Final Report Summary - CNKHIV (Characterization of NK cell distributions and functions in human tissues in HIV-1 pathogenesis) The main aim of this project was to characterize the different aspects of HIV pathogenesis and in particular the role of the tissue-specific cell populations as well as the tissue-specific environment that may contribute to a better understanding of the cellular and molecular aspects of the HIV infection and dissemination. Therefore, taking advantages of the possibility to obtain human intestine and renal tissue from cancer patients that underwent partial intestine tissue removal or radical nephrectomy following studies were performed:1. Characterization of a novel intestine-specific T gamma-delta/NKp46pos lymphocyte subset We characterized a novel subset of T gamma-delta/NKp46pos lymphocytes physiologically resident only within the intestinal epithelium that represent more than 15% of total T cells present in the intestinal epithelium. FACS analysis performed on lymphocytes resident in other tissues that are highly enriched with gamma-delta T cells including cervix, epidermis, liver and tonsils did not show the presence of this specific T gamma-delta/NKp46pos subset suggesting the intestinal-specific residence of this gamma-delta T subset. EDTA and DTT treatment that allow the specific separation of intestinal cripts from the lamina propria revealed that T gamma-delta/NKp46pos is predominantly resident in the intraepithelium (IEL) and not in lamina propria. In addition, our analysis showed that T gamma-delta/NKp46pos are primarily Vgamma1 restricted. Phenotypic characterization of this specific lymphocyte population show the characteristic receptor patterns expressions that include:CD16, CD56, NKG2D, NKp30, NKp46, NKp44, NKp30, DNAM-1, CD94, CD8, KIR2DL1, KIR2DL2, KIR3DL1. Microarray analysis in these cells identified the reduced expression of genes associated with TCR signaling suggesting that expression of NKp46 receptor in these cells may control the TCR activation. Indeed, we observed the anergic character of freshly purified T gamma-delta/NKp46pos cells with hypo-responsiveness in IFN-gamma secretion and cytolysis. Therefore, we believe that other then identifying a novel human immune cell subset physiologically resident within the intestinal epithelium, this is the first unexpected description in humans of a regulatory/inhibitory role for NKp46 receptor. We observed that the functional outcome of T gamma-delta/NKp46pos IELs in the mucosal immunity regulation may be relevant not only in human physiology but also in physiopathology. In fact, FACS analysis of T gamma-delta/NKp46pos lymphocytes isolated from IELs patients with different chronic inflammation diseases revealed a significant decreased frequency of this specific T gamma-delta/NKp46pos subset compared to the healthy counterpart of the same tissue. Therefore, we discovered a novel human, tissue-specific, T gamma-delta/NKp46pos lymphocyte subset physiologically resident in the intra-epithelium intestine involve in its homeostasis retention and which losing may be relevant in different human pathologies including HIV infection.2) Characterization of different cellular and molecular factors that contribute to the pathogenesis of kidney, tissue specific, HIV-associated nephropathy (HIVAN). In particular, following studies were performed:a) Characterization of human renal, tissue-specific, podocyte sub-population o expressing Dc-sign receptor.To study the expression of Dc-sign in human primary podocytes we used human renal tissue of patients affected by renal adenocarcinoma who underwent a radical nephrectomy. All tissue specimens used for this study were collected from the distal part of the pathological tissue and were free from any disease as was verified by haematoxylin-eosin-staining. Tissue analysis by using hybridization in situ and the specific anti-Dc-sign mRNA probe demonstrated that about 20% of renal glomerula express Dc-sign expression. By using the micro section technique we were able to analyze gene expression specifically from glomerular section of kidney tissue. We observed low but consistent Dc-sign expression in all analyzed samples contemporary with the specific markers of podocytes including Wilms’ tumor 1 (WT-1), synaptopodin, nephrin and podocin. Ex vivo cultured primary human podocytes obtained from human isolated glomeruli also expressed Dc-sign together with nephrin, podocin, synaptopodin and WT-1 by qPCR. In addition, the confocal microscopy analysis confirmed the co-expression of Dc-sign in a small sub-population of cultured podocytes with podocin. These data suggest the existence of a small sub-population of podocytes able to express Dc-sign receptor in vivo. Since previously, we have observed that human immortalized podocytes in vitro express DC-sign receptor, which efficiently captures and internalize infectious HIV we believe that also in vivo Dc-sign receptor may play a critical role in the establishment of HIV kidney reservoir and virus dissemination.b) Role of ApoL1 polymorphism in HIV internalization in human podocytesPatients with African ancestry carrying ApoL1 genetic variants (Vs) have been shown to develop HIV-associated nephropathy (HIVAN) at a 10-fold higher rate, if not treated with anti-viral therapy. Although podocytopathy in HIVAN has been attributed to the direct effect of HIV infection it still remains a question how ApoL1 Vs contribute to kidney injury. Studding the possible interaction of ApoL1 in HIV trafficking in human podocytes we observed that ApoL1 expression increased after incubation of podocytes with the virus. Activation of ApoL1 expression depended on the viral load since that augmented entry of virus due to the increased expression of Dc-sign after stimulation with IL-1beta, further enhanced ApoL1 expression. Interestingly, increased expression of ApoL1 in podocytes seems to induce anti-viral response. Podocytes over-expressing ApoL1 either through pre-treatment with IFN-gamma or through transfection with the specific ApoL1-expressing plasmid displayed diminished HIV accumulation in podocytes. On the other hand we observed that increased expression of ApoL1 HIVAN-associated variant was not only permissive for HIV entry but also displayed increased HIV concentration associated with decreased lysosome bioactivity. These findings indicate that ApoL1 polymorphism determinates HIV persistence that in the case of ApoL1-Vs and increased inflammatory renal environment might be relevant for the pathogenesis of HIVAN.c) Full-length Soluble Urokinase Plasminogen Activator Receptor Modulates Glomerular Permeability by Reducing Nephrin Expression in Podocytes.Increased plasma level of soluble urokinase-type plasminogen activator receptor (suPAR) is one of the putative causes of focal segmental glomerulosclerosis (FSGS). Plasma levels of suPAR have been also correlated with the progression and poor prognosis of different human disorders, such as HIV-1 infection, sharing systemic degrees of inflammation and chronic immune activation. However, the mechanistic insight(s) associated with suPAR-mediated podocytes dysfunctions have not yet been disclosed. We showed both in human primary podocytes and in conditional immortalized podocytes (CHIPs) that suPAR induces nephrin down-modulation. This phenomenon was mediated only by full-length suPAR, was time- and dose-dependent and was associated with the suppression of Wilms’ tumor 1 (WT-1) transcription factor. Moreover, antagonist of beta3 integrin RGDfv blocked suPAR-induced suppression of nephrin. These in vitro data were also confirmed in an in vivo suPAR knock out Plaur-/- mice model by demonstrating that the infusion of high dose of suPAR inhibits expressions of nephrin and WT-1 in podocytes and induces proteinuria. Finally, we showed that immune-depletion of suPAR both in plasma from HIV infected patients containing high levels of suPAR and in cell supernatants from human lymphoid organs infected in vitro with HIV reversed the down-modulation of nephrin in podocytes. Taken together, these data showed that increased concentration of plasma suPAR down-modulates nephrin in podocytes and affects glomerular permeability, thus being directly involved in the onset of different human pathologies including HIV-associated FSGS.