Nutritional immunology is a fascinating but highly complex and conflicted subject area. With almost every nutrient we consume having the ability to affect our immune response in one way or another and the activation of the immune system dramatically increasing nutrient requirements, understanding the genetic, cellular and metabolic mechanisms that interact, control and conflict with the immune system and how to manipulate them to our advantage, is fundamental to optimal health. We are thrilled to announce that we have linked up with Professor Phillip Calder, a world renowned and highly cited expert in nutritional immunology, with over 500 publications to his name. Professor Calder will be joining us as our guest speaker for our January Webinar to help us kick off what promises to be our most exciting year of clinical nutrition education yet. In this detailed Q&A session Professor Calder will shed light on a whole host of fascinating topics from the latest research into nutrition immunology, his projects involving nutrigenomics, probiotics and omega-3s, the real science behind effective clinical omega-3 interventions, his thoughts on the best forms of lipid supplementation, and doing some serious nutrition science myth busting.

1. A discussion with
Professor Philip Calder
Professor of Nutritional Immunology
University of Southampton
(pcc@soton.ac.uk)
1

2. My background
• Trained as a metabolic biochemist
• PhD in Biochemistry from University of Auckland in
New Zealand
• 1987 to 1995 at University of Oxford:
• Began studies of fatty acid functionality
particularly in the context of immune function
• Developed interest in the influence of other
nutrients on immune function
• Since1995 at University of Southampton:
• Expanded research into fatty acid metabolism
• Expanded research into inflammation and human
disease (mainly cardiometabolic)
• Developed translational approach (basic science ->
clinical trials -> public health & clinical
applications)
2

3. The current situation
• Run a research group of ~10 researchers (mainly in fatty
acids)
• Have many outside collaborations
• Many invitations to speak at conferences and educational
events
• Active in publishing
• Member of many editorial boards
• Previously Editor-in-Chief of British Journal of Nutrition
• Active in professional societies
• Previously President of the International Society for the
Study of Fatty Acids and Lipids
• Currently Chair of the Scientific Committee of the
European Society for Clinical Nutrition & Metabolism
• President-Elect of the Nutrition Society
• Advisor to food, supplements, infant nutrition, clinical
nutrition, pharmaceutical companies (EFSA too)
• ILSI Europe 3

4. 4

5. “Enjoyable” aspects
• Doing research that is well received is fulfilling
• A variety of research and research applications keeps
interest levels high
• Presenting research at conferences is very enjoyable
• Training young scientists is very rewarding
• I enjoy my work in publishing – interaction with editors
and authors to improve standards
• I also enjoy my work in professional societies –
interaction with colleagues; organising scientific events;
producing position papers etc.; social aspects are
important too as is the service element
• ILSI Europe - guidance documents
• Interaction with industry is always
interesting, especially where a high
value is placed upon science/
research/evidence 5

6. 6

7. 28 years of research on
omega-3 fatty acids & inflammation
7

8. Omega-3 (w-3, n-3) fatty acids
• a family of long chain & very long chain,
highly unsaturated fatty acids
• defined by the position of the methyl terminal
double bond
• include a-linolenic, stearidonic,
eicosapentaenoic, docosapentaenoic and
docosahexaenoic acids
• have different dietary sources
• are metabolically related to one another
8

9. Very long chain (“marine”) w-3
polyunsaturated fatty acids
Eicosapentaenoic acid (EPA) 20:5w-3
Docosapentaenoic acid (DPA) 22:5w-3
Docosahexaenoic acid (DHA) 22:6w-3
H3C COOH
H3C COOH
H3C
COOH
9

10. Pathway of
biosynthesis of
EPA, DPA and DHA
10

11. 11

12. EPA, DPA and especially DHA are
poorly synthesised in humans
-> dietary [i.e. preformed]
sources are important
12

13. Synthesised in plants
Found in green leaves, some seeds,
some nuts,
some plant oils
Found in seafood, especially oily
fish, and in fish oil supplements
Found in meat, eggs and offal
13

14. Effect on EPA + DHA intake by eating
oily fish or taking supplements
Grams per day
Normal
diet
+ one
concentrated
fish oil
capsule
+ one
standard
fish oil
capsule
+ one
pharma
capsule
One meal
of salmon
+ 4
pharma
capsules
14

15. In most cells and tissues the content of
EPA and DHA is low compared with the
content of w-6 PUFAs
15

16. w-6 and w-3 PUFA contents of phospholipids
of human white (mononuclear) cells
% of total fatty acids
Linoleic acid (18:2w-6) 10
DGLA (20:3w-6) 1.5
Arachidonic acid (20:4w-6) 20
a-Linolenic acid (18:3w-3) < 0.5
EPA 1.0
DHA 2.5
16

17. Direct link with inflammation:
Arachidonic acid (20:4w-6) gives rise to
eicosanoid mediators
ARA in various
membrane phospholipids
Free ARA
2-series PGs
2-series TXs
15-HPETE
15-HETE
Lipoxin A4
12-HPETE
12-HETE
5-HPETE
5-HETE
4-series LTs
2-AG AEA
COX pathway
12-LOXpathway
CYT P450 pathway
5-, 8-, 9-, 11-, 12-,
15-, 19-, 20-HETE
EETs
DHETs
20-carboxy-ARA
20-hydroxy-PGs
17

18. But increasing EPA+DHA intake [supply]
increases the EPA and DHA content of
blood lipids, blood cells, and many
tissues including liver, heart & skeletal
muscle – effect is dose, time and tissue
dependent
18

19. Browning et al. (2012) Am. J. Clin. Nutr. 96, 2012, 748-758
Dose and time dependent accretion of (oral) DHA
in plasma phospholipid in healthy subjects
19

20. Healy et al. (2000) Lipids 35, 763-768
Oral omega-3 in healthy subjects for 12 weeks
Neutrophil PL
20

21. Altered
EPA and DHA supply
Altered composition of
Inflammatory cell phospholipids
(more EPA & DHA; Less arachidonic acid)
Membrane
alterations: rafts; order;
trafficking
Lipid mediators
(e.g. less PGE2 and LTB4)
Signal transduction
pathways leading
to gene expression
Altered inflammatory cell phenotype
Altered inflammatory response
Working model:
21

22. Altered
EPA and DHA supply
Altered composition of
Inflammatory cell phospholipids
(more EPA & DHA; Less arachidonic acid)
Membrane
alterations: rafts; order;
trafficking
Lipid mediators
(e.g. less PGE2 and LTB4)
Signal transduction
pathways leading
to gene expression
Altered inflammatory cell phenotype
Altered inflammatory response
Working model:
22

23. ARA in various
membrane phospholipids
Free ARA
2-series PGs
2-series TXs
15-HPETE
15-HETE
Lipoxin A4
12-HPETE
12-HETE
5-HPETE
5-HETE
4-series LTs
2-AG AEA
COX pathway
12-LOXpathway
CYT P450 pathway
5-, 8-, 9-, 11-, 12-,
15-, 19-, 20-HETE
EETs
DHETs
20-carboxy-ARA
20-hydroxy-PGs
w-3 fatty acids
23

24. Fish oil-derived w-3 fatty acids exert anti-
inflammatory effects by inhibiting
production of arachidonic acid-derived
eicosanoids
24

25. EPA is also a substrate for
eicosanoid synthesis
Arachidonic acid EPA
2-series PGs 4-series LTs 3-series PGs 5-series LTs
25

26. Arachidonic acid EPA
2-series PGs 4-series LTs 3-series PGs 5-series LTs
STRONG WEAK
26

27. Nature Immunology 2001
27

28. Inflammation has two phases:
initiation and resolution
Initiation
phase
Resolution
phase
TIME
28

29. Inflammation has two phases:
initiation and resolution
Initiation
phase
Resolution
phase
TIME
Classical
AA derived
mediators
(PGE2, LTB4
etc.)
29

30. Inflammation has two phases:
initiation and resolution
Initiation
phase
Resolution
phase
TIME
Classical
AA derived
mediators
(PGE2, LTB4
etc.)
Novel pro-
resolving
mediators
30

31. EPA and DHA give rise to pro-resolving mediators:
Resolvins (E-series & D-series)
Protectins (Neuroprotectins)
Maresins
31

32. Tissue injury
Microbes
Acute inflammation
Chronic inflammation
Tissue damage
Persistent infection
Autoimmune disease
Resolution
Tissue repair
Healing
Omega-3
derived pro-
resolving
mediators
32

33. 33

34. Interesting projects
34
There are many!!

35. • Patients with MI within
the last 3 months
assigned to w-3 ethyl
esters vs placebo
• Follow up for 3.5 years
• 356 deaths and non-
fatal CV events in w-3
group vs. 414 in placebo
group
RRR in w-3 ethyl ester group
All fatal events -20%
CV death -30%
Coronary death -35%
Sudden death -45%
Other deaths -1%
35

36. Marchioli et al. (2002) Circulation 105, 1897-1903
36

37. Plaque rupture – an inflammatory event
37

38. Since long chain w-3 fatty acids are anti-
inflammatory we hypothesised that they
prevent cardiovascular events
by stabilising plaques
(i.e. making them less likely to rupture)
38

39. Carotid endarterectomy offers a model to provide
substantial numbers of advanced plaques and to
allow for pre-surgery interventions
39

40. We took advantage of this to study the
effect of w-3 fatty acids on plaque stability,
morphology and composition
40

41. • RCT of w-3 fatty acids (1.6 g
EPA+DHA per day) in patients
awaiting carotid
endarterectomy (n = 180)
• Median time of treatment: 42
days
• Patients given w-3 fatty acids
have higher w-3 fatty acid
content in plaques
• Patients given w-3 fatty acids
have fewer macrophages
within the plaque
Control Omega-3
35
30
25
20
15
10
5
0
P = 0.028
% of unstable plaques at surgery
41

42. Matrix metalloproteinases (MMPs) released from
macrophages, foam cells and smooth muscle cells
degrade the plaque cap making it
more vulnerable to rupture
42

43. Control group Omega-3 group
43

44. Increased omega-3 fatty acid availability
Incorporation of omega-3 fatty acids into plaque
Decreased number of macrophages, foam cells & T cells
Lower expression of selected inflammatory
cytokines and MMPs
Less plaque inflammation
Increased plaque stability
44

45. 45

46. What is new and exciting
in this field?
46
Three things

47. 1. Pro-resolving mediators are
produced from EPA and DHA
47
Initiation
phase
Resolution
phase
TIME
Novel pro-
resolving
mediators

48. 2. There are surface receptors
for omega-3 fatty acids that induce
anti-inflammatory signalling
48
TLR4
DHA
Inflammatory
Stimulus
GPR120
NFkB

49. 3. Membrane regions called “rafts”
can be modulated by EPA and DHA
49

50. Fatty acid exposure
Receptors Membrane composition
FluidityRaft assembly Substrates for
eicosanoids,
resolvins etc.
Signals
Cell responses
Altered
(patho)physiology
Health/
Clinical
outcome
50

51. 51

52. Omega-3 recommendations
• UK recommendation is for adults to consume
at least 450 mg of EPA + DHA per day
• This is based on eating one lean and one oily
fish meal per week
• Unless people eat oily fish or take omega-3
supplements their intake will be way below 450
mg/day
• Most studies show that 450 mg/day has a
limited effect on any health related outcomes
(but these studies are relatively short duration)
• UK recommendation allows for higher intakes
than 450 mg/day
52

53. Omega-3 recommendations
• Different people may need different amounts of
EPA and DHA
• Pregnancy
• Lactation
• Infancy/rapid growth
• Elderly
• Athletes
• People with disease
• Different dietary backgrounds
• Genetic influences
53

54. 54

55. EPA or DHA or both?
• A very good question, but one that is hard to
answer
• Nature puts them together in food
• EPA can (in theory) be converted to DHA (but
this seems limited) and DHA can be converted
to EPA (called retroconversion)
• Both EPA and DHA affect lipid mediators, cell
signalling and lipid rafts (i.e. both are
biologically active)
• The eye and brain have high contents of DHA
and little EPA -> specific role for DHA
55

56. 56
-> specific need for DHA pre-pregnancy,
during pregnancy, during lactation, in
infancy (but EPA still needed)
-> cannot differentiate need for EPA and
DHA at other times in the life course

57. 57

58. Fat digestion
From liver
and gall bladder
From
stomach
Fat droplets
(TGs)
Emulsified fat
Droplets (TGs)
From pancreas
FFA
+ 2MG
Micelles
Lipase
Bile salts,
cholesterol
& lecithin
58

59. Fat absorption
Micelles
2FA + 2-MG
TG Chlyomicrons
Assemble with
PL and apoproteins
Lymphatic
system
59

60. Overview of whole body fatty acid
metabolism
GUT
Digestion & absorption
Fatty acids in lipoproteins;
Non-esterified fatty acid
(Transport pools)
LIVER
Fatty acid
metabolism
ADIPOSE
Fatty acid
storage
CELLS AND
TISSUES
Fatty acids in cell
membranes
(Functional
pools)
DIET
(Mainly as TAG)
60

61. • Twenty healthy adults aged 50 to 65 y
• Consumed a test meal that included fish oil (2.3 g
EPA+DHA)
• Test meal: 55 g fat, 130 g CHO, 12 g protein
• Blood collected up to 6 hr
61

62. EPA in plasma TAG DHA in plasma TAG
62

63. 63

64. 64

65. Study design
• 5 group, parallel design, stratified by age and gender
• Control
• “1 portion oily fish” per week (2.8 g EPA+DHA on ONE
day = 2.8 g/week)
• “2 portions oily fish” per week (2.8 g EPA+DHA on TWO
days = 5.6 g/week)
• “2 portions oily fish” per week (taken daily – 5.6 g/week
as 0.8 g/day)
• “4 portions oily fish” per week (2.8 g EPA+DHA on FOUR
days = 11.2 g/week)
• 210 subjects (105 at each centre)
65

66. Randomisation (stratified by age and
gender)
Control
(Ctrl)
(n=42)
1 portion per
week (1P)
(n=42)
2 portions per
week (2P)
(n=42)
4 portions per
week (4P)
(n=42)
Fasting bloods at 0, 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months
Buccal wash at 0, 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 12 months
Adipose tissue biopsy at 0, 6 months, 12 months
Fatty acid composition analysis
Recruitment
healthy men and women, aged 20-80 years (n=210)
2 portions per
week continuous
(2P-D)
(n=42)
66

67. Overview of whole body fatty acid
metabolism
GUT
Digestion & absorption
Fatty acids in lipoproteins;
Non-esterified fatty acid
(Transport pools)
LIVER
Fatty acid
metabolism
ADIPOSE
Fatty acid
storage
CELLS AND
TISSUES
Fatty acids in cell
membranes
(Functional
pools)
DIET
(Mainly as TAG)
67

68. Analysed the fatty acid composition of:
• plasma PC, TAG, CE and NEFA
• platelets
• erythrocytes
• mononuclear cells
• buccal cells
• adipose tissue
From all subjects at
9 time points
From all subjects at
three time points
68

69. 69

70. 70

71. Can you have too much
EPA and DHA?
• Very high amounts may make people feel
nauseous and induce “stomach upsets”
• Bleeding problems were reported in the
Greenland Inuit – extremely high intakes
• Highly unlikely to be
a real problem
71

72. 72

73. 0 4 8 12 20
0
1
2
3
4
Time (weeks)
EPAinmononuclearcellPL(%)
0 4 8 12 20
1
2
3
4
Time (weeks)
DHAinmononuclearcellPL(%)
Effect of stopping intake of EPA and DHA
Healthy volunteers given fish oil for 12 weeks; then 8 week washout
Yaqoob et al. (2000) Eur. J. Clin. Invest. 30, 260-274
73

74. 74

75. Krill oil??
Some of the omega-3 fatty acids
are in phospholipid form
There has been an argument that this
will promote their uptake from the gut
75

76. • Acute study
• Twelve healthy young men
• Consumed a test meal plus omege-3 as rTG, EE
or Krill oil (~1.7 g EPA+DHA)
• Test meal: 30 g fat, 68 g CHO, 30 g protein
• Plasma PL fatty acids up to 72 hr
76

77. No difference between formulations
but EPA tended to be higher with krill oil
77

78. 78
Probably limited effect in
healthy people
But may have a role in
people who have problems
digesting or absorbing fat – but
this has not been tested yet

79. 79

80. Is there a role for
short chain,
plant-derived
omega-3?
Conversion
efficiency,
especially to
DHA is poor 80

81. 81

82. What about vegetarians
and vegans??
• EPA and DHA status often lower than in
omnivores and fish consumers
• But may be “good converters” – plant oils
may have a role
• May have mechanisms to retain EPA and
DHA
• Algal oil
82

83. 83

84. Important factors when chosing an
omega-3 source??
• Dose is important – how much EPA and
DHA do you get in a capsule
• Concentration – how much EPA and DHA
is there and how much other (not
necessary) fatty acids -> How much oil is
needed to get the desired dose of EPA
and DHA
= Dose and Purity
84

85. 85

86. Importance of EFSA
• Established process for substantiation of
claims on foods
• Consumer protection (against bogus or
unproven claims)
• Industry reward (for good science)
• Therefore a very useful process overall
• Some omega-3 claims are allowed (e.g.
blood triglycerides, blood pressure, brain
development)
• But in some areas (e.g. gut health &
immunity) few claims have been permitted
86

87. 87