Altered signaling through IL-12 receptor in children with very high serum IgE levels

An alteration of Th1/Th2 homeostasis may lead to diseases in humans. In this study, we investigated whether an impaired IL-12R signaling occurred in children with elevated serum IgE levels divided on the basis of the IgE levels (group A: >2000 kU/l; group B: <2000 kU/l). We evaluated the integrity of the IL-12R signaling through the analysis of phosphorylation/activation of STAT4, and mRNA expression and membrane assembly of the receptor chains. At a functional level, a proliferative defect of lymphocytes from group A patients was observed. In these patients, an abnormal IL-12R signaling was documented, and this finding was associated with abnormal expression of the IL-12Rb2 chain. Our data indicate that in patients with very high IgE levels the generation of Th1 response is impaired, and that this abnormality associates with abnormal IL-12R signaling

From Murine to Human Nude/SCID: The Thymus, T-Cell Development and the Missing Link

Primary immunodeficiencies (PIDs) are disorders of the immune system, which lead to increased susceptibility to infections. T-cell defects, which may affect T-cell development/function, are approximately 11% of reported PIDs. The pathogenic mechanisms are related to molecular alterations not only of genes selectively expressed in hematopoietic cells but also of the stromal component of the thymus that represents the primary lymphoid organ for T-cell differentiation. With this regard, the prototype of athymic disorders due to abnormal stroma is the Nude/SCID syndrome, first described in mice in 1966. In man, the DiGeorge Syndrome (DGS) has long been considered the human prototype of a severe T-cell differentiation defect. More recently, the human equivalent of the murine Nude/SCID has been described, contributing to unravel important issues of the T-cell ontogeny in humans. Both mice and human diseases are due to alterations of the FOXN1, a developmentally regulated transcription factor selectively expressed in skin and thymic epithelia

Abnormal cell-clearance and accumulation of autophagic vesicles in lymphocytes from patients affected with Ataxia-Teleangiectasia

Ataxia-Teleangiectasia (A-T) is a neurodegenerative disorder due to mutations in ATM gene. ATM in the nucleus ensures DNA repair, while its role in the cytosol is still poorly clarified. Abnormal autophagy has been documented in other neurodegenerative disorders, thus we evaluated whether alteration in this process may be involved in the pathogenesis of A-T by analyzing the autophagic vesicles and the genes implicated in the different stages of autophagy. Through transmission electron microscopy (TEM) and immunofluorescence analysis we observed an accumulation of APs associated with a LC3 puncta pattern, and a reduced number of ALs. We also documented an increased expression of genes involved in AP and lysosome biogenesis and function, and a decrease of Vps18 expression, involved in their vesicular trafficking and fusion. mTORC1-controlled proteins were hyperphosphorylated in A-T, in keeping with an increased mTOR inhibitory influence of autophagy. Betamethasone is able to promote the degradation of SQSTM1, a biomarker of autophagy. Collectively, our results indicate that in cells from A-T patients, the APs maturation is active, while the fusion between APs and lysosomes is inappropriate, thus implying abnormalities in the cell-clearance process. We also documented a positive effect of Betamethasone on molecules implicated in autophagosome degradation

Unbalanced Immune System: Immunodeficiencies and Autoimmunity

Increased risk of developing autoimmune manifestations has been identified in different primary immunodeficiencies (PIDs). In such conditions, autoimmunity and immune deficiency represent intertwined phenomena that reflect inadequate immune function. Autoimmunity in PIDs may be caused by different mechanisms, including defects of tolerance to self-antigens and persistent stimulation as a result of the inability to eradicate antigens. This general immune dysregulation leads to compensatory and exaggerated chronic inflammatory responses that lead to tissue damage and autoimmunity. Each PID may be characterized by distinct, peculiar autoimmune manifestations. Moreover, different pathogenetic mechanisms may underlie autoimmunity in PID. In this review, the main autoimmune manifestations observed in different PID, including humoral immunodeficiencies, combined immunodeficiencies, and syndromes with immunodeficiencies, are summarized. When possible, the pathogenetic mechanism underlying autoimmunity in a specific PID has been explained

Primary Immunodeficiencies: novel insights in pathogenesis and potential therapeutic approaches

The immune response is composed of a diverse network of defenses, including cellular components and soluble mediators. A proper immune response relies on the innate immunity, characterized by a rapid and nonspecific initial response to infections and later on the adaptive immunity, characterized by a specific response to a particular antigen. Failure of host defense may occur, causing the dysregulation of the immune system, in particular the onset of immunodeficiency, autoimmunity and cancer predisposition. Primary immunodeficiencies comprise more than 200 different disorders that affect the development and the functions of the immune system. Many scientific papers have been published on the molecular and cellular basis of the immune response and on the mechanisms involved in the correct development of immune system components. Although today the genetic and molecular basis of the principal mechanisms involved in the immune response are well known, some aspect in this field remain unclear. In this thesis, during the three years of my PhD program, I have contributed to elucidate “Primary Immunodeficiencies: novel insight in pathogenesis and potential therapeutic approach”, through the combination of clinical, cellular, functional and molecular approaches. In particular, my research work is focused on the deepening of the knowledge on thymic ontogeny, in particular on the study of the functional role of FOXN1 transcription factor in the development of the T-cell ontogeny and new strategy to develop an in vitro thymic organoid. Moreover, I participate to give a contribution to better define the regulatory mechanisms of the immune system, with particular regard to the central and peripheral tolerance, whom impairment function leads to autoimmunity. Finally, I also participated to better define the role of the immune system genes, whom alteration induce the development of cancer predisposition, endocrine system failure and neurodegeneration. In particular I studied the role of gc in cell cycle progression, strongly related to its cellular amount and GH-R signaling, defining the basis of the physiological interaction between endocrine and immune systems, and the role ATM in the progressive neurological dysfunction. Overall, all my studies were designed in order to clarify unsolved issues and unknown mechanisms underlying the functionality of the immune system. These results could be useful both in the clinical practice and in the basic research of immunedysregulation

Aberrant autophagic vesicles in the lymphocytes from patients affected with Ataxia-Telangiectasia

INTRODUCTION: Ataxia-Telangiectasia (AT) is a rare disorder mostly characterized by cerebellar neurodegeneration and immunodeficiency. AT is caused by mutations in the Ataxia-Telangiectasia-Mutated (ATM) gene encoding a kinase, mostly localized in the nucleus and involved in cell-cycle control and DNA repair. ATM also displays a cytoplasmic localization, where its role is still poorly defined. OBJECTIVE: To evaluate potential abnormalities in the autophagic vesicle formation, which could be responsible for an inappropriate cell-clearance. METHODS: The ultrastructure of autophagic structures, such as autophagosomes (AP) and autolysosomes (AL), was analyzed by transmission electron microscopy (TEM) in lymphocytes obtained from AT patients and healthy controls and cultured at basal conditions or in a serum-starved medium. A quantitative analysis was also performed. RESULTS: In the patients at basal conditions, we found that the number of APs was 7 times higher than in the controls (14.00 vs 2.00 average number/100µm2), while the number of ALs was more than two times lower compared to the healthy subjects (2.00 vs 4.80 average number/100µm2). Under basal conditions, APs/ALs ratio was much higher in the patients than in the controls (7 vs 0.5). Under starved conditions, in the patients the number of APs did not further increase, resulting in an APs/ALs ratio comparable to the controls. CONCLUSIONS: Our data suggest an aberrant pattern of autophagic structures in lymphocytes from AT patients, characterized by an imbalance between autolysosomes and autophagosomes, suggesting an impairment in the cell clearance network

FOXN1 in cell development and human diseases

FOXN1 gene belongs to the forkhead box gene family that comprises a diverse group of "winged-helix" transcription factors that have been implicated in a variety of biochemical and cellular processes, such as development, metabolism, aging and cancer. These transcription factors share the common property of being developmentally regulated and of directing tissue-specific transcription and cell-fate decisions. Foxn1 is selectively expressed in thymic and skin epithelial cells, where it acts through its molecular targets to regulate the balance between growth and differentiation. In particular, Foxn1 is required for thymic epithelial patterning and differentiation from the initial epithelial thymic anlage to a functional cortical and medullary thymic epithelial cells (TECs) meshwork necessary for the crosstalk with the lymphoid compartment. A mutation in FoxN1 generates alymphoid cystic thymic dysgenesis due to defective TECs, causing primary T-cell immunodeficiency, named Nude/SCID syndrome, and leads to a hairless "nude" phenotype in both mice and humans. This immune defect represents the first example of a Severe Combined Immunodeficiencies (SCID) phenotype not primarily related to an abnormality intrinsic of the hematopoietic cell, but rather to a peculiar alteration of the thymic epithelia cell. This review focuses on the key role of FOXN1 in cell development and its clinical implication in humans

Unraveling the link between ectodermal disorders and primary immunodeficiencies

Primary immunodeficiencies (PIDs) include a heterogeneous group of mostly monogenic diseases characterized by functional/developmental alterations of the immune system. Skin and skin annexa abnormalities may be a warning sign of immunodeficiency, since both epidermal and thymic epithelium have ectodermal origin. In this review, we will focus on the most common immune disorders associated with ectodermal alterations. Elevated IgE levels represent the immunological hallmark of hyper-IgE syndrome, characterized by severe eczema and susceptibility to infections. Ectodermal dysplasia (ED) is a group of rare disorders that affect tissues of ectodermal origin. Hypoidrotic ED (HED), the most common form, is inherited as autosomal dominant, autosomal recessive or X-linked trait (XLHED). HED and XLHED are caused by mutations in NEMO and EDA-1 genes, respectively, and show similarities in the cutaneous involvement but differences in the susceptibility to infections and immunological phenotype. Alterations in the transcription factor FOXN1 gene, expressed in the mature thymic and skin epithelia, are responsible for human and murine athymia and prevent the development of the T-cell compartment associated to ectodermal abnormalities such as alopecia and nail dystrophy. The association between developmental abnormalities of the skin and immunodeficiencies suggest a role of the skin as a primary lymphoid organ. Recently, it has been demonstrated that a co-culture of human skin-derived keratinocytes and fibroblasts, in the absence of thymic components, can support the survival of human haematopoietic stem cells and their differentiation into T-lineage committed cells

Altered regulatory mechanisms governing cell survival in children affected with clustering of autoimmune disorders

Clustering of Autoimmune Diseases (CAD) is now emerging as a novel clinical entity within monogenic immune defects with a high familial occurrence. Aim of this study is to evaluate the regulatory mechanisms governing cell survival, paying a particular attention to Fas-induced apoptosis, in a cohort of 23 children affected with CAD. In 14 patients, Fas stimulation failed to induce cell apoptosis and in 1 case it was associated with Fas gene mutation. Our study highlights the importance to evaluate cell apoptosis in the group of children with CAD, which, with this regard, represents a distinct clinical entity