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Autoantibodies represent a primary characteristic of many systemic autoimmune diseases [...]

Systemic sclerosis (SSc) is a rare autoimmune disease whose molecular mechanisms are not yet fully understood. There is no definitive cure, and the main causes of death are pulmonary fibrosis and pulmonary arterial hypertension. Here, we focus on the interferon regulators factor 8 (IRF8), a factor involved in the type I interferon (IFN-I) signature, which is present in about half of SSc patients. Variants of this factor may play a role in autoimmunity, but little is known regarding the role of IRF8 in SSc pathogenesis. We carried out a literature search to address the association between the IRF8 factor and SSc susceptibility and clinical manifestations. The current studies appear to confirm a possible association between the alteration of the gene for IRF8 and SSc susceptibility. A link between IRF8 mutations and expression of a pro-fibrotic phenotype at the cellular level also emerges. Additional investigations are needed to confirm the role of IRF8 in SSc. However, IRF8 is worth consideration as a possible new disease marker of fibrosis in SSc patients.

Psoriasis is a chronic skin disease evolving to psoriatic arthritis (PsA) in 30% of cases. LL37 is a psoriasis T-cell autoantigen and, in complex with self-DNA/RNA, a trigger of type I interferon (IFN-I) and pro-inflammatory factors in dendritic cells. LL37 can undergo irreversible post-translational modifications (PTMs), namely, citrullination and carbamylation, which are linked to a neutrophil-dominated inflammation. Notably, in PsA, carbamylated and citrullinated LL37 (carb-LL37 and cit-LL37) become antibody targets. Here, we analyze the presence of, and the T-cell and antibody reactivity to, cit-LL37 and carb-LL37, to address the occurrence and significance of these PTMs in psoriasis. The presence of modified LL37 in skin biopsies was assessed by laser scanner confocal microscopy (LSCM); T-cell responses to modified LL37 were assessed by Ki67 assay and intracellular cytokine staining using flow cytometry; serum autoantibodies to the same antigens were tested by enzyme-linked immunosorbent assay (ELISA). The results show that native and modified LL37 (both carb-LL37 and cit-LL37) are detectable in psoriatic skin, but not in healthy donors’ (HD) skin, where they colocalize with neutrophil infiltrates and neutrophil extracellular trap formation (NETosis). Psoriatic T cells and antibodies recognize native LL37, cit-LL37, and carb-LL37, but only CD4-T-cell responses to native LL37 and carb-LL37 correlate with psoriasis area severity index (PASI), whereas CD8-T-cell responses to the same peptides correlate with PASI in the HLA-Cw6*02-positive subgroup. CD4-T cells specific for modified LL37 express heterogeneous T-helper (Th) phenotypes: native/carb-LL37-specific T cells mainly manifest a Th1/Th17-like phenotype, whereas cit-LL37-specific T cells resemble Th-follicular (Thf)-like cells. In vitro T-cell polarization experiments suggest that distinct pro-inflammatory effects of LL37 and modified LL37, in complex with self-nucleic acids, may concur to these phenomena. This is the first evidence in psoriasis that PTMs of an autoantigen with innate immune cell stimulatory ability dictate autoreactive Th-cell polarization. These data, obtained using LL37 as a model autoantigen, indicate that citrullination and carbamylation pathways may play a role in the psoriasis course, generating epitopes to which immunological tolerance does not exist and potentially concur to PsA development.

Background: Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular and immunity alterations and skin/internal organ fibrosis. Aberrant levels of plasma CXCL4, CXCL4–RNA/DNA complexes, type I IFN (IFN-I) and anti-CXCL4 antibodies characterize SSc. These parameters influence each other: CXCL4–self-DNA/RNA complexes are triggers of IFN-I in plasmacytoid dendritic cells (pDCs), and anti-CXCL4 autoantibodies amplify this effect. Here, we assess the modulation over time of plasma CXCL4 and the related parameters of CXCL4–DNA/RNA complexes, anti-CXCL4 antibodies, IFN-α and TNF-α in an SSc cohort under the synthetic analogue of prostacyclin PGI2 (iloprost) treatment to address contribution of these parameters to pathogenesis and their role as biomarkers. Methods: We analyzed immunological parameters at baseline (T0) and after 3 (T3) and 6 (T6) months in 30 SSc patients. Responders were the patients that lowered their disease activity parameters after six months of treatment. Results: Anti-CXCL4 autoantibodies correlated with both IFN-α and TNF-α levels in SSc plasma. Responders significantly down-regulated serum IFN-α. In seven patients with a shorter disease duration, improvement coincides with a decrease in plasma IFN-α, CXCL4 and TNF-α. Iloprost efficiently blocks pDCs IFN-α production induced by CXCL4–DNA/RNA complexes in vitro. Conclusions: The data suggest a possible role of iloprost as a disease-modifying drug, mainly accompanied by down-regulation of plasma IFN-I levels. Since CXCL4, IFN-I and TNF-α down-modulation was evident and significant in improving SSc patients with a shorter disease duration, these results warrant future investigations on the early use of iloprost to slow SSc progression.

Chemokine (C-X-C motif) ligand 4 (CXCL4) is a biomarker of unfavorable prognosis in Systemic Sclerosis (SSc), a potentially severe autoimmune condition, characterized by vasculitis, fibrosis and interferon (IFN)-I-signature. We recently reported that autoantibodies to CXCL4 circulate in SSc patients and correlate with IFN-α. Here, we used shorter versions of CXCL4 and CXCL4-L1, the CXCL4 non-allelic variant, to search for autoantibodies exclusively reacting to one or the other CXCL4 form. Moreover, to address whether anti-CXCL4/CXCL4-L1 antibodies were present before SSc onset and predicted SSc-progression, we longitudinally studied two VEDOSS (Very Early Diagnosis of Systemic Sclerosis) patient cohorts, separating SSc-progressors from SSc-non-progressors. We found that anti-CXCL4-specific autoantibodies were present in both SSc and VEDOSS patients (both SSc-progressors and SSc-non-progressors). Anti-CXCL4-L1-specific autoantibodies were especially detected in long-standing SSc (lsSSc). Anti-CXCL4/CXCL4-L1 antibodies correlated with IFN-α and with specific SSc-skin features but only in lsSSc and not in early SSc (eaSSc) or VEDOSS. Thus, a broader antibody response, with reactivity spreading to CXCL4-L1, is characteristic of lsSSc. The early anti-CXCL4 autoantibody response seems qualitatively different from, and likely less pathogenic than, that observed in advanced SSc. Lastly, we confirm that anti-CXCL4 autoantibodies are SSc-biomarkers and uncover that also CXCL4-L1 becomes an autoantigen in lsSSc.

Systemic sclerosis (SSc) is an autoimmune disease characterized by specific autoantibodies, vasculopathy and fibrosis of the skin and internal organs. In SSc, chronic activation of the immune system is largely sustained by endogenous inflammatory mediators that act as damage-associated molecular patterns (DAMPs), which activate Toll-like receptors (TLRs). Major autoantigens are nucleic acids or molecules that are able to bind nucleic acids. It is important to identify solid and predictive biomarkers of both disease activity and disease subtype. CXCL4 has been regarded as a new biomarker for early SSc in recent years, and here, we discuss its modulation over the course of a disease and after pharmacological interventions. Moreover, we provide evidence that CXCL4, in addition to being a biomarker of SSc subtypes and a prognostic marker of disease severity, has a dual pathogenic role in SSc: on the one hand, in complex with self-nucleic acids, CXCL4 acts as a DAMP for IFN-I and pro-inflammatory cytokines’ release by innate immune cells (such as dendritic cells); on the other hand, CXCL4 is a target of both antibodies and T cells, functioning as an autoantigen. CXCL4 is certainly an interesting molecule in inflammation and autoimmunity, not only in SSc, and it may also be considered as a therapy target.

Biological drugs have revolutionized the treatment of many chronic diseases, starting with cancer. They normally consist of antibodies that are also effectively used to treat several autoimmune diseases, including psoriasis. These products, called biologics, work by selectively blocking the activity of certain targets, mainly cytokines, which play a crucial role in the pathogenic and inflammatory processes involved in a particular disease. Unfortunately, a reduction in response to these biological treatments may occur over time, and this phenomenon is often due to the development of antibodies against the therapeutic antibodies. The immune responses directed to these therapeutics range from transient anti-drug antibodies (ADA) formation, with no clinical significance, to the generation of high titers and persistence of ADA, causing loss of efficacy. Considering the costs associated with the use of biological drugs, there is growing interest in identifying biomarkers that can predict clinical response to personalize treatments.

CXCL4 is an important biomarker of systemic sclerosis (SSc), an incurable autoimmune disease characterized by vasculopathy and skin/internal organs fibrosis. CXCL4 contributes to the type I interferon (IFN-I) signature, typical of at least half of SSc patients, and its presence is linked to an unfavorable prognosis. The mechanism implicated is CXCL4 binding to self-DNA, with the formation of complexes amplifying TLR9 stimulation in plasmacytoid dendritic cells (pDCs). Here, we demonstrate that, upon binding to self-RNA, CXCL4 protects the RNA from enzymatic degradation. As a consequence, CXCL4-RNA complexes persist in vivo. Indeed, we show for the first time that CXCL4-RNA complexes circulate in SSc plasma and correlate with both IFN-I and TNF-α. By using monocyte-derived DCs (MDDCs) pretreated with IFN-α as a model system (to mimic the SSc milieu of the IFN-I signature), we demonstrate that CXCL4-RNA complexes induce MDDC maturation and increase, in particular, pro-inflammatory TNF-α as well as IL-12, IL-23, IL-8, and pro-collagen, mainly in a TLR7/8-dependent but CXCR3-independent manner. In contrast, MDDCs produced IL-6 and fibronectin independently in their CXCL4 RNA-binding ability. These findings support a role for CXCL4-RNA complexes, besides CXCL4-DNA complexes, in immune amplification via the modulation of myeloid DC effector functions in SSc and also during normal immune responses.

Systemic sclerosis (SSc) is characterized by skin/internal organ fibrosis, vasculopathy and autoimmunity. Chemokine (C-X-C motif) ligand 4 (CXCL4) is an SSc biomarker, predicting unfavorable prognosis and lung fibrosis. CXCL4 binds DNA/RNA and favors interferon (IFN)-α production by plasmacytoid dendritic cells (pDCs), contributing to the type I IFN (IFN-I) signature in SSc patients. However, whether CXCL4 is an autoantigen in SSc is unknown. Here, we show that at least half of SSc patients show consistent antibody reactivity to CXCL4. T-cell proliferation to CXCL4, tested in a limited number of patients, correlates with anti-CXCL4 antibody reactivity. Antibodies to CXCL4 mostly correlate with circulating IFN-α levels and are significantly higher in patients with lung fibrosis in two independent SSc cohorts. Antibodies to CXCL4 implement the CXCL4–DNA complex’s effect on IFN-α production by pDCs; CXCL4–DNA/RNA complexes stimulate purified human B-cells to become antibody-secreting plasma cells in vitro. These data indicate that CXCL4 is indeed an autoantigen in SSc and suggest that CXCL4, and CXCL4-specific autoantibodies, can fuel a harmful loop: CXCL4–DNA/RNA complexes induce IFN-α in pDCs and direct B-cell stimulation, including the secretion of anti-CXCL4 antibodies. Anti-CXCL4 antibodies may further increase pDC stimulation and IFN-α release in vivo, creating a vicious cycle which sustains the SSc IFN-I signature and general inflammation.

LL37 acts as T-cell/B-cell autoantigen in Systemic lupus erythematosus (SLE) and psoriatic disease. Moreover, when bound to “self” nucleic acids, LL37 acts as “danger signal,” leading to type I interferon (IFN-I)/pro-inflammatory factors production. T-cell epitopes derived from citrullinated-LL37 act as better antigens than unmodified LL37 epitopes in SLE, at least in selected HLA-backgrounds, included the SLE-associated HLA-DRB1*1501/HLA-DRB5*0101 backgrounds. Remarkably, while “fully-citrullinated” LL37 acts as better T-cell-stimulator, it loses DNA-binding ability and the associated “adjuvant-like” properties. Since LL37 undergoes a further irreversible post-translational modification, carbamylation and antibodies to carbamylated self-proteins other than LL37 are present in SLE, here we addressed the involvement of carbamylated-LL37 in autoimmunity and inflammation in SLE. We detected carbamylated-LL37 in SLE-affected tissues. Most importantly, carbamylated-LL37-specific antibodies and CD4 T-cells circulate in SLE and both correlate with disease activity. In contrast to “fully citrullinated-LL37,” “fully carbamylated-LL37” maintains both innate and adaptive immune-cells’ stimulatory abilities: in complex with DNA, carbamylated-LL37 stimulates plasmacytoid dendritic cell IFN-α production and B-cell maturation into plasma cells. Thus, we report a further example of how different post-translational modifications of a self-antigen exert complementary effects that sustain autoimmunity and inflammation, respectively. These data also show that T/B-cell responses to carbamylated-LL37 represent novel SLE disease biomarkers.