The Effects of Environmental and Dietary Factors on the Development of the Immune System in Children at Genetic Risk of Type 1 Diabetes

Type 1 diabetes (T1D) and allergies are considered autoimmune diseases. The exact mechanisms leading to these diseases are mostly unknown, but genetic and environmental factors are involved. Regulatory T cells (Tregs) have a crucial role in initiating and maintaining tolerance, limiting harmful autoantigen-specific inflammation processes. This doctoral thesis explores the effect of environmental factors on the development of the immune system by studying peripheral immunological responses in healthy infants at genetic risk of T1D in two follow-up studies. The first study investigated the development of peripheral Treg cells in infants from Estonia and Finland, neighboring countries with differing living standards, and the incidence of T1D and allergies. We demonstrated a two-step maturation process in the circulating Treg cell population in the first two years of life. First, a decrease in the proportion of Treg cells followed by an increase in the highly activated Treg cells (TregFOXP3high), with enhanced suppressive activity. The Treg cell maturation process coincided with the microbiota composition development from a Bifidobacteria dominated to one dominated by butyrate-producing species by 36 months. The abundance of Bifidobacteria and the relative abundance of B.longum alone at three months showed an inverse association with atopic sensitization. The switch in the microbiota happened earlier in Estonia and was associated with more highly activated TregFOXP3high cells with FOXP3 and CTLA-4 expression. This earlier maturation of the circulating Treg population is associated with a lower risk of allergic diseases in Estonian children. Additionally, we analyzed the common enteral virus infections in the first year of life. We observed a temporal association of virus infection with the expression of FOXP3 in regulatory T cells. Infants with enterovirus infection during the preceding 30 or 60 days had a significantly decreased FOXP3 expression in TregFOXP3high cells. In addition, we observed upregulation of Th1- and Th17 -related cytokines and a decreased activation of CCL22, associating with activation of proinflammatory pathways and reduced immune regulation. In the second study, we examined whether exposure to bovine insulin is a trigger of autoimmunity by studying the appearance of diabetes autoantibodies. We studied exclusively breastfed infants or infants after exposure to different infant formulas; cow-milk formula (CMF) with bovine insulin, whey-based hydrolyzed formula, or a virtually bovine insulin-free whey-based hydrolyzed FINDIA formula during the first six months of life, whenever breast milk was not available. The follow-up was up to at least three years of age. We showed that weaning to the FINDIA formula reduced the incidence of autoantibody positivity when compared with CMF-formula. This result might reflect a dysfunctional immune system. Bovine insulin peptides from the infant formula may activate autoreactivity against the primary autoantigen, i.e., human insulin, resulting in non-tolerogenic T cells. To conclude, the study shows that environmental factors may influence regulatory T cells and immunotolerance. Also, nutrition may influence the development of the immune system or autoantibodies to T1D in infants genetically at risk. Keywords: type 1 diabetes, allergy, regulatory T cells, autoantibodies, viruses, dietEpidemiologisissa tutkimuksissa on osoitettu yhteys immuunivälitteisten sairauksien, kuten allergioiden ja tyypin 1 diabeteksen (T1D), lisääntyneen esiintymisen ja parantuneen elintason ja hygienian välillä. Ympäristötekijöiden, kuten vähentyneen mikrobikuormituksen sekä muuttuneiden ravitsemustottumusten uskotaan vaikuttavan näiden sairauksien syntyyn. Autoimmuunisairauksien katsotaan johtuvan immunologisen toleranssin murtumisesta. Vaikka geneettisillä tekijöillä on merkitys, ne tuskin yksinään voivat selittää kummankin taudin nopeaa lisääntymistä. Tämän väitöskirjatutkimuksen tarkoituksena oli tutkia ympäristötekijöiden vaikutusta immuunijärjestelmän kehitykseen varhaislapsuudessa, tutkimalla ulostenäytteistä kuinka suoliston mikrobiota eli oma bakteerikanta ja virusinfektiot vaikuttavat sääteleviin veren T-auttajasoluihin (Treg-soluihin) geneettisen T1D-riskin omaavilla pikkulapsilla Suomesta ja Virosta. Maista joiden elintasot ja tyypin 1 diabeteksen ilmaantuvuus tutkimuksen aikaan erosivat toisistaan. Me seurasimme lapsia syntymästä kolmen vuoden ajan. Halusimme tutkia suoliston bakteerikannan kehitystä varhaislapsuudessa ja sen mahdollista vaikutusta T-auttajasoluihin. Tutkimus osoitti, että Treg solut kypsyvät kahden ensimmäisen elinvuoden aikana ja tämä kypsymisprosessi on samanaikainen suoliston bakteerikannan kehityksen kanssa. Bakteerikanta kehittyy Bifidobakteeri-valtaisesta bakteerikannasta aikuisen kaltaiseksi, jota hallitsevat Butyraattia tuottavat lajit. Kolmen kuukauden näytteiden Bifidobakteerien runsaus näytti vähentävän myöhempää atooppista herkistymistä ja allergian kehittymistä. Tämä bakteerikannan kehitys oli varhaisempi virolaisilla lapsilla ja heillä oli myös alhaisempi allergisten sairauksien riski verrattuna suomalaisiin lapsiin. Halusimme selvittää virustartuntojen vaikutusta varhaislapsuudessa. Havaitsimme, että enterovirustartunnan saaneilla imeväisillä oli vähentynyt Treg solujen aktivaatio, etenkin TregFOXP3high-soluissa ja näiden solujen muuntuminen samanaikaisesti Th1-, ja Th17-välittäjäaineita erittäviksi soluiksi. Tämä vaikutus kesti jopa 60 päivää infektion jälkeen. Tätä vähentymistä ei nähty muiden tutkittujen virusten osalta. Toisessa osatyössä tutkimme, varhaisen ruokavalion vaikutusta diabetekseen kytkettyjen autovasta-aineiden esiintymiseen ja etenemiseen diabetekseksi. Imeväiset satunnaistettiin erilaisiin äidinmaidonkorvikeryhmiin: lehmänmaitokorvike-, herapohjainen korvike-, ja naudan insuliinivapaa FINDIA-korvike joita käytettiin ensimmäisen kuuden kuukauden aikana, mikäli täysimetys ei onnistunut. Lapsia seurattiin vähintään kolmen vuoden ikään asti. Tutkimus osoitti, että FINDIA-korvikkeen käyttö vähensi autovasta-aineiden esiintyvyyttä verrattuna lehmänmaitokorvikkeeseen. Tämä tulos voi heijastaa immuunijärjestelmän häiriötä, jossa äidinmaitokorvikkeesta peräisin olevat naudan insuliinipeptidit saattavat aktivoida autoreaktiivisuuden ihmisen insuliinia vastaan ja johtaa ei-toleranssia omaaviin T-soluihin. Tutkimus osoitti, että ympäristötekijät, kuten suolen mikrobiotan kehitys, sekä sairastetut virusinfektiot voivat vaikuttaa säätelevien T-solujen ja immuunitoleranssin kehittymiseen varhaislapsuudessa. Myös ravitsemus, eli rintamaito tai äidinmaidonkorviketyyppi, voivat vaikuttaa diabeteksen autovasta-aineiden kehittymiseen näillä tyypin 1 diabeteksen geneettisen riskin omaavilla lapsilla. Avainsanat: tyypin 1 diabetes, allergia, säätelevät T-solut, autovasta-aineet, virukset, imeväisruokint

Impact of extensively hydrolyzed infant formula on circulating lipids during early life

Background: Current evidence suggests that the composition of infant formula (IF) affects the gut microbiome, intestinal function, and immune responses during infancy. However, the impact of IF on circulating lipid profiles in infants is still poorly understood. The objectives of this study were to (1) investigate how extensively hydrolyzed IF impacts serum lipidome compared to conventional formula and (2) to associate changes in circulatory lipids with gastrointestinal biomarkers including intestinal permeability. Methods: In a randomized, double-blind controlled nutritional intervention study (n = 73), we applied mass spectrometry-based lipidomics to analyze serum lipids in infants who were fed extensively hydrolyzed formula (HF) or conventional, regular formula (RF). Serum samples were collected at 3, 9, and 12 months of age. Child's growth (weight and length) and intestinal functional markers, including lactulose mannitol (LM) ratio, fecal calprotectin, and fecal beta-defensin, were also measured at given time points. At 3 months of age, stool samples were analyzed by shotgun metagenomics. Results: Concentrations of sphingomyelins were higher in the HF group as compared to the RF group. Triacylglycerols (TGs) containing saturated and monounsaturated fatty acyl chains were found in higher levels in the HF group at 3 months, but downregulated at 9 and 12 months of age. LM ratio was lower in the HF group at 9 months of age. In the RF group, the LM ratio was positively associated with ether-linked lipids. Such an association was, however, not observed in the HF group. Conclusion: Our study suggests that HF intervention changes the circulating lipidome, including those lipids previously found to be associated with progression to islet autoimmunity or overt T1D.Peer reviewe

Impact of Extensively Hydrolyzed Infant Formula on Circulating Lipids During Early Life

Background: Current evidence suggests that the composition of infant formula (IF) affects the gut microbiome, intestinal function, and immune responses during infancy. However, the impact of IF on circulating lipid profiles in infants is still poorly understood. The objectives of this study were to (1) investigate how extensively hydrolyzed IF impacts serum lipidome compared to conventional formula and (2) to associate changes in circulatory lipids with gastrointestinal biomarkers including intestinal permeability. Methods: In a randomized, double-blind controlled nutritional intervention study (n = 73), we applied mass spectrometry-based lipidomics to analyze serum lipids in infants who were fed extensively hydrolyzed formula (HF) or conventional, regular formula (RF). Serum samples were collected at 3, 9, and 12 months of age. Child's growth (weight and length) and intestinal functional markers, including lactulose mannitol (LM) ratio, fecal calprotectin, and fecal beta-defensin, were also measured at given time points. At 3 months of age, stool samples were analyzed by shotgun metagenomics. Results: Concentrations of sphingomyelins were higher in the HF group as compared to the RF group. Triacylglycerols (TGs) containing saturated and monounsaturated fatty acyl chains were found in higher levels in the HF group at 3 months, but downregulated at 9 and 12 months of age. LM ratio was lower in the HF group at 9 months of age. In the RF group, the LM ratio was positively associated with ether-linked lipids. Such an association was, however, not observed in the HF group. Conclusion: Our study suggests that HF intervention changes the circulating lipidome, including those lipids previously found to be associated with progression to islet autoimmunity or overt T1D.</p

Immunomodulatory Effects of Rhinovirus and Enterovirus Infections During the First Year of Life

Early childhood infections have been implicated in the development of immune-mediated diseases, such as allergies, asthma, and type 1 diabetes. We set out to investigate the immunomodulatory effects of early viral infections experienced before the age of one year on the peripheral regulatory T cell population (Treg) and circulating cytokines in a birth-cohort study of Estonian and Finnish infants. We show here a temporal association of virus infection with the expression of FOXP3 in regulatory T cells. Infants with rhinovirus infection during the preceding 30 days had a higher FOXP3 expression in Treg cells and decreased levels of several cytokines related to Th1 and Th2 responses in comparison to the children without infections. In contrast, FOXP3 expression was significantly decreased in highly activated (CD4+CD127-/loCD25+FOXP3high) regulatory T cells (TregFOXP3high) in the infants who had enterovirus infection during the preceding 30 or 60 days. After enterovirus infections, the cytokine profile showed an upregulation of Th1- and Th17-related cytokines and a decreased activation of CCL22, which is a chemokine derived from dendritic cells and associated with Th2 deviation. Our results reveal that immunoregulatory mechanisms are up-regulated after rhinovirus infections, while enterovirus infections are associated with activation of proinflammatory pathways and decreased immune regulation.Peer reviewe

Fungal Dysbiosis and Intestinal Inflammation in Children With Beta-Cell Autoimmunity

Although gut bacterial dysbiosis is recognized as a regulator of beta-cell autoimmunity, no data is available on fungal dysbiosis in the children at the risk of type 1 diabetes (T1D). We hypothesized that the co-occurrence of fungal and bacterial dysbiosis contributes to the intestinal inflammation and autoimmune destruction of insulin-producing beta-cells in T1D. Fecal and blood samples were collected from 26 children tested positive for at least one diabetes-associated autoantibody (IAA, GADA, IA-2A or ICA) and matched autoantibody-negative children with HLA-conferred susceptibility to T1D (matched for HLA-DQB1 haplotype, age, gender and early childhood nutrition). Bacterial 16S and fungal ITS2 sequencing, and analyses of the markers of intestinal inflammation, namely fecal human beta-defensin-2 (HBD2), calprotectin and secretory total IgA, were performed. Anti-Saccharomyces cerevisiae antibodies (ASCA) and circulating cytokines, IFNG, IL-17 and IL-22, were studied. After these analyses, the children were followed for development of clinical T1D (median 8 years and 8 months). Nine autoantibody positive children were diagnosed with T1D, whereas none of the autoantibody negative children developed T1D during the follow-up. Fungal dysbiosis, characterized by high abundance of fecal Saccharomyces and Candida, was found in the progressors, i.e., children with beta-cell autoimmunity who during the follow-up progressed to clinical T1D. These children showed also bacterial dysbiosis, i.e., increased Bacteroidales and Clostridiales ratio, which was, however, found also in the non-progressors, and is thus a common nominator in the children with beta-cell autoimmunity. Furthermore, the progressors showed markers of intestinal inflammation detected as increased levels of fecal HBD2 and ASCA IgG to fungal antigens. We conclude that the fungal and bacterial dysbiosis, and intestinal inflammation are associated with the development of T1D in children with beta-cell autoimmunity

Maturation of Gut Microbiota and Circulating Regulatory T Cells and Development of IgE Sensitization in Early Life

Recent studies suggest that the cross-talk between the gut microbiota and human immune system during the first year of life is an important regulator of the later development of atopic diseases. We explored the changes in the gut microbiota, blood regulatory T cells, and atopic sensitization in a birth-cohort of Estonian and Finnish children followed from 3 to 36 months of age. We describe here an infant Treg phenotype characterized by high Treg frequency, the maturation of Treg population characterized by a decrease in their frequency accompanied with an increase in the highly activated Treg cells. These changes in Treg population associated first with the relative abundance of Bifidobacterium longum followed by increasing colonization with butyrate producing bacteria. High bifidobacterial abundance in the neonatal microbiota appeared to be protective, while colonization with Bacteroides and E. coli was associated with later risk of allergy. Estonian children with lower risk of IgE mediated allergic diseases than Finnish children showed an earlier maturation of the gut microbiota, detected as earlier switch to an increasing abundance of butyrate-producing bacteria, combined with an earlier maturation of Treg cell phenotype and total IgE production. The children with established allergic diseases by age 3 showed a decreased abundance of butyrate producing Faecalibacterium. These results suggest that as well as the maintenance of a bifidobacterial dominated gut microbiota is important during the first weeks of life, the overtake by butyrate producing bacteria seems to be a beneficial shift, which should not be postponed.Peer reviewe

Rhinoviruses in infancy and risk of immunoglobulin E sensitization

Previous data about the role of viruses in the development of allergic immunoglobulin E (IgE) sensitization are contradictory. The aim of this study was to determine the possible associations between exposure to different viruses (rhinovirus, enterovirus, norovirus, and parechovirus) during the first year of life and IgE sensitization. Viruses were analyzed from stool samples collected monthly from infants participating in a prospective birth cohort study. From that study, 244 IgE sensitized case children and 244 nonsensitized control children were identified based on their allergen-specific IgE antibody levels at the age of 6, 18, and 36 months. Stool samples (n = 4576) from the case and control children were screened for the presence of rhinovirus, enterovirus, norovirus, and parechovirus RNA by reverse transcription quantitative polymerase chain reaction. The study showed that rhinovirus was the most prevalent virus detected, present in 921 (20%) samples. None of the viruses were associated with IgE sensitization in the full cohort but after stratifying by sex, the number of rhinovirus positive samples was inversely associated with IgE sensitization in boys (odds ratio [OR]: 0.81; 95% confidence interval [CI]: 0.69-0.94; P = 0.006). There was also a temporal relation between rhinoviruses and IgE sensitization, as rhinovirus exposure during the first 6 months of life was associated with a reduced risk of subsequent IgE sensitization in boys (OR: 0.76; 95% CI: 0.6-0.94; P = 0.016). In conclusion, early exposure to rhinoviruses was inversely associated with IgE sensitization but this protective association was restricted to boys.Peer reviewe

Standard of hygiene and immune adaptation in newborn infants

Peer reviewe

Effect of Early Feeding on Intestinal Permeability and Inflammation Markers in Infants with Genetic Susceptibility to Type 1 Diabetes : A Randomized Clinical Trial

Objectives To assess whether weaning to an extensively hydrolyzed formula (EHF) decreases gut permeability and/or markers of intestinal inflammation in infants with HLA-conferred diabetes susceptibility, when compared with conventional formula. Study design By analyzing 1468 expecting biological parent pairs for HLA-conferred susceptibility for type 1 diabetes, 465 couples (32 %) potentially eligible for the study were identified. After further parental consent, 332 babies to be born were randomized at 35th gestational week. HLA genotyping was performed at birth in 309 infants. Out of 87 eligible children, 73 infants participated in the intervention study: 33 in the EHF group and 40 in the control group. Clinical visits took place at 3, 6, 9, and 12 months of age. The infants were provided either EHF or conventional formula whenever breastfeeding was not available or additional feeding was required over the first 9 months of life. The main outcome was the lactulose to mannitol ratio (L/M ratio) at 9months. The secondary outcomes were L/M ratio at 3, 6, and 12 months of age, and fecal calprotectin and human beta-defensin 2 (HBD-2) levels at each visit. Results Compared with controls, the median L/M ratio was lower in the EHF group at 9 months (.006 vs.028; P = .005). Otherwise, the levels of intestinal permeability, fecal calprotectin, and HBD-2 were comparable between the two groups, although slight differences in the age-related dynamics of these markers were observed. Conclusions It is possible to decrease intestinal permeability in infancy through weaning to an extensively hydrolyzed formula. This may reduce the early exposure to dietary antigens.Peer reviewe