CXCL4 assembles DNA into liquid crystalline complexes to amplify TLR9-mediated interferon-α production in systemic sclerosis

Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by fibrosis and vasculopathy. CXCL4 represents an early serum biomarker of severe SSc and likely contributes to inflammation via chemokine signaling pathways, but the exact role of CXCL4 in SSc pathogenesis is unclear. Here, we elucidate an unanticipated mechanism for CXCL4-mediated immune amplification in SSc, in which CXCL4 organizes “self” and microbial DNA into liquid crystalline immune complexes that amplify TLR9-mediated plasmacytoid dendritic cell (pDC)-hyperactivation and interferon-α production. Surprisingly, this activity does not require CXCR3, the CXCL4 receptor. Importantly, we find that CXCL4-DNA complexes are present in vivo and correlate with type I interferon (IFN-I) in SSc blood, and that CXCL4-positive skin pDCs coexpress IFN-I-related genes. Thus, we establish a direct link between CXCL4 overexpression and the IFN-I-gene signature in SSc and outline a paradigm in which chemokines can drastically modulate innate immune receptors without being direct agonists.


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Policy information about availability of computer code Data collection Images were taken with the built-in softwares of Leica TCS SP2, FV1000 Olympus, Zeiss 510 confocal microscopes as described in Methods. Flow cytometry data were collected with a BD FACSDiva sofware. SAXS experiments were performed at the Stanford Synchrotron Radiation Lightsource (SSRL. Beamline 4-2).

Data analysis
Confocal images were analysed by a Leica software 2.6 and by ZEISS 510 software. Flow cytometry data were analysed by FlowJo 7.6.5. Xray data was analysed using Igor Pro 7 and Mathematica 11 as detailed in Methods.
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Sample size
We did not use any statistical method to predetermine sample size for patients because the patients' cohorts were composed of previously enrolled subjects by the clinicians. Sample sizes are similar to those used in previous published clinical studies.
Data exclusions No data were excluded Replication About SAXS data, Independent identical samples were prepared and measured over multiple separate experiments (n ≥ 3) as detailed in Methods. CXCL4 levels, and related correlations, were measured by ELISA in the plasma of SSc (Discovery cohort) and replicated in the sera of a second cohort of SSc patients (Replication cohort) as specified in the figure legends. In vitro experiments were confirmed by multiple replicates as detailed in the figure legends.

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Validation anti-S100A8/MRP8 (ABF125, Merck Millipore) was validated for use in WB, IHC and Activity Assay by the manufacturer. anti-CXCR3 clone 49801 (R&D) was validated for neutralization activity by the manufacturer. anti-CXCL4 rabbit polyclonal (ab9561, Abcam) is validated by the manufacturer for WB, ELISA, inhibition of the biological activity of PF4, immunostaining on paraffinated tissue sections. Anti-CD32 (clone AT10, Abcam) is validated for blocking and neutralising assay by the manufacturer. Fluorochrome-conjugated anti-CXCR3 (clone 1C6, BD Biosciences) is validated for flow cytometry. Anti-BDCA2 (AF1376, R&D) is validated for WB, flow cytometry and immunostaining on tissues. Anti-Mx1 (clone OTI2G12, Novus Biologicals) is validated for WB, immunofluorescence and immunoistochemistry Population characteristics characteristics of these subjects are detailed in Supplementary Table 1. Patients with SSc satisfied the ACR/EULAR 2013 classification criteria for SSc. This study was approved by the ethical committee of the institutions involved ("Commission cantonale d'éthique de la recherche", Geneva, Switzerland; the Ethics Committee of the Sapienza University, Rome, and University Hospital of Bordeaux, France) and was conducted according to the Declaration of Helsinki. Informed, written consent was obtained from all participants according to the declaration of Helsinki.

Ethics oversight
This study was approved by the ethical committee of the institutions involved ("Commission cantonale d'éthique de la recherche", Geneva, Switzerland; the Ethics Committee of the Sapienza University, Rome, and University Hospital of Bordeaux, France) and was conducted according to the Declaration of Helsinki. Informed, written consent was obtained from all participants according to the declaration of Helsinki.
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Flow Cytometry
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Methodology
Sample preparation -Buffy coat from healthy donors were provided by the Blood center in Lausanne (CH) and Rome (IT). Plasmacytoid cells (PDC) were purified from buffy coat by using Diamond Plasmacytoid Dendritic Cell Isolation II Kit (Miltenyi Biotec) to obtain 99% of purity.
-For SAXS experiments, E. coli DNA was ethanol precipitated and resuspended in physiological buffer (140 mM NaCl + 10 mM HEPES, pH 7.4) to 5 mg/ml. HuDNA was used directly unfragmented or fragmented by sonication. Self-assembled protein-DNA complexes were formed by incubating the CXCL4 or LL37 with DNA at isoelectric peptideto-DNA charge ratios (P/DNA = 1/1) in microcentrifuge tubes. Complexes were vortexed at low speeds (900 RPM) for 1 hour or until strong precipitates formed. After thorough mixing and centrifugation, precipitated complexes are transferred to 1.5 mm quartz capillaries (Hilgenberg GmbH, Mark-tubes) and hermetically sealed using an oxygen torch. The structures of CXCL4-DNA and related complexes were solved using SAXS.

Instrument
Flow cytometry data were collected by FACSCanto (BD). For confocal microscopy images a FV1000 Olympus (Tokyo, Japan) and a Leica TCS SP2 apparatus were used.
For SAXS experiments we used the Stanford Synchrotron Radiation Lightsource (SSRL, Beamline 4-2) using monochromatic X-rays with an energy of 9 keV. A Rayonix MX225-HE detector (pixel size 73.2 μm) was used to measure the scattered radiation Software Flow cytometry data were acquired by a FACSDiva software (BD), and analyzed by FlowJo (version 10.0.7, BD) Cell population abundance Plasmacytoid cells were purified from buffy coat by using Diamond Plasmacytoid Dendritic Cell Isolation Kit (Miltenyi Biotec) to obtain 99% pure pDCs. Purity was evaluated by staining with fluorochrome-conjugated anti-CD123 and anti-BDCA2 antibodies.

Gating strategy
All cells purified by Diamond Plasmacytoid Dendritic Cell Isolation Kit were plasmacytoid dendritic cells as previously described.
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