AIM OF THE STUDY Systemic sclerosis (SSc) is a chronic autoimmune condition characterized by excessive tissue fibrosis, microvascular alterations and immune dysfunction with the production of characteristic autoantibodies. These autoantibodies are highly specific for SSc diagnosis, and provide the most reliable tool to predict disease subset and the pattern of internal organ involvement. Despite such diagnostic and prognostic role, no evidence supporting the pathogenic potential of these autoantibodies has to date been raised. The working hypothesis of this study envisaged that immune complexes (ICs) containing scleroderma specific autoantibodies -rather than the mere antibody- might be able to elicit a proinflammatory and pro-fibrotic signaling cascade in target cells, thus contributing to SSc multifaceted etiopathogenesis. Since scleroderma autoantibodies bind –either directly or via bridge proteins- to nucleic acids, it was also postulated that the effects induced by SSc-ICs might be mediated by Toll-like Receptors (TLR). MATERIALS AND METHODS Fibroblasts have been isolated from skin biopsies from healthy controls and then cultured in adequate conditions. ICs have been purified from sera of scleroderma patients bearing different autoantibody specificities (antibodies against centromeric proteins [ACA], DNA topoisomerase I [ATA], RNA polymerase [ARA] and Th/To [anti-Th/To]) or of healthy controls using polyethylen glycol precipitation. Fibroblasts were transiently silenced for tlr3 using a specific small interfering RNA (siRNA); silencing was confirmed by RT-PCR and Western Blotting. Naïve and tlr3-silenced cells have been incubated with pathologic and control ICs and with TLR3 [Poly(I:C)] and TLR4 (LPS) agonists. Several parameters of cell activation have been assessed in the different experimental conditions. In particular, mRNA levels of type I interferons (IFN-α and IFN-β) and TLR (TLR3 and TLR9) have been investigated by real-Time PCR; ICAM-1 expression has been evaluated by cell-ELISA and the secretion of IL-6 and IL-8 in culture supernatants has been measured by commercial ELISA kits. Furthermore, the involvement of intracellular signaling pathways culminating with the activation of p38 MAPK and NFκB has been assessed. RESULTS Stimulation of normal skin fibroblasts with pathologic ICs induced a significant increase in the gene expression levels of both IFN-α and IFN-β; similar results have been reported in the presence of TLR agonists but not of control ICs and medium alone. In addition, ICAM-1 expression and IL-6 and IL-8 secretion were up-regulated by Poly(I:C), LPS and ICs from scleroderma patients but not healthy controls and medium alone. Further, pathologic ICs induced the activation of both p38 MAPK and NFκB. The expression levels of TLR3 and -to a greater extent- TLR9 were significantly increased in cells treated with TLR3 agonist and ICs from SSc patients but not healthy controls. The efficiency of tlr3 silencing in skin fibroblasts was confirmed at both mRNA and protein levels. tlr3 silencing significantly affected ICAM-1 expression and IL-6, but not IL-8, secretion in cells treated with both TLR agonists and ICs from SSc patients but not healthy controls and medium alone. CONCLUSIONS These data provide the first demonstration of the pathogenic role of ICs isolated from scleroderma patients with different autoantibody specificities in the inductor phase of SSc. Indeed, pathologic ICs can interact with normal skin fibroblasts, inducing a pro-inflammatory phenotype mediated by p38 MAPK and NFκB. In particular, ICs isolated from SSc patients have been shown to recruit TLR3 leading to fibroblast activation, with upregulation of adhesion molecules and secretion of pro-inflammatory interleukins. Other TLRs, such as TLR7, TLR8 and TLR9, might be implicated in the cell response to scleroderma ICs. These evidences fit well with the diagnostic and prognostic role of scleroderma-specific autoantibodies.

THE PATHOGENIC ROLE OF IMMUNE COMPLEXES CONTAINING SCLERODERMA-SPECIFIC AUTOANTIBODIES / C.b. Chighizola ; tutor: P. L. Meroni ; coordinatore: M. Locati. - : . DIPARTIMENTO DI SCIENZE CLINICHE E DI COMUNITA', 2015 Feb 10. ((27. ciclo, Anno Accademico 2014. [10.13130/chighizola-cecilia-beatrice_phd2015-02-10].

THE PATHOGENIC ROLE OF IMMUNE COMPLEXES CONTAINING SCLERODERMA-SPECIFIC AUTOANTIBODIES

C.B. Chighizola
2015-02-10

Abstract

AIM OF THE STUDY Systemic sclerosis (SSc) is a chronic autoimmune condition characterized by excessive tissue fibrosis, microvascular alterations and immune dysfunction with the production of characteristic autoantibodies. These autoantibodies are highly specific for SSc diagnosis, and provide the most reliable tool to predict disease subset and the pattern of internal organ involvement. Despite such diagnostic and prognostic role, no evidence supporting the pathogenic potential of these autoantibodies has to date been raised. The working hypothesis of this study envisaged that immune complexes (ICs) containing scleroderma specific autoantibodies -rather than the mere antibody- might be able to elicit a proinflammatory and pro-fibrotic signaling cascade in target cells, thus contributing to SSc multifaceted etiopathogenesis. Since scleroderma autoantibodies bind –either directly or via bridge proteins- to nucleic acids, it was also postulated that the effects induced by SSc-ICs might be mediated by Toll-like Receptors (TLR). MATERIALS AND METHODS Fibroblasts have been isolated from skin biopsies from healthy controls and then cultured in adequate conditions. ICs have been purified from sera of scleroderma patients bearing different autoantibody specificities (antibodies against centromeric proteins [ACA], DNA topoisomerase I [ATA], RNA polymerase [ARA] and Th/To [anti-Th/To]) or of healthy controls using polyethylen glycol precipitation. Fibroblasts were transiently silenced for tlr3 using a specific small interfering RNA (siRNA); silencing was confirmed by RT-PCR and Western Blotting. Naïve and tlr3-silenced cells have been incubated with pathologic and control ICs and with TLR3 [Poly(I:C)] and TLR4 (LPS) agonists. Several parameters of cell activation have been assessed in the different experimental conditions. In particular, mRNA levels of type I interferons (IFN-α and IFN-β) and TLR (TLR3 and TLR9) have been investigated by real-Time PCR; ICAM-1 expression has been evaluated by cell-ELISA and the secretion of IL-6 and IL-8 in culture supernatants has been measured by commercial ELISA kits. Furthermore, the involvement of intracellular signaling pathways culminating with the activation of p38 MAPK and NFκB has been assessed. RESULTS Stimulation of normal skin fibroblasts with pathologic ICs induced a significant increase in the gene expression levels of both IFN-α and IFN-β; similar results have been reported in the presence of TLR agonists but not of control ICs and medium alone. In addition, ICAM-1 expression and IL-6 and IL-8 secretion were up-regulated by Poly(I:C), LPS and ICs from scleroderma patients but not healthy controls and medium alone. Further, pathologic ICs induced the activation of both p38 MAPK and NFκB. The expression levels of TLR3 and -to a greater extent- TLR9 were significantly increased in cells treated with TLR3 agonist and ICs from SSc patients but not healthy controls. The efficiency of tlr3 silencing in skin fibroblasts was confirmed at both mRNA and protein levels. tlr3 silencing significantly affected ICAM-1 expression and IL-6, but not IL-8, secretion in cells treated with both TLR agonists and ICs from SSc patients but not healthy controls and medium alone. CONCLUSIONS These data provide the first demonstration of the pathogenic role of ICs isolated from scleroderma patients with different autoantibody specificities in the inductor phase of SSc. Indeed, pathologic ICs can interact with normal skin fibroblasts, inducing a pro-inflammatory phenotype mediated by p38 MAPK and NFκB. In particular, ICs isolated from SSc patients have been shown to recruit TLR3 leading to fibroblast activation, with upregulation of adhesion molecules and secretion of pro-inflammatory interleukins. Other TLRs, such as TLR7, TLR8 and TLR9, might be implicated in the cell response to scleroderma ICs. These evidences fit well with the diagnostic and prognostic role of scleroderma-specific autoantibodies.
LOCATI, MASSIMO
LOCATI, MASSIMO
Settore MED/16 - Reumatologia
THE PATHOGENIC ROLE OF IMMUNE COMPLEXES CONTAINING SCLERODERMA-SPECIFIC AUTOANTIBODIES / C.b. Chighizola ; tutor: P. L. Meroni ; coordinatore: M. Locati. - : . DIPARTIMENTO DI SCIENZE CLINICHE E DI COMUNITA', 2015 Feb 10. ((27. ciclo, Anno Accademico 2014. [10.13130/chighizola-cecilia-beatrice_phd2015-02-10].
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/259694
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