Regulatory Mechanisms involved in Th2 Cell Differentiation. A Proteomics Approach.

Th2-solujen erilaistumista ohjaavat säätelyverkostot ja niiden tutkiminen proteomiikan avulla Astma ja allergiat ovat laajalle levinneitä ja vakavia sairauksia, joista kärsivät miljoonat ihmiset ympäri maailmaa. Koe-eläimillä tehdyt tutkimukset osoittavat, että interleukiini-4 (IL-4) on tärkeä allergisen astman ja allergioiden kehittymiselle ja kroonistumiselle. Se ohjaa T-auttajasolujen (Th-solujen) kehittymistä Th2-tyypin soluiksi, joilla on merkittävä rooli näiden tautien puhkeamisessa. Th2-solut tuottavat myös itse IL-4:ä, joka edesauttaa taudin seuraavien vaiheiden kehittymistä. Erityisesti STAT6-proteiini, joka aktivoituu IL-4-stimulaation seurauksena, on tarpeen Th2- vasteen syntymiselle ja kroonistumiselle antigeenin aiheuttamassa keuhkoputkien astmaattisessa tulehduksessa. Väitöskirjatyöni tarkoituksena oli käyttää kaksidimensionaaliseen elektroforeesiin (2- DE) perustuvaa proteomiikkaa ja massaspektrometriaa uusien Th2-solujen erilaistumista säätelevien proteiinien tunnistamiseksi. Erilaistumattomat Th-solut eristettiin vastasyntyneen napaverestä tai hiiren pernasta. Solut aktivoitiin Tsolureseptorin ja ns. ko-stimulatoristen reseptorien kautta ja erilaistettiin joko Th1- tai Th2-suuntaan vastaavasti erilaistavien IL-12- ja IL-4-sytokiinien avulla. Ensimmäisessä tutkimuksessa in vitro -erilaistettujen Th1- ja Th2-solujen proteomeja verrattiin keskenään proteiinien ilmenemisessä tai proteiinimodifikaatioissa olevien erojen tunnistamiseksi. Kaksi muuta päätutkimusta keskittyivät IL-4:n aiheuttamaan proteiinitason säätelyyn ensimmäisen vuorokauden aikana T-soluaktivaation jälkeen. Näistä ensimmäisessä IL-4:n aiheuttamia eroja tunnistettiin aktivoiduista ihmisen Thsoluista. IL-4:n todettiin säätelevän useita proteiineja kaspaasien välittämissä signalointiteissä sekä lisäävän T-solujen elävyyttä ja aktivoitumista. Toisessa tutkimuksessa STAT6-poistogeenisten hiirien lymfosyyttien proteomia verrattiin villityypin kontrollisoluihin T-soluaktivaation ja IL-4-stimulaation jälkeen. Näissä tutkimuksissa karakterisoitiin useita uusia IL-4:n ja STAT6:n kohdeproteiineja ja löydettiin uusia säätelyverkostoja. Tutkimustulokset ovat johtaneet uusiin Th2-erilaistumismekanismeja koskeviin hypoteeseihin.

Immunopathogenesis of Asthma and Atopic Diseases – The specific Role of a selected Panel of Genes in human T helper Cell Differentiation

T helper cell (Th) functions are crucial for proper immune defence against various intra- and extracellular pathogens. According to the specific immune responses, Th cells can be classified into subtypes, Th1 and Th2 cells being the most frequently characterized classes. Th1 and Th2 cells interact with other immune cells by regulating their functions with specific cytokine production. IFN, IL-2 and TNF- are the cytokines predominantly produced by Th1 cells whereas Th2 cells produce Th2-type cytokines, such as IL-4, IL-5 and IL-13. Upon TCR activation and in the presence of polarizing cytokines, Th cells differentiate into effector subtypes from a common precursor cell. IFN and IL-12 are the predominant Th1 polarizing cytokines whereas IL-4 directs Th2 polarization. The cytokines mediate their effects through specific receptor signalling. The differentiation process is complex, involving various signalling molecules and routes, as well as functions of the specific transcription factors. The functions of the Th1/Th2 cells are tightly regulated; however, knowledge on human Th cell differentiation is, as yet, fairly poor. The susceptibility for many immune-mediated disorders often originates from disturbed Th cell responses. Thus, research is needed for defining the molecular mechanisms involved in the differentiation and balanced functions of the Th cells. Importantly, the new information obtained will be crucial for a better understanding of the pathogenesis of immune-mediated disorders, such as asthma or autoimmune diseases. In the first subproject of this thesis, the role of genetic polymorphisms in the human STAT6, GATA3 and STAT4 genes were investigated for asthma or atopy susceptibility in Finnish asthma families by association analysis. These genes code for key transcription factors regulating Th cell differentiation. The study resulted in the identification of a GATA3 haplotype that associated with asthma and related traits (high serum IgE level). In the second subproject, an optimized method for human primary T cell transfection and enrichment was established. The method can be utilized for functional studies for the selected genes of interest. The method was also utilized in the third subproject, which aimed at the identification of novel genes involved in early human Th cell polarization (0-48h) using genome-wide oligonucleotide arrays. As a result, numerous genes and ESTs with known or unknown functions were identified in the study. Using an shRNA knockdown approach, a panel of novel IL-4/STAT6 regulated genes were identified in the functional studies of the genes. Moreover, one of the genes, NDFIP2, with a previously uncharacterized role in the human Th differentiation, was observed to promote IFN production of the differentiated Th1 cells. Taken together, the results obtained have revealed potential new relevant candidate genes serving as a basis for further studies characterizing the detailed networks involved in the human Th cell differentiation as well as in the genetic susceptibility of Th-mediated immune disorders.

Quantitative proteomics in the characterization of T helper lymphocyte differentiation

The term proteome is used to define the complete set of proteins expressed in cells or tissues of an organism at a certain timepoint. Respectively, proteomics is used to describe the methods, which are used to study such proteomes. These methods include chromatographic and electrophoretic techniques for protein or peptide fractionation, mass spectrometry for their identification, and use of computational methods to assist the complicated data analysis. A primary aim in this Ph.D. thesis was to set-up, optimize, and develop proteomics methods for analysing proteins extracted from T-helper (Th) lymphocytes. First, high-throughput LC-MS/MS and ICAT labeling methods were set-up and optimized for analysing the microsomal fraction proteins extracted from Th lymphocytes. Later, iTRAQ method was optimized to study cytokine regulated protein expression in the nuclei of Th lymphocytes. High-throughput LC-MS/MS analyses, like ICAT and iTRAQ, produce large quantities of data and robust software and data analysis pipelines are needed. Therefore, different software programs used for analysing such data were evaluated. Moreover, a pre-filtering algorithm was developed to classify good-quality and bad-quality spectra prior to the database searches. Th-lymphocytes can differentiate into Th1 or Th2 cells based on surrounding antigens, co-stimulatory molecules, and cytokines. Both subsets have individual cytokine secretion profiles and specific functions. Th1 cells participate in the cellular immunity against intracellular pathogens, while Th2 cells have important role in the humoral immunity against extracellular parasites. An abnormal response of Th1 and Th2 cells and imbalance between the subsets are charasteristic of several diseases. Th1 specific reactions and cytokines have been detected in autoimmune diseases, while Th2 specific response and cytokine profile is common in allergy and asthma. In this Ph. D. thesis mass spectrometry-based proteomics was used to study the effects of Th1 and Th2 promoting cytokines IL-12 and IL-4 on the proteome of Th lymphocytes. Characterization of microsomal fraction proteome extracted from IL-12 treated lymphobasts and IL-4 stimulated cord blood CD4+ cells resulted in finding of cytokine regulated proteins. Galectin-1 and CD7 were down-regulated in IL-12 treated cells, while IL-4 stimulation decreased the expression of STAT1, MXA, GIMAP1, and GIMAP4. Interestingly, the transcription of both GIMAP genes was up-regulated in Th1 polarized cells and down-regulated in Th2 promoting conditions.

New mechanisms regulating human Th1 and Th2 cell differentiation

Selective development of human T helper (Th) cells into functionally distinct Th1 and Th2 subtypes plays an essential role in the host immune response towards pathogens. However, abnormal function or differentiation of these cells can lead to development of various autoimmune diseases as well as asthma and allergy. Therefore, identification of key factors and the molecular mechanisms mediating Th1 and Th2 cell differentiation is important for understanding the molecular mechanisms of these diseases. The goal of this study was to identify novel factors involved in the regulation of Th1 and Th2 differentiation processes. A new method was optimized for enrichment of transiently transfected resting human primary T lymphocytes, that allowed the study of the influence of genes of interest in human Th1/Th2 cell differentiation and other primary Th cell functions. Functional characterization of PRELI, a novel activation-induced protein in human Th cells, identified it as a mitochondrial protein involved in the regulation of Th cell differentiation and apoptosis. By influencing the intracellular redox state, PRELI induces mitochondrial apoptosis pathway and downregulates STAT6 and Th2 differentiation. The data suggested that Calpain, an oxidative stress induced cysteine protease, is involved as a mediator in PRELI-induced downregulation of STAT6. PIM serine/threonine-specific kinases were identified as new regulators of human Th1 cell differentiation. PIM1 and PIM2 kinases were shown to be preferentially expressed in Th1 cells as compared to Th2 cells. RNA interference studies showed that PIM kinases enhance the production of IFN, the hallmark cytokine produced by Th1 cells. They also induce the expression of the key Th1-driving factor T-bet and the IL-12 signaling pathway during early phases of Th1 cell differentiation. Taken together, new regulators of human T helper cell differentiation were identified in this study, which provides new insights into the signaling mechanisms controlling the selective activation of human Th cell subsets.

System-wide approaches to uncover Th2 cell lineage commitment

T helper (Th) cells are vital regulators of the adaptive immune system. When activated by presentation of cognate antigen, Th cells demonstrate capacity to differentiate into functionally distinct effector cell subsets. The Th2 subset is required for protection against extracellular parasites, such as helminths, but is also closely linked to pathogenesis of asthma and allergies. The intracellular molecular signal transduction pathways regulating T helper cell subset differentiation are still incompletely known. Moreover, great majority of studies regarding Th2 differentiation have been conducted with mice models, while studies with human cells have been fewer in comparison. The goal of this thesis was to characterize molecular mechanisms promoting the development of Th2 phenotype, focusing specifically on human umbilical cord blood T cells as an experimental model. These primary cells, activated and differentiated to Th2 cells in vitro, were investigated by complementary system-wide approaches, targeting levels of mRNA, proteins, and lipid molecules. Specifically, the results indicated IL4-regulated recruitment of nuclear protein, and described novel components of the Th2-promoting STAT6 enhanceosome complex. Furthermore, the development of the activated effector cell phenotype was found to correlate with remodeling of the cellular lipidome. These findings will hopefully advance the understanding of human Th2 cell lineage commitment and development of Th2-associated disease states.

System-level characterization of Th2 cell development and immune cell responses to ZnO and TiO2 nanoparticles

Asthma and allergy are common diseases and their prevalence is increasing. One of the hypotheses that explains this trend is exposure to inhalable chemicals such as traffi c-related air pollution. Epidemiological research supports this theory, as a correlation between environmental chemicals and allergic respiratory diseases has been found. In addition to ambient airborne particles, one may be exposed to engineered nanosized materials that are actively produced due to their favorable physico-chemical properties compared to their bulk size counterparts. On the cellular level, improper activity of T helper (Th) cells has been connected to allergic reactions. Th cells can differentiate into functionally different effector subsets, which are identifi ed according to their characteristic cytokine profi les resulting in specifi c ability to communicate with other cells. Th2 cells activate humoral immunity and stimulate eradication of extracellular pathogens. However, persistent predominance of Th2 cells is involved in a development of number of allergic diseases. The cytokine environment at the time of antigen recognition is the major factor determining the polarization of a naïve Th cell. Th2 cell differentiation is initiated by IL4, which signals via transcription factor STAT6. Although the importance of this pathway has been evaluated in the mouse studies, the signaling components involved have been largely unknown. The aim of this thesis was to identify molecules, which are under the control of IL4 and STAT6 in Th cells. This was done by using system-level analysis of STAT6 target genes at genome, mRNA and protein level resulting in identifi cation of various genes previously not connected to Th2 cell phenotype acquisition. In the study, STAT6-mediated primary and secondary target genes were dissection from each other and a detailed transcriptional kinetics of Th2 cell polarization of naïve human CD4+ T cells was collected. Integration of these data revealed the hierarchy of molecular events that mediates the differentiation towards Th2 cell phenotype. In addition, the results highlighted the importance of exploiting proteomics tools to complement the studies on STAT6 target genes identifi ed through transcriptional profi ling. In the last subproject, the effects of the exposure with ZnO and TiO2 nanoparticles was analyzed in Jurkat T cell line and in primary human monocyte-derived macrophages and dendritic cells to evaluate their toxicity and potential to cause infl ammation. Identifi cation of ZnO-derived gene expression showed that the same nanoparticles may elicit markedly distinctive responses in different cell types, thus underscoring the need for unbiased profi ling of target genes and pathways affected. The results gave additional proof that the cellular response to nanosized ZnO is due to leached Zn2+ ions. The approach used in ZnO and TiO2 nanoparticle study demonstrated the value of assessing nanoparticle responses through a toxicogenomics approach. The increased knowledge of Th2 cell signaling will hopefully reveal new therapeutic nodes and eventually improve our possibilities to prevent and tackle allergic infl ammatory diseases.

Herpes simplex virus type 1 (HSV-1) pathogenesis and HSV gene therapy of experimental autoimmune encephalomyelitis

The aim of this thesis was to develop new herpes simplex virus (HSV) vectors for gene therapy of experimental autoimmune encephalomyelitis (EAE), the principal model of multiple sclerosis (MS), and to study the pathogenesis of wild-type HSV-1 and HSV-1 vectors in vivo. By introducing potential immunomodulatory factors into mice with EAE we strived to develop therapies and possibly find molecules improving recovery from EAE. We aimed at altering the immune response by inducing favorable Th2-type cytokines, thus shifting the immune response from a Th1- or a Th17-response. Our HSV vector expressing interleukin (IL)-5 modulated the cytokine responses, decreased inflammation and alleviated EAE. The use of a novel method, bacterial artificial chromosome (BAC), for engineering recombinant HSV facilitated the construction of a new vector expressing leukemia inhibitory factor (LIF). LIF is a neurotropic cytokine with broad functions in the central nervous system (CNS). LIF promotes oligodendrocyte maturation and decreases demyelination and oligodendrocyte loss. The BAC-derived HSV-LIF vector alleviated the clinical symptoms, induced a higher number of oligodendrocytes and modulated T cell responses. By administering HSV via different infection routes, e.g. peripherally via the nose or eye, or intracranially to the brain, the effect of the immune response on HSV spread at different points of the natural infection route was studied. The intranasal infection was an effective delivery route of HSV to the trigeminal ganglion and CNS, whereas corneal infection displayed limited spread. The corneal and intranasal infections induced different peripheral immune responses, which might explain the observed differences in viral spread.

Blackcurrant seed oil for prevention of atopic dermatitis

One hypothesis for the increased incidence of atopic diseases has been that it is associated with changing dietary habits, especially the changed intake of essential fatty acids (EFAs). The metabolism of EFAs produces eiconasoids, prostaglandins and leukotrienes, which are essential to organs and play a major role in regulation of inflammation and immune response. In some studies persons with atopic dermatitis have been found to have reduced levels of EFAs. The first year of infancy as well as the foetal period are crucial for the development of atopic immune response. The composition of blackcurrant seed oil (BCSO) corresponds to the recommended ratio of EFAs n-3 and n-6 in the diet (1/3-1/4) and as a dietary supplement could, even in small doses, modify the unbalance of EFAs in an efficient way. The purpose of this study was to find out whether atopic allergies can be prevented by supplementing the diet of pregnant mothers with blackcurrant seed oil and whether it could affect the immunological balance of a child. We also sought to find out whether a blackcurrant seed oil supplementation can affect the composition of breast milk to suppress the T helper 2 lymphocyte (Th2) responses in infants. 313 pregnant mothers were randomly assigned to receive BCSO (n=151) or olive oil as placebo (n=162). Supplementation was started at the 8th to 16th weeks of pregnancy, 6 capsules per day (dose of 3 g), and continued until the cessation of breastfeeding. It was thereafter followed by direct supplementation to infants of 1 ml (1 g) of oil per day until the age of two years. Atopic dermatitis and its severity (SCORAD index) were evaluated, serum total IgE was measured and skin prick tests were performed at the age of 3, 12 and 24 months. Peripheral blood mononuclear cell (PBMC) samples were taken at the age of 3 and 12 months and breast milk samples were collected during the first 3 months of breastfeeding. Parental atopy was common (81.7%) in the studied infants, making them infants with increased atopy risk. There was a significantly lower prevalence of atopic dermatitis in the BCSO group (33%) than in the olive oil group (47%) at the age of 12 months. Also, SCORAD was lower in the BCSO group than in the olive oil group. Dietary intervention with BCSO had immunomodulatory effects on breast milk, inducing cytokine production from Th2 to Th1 immunodeviation. It decreased the level of IL-4 and elevated the level of IFN-γ. BCSO intervention did not affect cytokines in the children’s PBMC. However, children of smoking parents in the combined BCSO and olive oil group had significantly elevated levels of Th2 type cytokines IL-4, IL-5 and the proinflammator cytokine TNF. Dietary supplementation with BCSO is safe. It is well tolerated and transiently reduces the prevalence of atopic dermatitis at the age of 12 months. It can possibly become a potential tool in prevention of atopic symptoms when used at the early stages of life.

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.