The role of CD8+CCR4+ T-cells in axial spondyloarthritis

Axial spondyloarthritis (AxSpA) is an inflammatory disease affecting the axial skeleton. The infiltrate of T-cells in the structural lesions has been found to contribute to bone remodeling, but consensus relating the functional contribution of different T-cell subsets to pathogenesis has not been reached yet. Aim of the project was to characterize circulating T-cells and their homing markers from axSpA patients in order to identify cellular populations that could migrate to inflamed tissues and be implicated in axSpA. We found an altered proportion of circulating naïve and memory T-cells in axSpA patients, and a skew in favor of CD8+ T-cells expressing the chemokine receptor CCR4. Since CCL17 and CCL22, the two ligands for CCR4, are found to be elevated in the sera of axSpA patients, we investigated in details the role of CD8+CCR4+ T cells in axSpA. Our data showed that circulating CD8+CCR4+ T-cells display an effector memory phenotype and express homing markers for tissues that are target of the disease. Noteworthy, CD8+CCR4+ T cells from axSpA patients were activated, expressed markers of proliferation and acquired a cytotoxic phenotype, as demonstrated by the increased production of granzyme and perforin. CD8+CCR4+ T cells from axSpA patients upregulate the transcription of genes involved in bone mineralization and downregulate genes involved in osteoclast differentiation, indicating their possible involvement in bone remodeling. Furthermore, CD8+CCR4+ T cells stimulated with PMA and ionomycin were able to produce and release TNF and IL-8, two cytokines involved in osteoclastogenesis, indicating that CD8+CCR4+ T-cells after stimulation would be able to promote osteoclasts differentiation and neutrophils recruitment. Taken together our data suggest that CD8+CCR4+ T cells might exert a pathogenic role in axSpA, by releasing mediators of tissue damage, bone remodeling and recruitment of other pro inflammatory cells.

Evaluation of bone strength: microtomographic tecniques

This thesis is a part of a larger study about the characterization of mechanical and histomorphometrical properties of bone. The main objects of this study were the bone tissue properties and its resistance to mechanical loads. Moreover, the knowledge about the equipment selected to carry out the analyses, the micro-computed tomography (micro-CT), was improved. Particular attention was given to the reliability over time of the measuring instrument. In order to understand the main characteristics of bone mechanical properties a study of the skeletal, the bones of which it is composed and biological principles that drive their formation and remodelling, was necessary. This study has led to the definition of two macro-classes describing the main components responsible for the resistance to fracture of bone: quantity and quality of bone. The study of bone quantity is the current clinical standard measure for so-called bone densitometry, and research studies have amply demonstrated that the amount of tissue is correlated with its mechanical properties of elasticity and fracture. However, the models presented in the literature, including information on the mere quantity of tissue, have often been limited in describing the mechanical behaviour. Recent investigations have underlined that also the bone-structure and the tissue-mineralization play an important role in the mechanical characterization of bone tissue. For this reason in this thesis the class defined as bone quality was mainly studied, splitting it into two sub-classes of bone structure and tissue quality. A study on bone structure was designed to identify which structural parameters, among the several presented in the literature, could be integrated with the information about quantity, in order to better describe the mechanical properties of bone. In this way, it was also possible to analyse the iteration between structure and function. It has been known for long that bone tissue is capable of remodeling and changing its internal structure according to loads, but the dynamics of these changes are still being analysed. This part of the study was aimed to identify the parameters that could quantify the structural changes of bone tissue during the development of a given disease: osteoarthritis. A study on tissue quality would have to be divided into different classes, which would require a scale of analysis not suitable for the micro-CT. For this reason the study was focused only on the mineralization of the tissue, highlighting the difference between bone density and tissue density, working in a context where there is still an ongoing scientific debate.