Diabetes Research Institute hosts first PreDiRe T1D Symposium to focus on preventing type 1 diabetes and its recurrence.

1. Omega-3 fatty acids; affects on inflammation
and impact on autoimunity
Michael J. Clare-Salzler, M.D.
Professor and Chair
Department of Pathology, Immunology and Laboratory Medicine

2. Presentation outline
• Type 1 diabetes (T1D) pathogenesis
• Reduced N-3 fatty acid content of Western diets in
the last century
• How do fatty acids suppress inflammation?
• Studies showing increased N-3 fatty acid intake
reduces risk for autoimmune disease
• Animal studies demonstrating reduced inflammation,
reduced risk for autoantibodies in autoimmune
disease

3. Genetic, environmental and immunologic interactions in the
pathogenesis of T1D
Environment
Viruses
(Coxsackievirus)
Immune/b cell
response
PDC
Genetics
Interferon
response
MDA-5, TLR7,8
IRF7, PTPN22,
Tyk2,
b cell expression
dysfunction/destruction
IFNa/b/l
Immune activation/modulation
(APC, CD4, CD8 T cells (Tscm, Texh)

4. b cell
mass
Genetic Risk
Relative
(MHC class II
40+ susceptibility
loci)
Islet cell autoantibodies
GAD65, IA2, insulin, T cell responses
Loss of first phase insulin release and
Glucose stimulated insulin secretion
Glucose intolerance
Clinical diabetes
Years
Natural History of T1D in humans
Environmental trigger; virus
Environmental modulator; diet
Screening for disease
• Hi-risk infant screening
• First-degree relative screening
• Impaired Glucose Tolerance

5. b Cells
The quick and dirty;T1D pathogenesis
Macrophage
1. Inflammatory
factors (IL-1b)
2. Toxic to b cells
3. Attract T cells
CD4+ and CD8+
T cells infiltrate and
kill b cells
X
X

6. The chronic inflammatory lesion of T1D;insulitis
Blue= insulin, Green= T cells, Red= Macrophages Blue=insulin, Green= CVB viral protein,
Red= viral sensing molecule

7. Underlying heightened inflammatory response in
T1D patients
• Early events; islet inflammation
• Immune cells in T1D patients produce high-levels of IL-1b,
IL-6, TNF-a
• Increased expression of inflammatory Cox-2 in human T1D
monocyte/macrophages
• Limited production of anti-inflammatory cytokine IL-10
and reduced suppressive effects
• Increased incidence of T1D in the last few decades

8. • Genetics cannot explain the increase
• Environmental factors possible, e.g. changes in virus
genetic/biology, toxins, dietary changes
Diabetes incidence is increasing over the last few decades;
So what’s up ?

9. “Thanksgiving Dinner”; Norman Rockwell 1943
? Decreased n-3 fatty acids in Western diets

10. Are we what we eat?
Trans and n-6 fatty acids promote inflammation

11. Omega-3 fatty acid intake – is the Western diet an
environmental factor for inflammatory diseases?
High n-3 diet: anti-inflammatory
High n-6 and Trans fat diets: pro-inflammatory
Changes in Western diet ratios of n-6 to n-3 Fatty Acids
1800’s = 1 or 2 (n-6) to 1 (n-3)
Present = 20 or 30 (n-6) to 1 (n-3)

12. Algae, Fish, Flax seed, Plants/
Cannola oil
-3 PUFA aLinolenic -6 PUFA
(a LA)
Linoleic
EPA
+
DHA Arachidonic (animal fat)
Resolvins prostanoids, leukotrienes
(Anti-inflammatory) (Inflammatory)
• Mammals cannot synthesize aLA (the predominant PUFA in
Western diets) and therefore these fatty acids are “essential” and
must be ingested. Mammals, unlike plants, cannot interconvert n-9,
n-6, n-3 fatty acids

13. Fig. 1
Prostaglandins, Leukotrienes and Essential Fatty Acids 2015 99, 19-23DOI: (10.1016/j.plefa.2015.04.005)
Bubis and Lands Prost and Ess Fatty Acids 2017
n-6 fatty acids compete with n-3 fatty acids
n-6
n-3

14. Ancient understanding of
inflammation

15. How do n-3 fatty acids suppress inflammation
• Enzymes convert n-3 fatty acids to regulatory metabolites that
are;
– Anti-inflammatory
– Resolve cellular and molecular aspects of inflammation
– Provide protection of tissues from inflammation
– Promote tissue regeneration
• Metabolites of n-3 fatty acid
– Resolvins
– Protectins
– Maresin

16. Effects of n-3 fatty acid metabolites
Serhan, Chiang, Dalli Molec. Aspects of Med. 2017

17. EPA metabolism to specialized pro-resolving mediators
(SPM) *
*
*
*
*

18. DHA metabolized to specialized pro-resolving mediators
(SPM) *
**
*
*
* *
*
* *
*
*
*
*

19. Omega-3 fatty acids (DHA) are converted to potent anti-
inflammatory resolvin molecules at sites of inflammation
DHA
5-lipoxgenase
15-lipoxygenase
cyclooxygenase-2
Inflammatory T cells
Inflammation; IL-1b/a, IL-6, TNF-a
Resolvins
Polyvalent anti-inflammatory
action
Inflammatory stimulus in the islet
leads to production of anti-inflammatory
resolvins only at the site of inflammation
Immune cells infiltrating the islet contain high-levels
of DHA as a result of dietary supplementation
Dietary
DHA
incorporated
into cell
membranes

20. n-3 feeding and Lipoxin/Resolvin analogues prevents
diabetes in NOD mice
0 5 10 15 20 25
0
10
20
30
40
50
60
70
80
90
100
Trienyne
(12.5ug)
High Dose Trienyne Compound ZK-224
Delays Onset of Diabetes in the NOD Mouse.
p=0.0199
Vehicle (20%)
(67%)
Time (weeks)
D
i
a
b
e
t
e
s
S
u
r
v
i
v
a
l
Rx
JCI Xinyun 2017

21. Risk of Developing the Outcome of Multiple Autoantibodies
or Type 1 Diabetes by Dietary Intake of PUFAs (Study 1)a
Adjusted HR (95% CI) P Value
Total omega-3 FA intake 0.23 (0.09-0.58) .002
Total omega-6 FA intake 1.50 (0.67-3.35) .32
Norris JM JAMA 2007

22. Association Between Omega-3 and Omega-6 Fatty Acids in
Erythrocyte Membranes and Risk of IA (Study 2)a
Fatty Acids Adjusted HR (95% CI) P Value
Total omega-3 fatty acid 0.63 (0.41-0.96) .03
Marine PUFA 0.87 (0.53-1.43) .59
Total omega-6 fatty acid 1.02 (0.68-1.53) .92
Arachidonic acid 0.79 (0.52-1.21) .28
Total n-3 in RBC membranes is associated with a lower risk of
developing islet autoantibodies
JAMA 2007

23. Unadjusted HR Adjusted HR
95% CI
†
p 95% CI
†
,
‡
p
Total omega-3 fatty
acids
0.99 (0.70–1.41) 0.96 1.06 (0.75–1.50) 0.73
ALA 1.02 (0.79–1.31) 0.89 0.99 (0.73–1.34) 0.96
EPA 1.10 (0.83–1.47) 0.52 1.14 (0.85–1.52) 0.39
DHA 1.13 (0.84–1.53) 0.43 1.21 (0.90–1.65) 0.21
DPA 0.81 (0.49–1.33) 0.41 0.86 (0.53–1.39) 0.54
Total omega-6 fatty
acids
1.28 (0.89–1.84) 0.18 1.25 (0.88–1.77) 0.22
LA 1.25 (0.90–1.73) 0.18 1.19 (0.87–1.63) 0.29
GLA 0.97 (0.58–1.62) 0.91 0.96 (0.62–1.50) 0.87
ARA 1.13 (0.81–1.56) 0.48 1.16 (0.84–1.60) 0.38
Association between erythrocyte membrane omega-3 or omega-6
fatty acid content and risk of developing type 1 diabetes in children
with islet autoantibodies
Norris JM, JAMA 2007

24. Association between omega-3 fatty acid
biomarkers and prevalent inflammatory arthritis in
CCP3+ subjects
n-3FA% in RBC OR(95% CI) P-value
ALA (18:3n-3) 2.76 (0.83, 9.18) 0.10
EPA (20:5n-3) 0.25 (0.03, 2.09) 0.20
DCPA (22:5n-3) 0.19 (0.04, 0.94) 0.04
DHA (22:6n-3) 0.24 (0.06, 0.94) 0.04
EPA+DHA 0.20 (0.04, 0.98) 0.05
Total n-3 FA 0.09 (0.01, 0.85) 0.03
• Omega-3 fatty acids were associated with lower prevalence of inflammatory
arthritis in CCP positive subjects.
• Docosapentaenoic acid reduced risk of inflammatory arthritis in CCP positive
subjects.
• Longer-chain omega-3 fatty acids may protect against development of
inflammatory arthritis in CCP positive persons
Summary of the study
Studies in subjects at high-risk for Inflammatory Arthritis
Gan RW, Rheumatology 2017

25. NIP Study Hypothesis
• The infiltration of macrophages and production of
inflammatory mediators are early key events that initiate
insulitis and are essential for the development of T1D.
• Factors which interrupt macrophage infiltration or resolve
inflammation will reduce or prevent autoimmunity
(autoantibodies) or T1D
• These factors will be most effective when applied at the initial
phase of disease (infants and young children)

26. NIP Study Design
Chase et. al.
Pediatric Diabetes 2014
All infants off formula and
Breast feeding; oral supplement
Dose;
38mg/kg/day

27. Study Endpoints
• 20% increase in DHA RBC membrane concentrations in
treated vs. control subjects in 1 year old infants receiving DHA
for 6 months
• 20% reduction in IL-1b in LPS (1mcg/ml) stimulated PBMC in
subjects treated with DHA vs. controls in 1 year old receiving
DHA for 6 months

28. p<0.001 at each age
Supplementation leads to significant increase RBC membrane
DHA concentrations
Breast and bottle feeding
supplementation
Chase et. al.
Ped. Diabetes, 2014

29. Figure 1
Immunity 2012 37, 771-783DOI: (10.1016/j.immuni.2012.10.014)
Kollmann and Levy Nature Immun. Reviews
Dynamic changes in immune response in humans during
the aging process
? Age related changes in metabolism of n-3 fatty acids

30. Assays for inflammation and DHA metabolites
• LPS stimulation of peripheral blood sample
• Induces strong inflammation
• Measure cellular production of inflammatory factors, e.g. IL-
1b
• Measure resolvins
• Correlate DHA- based resolvins with inflammatory factors e.g.,
increased resolvins with reduced inflammation in DHA
supplemented infants

31. • 15-LOX pathway (monocyte/some PMN)
• 15-HETE
• 17-HDHA
• LXA4
• NPD1
• w-22 PD1
• RvD1
• 5-LOX pathway (monocyte/PMN)
• 5-HETE
• LTB4
• 4-HDHA
• 7-HDHA
• Anti-inflammatory/Pro-resolving/Counter-balancing
• LXA4
• NPD1
• w-22 PD1
• RvD1
• 17-HDHA
• 7-HDHA
• 4-HDHA
• 15-HETE
• DHA*
• EPA*
DHA/EPA Metabolome
AA
DHA
EPA
12 HETE
12HEPE
13 HODE
14HDHA
15HEPE
15HETE
17 HDHA
4HDHA
5HETE
6LTB4
7HDHA
LTB4
LXA4
MARESIN
NPD1
PGD2
PGE2
PGF2a
RvD1
TXB2
W22PD1
Pathway analysis
• All measured in Cx
Supernatants
• LC-MS/MS method (Gronert)
• * Correlation with RBC
Membrane concentrations

32. Dynamic changes in IL-1β cytokine production
as infants age
0
100
200
300
400
500
600
700
800
900
1000
6 M 12 M 18M
DHA
control
18M
38% suppression; p=0.06
60% increase in DHA Tx group; p<0.0001
DHA conc. Tx 46.9ug/ml
DHA conc. Cont. 29.2ug/ml
11.4% suppression; p=0.57
48.6% increase in DHA Tx group; p<0.0001
DHA conc. 75.5ug/ml
DHA conc. Cont. 50.8ug/ml
6M
17.2% suppression; p=0.38
43.8% increase in DHA Tx; p=0.0007
DHA conc. Tx group 72.9 ug/ml
DHA conc. cont. 50.7ug/ml
12M

33. Dynamic changes in TNF-α cytokine production
as infants age
0
100
200
300
400
500
600
700
800
900
6 M 12 M 18 M
DHA
Control18M
28% suppression; p=0.15
60% increase in DHA Tx group; p<0.0001
DHA conc. Tx 46.9ug/ml
DHA conc. Cont. 29.2ug/ml
12M
30.2% suppression; p=0.11
48.6% increase in DHA Tx group; p<0.0001
DHA conc. 75.5ug/ml
DHA conc. Cont. 50.8ug/ml
6M
5.7% suppression; p=0.78
43.8% increase in DHA Tx; p=0.0007
DHA conc. Tx group 72.9 ug/ml
DHA conc. cont. 50.7ug/ml

34. DHA+EPA v Cytokines and PGE2
Regression
6-Months
Metabolome
Il1B1 t-
Statistic
Il1B1 p-
value
TnF-a t-
Statistic
TnF-a
p-value
GMCSF
t-Statistic
GMCSF
p-value
IL6 t-
Statistic
IL6 p-
value
IL10 t-
Statistic
IL10 p-
value
PGE2 t-
Statistic
PGE2
p-value
DHA+EPA 0.22 0.828 -0.01 0.990 0.23 0.821 -0.25 0.804 -0.92 0.362 1.99 0.051
12-Months
Metabolome
Il1B1 t-
Statistic
Il1B1 p-
value
TnF-a t-
Statistic
TnF-a
p-value
GMCSF
t-Statistic
GMCSF
p-value
IL6 t-
Statistic
IL6 p-
value
IL10 t-
Statistic
IL10 p-
value
PGE2 t-
Statistic
PGE2
p-value
DHA+EPA -0.38 0.703 -1.24 0.221 -0.37 0.716 -0.35 0.727 -1.09 0.282 -0.61 0.547
18-Months
Metabolome
Il1B1 t-
Statistic
Il1B1 p-
value
TnF-a t-
Statistic
TnF-a
p-value
GMCSF
t-Statistic
GMCSF
p-value
IL6 t-
Statistic
IL6 p-
value
IL10 t-
Statistic
IL10 p-
value
PGE2 t-
Statistic
PGE2
p-value
DHA+EPA -1.47 0.148 -0.87 0.389 -0.59 0.557 -0.12 0.907 0.10 0.921 -1.43 0.159
24-Months
Metabolome
Il1B1 t-
Statistic
Il1B1 p-
value
TnF-a t-
Statistic
TnF-a
p-value
GMCSF
t-Statistic
GMCSF
p-value
IL6 t-
Statistic
IL6 p-
value
IL10 t-
Statistic
IL10 p-
value
PGE2 t-
Statistic
PGE2
p-value
DHA+EPA 1.04 0.305 -0.00 1.000 0.97 0.338 0.03 0.975 -0.47 0.643 -0.51 0.609
30-Months
Metabolome
Il1B1 t-
Statistic
Il1B1 p-
value
TnF-a t-
Statistic
TnF-a
p-value
GMCSF
t-Statistic
GMCSF
p-value
IL6 t-
Statistic
IL6 p-
value
IL10 t-
Statistic
IL10 p-
value
PGE2 t-
Statistic
PGE2
p-value
DHA+EPA 1.98 0.054 2.60 0.013 0.74 0.465 1.43 0.159 0.12 0.906 -0.85 0.400
36-Months
Metabolome
Il1B1 t-
Statistic
Il1B1 p-
value
TnF-a t-
Statistic
TnF-a
p-value
GMCSF
t-Statistic
GMCSF
p-value
IL6 t-
Statistic
IL6 p-
value
IL10 t-
Statistic
IL10 p-
value
PGE2 t-
Statistic
PGE2
p-value
DHA+EPA 2.53 0.016 2.01 0.052 1.59 0.121 2.33 0.026 -0.11 0.914 0.02 0.983
*p-value for test of slope not equal to zero.
(LN transformations used on metabolome and Cytokines)
significant suppression of SIL-1b, TNF-a and IL6 at 30-36m

35. 14HDHA+17HDHA+4HDHA+7HDHA v Cytokines and PGE2
Regression
6-Months
Metabolome
Il1B1 t-
Statistic
Il1B1 p-
value
TnF-a t-
Statistic
TnF-a
p-value
GMCSF
t-Statistic
GMCSF
p-value
IL6 t-
Statistic
IL6 p-
value
IL10 t-
Statistic
IL10 p-
value
PGE2 t-
Statistic
PGE2
p-value
SUM
HDHA
0.76 0.452 0.80 0.428 0.63 0.532 0.22 0.827 -0.12 0.904 2.02 0.048
12-Months
Metabolome
Il1B1 t-
Statistic
Il1B1 p-
value
TnF-a t-
Statistic
TnF-a
p-value
GMCSF
t-Statistic
GMCSF
p-value
IL6 t-
Statistic
IL6 p-
value
IL10 t-
Statistic
IL10 p-
value
PGE2 t-
Statistic
PGE2
p-value
SUM
HDHA
-0.38 0.707 -1.28 0.207 0.31 0.757 -0.84 0.405 0.27 0.791 -0.14 0.888
18-Months
Metabolome
Il1B1 t-
Statistic
Il1B1 p-
value
TnF-a t-
Statistic
TnF-a
p-value
GMCSF
t-Statistic
GMCSF
p-value
IL6 t-
Statistic
IL6 p-
value
IL10 t-
Statistic
IL10 p-
value
PGE2 t-
Statistic
PGE2
p-value
SUM
HDHA
-0.55 0.583 -1.25 0.218 0.72 0.476 0.42 0.673 1.25 0.216 1.11 0.272
24-Months
Metabolome
Il1B1 t-
Statistic
Il1B1 p-
value
TnF-a t-
Statistic
TnF-a
p-value
GMCSF
t-Statistic
GMCSF
p-value
IL6 t-
Statistic
IL6 p-
value
IL10 t-
Statistic
IL10 p-
value
PGE2 t-
Statistic
PGE2
p-value
SUM
HDHA
1.53 0.133 -0.21 0.834 1.80 0.078 1.18 0.242 1.57 0.122 1.32 0.193
30-Months
Metabolome
Il1B1 t-
Statistic
Il1B1 p-
value
TnF-a t-
Statistic
TnF-a
p-value
GMCSF
t-Statistic
GMCSF
p-value
IL6 t-
Statistic
IL6 p-
value
IL10 t-
Statistic
IL10 p-
value
PGE2 t-
Statistic
PGE2
p-value
SUM
HDHA
2.04 0.048 1.46 0.151 1.51 0.137 1.79 0.080 0.43 0.671 0.21 0.834
36-Months
Metabolome
Il1B1 t-
Statistic
Il1B1 p-
value
TnF-a t-
Statistic
TnF-a
p-value
GMCSF
t-Statistic
GMCSF
p-value
IL6 t-
Statistic
IL6 p-
value
IL10 t-
Statistic
IL10 p-
value
PGE2 t-
Statistic
PGE2
p-value
SUM
HDHA
2.95 0.006 1.37 0.178 0.80 0.428 2.88 0.007 1.54 0.133 0.91 0.370
*p-value for test of slope not equal to zero.
(LN transformations used on metabolome and Cytokines)
significant suppression of IL-1b, TNF-a and IL6 at 30-36m

36. Conclusions from NIP study
• Significant levels of DHA are achieved in PBMC
membranes which is released upon LPS activation
• SPM are produced in significant quantities
• There are age related changes in N-3 metabolism
• Pro-resolving/anti-inflammatory activity of SPM is not
present until 30-36 months of age, suggesting
– Modification of receptor expression or signaling
– ? Metabolism defect of 17HDHA to 7HDHA (reduced RvD1,2,5)
– Modification of inflammatory response that allows SPM anti-
inflammatory action
– Correlates with effect of DHA/EPA on reduced risk of islet
autoimmunity in children beginning at 4.25 years
– IL-1b/IL-6 vs. TNF-a are modulated by different SPMs

37. Questions
• Doses of DHA (omega-3 fatty acid); more??
• When to dose (what age)
• Does vitamin D promote fatty acid metabolism or resolving
metabolism
• Do we need to control diet, e.g., n-6 fatty acid intake
• Is the metabolism of N-3 fatty acids in infants at high-risk
different that healthy controls (genetic regulation, absorption,
immune response)

38. Acknowledgements
UF
MCS lab
Michelle Rodriguez
Sally Litherland
Ron Ferguson
U of Colorado
Peter Chase (Co-PI)
Sonia Cooper
Jill Norris
Mike Holers
Participating NIP Centers
UCSF
University of Utah
Indiana University
Children’s Hospital of Orange County
University of Minnesota
Joslin Diabetes Center, Harvard
University of Missouri, Kansas City
Children’s Hospital of Los Angeles
Scientific
collaborations
Harvard
Charlie Serhan
Berlex
John Parkinson
UC Berkley
Karsten Gronert**

44. Infant screening;
•Screening modality;DR3/4, DQ2/8; (hi-risk HLA)
•1rst degree relatives
•Risk; 20% develop autoantibodies
insulin, GAD, ICA512 within first few years of life
First-degree relative screening;
• 2 or more biochemical autoantibodies
•loss of 1rst phase insulin secretion (IVGTT)
•HLA DQb0602 negative (protective MHC allele)
•70% develop T1D within 5 years
Identifying subjects at high-risk for T1D

45. Omega-3 Fatty Acids and modify risk for autoimmunity
in at risk infants
• Stene; administration of cod liver oil in the first year
of life reduces risk for T1D in infants (Am. J. Clin.
Nutr., 2003)
• Norris; Dietary intake of omega-3 fatty acids is
associated with reduced risk of IA in children at
increased genetic risk for type 1 diabetes (JAMA,
2007)
• Norris;Dietary intake of omega-3 fatty acids is
associated with reduced risk of IA in children at
increased genetic risk for type 1 diabetes (Ped.
Diabetes 2011)

46. Modified TLR signaling pathways in infancy

47. Inflammatory response varies with age
Yerkovitch et. al.
Ped. Res. 2007

48. DHA levels
*
* * *
*

49. Pro-resolving 15-Lox metabolite 17HDHA

50. Pro-resolving 5-Lox metabolite 4HDHA
*
**

51. Pro-inflammatory mediators
12 HETE

52. Pro-inflammatory PGE-2