Anti-CXCL4 Antibody Reactivity Is Present in Systemic Sclerosis (SSc) and Correlates with the SSc Type I Interferon Signature

Abstract

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.

New Autoantibody Specificities in Systemic Sclerosis and Very Early Systemic Sclerosis

“…(See Table 1, for SSc patients, VEDOSS patients, and control HD studied). In keeping with previously published work, anti-CXCL4-autoantibody reactivity was detectable in SSc and not in HD (Figure 1a) [15]. SSc subtype analysis indicated that anti-CXCL4 autoantibodies were present in both eaSSc and lsSSc.…”

“…In contrast, the eaSSc patients with pitting scars were characterized by lower anti-CXCL4 antibody reactivity, as compared to those with no pitting scars (Figure S3). These results suggest that anti-CXCL4 autoantibodies in lsSSc, but not in eaSSc, may be markers of skin inflammation, in addition to being associated with lung fibrosis, as previously reported [15]. The results seem also suggest that anti-CXCL4-, and possibly anti-CXCL4-L1-autoantibodies, may acquire pathogenic functions in the skin inflammation and lung fibrosis at late SSc-phases.…”

“…We measured the anti-CXCL4 and anti-CXCL4-L1 antibodies by ELISA, as described [15]. Briefly, 96-well flat-bottom plates (non-binding surface polystyrene, Corning, Corning, NY, USA) were coated with 2 µg/mL CXCL4, or CXCL4 L1, or with short CXCL4/CXCL4-L1 27-mer peptides (all at the same mMolar concentrations as CXCL4/CXCL4-L1) in carbonate buffer (0.1 M NaHCHO3, pH 9), for 2 h (or overnight), and subsequently washed four times with PBS 1× + 0.1% Tween-20.…”

“…To address the presence of anti-CXCL4 and anti-CXCL4-L1 antibodies in SSc, as compared to VEDOSS, we took advantage of an in house-ELISA test that we had previously set-up [15]. As control, we assessed the anti-CXCL4/CXCL4-L1 antibody reactivity in healthy donors (HD).…”

“…The CXCL4 capacity to condense DNA/RNA in nanocrystalline structures could facilitate anti-CXCL4 autoantibody generation in SSc, as particulate structures confer antigenicity to otherwise poorly immunogenic molecules [13,14], and in fact, we have demonstrated that CXCL4 acts as an autoantigen in a consistent proportion of SSc patients. Most importantly, anti-CXCL4 autoantibodies correlate with the IFN-α signature [15].…”