Gene Regulation in The Human Immune System. Gene Expression Signatures of Th2 Cell Differentiation, Type 1 Diabetes, and Intrauterine Immune Adaptation

The human immune system is constantly interacting with the surrounding stimuli and microorganisms. However, when directed against self or harmless antigens, these vital defense mechanisms can cause great damage. In addition, the understanding the underlying mechanism of several human diseases caused by aberrant immune cell functions, for instance type 1 diabetes and allergies, remains far from being complete. In this Ph.D. study these questions were addressed using genome-wide transcriptomic analyses. Asthma and allergies are characterized by a hyperactive response of the T helper 2 (Th2) immune cells. In this study, the target genes of the STAT6 transcription factor in naïve human T cells were identified with RNAi for the first time. STAT6 was shown to act as a central activator of the genes expression upon IL-4 signaling, with both direct and indirect effects on Th2 cell transcriptome. The core transcription factor network induced by IL-4 was identified from a kinetic analysis of the transcriptome. Type 1 diabetes is an autoimmune disease influenced by both the genetic susceptibility of an individual and the disease-triggering environmental factors. To improve understanding of the autoimmune processes driving pathogenesis in the prediabetic phase in humans, a unique series of prospective whole-blood RNA samples collected from HLA-susceptible children in the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study was studied. Changes in different timewindows of the pathogenesis process were identified, and especially the type 1 interferon response was activated early and throughout the preclinical T1D. The hygiene hypothesis states that allergic diseases, and lately also autoimmune diseases, could be prevented by infections and other microbial contacts acquired in early childhood, or even prenatally. To study the effects of the standard of hygiene on the development of neonatal immune system, cord blood samples from children born in Finland (high standard of living), Estonia (rapid economic growth) and Russian Karelia (low standard of living) were compared. Children born in Russian Karelia deviated from Finnish and Estonian children in many aspects of the neonatal immune system, which was developmentally more mature in Karelia, resembling that of older infants. The results of this thesis offer significant new information on the regulatory networks associated with immune-mediated diseases in human. The results will facilitate understanding and further research on the role of the identified target genes and mechanisms driving the allergic inflammation and type 1 diabetes, hopefully leading to a new era of drug development.

Early nutritional determinants in cardio-metabolic programming – A prospective randomized controlled dietary intervention study

Western societies have been faced with the fact that overweight, impaired glucose regulation and elevated blood pressure are already prevalent in pediatric populations. This will inevitably mean an increase in later manifestations of cardio-metabolic diseases. The dilemma has been suggested to stem from fetal life and it is surmised that the early nutritional environment plays an important role in the process called programming. The aim of the present study was to characterize early nutritional determinants associating with cardio-metabolic risk factors in fetuses, infants and children. Further, the study was designated to establish whether dietary counseling initiated in early pregnancy can modify this cascade. Healthy mother-child pairs (n=256) participating in a dietary intervention study were followed from early pregnancy to childhood. The intervention included detailed dietary counseling by a nutritionist targeting saturated fat intake in excess of recommendations and fiber consumption below recommendations. Cardio-metabolic programming was studied by characterizing the offspring’s cardio-metabolic risk factors such as over-activation of the autonomic nervous system, elevated blood pressure and adverse metabolic status (e.g. serum high split proinsulin concentration). Fetal cardiac sympathovagal activation was measured during labor. Postnatally, children’s blood pressure was measured at six-month and four-year follow-up visits. Further, infants’ metabolic status was assessed by means of growth and serum biomarkers (32-33 split proinsulin, leptin and adiponectin) at the age of six months. This study proved that fetal cardiac sympathovagal activity was positively associated with maternal pre-pregnancy body mass index indicating adverse cardio-metabolic programming in the offspring. Further, a reduced risk of high split proinsulin in infancy and lower blood pressure in childhood were found in those offspring whose mothers’ weight gain and amount and type of fats in the diet during pregnancy were as recommended. Of note, maternal dietary counseling from early pregnancy onwards could ameliorate the offspring’s metabolic status by reducing the risk of high split proinsulin concentration, although it had no effect on the other cardio-metabolic markers in the offspring. At postnatal period breastfeeding proved to entail benefits in cardio-metabolic programming. Finally, the recommended dietary protein and total fat content in the child’s diet were important nutritional determinants reducing blood pressure at the age of four years. The intrauterine and immediate postnatal period comprise a window of opportunity for interventions aiming to reduce the risk of cardio-metabolic disorders and brings the prospect of achieving health benefits over one generation.

The effects of maternal hyperglycemia on human umbilical vascular gene expression and neonatal rat lung development

Background: Maternal diabetes affects many fetal organ systems, including the vasculature and the lungs. The offspring of diabetic mothers have respiratory adaptation problems after birth. The mechanisms are multifactorial and the effects are prolonged during the postnatal period. An increasing incidence of diabetic pregnancies accentuates the importance of identifying the pathological mechanisms, which cause the metabolic and genetic changes that occur in offspring, born to diabetic mothers. Aims and methods: The aim of this thesis was to determine changes both in human umbilical cord exposed to maternal type 1 diabetes and in neonatal rat lungs after streptozotocin-induced maternal hyperglycemia, during pregnancy. Rat lungs were used as a model for the potential disease mechanisms. Gene expression alterations were determined in human umbilical cords at birth and in rat pup lungs at two week of age. During the first two postnatal weeks, rat lung development was studied morphologically and histologically. Further, the effect of postnatal hyperoxia on hyperglycemia-primed rat lungs was investigated at one week of age to mimic the clinical situation of supplemental oxygen treatment. Results: In the umbilical cord, maternal diabetes had a major negative effect on the expression of genes involved in blood vessel development. The genes regulating vascular tone were also affected. In neonatal rat lungs, intrauterine hyperglycemia had a prolonged effect on gene expression during late alveolarization. The most affected pathway was the upregulation of extracellular matrix proteins. Newborn rat lungs exposed to intrauterine hyperglycemia had thinner saccular walls without changes in airspace size, a smaller relative lung weight and lung total tissue area, and increased cellular apoptosis and proliferation compared to control lungs, possibly reflecting an aberrant maturational adaptation. At one and two weeks of age, cell proliferation and secondary crest formation were accelerated in hyperglycemia-exposed lungs. Postnatal hyperoxic exposure, alone caused arrested alveolarization with thin-walled and enlarged alveoli. In contrast, the dual exposure of intrauterine hyperglycemia and postnatal hyperoxia resulted in the phenotype of thick septa together with arrested alveolarization and decreased number of small pulmonary arteries. Conclusions: Maternal diabetic environment seems to alter the umbilical cord gene expression profile of the regulation of vascular development and function. Fetal hyperglycemia may additionally affect the genetic regulation of the postnatal lung development and may actually induce prolonged structural alterations in neonatal lungs together with a modifying effect on the deleterious pulmonary exposure of postnatal hyperoxia. This, combined with the novel human umbilical cord gene data could serve as stepping stones for future therapies to curb developmental aberrations.

The Association between Growth and Neurodevelopment in Very Preterm Infants – A Follow-up Study in the PIPARI Study

Placental insufficiency is one major cause of intrauterine growth restriction and also relates to neurodevelopment. Preterm infants with very low birth weight are at risk of postnatal growth restriction as well as neurodevelopmental impairments. However, the optimal postnatal growth for long-term neurodevelopment is still unclear. The objective of this study was thus to investigate the association between growth and neurodevelopment in very preterm infants. The study populations consisted of 83 (I), 55 (II), 36 (III) and 181 (IV) infants with very low birth weight (below 1501 grams), and very or extremely low gestational age (below 32 and 26 weeks). Foetal blood circulation in relation to two-year neurodevelopment and the association between early growth and brain maturation at term age were studied. Postnatal growth, and its association with five-year cognitive outcome, was analysed. Changes in foetal blood circulation related to placental insufficiency associated with an adverse two-year cognitive outcome. Early postnatal growth in extremely preterm infants was comparable to a similar Swedish cohort. Preterm infants with slow intrauterine growth had less mature brains at term age; rapid catch-up growth until term age did not eliminate this difference. Weight gain and head circumference growth from birth until two years of age associated positively with five-year cognitive outcome in appropriate for gestational age infants. In small for gestational age infants, head circumference growth from term age to four months (corrected age) associated positively with their five-year cognitive outcome. The association between postnatal growth and neurodevelopment was different for prenatally normally grown versus slow grown preterm infants.

Biomarkers, medical treatment and fetal intrapartum surveillance in intrahepatic cholestasis of pregnancy

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific disorder characterized by maternal pruritus and elevated liver enzymes. It usually begins in the third trimester of pregnancy and resolves spontaneously after delivery. ICP is considered benign for the pregnant woman, but it is associated with an increased risk for unexplained term stillbirth and preterm delivery. There are no specific laboratory markers to diagnose ICP. The diagnosis is currently based on the presence of maternal pruritus and elevated values of alanine aminotransaminases (ALT) and serum bile acids (BA). Recently, ursodeoxycholic acid (UDCA) has been used for treatment. Mechanisms leading to intrauterine fetal death (IUFD) may be multifactorial and are unknown at present. For this thesis, 415 pregnant women with ICP were studied. The aim was to evaluate the value of the liver enzyme glutathione S-transferase alpha (GSTA) as a specific marker of ICP and to assess the effect of maternal UDCA therapy on maternal laboratory values and fetal outcome. The specific markers predisposing the fetus to heart arrhythmia were studied by comparing waveform analysis of fetal electrocardiograms (FECG) during labor in pregnancies complicated by ICP with controls. The levels of maternal GSTA were high and the values correlated with the value of ALT in patients with ICP. UDCA therapy reduced the values of the liver enzymes and alleviated maternal pruritus, but it did not influence maternal hormonal values. Although the newborns experienced an uneventful perinatal outcome, severe ICP was still associated with preterm birth and admission to the neonatal intensive care unit (NICU). There were no significant differences in intrapartum FECG findings between fetuses born to ICP women and controls.

Diet and Probiotics during Pregnancy - Endorsing developmental Programming

Maternal obesity has been shown to increase the risk for adverse reproductive health outcomes such as gestational diabetes, hypertension, and preeclampsia. Moreover, several studies have indicated that overnutrition and maternal obesity adversely program the development of offspring by predisposing them to obesity and other chronic diseases later in life. The exact molecular mechanisms leading to developmental programming are not known, but it has recently been suggested that obesity-related low-grade inflammation, gut microbiota and epigenetic gene regulation (in particularly DNA methylation) participate in the developmental programming phenomenon. The aim of this thesis was to evaluate the effect of diet, dietary counseling and probiotic intervention during pregnancy in endorsing favorable developmental programming. The study population consisted of 256 mother-child pairs participating in a prospective, double-blinded dietary counselling and probiotic intervention (Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb12) NAMI (Nutrition, Allergy, Mucosal immunology and Intestinal microbiota) study. Further overweight women were recruited from maternal welfare clinics in the area of Southwest Finland and from the prenatal outpatient clinic at Turku University Hospital. Dietary counseling was aimed to modify women’s dietary intake to comply with the recommended intake for pregnant women. Specifically, counseling aimed to affect the type of fat consumed and to increase the amount of fiber in the women’s diets. Leptin concentration was used as a marker for obesity-related low-grade inflammation, antioxidant vitamin status as an efficiency marker for dietary counselling and epigenetic DNA methylation of obesity related genes as a marker for probiotics influence. Results revealed that dietary intake may modify obesity-associated low-grade inflammation as measured by serum leptin concentration. Specifically, dietary fiber intake may lower leptin concentration in women, whereas the intakes of saturated fatty acids and sucrose have an opposite effect. Neither dietary counselling nor probiotic intervention modified leptin concentration in women, but probiotics tended to increase children’s leptin concentration. Dietary counseling was an efficient tool for improving antioxidant vitamin intake in women, which was reflected in the breast milk vitamin concentration. Probiotic intervention affected DNA methylation of dozens of obesity and weight gain related genes both in women and their children. Altogether these results indicate that dietary components, dietary counseling and probiotic supplementation during pregnancy may modify the intrauterine environment towards favorable developmental programming.