL-1b, IL-6, IL-8) are
largely, if not completely, responsible for the
clinical signs and symptoms of the septic
response to bacterial infection. Vervloet et
al. (1998) Sem. Thromb. Hemostasis 24, 33-44
IL-8
Sepsis-Induction
Hyper
IL-6
IL-1
TNF
physiologicalRange
Inflammation
IL-10
TGFb
Hypo
IL-13
IL-4
Mayer et al. (1998) Clinical use of lipids to
control inflammatory disease. Curr. Opin. Clin.
Nutr. Metab. Care 1, 179-184.
13
Our arsenals for fighting off bacteria are so
powerful, and involve so many different defence
mechanisms, that we are more in danger from them
than from the invaders. We live in the midst of
explosive devices we are mined. L. Thomas. New
England Journal of Medicine 287, 553-555
14
Balances within the immune system
Pro-inflammatory cytokines (TNF, IL-1) vs.
anti-inflammatory cytokines (IL-10, IL-1ra) Pro-
and anti-inflammatory effects of IL-6 Th-1
cytokines (IL-2, IFN-g) vs. Th-2 cytokines (IL-4,
IL-10)
15
Nutritional Immunomodulation
Nutrients to enhance immune function
Nutrients to modulate or modify immune disease
Nutrient status playing a role in predisposing
to immune disease
16
.
Nutritional immunomodulation applied to
post-surgery, critical illness, burns etc. has
been termed IMMUNONUTRITION
Improved barrier function
Improved immune function (?decreased
hyperinflammation)
Immunonutrition
Improved wound healing
17
Immunonutrients
Amino acids - glutamine (glutamine precursors) -
arginine (arginine precursors) - cysteine
(cysteine precursors) Antioxidants - vitamins C
and E - b-carotene - trace elements (Zn, Cu, Se,
Fe, Mn) - taurine Fatty acids - long-chain n-3
fatty acids
18
Glutamine homeostasis
Liver
Bloodstream
Skeletal muscle
Glutamine
Kidney
Glutamine
Immune system
Diet
Glutamine
Intestinal tract
19
Metabolic roles of glutamine
Protein synthesis
Glutamine
Glutathione synthesis
NH3 donor (acid/base balance)
N transport in bloodstream
Carbon skeleton
Urea synthesis
N donor
Energy
Glucose
Nucleotide synthesis
20
Glutamine and the immune system

• The activity of glutaminase is high in lymphoid
  tissues and in cells of the immune system
• Cells of the immune system use glutamine in
  culture
• The rate of glutamine utilisation is greater than
  the rate of glucose utilisation
• The rate of glutamine utilisation is increased
  when the cells are stimulated
• Glutamine appears to be used by the immune system
  under stress in vivo

21
Glutamine and immune function cell culture
studies 1

• Lymphocyte division is dependent upon a supply of
  glutamine
• Lymphocyte division is maximal at the normal
  physiological glutamine concentration
• CD4 cells appear to divide preferentially in
  response to glutamine

Physiological concentration
range
Thymidine incorporation (dpm)
Glutamine in culture medium (mM)
22
Glutamine and immune function cell culture
studies 2
Glutamine concentration determines - IL-2 and
IFN-g production by T cells - IL-2 receptor
expression on T cells - B cell differentiation
to antibody producing cells - Expression of
phagocytic receptors on monocytes - Expression
of MHCII on monocytes - Phagocytosis by
monocytes, macrophages and neutophils - Antigen
presentation by monocytes
Level of expression on human monocytes
0.05 mM 2 mM FcgR1 35 (11) 95
(8) CR3 99 (16) 254 (21) HLA-DR
125 (38) 295 (49)
Spittler et al. (1997) Clin. Nutr. 16, 97-99
23

NK
B
Th
Activate
Activate
Activate
Tc


Antigen presentation
Activate
Antibodies
Antigen presentation
Lysis

Phagocyte
Bacteria
Virus infected cell
24
Glutamine and immune function animal feeding
studies (non-infection)
Control Extra Gln T cell proliferation
6.7 21.4 IL-2R ( ) 31.1
61.5 IL-2 106 189 IFN-g
15.4 17.9 IL-4 15.9 16.7 IL-10
123 158
Increasing the amount of glutamine in the diet
(of mice) increased - T cell proliferation -
IL-2 receptor expression - IL-2 (but not IFN-g)
production
Kew et al. (1999) J. Nutr. 129, 1524-1531
25
Glutamine and immune function animal feeding
studies (infection models)
E. coli infected piglets Blood T cell
proliferation higher if glutamine included in the
diet. Yoo et al. (1997). Septic rats Blood T
cell proliferation higher with glutamine
infusion. Yoshida et al. (1992). Influenza virus
infected mice Lymphocyte yield in Peyers patches
and intestinal integrity improved with glutamine
infusion. Li et al. (1997, 1998).
26
Glutamine and infection (animal studies)
Mortality () Model Control Gln Author
Parenteral Gln CLP (rat) 75 25 Ardawi
(1991) i.p. E. coli (rat) 45 8 Inoue
(1993) i.p. E. coli (rat) 66 14 Naka
(1996) i.t. Pseudomonas (rat) 75 30 DeWitt
(1999) Oral Gln i.v. S. aureus (mice)
80 60, 30 Suzuki (1993)
27
Survival after i.v. Staphylococcus aureus Adjei
et al. (1994) Nutr. Res. 14, 1591-1599
100 80 60 40 20 0
Survival
GLN
CONTROL
0 5 10 15 20
Days
28
Glutamine and catabolic states (human studies)
Controls
Patients
Muscle glutamine (mM)
Plasma glutamine (mM)
29
Lowered muscle and plasma glutamine in catabolic
states may indicate an imbalance between supply
and demand Glutamine may be conditionally
essential in catabolic states Insufficient
supply of glutamine may contribute to increased
infection rates and complications in certain
patient groups
30
There has been a long term interest in the
parenteral supply of - glutamine - glutamine
dipeptides - glutamine precursors (e.g. BCAA) to
catabolic patients There has been some interest
in enteral supply of glutamine to such patients
31
Some studies show that parenteral or enteral
glutamine increases blood lymphocyte numbers in
post-surgical patients (e.g. Morlion et al. 1998
Yoshida et al., 1998). Others do not show such an
effect (e.g. Spittler et al., 2001). Jensen et
al. (1996) reported increased CD4 to CD8 ratio
after enteral glutamine in ICU patients. ORiorda
in et al. (1994) reported increased lymphocyte
proliferation in post-surgery patients receiving
parenteral glutamine.
32
Parenteral glutamine and HLA-DR expression on
monocytes in post-surgical patients
Immediately post-op 48 hr Control
Gly-Gln Control Gly-Gln HLA-DR ( )
97.5 95.0 90.0 92.0 HLA-DR (MFI)
21.0 19.5 8.0 11.0
Spittler et al. (2001) 20, 37-42
33
Glutamine and infection (human studies)
1. Parenteral glutamine decreased infection rates
(12 vs. 42) in bone marrow transplantation
patients Ziegler et al., 1992. These patients
had increased numbers of lymphocytes, T
lymphocytes and CD4 lymphocytes in the
bloodstream Ziegler et al., 1998. 2. Very low
birthweight babies who received enteral glutamine
had lower rates of sepsis (11 vs. 31) Neu et
al., 1997. 3. Glutamine decreased (late)
mortality among intensive care patients
Griffiths et al., 1997. 4. Enteral glutamine
decreased incidence of pneumonia (17 vs. 42),
bacteraemia (7 vs. 42) and severe sepsis (4
vs. 26) in trauma patients Houdijk et al.,
1998. There was no effect on mortality.
34
Recent meta-analyses of glutamine
Novak et al. (2002) Crit. Care Med. 30,
2022-2029 - surgical or critically ill (BMT and
prem. Infants excluded) - Gln alone only -
parenteral or enteral - 14 studies (751
patients)
Mortality RR 0.78 (0.58 - 1.04) Mortality - hi
dose Gln (gt 0.2 g/kg/d) RR 0.73 (0.53 -
1.0) Mortality - lo dose Gln (lt 0.2 g/kg/d) RR
1.02 (0.52 - 2.0) Mortality - Parenteral RR 0.71
(0.51 - 0.99) Mortality - Enteral RR 1.08 (0.57
- 2.01)
35
Infections RR 0.81 (0.64 - 1.0) Hosp. stay
-2.6 d (-4.5 - -0.7) Hosp. Stay (surg.
pts.) -3.5 d (-5.3 - -1.7) Hosp. Stay (crit. Ill
pts.) 0.9 d (-4.9 - 6.8) Sub-group analyses
favoured benefit of high dose or parenteral Gln
on length of stay and infections
36
CONCLUSIONS FROM NOVAK ET AL. Surgical patients
Gln associated with decreased infectious
complications and length of hospital stay without
any significant effect on mortality Critically
ill patients Gln associated with decreased
infectious complications and mortality,
especially when high dose, parenteral route used.
37
Recent meta-analysis of glutamine in BMT
Murray and Pindora (2002) Cochrane
Library Parenteral Gln vs. Standard
parenteral Length of stay -6.62 d (-9.77 -
-3.47) Development of positive blood culture OR
0.23 (0.08 - 0.65)
CONCLUSIONBMT patients with GI failure should
receive parenteral nutrition supplemented with Gln
38
Glutamine Conclusions
1. Glutamine is used by cells of the immune
system in vitro and in vivo. 2. Glutamine is
essential for maximal immune cell functioning in
vitro. 3. Dietary glutamine improved ex vivo
immune cell function in a number of animal
studies including several involving
infections. 4. Parenteral or dietary glutamine
improves survival of experimental animals
following challenge with pathogens. 5.
Parenteral or enteral glutamine decreases
infection rates in some patient groups at risk of
infection. Improved immune function may be a
contributor to this. Improved intestinal
integrity important too.
39
Importance of glutathione

• In vitro glutathione depletion is associated with
  decreased IFN-g and IL-12 production by antigen
  stimulated lymph node cells
• Mediated by APC
• Suggest glutathione acts via inducing IL-12
  production by APC

of Control
IL-12 IFN-g
GSH (pmol/million cells)
Peterson et al. (1998) PNAS 95, 3071-3076
40
Glutamine TPN and glutathione
Rats given saline,
Rats given saline,
ä
ä
standard TPN or
standard TPN or


glutamine-enriched TPN
glutamine-enriched TPN
at 2.2 ml/hr for 4 days
at 2.2 ml/hr for 4 days
Saline group also had
Saline group also had
Plasma total glutathione
Plasma total glutathione
ä
ä
access to food (chow)
access to food (chow)
Denno et al. (1996) J.
Denno et al. (1996) J.
ä
ä
Surg. Res. 61, 35-38.
Surg. Res. 61, 35-38.
41
Arginine

• Non-essential amino acid
• Involved in protein, urea and nucleotide
  synthesis
• Precursor of nitric oxide (immunoregulatory
  cytotoxic to tumour cells and some
  microorganisms)
• Precursor for polyamines (DNA replication, cell
  cycle, cell division)
• Secretagogue for hormones that are
  immunoregulatory (prolactin, GH, IGF-1)

42
Gram -ve bacteria
Th1 cells
LPS
IFN-g
Arg



Arg
iNOS
NO
Arginase
-
Host defence
Ornithine
T cell derived cytokines
43
Arg

Arg
iNOS
NO
Arginase

Ornithine
Mitogenic signals
Polyamines
Proliferation
44
Arginine vs. glutamine lymphocyte proliferation
45
Arginine, immunity and infection animal studies

• Prevents trauma-induced thymic involution
• Promotes thymus repopulation and cellularity
• Increases lymphocyte proliferation, IL-2
  production, NK cell activity and macrophage
  cytotoxicity
• Improves DTH (cell-mediated immunity)
• Increases resistance to infections
• Increases survival in sepsis models
• Promotes wound healing

46

NK
B
Th
Activate
Activate
Activate
Tc

Antigen presentation
Activate

Antibodies

Antigen presentation
Lysis
Phagocyte
Bacteria
Virus infected cell
47
Survival after i.v. Staphylococcus aureus Adjei
et al. (1994) Nutr. Res. 14, 1591-1599
100 80 60 40 20 0
GLN
Survival
ARG
CONTROL
0 5 10 15 20
Days
48
Survival after i.v. Staphylococcus aureus Adjei
et al. (1994) Nutr. Res. 14, 1591-1599
100 80 60 40 20 0
GLN ARG
GLN
Survival
ARG
CONTROL
0 5 10 15 20
Days
49
Arginine and lymphocytes Studies in humans
45 40 35 30 25 20

• In healthy humans 30 g/day increased lymphocyte
  proliferation, increased CD4/CD8 ratio and
  promoted wound healing (Barbul et al. 1981, 1990)
• In surgical patients 25 g/day enterally enhanced
  lymphocyte proliferation and increased CD4 number
  (Daly et al. 1988)

Arg Control
CD4
Pre-op 1 4 7
100 75 50 25 0
Arg Control
Lymphocyte proliferation
Pre-op 1 4 7
50
Enteral feeds including arginine (and other
nutrients) have been developed
51
Studies using IMPACT and similar enteral formulae
in intensive care or surgical patients

• gt 20 controlled trials performed
• Several report improved phagocytosis, lymphocyte
  proliferation, HLA-DR expression
• Many report clinical benefit - decreased
  infection, decreased wound complications,
  decreased need for ventilation, decreased
  progression to SIRS, decreased length of ICU /or
  hospital stay
• Meta-analyses confirm reduced infection rate,
  decreased number of ventilator days, decreased
  length of stay but no effect on mortality (Heys
  et al. 1999 Beale et al. 1999 Heyland et al.
  2001)
• Benefits are greater in post-surgical than in
  critically ill patients
• Arginine or other nutrients or the combination?

52
Arginine Conclusions
1. Arginine is used by cells of the immune system
in vitro and in vivo. 2. Arginine appears to be
essential for maximal immune cell functioning in
vitro. 3. Dietary arginine improved ex vivo
immune cell function in a number of animal
studies including several involving
infections. 4. Dietary arginine improves
survival of experimental animals following
challenge with pathogens. 5. Enteral arginine
may decrease infection rates in some patient
groups at risk of infection (e.g. post-surgery).
Improved immune function may be a contributor to
this.
53
Antioxidants
Cell-mediated immunity
Oxidative stress
Inflammation
54
Antioxidant defence systems
Dehydro Vit. C
2GSH
e.
Vit. E
Glutathione reductase (Riboflavin)
Ox. Vit. E
Vit. C
GSSG
ANTIOXIDANT ENZYMES Superoxide dismutase
(Cu/Zn Mn) Catalase (Fe) Glutathione peroxidase
(Se)
55
Cysteine
Glutamate
Gln
Glycine
Dehydro Vit. C
2GSH
Vit. E
e.
Ox. Vit. E
Vit. C
GSSG
56
Polyamines
Methionine
Protein
THF
SAM
CH3 donor
SAH
5MTHF
Taurine
HCy
Glutathione
Cystathionine
Cysteine
Protein
57
Meydani et al. (1997) JAMA 277, 1380-1386
Antioxidants and related nutrients and
metabolites shown to improve the immune
response Folic acid Vitamin B6 Vitamin
B12 Riboflavin Vitamin E Vitamin C b-carotene
Zn Cu Fe Se N-acetylcysteine Taurine
Increase in induration index (mm)
Fold increase in antibody titre to Hepatitis B
Vitamin E intake group
58
Multivitamin mixtures, immune function and
infection in humans
Healthy elderly subjects Received placebo or a
multivitamin/mineral supplement - US
recommended amounts of vitamins A, B6, B12, and
D, thiamin, niacin, folate, iron, zinc, copper,
selenium, iodine, calcium and magnesium and
higher than the US recommended amounts of
vitamins C and E and beta carotene. for 12
months Increased circulating T cell
numbers Increased natural killer cell
activity Increased IL-2 production Improved
antibody responses to influenza vaccine Less
infection-related illness Chandra (1992) Lancet
340, 1124-1127
59
Study of enteral fish oil in ARDS Gadek et al.
(1999) Crit. Care Med. 27, 1409-1420

• Prospective, randomised, placebo controlled,
  multi-centre
• About 50 evaluable patients in each group
• Groups well matched at study entry
• 20 surgical trauma 50 sepsis/pneumonia
• 100 respiratory failure 33 failure of at least
  one other organ system
• Moderate and severe ARDS
• Enteral formulae
• identical amounts and types of protein (amino
  acids) and CHO
• identical amounts of fat (55 energy)
• Control 97 corn oil 3 soybean lecithin
• Experimental 32 canola oil 25 MCT 20
  borage oil 20 fish oil 3 soybean lecithin
  extra vitamin E and C b-carotene taurine
  carnitine
• 7 days intervention

60
Study of enteral fish oil in ARDSGadek et al.
(1999) Crit. Care Med. 27, 1409-1420

• By day 4 of treatment
• decreased numbers of leukocytes and neutrophils
  in alveolar fluid
• improved arterial oxygenation
• improved gas exchange
• decreased requirement for supplemental oxygen
• Decreased time on ventilator
• Reduced ICU stay (12.8 1.1 vs. 17.5 1.7 days)
• Reduced development of new organ failure (4/51
  vs. 13/47)
• Reduced hospital stay (29.4 2.6 vs. 34.6 3.3
  days) NS
• Reduced mortality (19 vs. 12) NS
• n-3 PUFA or GLA or MCT or antioxidants or the
  combination or less n-6 PUFA??

61
Antioxidants Conclusions
1. A wide range of fat and water soluble
vitamins, trace elements and other dietary
antioxidants, antioxidant precursors and
antioxidant derivatives have been shown to
improve immune function in animal studies and in
trials in healthy humans. 2. Some of these
compounds have been demonstrated to improve
clinically-related outcomes and/or survival of
experimental animals following challenge with
pathogens. 3. Parenteral and enteral formulas
contain classic and emerging antioxidants. 4.
Some of the immune and clinical benefits of
enteral formulas may relate to the quantity and
quality of antioxidants they contain.
62
Summary

• A number of nutrients are able to modulate immune
  and inflammatory responses in ways that may be of
  clinical benefit
• The roles of arginine and glutamine have been
  examined and confirmed in cell culture, animal
  feeding, healthy human and clinical studies
• Evidence for benefit of glutamine (especially
  parenteral) is strong in both post-surgery and
  critically ill patients
• Evidence for benefit of enteral feeds containing
  arginine (but also other nutrients) is strong in
  post-surgery patients, but is weak in critically
  ill patients
• There is a role for antioxidants in parenteral
  and enteral formulas

63
Immunonutrition
Improved barrier function
Improved immune function
Improved wound healing
Decreased hyperinflammation
Improved clinical outcome