Role of CD73/adenosine/A2A pathway in the modulation of inflammatory processes

Manipulation of the course or of the extension of an inflammatory reaction may include the effort not only to inhibit pro-inflammatory pathways but also through an approach able to enhance endogenous anti-inflammatory processes. Thus, it appears of fundamental importance to identify those of metabolic pathways that sense excessive damage and contribute to terminate inflammation. Adenosine has been recognized to be a molecule with autocrine/paracrine functions, acting as a signal molecule to preserve host defence and tissue integrity during inflammation and trauma. Adenosine is a nucleoside always present both within and outside cells in nanomolar concentration (10–100nM) under physiological conditions, deriving by the breakdown of intra – or –extra cellular adenine nucleotides. Following trauma or cellular stress, such as during hypoxia, ischemia or inflammation, its levels increase rapidly following ATP degradation. Levels of extracellular adenosine from ATP are controlled by ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase/CD39) and ecto-5’-nucleotidase (e-5’NT/CD73); the latter converting AMP to adenosine and inorganic phosphate (Pi) and represents the rate limiting step reaction controlling the ratio between extracellular proinflammatory ATP and anti-inflammatory adenosine. Extracellular adenosine accumulation represents an endogenous signal controlling inflammation and immune responses; its effects are mediated through the interaction with four G-protein coupled receptors, indicated as A1, A2A, A2B, A3, among which A2A plays a role in mediating adenosine antinflammatory effects. Evidence that cellular expression and activity of CD39 and CD73 may be subject to changes under pathophysiological conditions has led to identify this pathway as an endogenous modulator in several immune/inflammatory diseases. In particular, CD73 was shown to play an important role in regulating vascular permeability and leukocyte trafficking in inflammatory diseases; and a crucial role in the regulation of immune/inflammatory cell function. There is evidence that well known anti-inflammatory drugs act through adenosine signalling. Indeed, CD73 is required for the anti-inflammatory effect of methotrexate; furthermore, the beneficial effect of interferon (IFN)-β in patients affected by multiple sclerosis has been associated to CD73 upregulation at the blood-brain barrier; it has been demonstrated that the anti-inflammatory effect of aspirin involves also adenosine accumulation, independently from cyclooxygenase (COX) inhibition. The objective of our study is to investigate on the role of CD73/adenosine pathway in the development of inflammation rat and to evaluate whether CD73/adenosine pathway is affected by cyclooxygenase inhibitors in our models. Experiments will be performed in two standard animal models of acute and chronic inflammation such as carrageenan-induced pleurisy in rats and adjuvant induced arthritis in mice, respectively. In particular, we will evaluate changes in expression and activity of CD73 following pleurisy compared to non- inflamed animals; the effect of animal treatment with soluble enzyme (CD73) and relative inhibitors, or with A2A agonist and A2A antagonist on the development of inflammation; we will also evaluate whether antinflammatory treatment with a classical FAN, such as indomethacin, or with celecoxib, as representative of coxibs, affect CD73/adenosine/A2A pathway. In a second phase of our study, we will evaluate the role of CD73/adenosine/A2A signalling in the development of adjuvant arthritis in mice, representing a model involving immune system. The effect of treatments, already described for pleurisy, will be evaluated; however, in this model we will treat mice by using different dosing protocols that take in account the 3 phases of arthritis development: 1) preventive; 2) during the development of immune cell proliferation; 3) when immune inflammation is established. Furthermore, this model will be helpful to investigate whether CD73/adenosine signalling is involved in the antinflammatory effect of indomethacin and celecoxib two anti-inflammatory drugs widely used for arthritis and osteoarthritis respectively. In the end, the model of arthritis will be developed in CD73 knockout mice. Results of this study could offer a scientific rationale based on pharmacological studies to project drugs that might have an antinflammatory effect by targeting an endogenous antinflammatory pathway.