Potenziale coinvolgimento dell’NGF nell’infiammazione e nel rimodellamento tissutale della superficie oculare

Inflammation is by definition a local reaction against a tissue damage, trying to solve the traumatic effects with physiological or rarely pathological behaviours. The fibroblasts (FBs) are the key cells of tissue remodelling process, interacting both with inflammatory and structural cells. The activated FBs differentiate into myofibroblasts (myoFbs), characterized by the expression of the alpha-Smooth Muscle Actin (alpha-SMA) marker. In the fibrogenic factors set, the Nerve Growth Factor (NGF), seems to be associated with the tissue remodelling process via the two receptors trkA and p75. We have studied two ocular pathologies with different chronic inflammatory courses associated with fibrosis: the Vernal Keratocongiuntivitis (VKC, an allergic inflammation, Th2) and the Ocular Cicatricial Pemphigoid (OCP, an auto-immune disorder). Both these pathologies share the common feature of an increased deposition of Extra-Cellular Matrix (ECM). In this study in particular we have analyzed the following points: 1. the NGF effects on FBs/myoFBs phenotype of VKC and OCP; 2. the relation between NGF and FBs/myoFBs phenotypes; 3. the possible modulation effects by NGF in this remodelling process in relation with TGF-beta1 and other inflammatory cytokines (Th1/Th2); 4. The possible involvement of NGF in the innate immunity (TLRs). To this aim the we have analysed conjunctival biopsies of VKC, OCP and of healthy subjects. Primary FBs cultures have been created, characterized, NGF treated and finally evaluated at cellular (confocal), biochemical (Western Blot, ELISA, FACS) and molecular (Real-Time PCR) levels. Both VKC and OCP FBs data showed that a large part of these cells are myoFBs, expressing the NGF receptors and releasing high NGF levels. In VKC, the NGF treatment results in an increase of the ECM production but does not influence alpha-SMA expression. On the other hand, in OCP FBs the alpha-SMA expression is strictly connected with the pathological status of the disease. In accordance with VKC data, these FBs result also p75 positive, with a larger expression in the advanced stage of the pathology. Differently, TrkA is present in the in initial stage of the disease and decreases in the advanced one. The NGF treatment inverts this trend in FBs from patients at early stages of the disease. Moreover the NGF effect on synthesis and release of the pro/anti-inflammatory cytokines of the Th1 response, by FBs under exam, has been evaluated. The IL-2, IL-4 and TGF-beta1 levels, which were high in first pathological phase, decreased in the more severe conditions of the disease and resulted to be relevantly modulated by NGF. Probably, NGF might act both on survival/apoptosis of lymphocytes and on the activation of FBs/myoFBs. In order to evaluate if the NGF effect could be independent by TGF-beta1, we have stimulated non pathologic FBs with NGF and/or TGF-beta1 or neutralizing antibodies, and we have observed that both these factors stimulate each other on alpha-SMA phenotype. A possible role of microflora (biofilm) and TLRs in the remodelling process has been also studied. The evaluation of TLR4 and TLR9 expression in VKC and OCP FBs/myoFBs demonstrates that the expression is different in these two pathologies: while in FBs with OCP, being both Toll, these are augmented, in those of VKC these are down regulated with respect to normal controls. This fact is probably due to a different immunological history in the two pathologies. The NGF treatment seems to modulate the expression of these receptors making the cells more or less reactive in recognizing bacteria. From these studies it appeared that the NGF pro-fibrogenic effect could be partially dependent by the expression ratio of trkA and p75 on the FBs/myoFBs, by the TGF-beta1 and TLRs. In particular in the study of OCP it is possible that these ratio could be altered and this would explain the different FBs/myoFBs behaviour in relation with different NGF stimuli and with the different levels of the pathology.