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.

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.

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.

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.