Riccardo Navone

Systemic sclerosis immunoglobulin G autoantibodies bind the human cytomegalovirus late protein UL94 and induce apoptosis in human endothelial cells

Systemic sclerosis is an autoimmune disease characterized by immunological and vascular abnormalities. Autoantibodies against intracellular antigens are associated with particular clinical features of the disease, whereas autoantibodies against cell surface antigens may be pathogenic by inducing endothelial cell damage, considered the primary event in the pathogenesis of the disease. Latent human cytomegalovirus infection may contribute to progression of systemic sclerosis through its ability to infect endothelial cells; however, direct links between human cytomegalovirus infection and systemic sclerosis are still lacking. Molecular mimicry is one of the mechanisms that account for the link between infection and autoimmunity. Here we have identified an immunodominant peptide using systemic sclerosis serum screening of a random peptide library; such peptide shares homology with autoantigens and with the human cytomegalovirus late protein UL94 (ref. 9). Immunoglobulin G antibodies against the peptide affinity-purified from the sera of patients with systemic sclerosis specifically recognized the viral product and autoantigens; moreover, such antibodies induced endothelial cell apoptosis through specific interaction with the cell surface integrin–NAG-2 protein complex. Our results provide evidence that antibodies against human cytomegalovirus cause apoptosis of endothelial cells, considered the initial pathogenic event of systemic sclerosis, and indicate a previously unknown mechanism for the etiological link between human cytomegalovirus infection and autoimmunity.

In Celiac Disease, a Subset of Autoantibodies against Transglutaminase Binds Toll-Like Receptor 4 and Induces Activation of Monocytes

Background Celiac disease is a small intestine inflammatory disorder with multiple organ involvement, sustained by an inappropriate immune response to dietary gluten. Anti-transglutaminase antibodies are a typical serological marker in patients with active disease, and may disappear during a gluten-free diet treatment. Involvement of infectious agents and innate immunity has been suggested but never proven. Molecular mimicry is one of the mechanisms that links infection and autoimmunity. Methods and Findings In our attempt to clarify the pathogenesis of celiac disease, we screened a random peptide library with pooled sera of patients affected by active disease after a pre-screening with the sera of the same patients on a gluten-free diet. We identified a peptide recognized by serum immunoglobulins of patients with active disease, but not by those of patients on a gluten-free diet. This peptide shares homology with the rotavirus major neutralizing protein VP-7 and with the self-antigens tissue transglutaminase, human heat shock protein 60, desmoglein 1, and Toll-like receptor 4. We show that antibodies against the peptide affinity-purified from the sera of patients with active disease recognize the viral product and self-antigens in ELISA and Western blot. These antibodies were able to induce increased epithelial cell permeability evaluated by transepithelial flux of [3H] mannitol in the T84 human intestinal epithelial cell line. Finally, the purified antibodies induced monocyte activation upon binding Toll-like receptor 4, evaluated both by surface expression of activation markers and by production of pro-inflammatory cytokines. Conclusions Our findings show that in active celiac disease, a subset of anti-transglutaminase IgA antibodies recognize the viral protein VP-7, suggesting a possible involvement of rotavirus infection in the pathogenesis of the disease, through a mechanism of molecular mimicry. Moreover, such antibodies recognize self-antigens and are functionally active, able to increase intestinal permeability and induce monocyte activation. We therefore provide evidence for the involvement of innate immunity in the pathogenesis of celiac disease through a previously unknown mechanism of engagement of Toll-like receptor 4.

Endothelial Cells' Activation and Apoptosis Induced by a Subset of Antibodies against Human Cytomegalovirus: Relevance to the Pathogenesis of Atherosclerosis

Background Human cytomegalovirus (hCMV) is involved in the pathogenesis of atherosclerosis. We have previously shown in patients with atherosclerosis that antibodies directed against the hCMV-derived proteins US28 and UL122 are able to induce endothelial cell damage and apoptosis of non-stressed endothelial cells through cross-rection with normally expressed surface molecules. Our aim was to dissect the molecular basis of such interaction and to investigate mechanisms linking innate immunity to atherosclerosis. Methodology/Principal Findings We analysed the gene expression profiles in endothelial cells stimulated with antibodies affinity-purified against either the UL122 or the US28 peptides using the microarray technology. Microarray results were validated by quantitative PCR and by detection of proteins in the medium. Supernatant of endothelial cells incubated with antibodies was analysed also for the presence of Heat Shock Protein (HSP)60 and was used to assess stimulation of Toll-Like Receptor-4 (TLR4). Antibodies against UL122 and US28 induced the expression of genes encoding for adhesion molecules, chemokines, growth factors and molecules involved in the apoptotis process together with other genes known to be involved in the initiation and progression of the atherosclerotic process. HSP60 was released in the medium of cells incubated with anti-US28 antibodies and was able to engage TLR4. Conclusions/Significance Antibodies directed against hCMV modulate the expression of genes coding for molecules involved in activation and apoptosis of endothelial cells, processes known to play a pivotal role in the pathogenesis of atherosclerosis. Moreover, endothelial cells exposed to such antibodies express HSP60 on the cell surface and release HSP60 in the medium able to activate TLR4. These data confirm that antibodies directed against hCMV-derived proteins US28 and UL122 purified from patients with coronary artery disease induce endothelial cell damage and support the hypothesis that hCMV infection may play a crucial role in mediating the atherosclerotic process.

DNase I behaves as a transcription factor which modulates Fas expression in human cells

DNase I is the major nuclease present in biological fluids and is ubiquitously expressed in mammalian tissues. It is responsible for the removal of DNA from nuclear antigens, and consistently with this function, DNase I‐deficient mice show features of autoimmunity. The enzyme seems also to be involved in apoptosis (programmed cell death). We demonstrate that DNase I is internalized by human cells upon binding mannose 6‐phosphate receptor and gains access into the cells. Following internalization of the enzyme, the cells show an increased surface expression of Fas molecule, a key regulator of apoptosis. Here we show that DNase I up‐regulates fas transcription upon interaction with the fas gene promoter. Moreover, overexpression of the DNase I gene in human cells results in a similar modulation of the fas gene expression. Our data provide the first evidence that the endonuclease DNase I behaves as a transcription factor which selectively regulates cell surface Fas expression in human cells and point towards a fundamental role of DNase I in the regulation of the apoptotic machinery.

Antiflagellin antibodies recognize the autoantigens Toll‐Like Receptor 5 and Pals 1‐associated tight junction protein and induce monocytes activation and increased intestinal permeability in Crohn’s disease

Background and objectives.  Bacterial flagellin is considered an important antigen in Crohn’s disease (CD) as it activates innate immunity through Toll‐Like Receptor 5 (TLR5) engagement and induces an elevated adaptive immune response. Little is known about the presence of an autoimmune process in CD. We aimed to identify pathogenically relevant autoantigen targets in CD.