Elena Gianchecchi

Recent insights into the role of the PD-1/PD-L1 pathway in immunological tolerance and autoimmunity.

Autoimmune diseases represent a heterogeneous group of conditions whose incidence is increasing worldwide. This has stimulated studies on their etiopathogenesis, derived from a complex interaction between genetic and environmental factors, in order to improve prevention and treatment of these disorders. The relevance of T regulatory cells and of the PD-1/PD-L1 pathway in controlling immune responses has been highlighted. Recent studies have in particular elucidated the putative role of the PD-1/PD-L1 pathway in regulating T cell responses and its effects on immunological tolerance and immune-mediated tissue damage. The role of the PD-1/PD-L1 pathway in autoimmunity has been already investigated in vivo in several experimental animal models including insulin-dependent diabetes mellitus, systemic lupus erythematosus, myocarditis, encephalomyelitis, rheumatoid arthritis and inflammatory bowel diseases. With the advent of candidate gene and genome-wide association studies, single nucleotide polymorphisms (SNPs) in PD-1 gene in humans have demonstrated relevant associations with a higher risk of developing autoimmune diseases in certain ethnic groups. In this review we present recent insights into the role of the PD-1/PD-L1 pathway in regulating lymphocyte activation, promotion of T regulatory cell development and function, breakdown of tolerance and development of autoimmunity. We finally speculate on the possible development of novel therapeutic treatments in human autoimmunity by modulating the PD-1/PD-L1 pathway.

The putative role of the C1858T polymorphism of protein tyrosine phosphatase PTPN22 gene in autoimmunity

Autoimmune diseases represent a heterogeneous group of conditions whose incidence is increasing worldwide. This has stimulated studies on their etiopathogenesis, derived from a complex interaction between genetic and environmental factors, in order to improve prevention and treatment of these diseases. An increasing amount of epidemiologic investigations has associated the presence of the C1858T polymorphism in the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene to the onset of several autoimmune diseases including insulin-dependent diabetes mellitus (Type 1 diabetes). PTPN22 encodes for the lymphoid tyrosine phosphatase Lyp. This belongs to non-receptor-type protein tyrosine phosphatases involved in lymphocyte activation and differentiation. In humans, Lyp may have a role in the negative regulation of T cell receptor signaling. The single nucleotide polymorphism C1858T encodes for a more active phosphatase Lyp R620W. This has the ability to induce a higher negative regulation of T cell receptor signaling. Thus, C1858T could play an important role at the level of thymocyte polarization and escape of autoreactive T lymphocytes, through the positive selection of otherwise negatively selected autoimmune T cells. In this review we discuss the physiological role exerted by the PTPN22 gene and its encoded Lyp product in lymphocyte processes. We highlight the pathogenic significance of the C1858T PTPN22 polymorphism in human autoimmunity with special reference to Type 1 diabetes. Recently the genetic variation in PTPN22 was shown to induce altered function of T and B-lymphocytes. In particular BCR signaling defects and alterations in the B cell compartment were reported in T1D patients. We finally speculate on the possible development of novel therapeutic treatments in human autoimmunity aiming to selectively target the variant Lyp protein in autoreactive T and B lymphocytes.

Gene/environment interactions in the pathogenesis of autoimmunity: new insights on the role of Toll-like receptors.

Autoimmune disorders are increasing worldwide. Although their pathogenesis has not been elucidated yet, a complex interaction of genetic and environmental factors is involved in their onset. Toll-like receptors (TLRs) represent a family of pattern recognition receptors involved in the recognition and in the defense of the host from invading microorganisms. They sense a wide range of pathogen associated molecular patterns (PAMPs) deriving from metabolic pathways selective of bacterial, viral, fungal and protozoan microorganisms. TLR activation plays a critical role in the activation of the downstream signaling pathway by interacting and recruiting several adaptor molecules. Although TLRs are involved in the protection of the host, several studies suggest that, in certain conditions, they play a critical role in the pathogenesis of autoimmune diseases. We review the most recent advances showing a correlation between some single nucleotide polymorphisms or copy number variations in TLR genes or in adaptor molecules involved in TLR signaling and the onset of several autoimmune conditions, such as Type I diabetes, autoimmune polyendocrinopathy candidiasis-ectodermal dystrophy, rheumatoid arthritis, systemic lupus erythematosus and systemic sclerosis. In light of the foregoing we finally propose that molecules involved in TLR pathway may represent the targets for novel therapeutic treatments in order to stop autoimmune processes.

Recent insights on the putative role of autophagy in autoimmune diseases.

The incidence of autoimmune pathologies is increasing worldwide. This has stimulated interest on their etiopathogenesis, caused by a complex interaction of genetic and environmental factors. With the advent of genome-wide linkage, candidate gene and genome wide association studies, risk polymorphisms in autophagy-related genes were discovered in several autoimmune conditions suggesting the possible contribution of autophagy to their etiopathogenesis. Autophagy represents the principal catabolic process mediated by lysosomes used by eukaryotic cells and is strictly regulated by proteins belonging to the Atg family. The function of autophagy has been well characterized in various tissues and systems, but its role in the regulation of innate and adaptive immune systems has been only recently discovered. It plays a fundamental role in the modulation of thymocyte selection and in the generation of T lymphocyte repertoire by participating in the intracellular antigen presentation on MHC class-II molecules by thymic epithelial cells. Furthermore, the generation of mice with knockout for specific autophagy-related genes induced several immunological alterations, including defects in B and T cell compartments and in T cell activation. In this review we report recent evidence on the role of autophagy in autoimmunity and discuss its relevance to the pathogenesis of these diseases. We finally highlight that future research may disclose potential new therapeutic targets for the treatment of this category of disorders by modulating the autophagic pathway.

Altered B cell homeostasis and toll-like receptor 9-driven response in type 1 diabetes carriers of the C1858T PTPN22 allelic variant: implications in the disease pathogenesis.

Type 1 diabetes is an autoimmune disease caused by the destruction of pancreatic beta cells by autoreactive T cells. Among the genetic variants associated with type 1 diabetes, the C1858T (Lyp) polymorphism of the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene alters the function of T cells but also of B cells in innate and adaptive immunity. The Lyp variant was shown to diminish interferon production and responses upon Toll-like receptor stimulation in macrophages and dendritic cells, possibly leading to uncontrolled infections as triggers of the diabetogenic process. The aim of this study was to unravel the yet uncharacterized effects that the variant could exert on the immune and autoimmune responses, particularly regarding the B cell phenotype, in the peripheral blood lymphocytes of diabetic patients and healthy controls in basal conditions and after unmethylated bacterial DNA CpG stimulation. The presence of the Lyp variant resulted in a significant increase in the percentage of transitional B cells in C/T carriers patients and controls compared to C/C patients and controls, in C/T carrier patients compared to C/C controls and in C/T carrier patients compared to C/C patients. A significant reduction in the memory B cells was also observed in the presence of the risk variant. After four days of CpG stimulation, there was a significant increase in the abundance of IgM+ memory B cells in C/T carrier diabetics than in C/C subjects and in the groups of C/T carrier individuals than in C/C individuals. IgM− memory B cells tended to differentiate more precociously into plasma cells than IgM+ memory B cells in heterozygous C/T subjects compared to the C/C subjects. The increased Toll-like receptor response that led to expanded T cell-independent IgM+ memory B cells should be further investigated to determine the putative contribution of innate immune responses in the disease pathogenesis.

Analysis of the autoimmune regulator gene in patients with autoimmune non-APECED polyendocrinopathies.

The pathogenesis of autoimmunity was derived from a complex interaction of genetic and environmental factors. Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy is a rare autosomal recessive disease caused by mutations in the autoimmune regulator (AIRE) gene. AIRE gene variants and, in particular, heterozygous loss-of-function mutations were also discovered in organ-specific autoimmune disorders, possibly contributing to their etiopathogenesis. It was suggested that even predisposition to develop certain autoimmune conditions may be derived from AIRE gene polymorphisms including S278R and intronic IVS9+6 G>A. In this study we unravel the hypothesis on whether AIRE gene variants may predispose individuals to associated autoimmune conditions in 41 Italian patients affected by non-APECED autoimmune polyendocrinopathies. We could not detect any heterozygous mutations of the AIRE gene. Although a trend of association was observed, heterozygous polymorphisms S278R and IVS9+6 G>A were detected in patients without statistically significant prevalence than in controls. Their putative contribution to autoimmune polyendocrinopathies and their predictive value in clinical strategies of disease development could be unravelled by analysing a larger sample of diseased patients and healthy individuals.

On the pathogenesis of insulin-dependent diabetes mellitus: the role of microbiota

Type 1 diabetes (T1D) is an autoimmune disorder characterized by the selective destruction of insulin-producing β cells as result of a complex interplay between genetic, stochastic and environmental factors in genetically susceptible individuals. An increasing amount of experimental data from animal models and humans has supported the role played by imbalanced gut microbiome in T1D pathogenesis. The commensal intestinal microbiota is fundamental for several physiologic mechanisms, including the establishment of immune homeostasis. Alterations in its composition have been correlated to changes in the gut immune system, including defective tolerance to food antigens, intestinal inflammation and enhanced gut permeability. Early findings reported differences in the intestinal microbiome of subjects affected by prediabetes or overt disease compared to healthy individuals. The present review focuses on microbiota-host homeostasis, its alterations, factors that influence microbiome composition and discusses their putative correlation with T1D development. Further studies are necessary to clarify the role played by microbiota modifications in the processes that cause enhanced permeability and the autoimmune mechanisms responsible for T1D onset.

Altered B cell homeostasis and Toll-like receptor 9-driven response in patients affected by autoimmune polyglandular syndrome Type 1: Altered B cell phenotype and dysregulation of the B cell function in APECED patients.

APECED is a T-cell mediated disease with increased frequencies of CD8+ effector and reduction of FoxP3+ T regulatory cells. Antibodies against affected organs and neutralizing to cytokines are found in the peripheral blood. The contribution of B cells to multiorgan autoimmunity in Aire-/- mice was reported opening perspectives on the utility of anti-B cell therapy. We aimed to analyse the B cell phenotype of APECED patients compared to age-matched controls. FACS analysis was conducted on PBMC in basal conditions and following CpG stimulation. Total B and switched memory (SM) B cells were reduced while IgM memory were increased in patients. In those having more than 15 years from the first clinical manifestation the defect included also mature and transitional B cells; total memory B cells were increased, while SM were unaffected. In patients with shorter disease duration, total B cells were unaltered while SM and IgM memory behaved as in the total group. A defective B cell proliferation was detected after 4day-stimulation. In conclusion APECED patients show, in addition to a significant alteration of the B cell phenotype, a dysregulation of the B cell function involving peripheral innate immune mechanisms particularly those with longer disease duration.

Use of short interfering RNA delivered by cationic liposomes to enable efficient down-regulation of PTPN22 gene in human T lymphocytes

Type 1 diabetes and thyroid disease are T cell-dependent autoimmune endocrinopathies. The standard substitutive administration of the deficient hormones does not halt the autoimmune process; therefore, development of immunotherapies aiming to preserve the residual hormonal cells, is of crucial importance. PTPN22 C1858T mutation encoding for the R620W lymphoid tyrosine phosphatase variant, plays a potential pathophysiological role in autoimmunity. The PTPN22 encoded protein Lyp is a negative regulator of T cell antigen receptor signaling; R620W variant, leading to a gain of function with paradoxical reduced T cell activation, may represent a valid therapeutic target. We aimed to develop novel wild type PTPN22 short interfering RNA duplexes (siRNA) and optimize their delivery into Jurkat T cells and PBMC by using liposomal carriers. Conformational stability, size and polydispersion of siRNA in lipoplexes was measured by CD spectroscopy and DLS. Lipoplexes internalization and toxicity evaluation was assessed by confocal microscopy and flow cytometry analysis. Their effect on Lyp expression was evaluated by means of Western Blot and confocal microscopy. Functional assays through engagement of TCR signaling were established to evaluate biological consequences of down-modulation. Both Jurkat T cells and PBMC were efficiently transfected by stable custom lipoplexes. Jurkat T cell morphology and proliferation was not affected. Lipoplexes incorporation was visualized in CD3+ but also in CD3- peripheral blood immunotypes without signs of toxicity, damage or apoptosis. Efficacy in affecting Lyp protein expression was demonstrated in both transfected Jurkat T cells and PBMC. Moreover, impairment of Lyp inhibitory activity was revealed by increase of IL-2 secretion in culture supernatants of PBMC following anti-CD3/CD28 T cell receptor-driven stimulation. The results of our study open the pathway to future trials for the treatment of autoimmune diseases based on the selective inhibition of variant PTPN22 allele using lipoplexes of siRNA antisense oligomers.

Type 1 Diabetes and Its Multi-Factorial Pathogenesis: The Putative Role of NK Cells

Type 1 diabetes (T1D) affects millions of people worldwide and is the prevalent form of all pediatric diabetes diagnoses. T1D is recognized to have an autoimmune etiology, since failure in specific self-tolerance mechanisms triggers immune reactions towards self-antigens and causes disease onset. Among all the different immunocytes involved in T1D etiopathogenesis, a relevant role of natural killer cells (NKs) is currently emerging. NKs represent the interface between innate and adaptive immunity; they intervene in the defense against infections and present, at the same time, typical features of the adaptive immune cells, such as expansion and generation of memory cells. Several recent studies, performed both in animal models and in human diabetic patients, revealed aberrations in NK cell frequency and functionality in the peripheral blood and in damaged tissues, suggesting their possible redirection towards affected tissues. NKs oscillate from a quiescent to an activated state through a delicate balance of activating and inhibitory signals transduced via surface receptors. Further accurate investigations are needed to elucidate the exact role of NKs in T1D, in order to develop novel immune-based therapies able to reduce the disease risk or delay its onset.