Bioactive Plasma Mitochondrial DNA Is Associated With Disease Progression in Scleroderma-Associated Interstitial Lung Disease.

OBJECTIVE: Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is characterized by variable clinical outcomes, activation of innate immune pattern-recognition receptors (PRRs), and accumulation of α-smooth muscle actin (α-SMA)-expressing myofibroblasts. The aim of this study was to identify an association between these entities and mitochondrial DNA (mtDNA), an endogenous ligand for the intracellular DNA-sensing PRRs Toll-like receptor 9 (TLR-9) and cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING), which has yet to be determined. METHODS: Human lung fibroblasts (HLFs) from normal donors and SSc-ILD explants were treated with synthetic CpG DNA and assayed for α-SMA expression and extracellular mtDNA using quantitative polymerase chain reaction for the human MT-ATP6 gene. Plasma MT-ATP6 concentrations were evaluated in 2 independent SSc-ILD cohorts and demographically matched controls. The ability of SSc-ILD and control plasma to induce TLR-9 and cGAS/STING activation was evaluated with commercially available HEK 293 reporter cells. Plasma concentrations of type I interferons (IFNs), interleukin-6 (IL-6), and oxidized DNA were measured using electrochemiluminescence and enzyme-linked immunosorbent assay-based methods. Extracellular vesicles (EVs) precipitated from plasma were evaluated for MT-ATP6 concentrations and proteomics via liquid chromatography mass spectrometry. RESULTS: Normal HLFs and SSc-ILD fibroblasts developed increased α-SMA expression and MT-ATP6 release following CpG stimulation. Plasma mtDNA concentrations were increased in the 2 SSc-ILD cohorts, reflective of ventilatory decline, and were positively associated with both TLR-9 and cGAS/STING activation as well as type I IFN and IL-6 expression. Plasma mtDNA was not oxidized and was conveyed by EVs displaying a proteomics profile consistent with a multicellular origin. CONCLUSION: These findings demonstrate a previously unrecognized connection between EV-encapsulated mtDNA, clinical outcomes, and intracellular DNA-sensing PRR activation in SSc-ILD. Further study of these interactions could catalyze novel mechanistic and therapeutic insights into SSc-ILD and related disorders.

Serum biomarker for diagnostic evaluation of pulmonary arterial hypertension in systemic sclerosis.

BACKGROUND: Systemic sclerosis-associated pulmonary arterial hypertension (SSc-PAH) is one of the leading causes of death in SSc. Identification of a serum-based proteomic diagnostic biomarker for SSc-PAH would allow for rapid non-invasive screening and could positively impact patient survival. Identification and validation of novel proteins could potentially facilitate the identification of SSc-PAH, and might also point to important protein mediators in pathogenesis. METHODS: Thirteen treatment-naïve SSc-PAH patients had serum collected at time of diagnosis and were used as the discovery cohort for the protein-expression biomarker. Two proteins, Midkine and Follistatin-like 3 (FSTL3) were then validated by enzyme-linked immunosorbent assays. Midkine and FSTL3 were tested in combination to identify SSc-PAH and were validated in two independent cohorts of SSc-PAH (n = 23, n = 11). RESULTS: Eighty-two proteins were found to be differentially regulated in SSc-PAH sera. Two proteins (Midkine and FSTL3) were also shown to be elevated in publicly available data and their expression was evaluated in independent cohorts. In the validation cohorts, the combination of Midkine and FSTL3 had an area under the receiver operating characteristic curve (AUC) of 0.85 and 0.92 with respective corresponding measures of sensitivity of 76% and 91%, and specificity measures of 76% and 80%. CONCLUSIONS: These findings indicate that there is a clear delineation between overall protein expression in sera from SSc patients and those with SSc-PAH. The combination of Midkine and FSTL3 can serve as an SSc-PAH biomarker and are potential drug targets for this rare disease population.

A longitudinal biomarker for the extent of skin disease in patients with diffuse cutaneous systemic sclerosis.

OBJECTIVE: To define a pharmacodynamic biomarker based on gene expression in skin that would provide a biologic measure of the extent of disease in patients with diffuse cutaneous systemic sclerosis (dcSSc) and could be used to monitor skin disease longitudinally. METHODS: Skin biopsy specimens obtained from a cohort of patients with dcSSc (including longitudinal specimens) were analyzed by microarray. Expression of genes correlating with the modified Rodnan skin thickness score (MRSS) were examined for change over time using a NanoString platform, and a generalized estimating equation (GEE) was used to define and validate longitudinally measured pharmacodynamic biomarkers composed of multiple genes. RESULTS: Microarray analysis of genes parsed to include only those correlating with the MRSS revealed prominent clusters of profibrotic/transforming growth factor ß-regulated, interferon-regulated/proteasome, macrophage, and vascular marker genes. Using genes changing longitudinally with the MRSS, we defined 2 multigene pharmacodynamic biomarkers. The first was defined mathematically by applying a GEE to longitudinal samples. This modeling method selected cross-sectional THBS1 and longitudinal THBS1 and MS4A4A. The second model was based on a weighted selection of genes, including additional genes that changed statistically significantly over time: CTGF, CD163, CCL2, and WIF1. In an independent validation data set, biomarker levels calculated using both models correlated highly with the MRSS. CONCLUSION: Skin gene expression can be used effectively to monitor changes in SSc skin disease over time. We implemented 2 relatively simple models on a NanoString platform permitting highly reproducible assays that can be applied directly to samples from patients or collected as part of clinical trials.