Early Supplementation of Human Milk Oligosaccharides Suppresses Spontaneous Autoimmune Diabetes in Non-obese Diabetic Mice Later in Life Ling Xiao Utrecht University, The Netherlands L.XIAO@UU.NL IPC 2016, Budapest
Script of The Presentation Human Milk Oligosaccharides (HMOS) Structures Pharmaceutical properties HMOS decrease type 1 diabetes incidence in NOD mice Type 1 diabetes HMOS modulate immune system HMOS alter gut microbiota Hypothesis: HMOS-induced interplay between gut microbiota and intestinal immune system influences the development of T1D 2
Protection by Breast Milk From a protected environment to... A challenging extra-uterine world Breast milk: The best nutritional option for growth and healthy development of the newborn 3
Breast Milk: What Makes It Unique? Each drop of breast milk contains thousands of different molecules that act together like musicians in an orchestra, such as : 4
Human Milk Oligosaccharides (HMOS) A family of structurally diverse unconjugated glycans The third most abundant in human milk Unique to human milk: types, amount, functionalities Estimated 1000 structures Basic structure contains the disaccharide lactose at their reducing end HMOS Day matter of human milk Basic structure of human milk oligosaccharides (adapted from Boehm & Stahl 2003) 5
HMOS: Prebiotics and Beyond Human milk reduces the risk of developing respiratory infections HMOS may reduce the risk of developing respiratory infections Human milk reduces the risk of developing Otitis media HMOS inhibit common pathogens linked to the development of otitis media Human milk reduces the risk of developing urinary tract infections HMOS inhibit the binding of uropathogenic Escherichia coli to bladder epothelium HMOS can inhibit the binding of Helicobacter pylori to the mucosal receptors in the stomach HMOS (2 FL and 6 SL) can attenuate food allergy symptoms The microbiome composition of infants receiving human milk contains high amount of beneficial bifidobacteria, which is supported by HMOS Human milk protects infants against type 1 diabetes HMOS and type 1 diabetes? K.Borch-Johnsen,et,al. The Lacncet. 1984 Ling Xiao, et, al. Book chapter ACCEPTED. 6
Insulin-dependent Diabetes Islet β cell CTL Kill 7
Factors Contribute to Type 1 Diabetes Genetics Immune T1D Adaptive Innate System Environmental Factors Gut Microbiota 8
Our Job: Find A Firefighter Pre-diabetic stage Severe-diabetic stage 9
Hypothesis Early exposure to HMOS can influence development of T1D in later life 10
Experimental Setup NOD Mouse: model of spontaneous T1D, sensitive to dietary influences Total Fraction of HMOS: isolated from a pooled mature human milk sample and reduced in lactose (84% HMOS, 16% lactose; method: Geisser et al, J Chromatogr A, 2005) Comparison: AIN-93M control diet versus AIN-93M + 1% (w/w) HMOS diet
B lo o d g lu c o s e (m M ) D ia b e te s -fr e e s u r v iv a l (% ) Reduces Diabetes Incidence in Later Life Diabetes development Endpoint measurements Urine glucose score results 1 0 0 8 0 6 0 p = 0.0 2 6 H M O S (n = 2 0 ) C o n tr o l (n = 1 9 ) 4 0 2 0 D ia te r y in t e r v e n t io n Blood glucose score results 0 0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 2 6 2 8 3 0 W e e k s o f fo llo w -u p 4 0 3 0 C o n tro l H M O S 2 0 1 0 Food intake & body weight were similar between experimental groups 0 D ia b e t ic N o n - d ia b e t ic 12
Scoring Method to Quantify Pancreas Inflammatory Islet Infiltration Each islet of each section was scored by this system: 0 = No Insulitis 1 = Peri Insulitis No insulitis Insulitis >50% Peri-insulitis Complete insulitis 2 = Insulitis affecting less than 50% of the islet area 3 = Insulitis affecting more than 50% of the islet area 4 = Complete Insulitis Average of 46 islets per animal were analyzed 13
Decreased Insulitis in HMOS Group Most prevalent insulitis score per animal Mean normalized insulitis score (range 0-4) 5 In s u litis s c o re in % 1 0 0 C o m p le te in s u litis ** In s u litis > 5 0 % p < 0.0 0 1 5 0 In s u litis < 5 0 % ** P e ri-in s u litis ** N o in s u litis *** M e a n n o rm a liz e d in s u litis s o c o re 4 3 2 1 0 C o n t r o l H M O S 0 C o n t r o l H M O S Insulitis scores showed a correlation with urine glucose values 14
What Are The Possible Mechanims? Genetics Immune T1D System? Adaptive Innate HMOS Gut Microbiota 15
T cells Balance & T1D Homeostasis Autoimmunity Pathogenic T cells (Mediate disease) Regulatory T cells (Control immunity) Pathogenic T cells (Mediate disease) Regulatory T cells (Control immunity) An increase in regulatory T cells (Tregs and Th2) and a decrease in pathogenic T cells (Th1 and Th17) could have been expected in an effective treatment 16
% C D 6 9 + T -b e t o f C D 4 + c e lls % C D 6 9 + T -b e t o f C D 4 + c e lls % C D 2 5 + F o x p 3 o f C D 4 + c e lls Reduced Activated Th1 Cells and Tregs of Spleen CD4 T-cells Reduced T-regulatory cells 2 5 p = 0.0 2 2 0 1 5 1 0 Alterations of splenic Tregs are NOT the causual factor of the protective HMOS effect. 5 0 C o n t r o l H M O S Decreased activated Th1 cells in non-diabetic mice of HMOS group 1.5 p = 0.0 5 1.5 p = 0.0 2 1.0 1.0 0.5 0.5 0.0 0.0 C o n t r o l H M O S C o n t r o l- N o n d ia b e t ic H M O S - N o n d ia b e t ic Suppressive effect is associated with lower induction of Th1 cells. No significant differences were observed in % of Th2 or Th17 cells between dietary groups 17
% C D 6 9 o f C D 4 c e lls % C D 2 5 o f C D 4 c e lls Reduced Overall Immune Activation Reduced overall activated T-helper cells % C D 6 9 o f C D 4 c e lls 2 0 1 5 1 0 5 p = 0.0 3 % C D 2 5 o f C D 4 c e lls 2 0 1 5 1 0 5 p = 0.0 1 0 c o n t r o l H M O S 0 C o n t r o l H M O S Related to decreased overall immune activation marker expression? 1 5 p < 0.0 0 1 2 0 p = 0.3 2 1 0 1 5 1 0 5 5 0 D ia b e t ic N o n - d ia b e t ic 0 D ia b e t ic N o n - d ia b e t ic Protective effect is associated with lower overall immune activation! 18
S e r u m IL -1 7 (p g /m L ) Decreased Levels of IL-17 in Serum Reduced serum IL-17 levels Lower IL-17 levels in HMOS- Nondiabetic mice 4 0 p = 0.0 2 5 0 3 0 2 0 1 0 S e ru m IL -1 7 (p g /m L ) 4 0 3 0 2 0 1 0 p = 0.0 2 C o n tr o l H M O S 0 C o n t r o l H M O S 0 D ia b e t ic N o n - d ia b e t ic Suppressive effect is associated with down-regulation of IL-17! No significant differences were seen on IL-4,IL-6,IL-10, and IFNγ production. 19
What Are the Possible Mechanims? Genetics Immune T1D System Adaptive Innate? HMOS Gut Microbiota 20
Time Points of Fecal And Cecal Sample Collection 9 14 Feces Feces Feces Feces Cecum 21
B u ty ric a c id (m m o l/l) Increased SCFAS in Fecal Samples (Week 9) F e c a l S C F A s 2 5 p < 0.0 0 0 1 T o ta l S C F A s (m m o l/l) 2 0 1 5 1 0 5 0 C o n t r o l H M O S A c e tic a c id P r o p io n ic a c id B u ty r ic a c id A c e tic a c id (m m o l/l) 2 5 2 0 1 5 1 0 5 0 C o n t r o l p < 0.0 0 0 1 H M O S P r o p io n ic a c id (m m o l/l) 2.0 1.5 1.0 0.5 0.0 C o n t r o l p = 0.0 0 8 H M O S 0.6 0.4 0.2 0.0 C o n t r o l p < 0.0 5 H M O S 22
SCFAs in Cecal Samples 5 5 C e c a l s h o rt c h a in fa tty a c id s (m m o l/l) 5 0 4 5 4 0 3 5 3 0 2 5 2 0 1 5 1 0 5 V a le r ic a c id B u ty r ic a c id P ro p io n ic a c id A c e tic a c id 0 C o n tr o l H M O S 23
Real-time PCR Quantification Of Microbiota Phyla In Healthy And Diebetic Children Bacteriodetes is defined as potential contributors of T1D because such species are observed to be more common in the diabetic than in healthy individuals. Conversely, a successive decline in Firmucutes abundance in the gut microbiota over time in the diabetic individuals has been observed. Mora Murri, 2013, BMC Medicine 23
Changes Of Phylum Distribution Of Fecal Samples Control diet HMOS diet Wk 4 p=0.05 p=0.01 Wk14 p=0.02 p=0.04 p=0.06 p=0.01 p=0.03 End point Gut microbiota was dominated by four main phyla; Firmicutes and Bacteriodetes are the most abundant. In HMOS treated group, Bacteroidetes was significanty decreased overtime while Firmicutes was increased overtime. At wk14, Bacteriodetes in HMOS groups was significantly lower than that of control group. The difference was even greater at the end point. Firmicutes appear to be higher in HMOS group than that of control group.
Ratio of Firmicutes/Bacteroidetes in T1D The Ratio of Firmicutes/Bacteroidetes is critical in the setting of T1D and has been shown to decline over time in the individuals who developed T1D, while the inverse pattern was observed in the healthy individuals. Negative correlation between this ratio and the glucose levels in children with T1D. Mora Murri, 2013, BMC Medicine 25
F F irm ic u u te s s /B a a c c te ro id e te s ra tio HMOS Changed The Ratio of Firmicutes/Bacteroidetes Over Time 2.5 2.0 C o n tro l H M O S 1.5 1.0 0.5 0.0 W k 4 W k 1 4 E n d p o in t Ratio of control diet remianed unchanged; Ratio of HMOS diet changes over time. HMOS group has higher Firmicutes/Bacteroidetes ratio than control group at each time point. HMOS Supplementation beneficially modulates the gut microbiota composition! 26
Work in Progress Bacterial diversity Correlation between fancy species and insulitis severity and incidence of T1D Insights of how HMOS exert protective effects Define autoimmune microbiome for T1D 27
Conclusions Early HMOS diet suppresses autoimmune diabetes development in NOD mice later in life. Associated with milder insulitis, lower induction of Th1 cells, overall immune activation markers expression and downregulation of serum IL-17. Likely related to regulation of intestinal microbiota composition. HMOS in early life modulate T1D in later life: an example of immunological programming. 29
Script of The Presentation Human Milk Oligosaccharides (HMOS) Structures Pharmaceutical properties HMOS decrease T1D incidence in NOD mice Type 1 diabetes HMOS modulate immune system HMOS alter gut microbiota Hypothesis: HMOS-induced interplay between gut microbiota and intestinal immune system influences the development of T1D 30
A Trio of Factors That Create A Perfect Storm of Events Leading to Autoimmunity in T1D Type 1 Diabetes Facet 1- Altered Microbiota Facet 2- Leaky Gut Facet 3- Altered Mucosal Immunity This model provides important insights toward strategies for prevention of T1D. Outi Vaarala, 2008, Diabetes 31
Hypothetical Interplay Between Gut Microbiota and Intestinal Immune System Induced by HMOS HMOS influence the mucosal innate immune system by altering intestinal epthelial cells and DCs differentiation, which contributes to expansion and differentiation of Tregs that regulate and suppress other immune cells. HMOS modulate the adaptive immune system by promotion of regulatory T cells via GPR signaling in epithelial cells and DCs. HMOS maintain a healthy microbiota in the gut by inhibiting pathogen adhension and flourishing benefitial bacteria. HMOS maintain gut intergrity thus decreasing antigen exposure to the mucosal immune system. Ling Xiao, et, al. Review in preparation. 32
Firefighters---HMOS Pre-diabetic stage Severe-diabetic stage 33
Acknowledgement Gert Folkerts Johan Garssen Angie Nato Ingrid an Ark Thea Leusink Gemma Dingjan Mara Diks Belinda Van t Land Bernd Stahl Jacqueline Bastiaanse Paul Vos Eline Voogd Ali Keshavarzian Phillip Engen