Major Grant
on ILD

Project Title: Circulating Cell-Free DNA as a Biomarker for Assessing Interstitial Lung Disease in Systemic Sclerosis Patients

Projcet Summary : Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is a leading cause of morbidity and mortality in systemic sclerosis, marked by progressive lung inflammation and fibrosis. Current tools to monitor disease activity, such as high-resolution CT (HRCT) and pulmonary function tests (PFTs), are limited by low sensitivity to early changes, radiation exposure, and interobserver variability. There is an urgent need for dynamic, minimally invasive biomarkers that can capture real-time disease evolution and guide treatment decisions. We hypothesize that circulating cell-free DNA (ccfDNA), particularly lung-derived fragments, reflects ongoing lung inflammation and fibrosis in SSc-ILD and correlates with disease severity, progression, and response to therapy. By analysing ccfDNA fragmentation patterns and epigenetic signatures, we aim to identify specific biomarkers to enable real-time monitoring and refined risk stratification. The main objectives of this project are to quantify lung-derived ccfDNA levels in SSc-ILD patients compared to healthy controls, evaluate its correlation with HRCT and PFT parameters, and potentially determine its predictive value for disease progression. Additionally, we aim to assess dynamic changes in ccfDNA during immunosuppressive and/or anti-fibrotic treatment, evaluating whether these changes reflect clinical response. If successful, this project could pave the way for incorporating lung-derived ccfDNA into both clinical practice and future SSc-ILD trials, offering a non-invasive tool to guide personalized treatment strategies, reduce reliance on imaging, and improve patient outcomes through more timely and precise interventions.

Project Title: IL-6 regulation of the CCL18 basic helix-loop-helix enhancer in profibrotic macrophages in scleroderma-interstitial lung
disease

Project Summary:
Multiple studies, including our own, suggest that a subset of lung macrophages, marked by expression of SPP1 and
CCL18, release of soluble mediators that promote fibrosis in scleroderma-associated interstitial lung disease (SSc-ILD).
These SPP1-expressing macrophages drive myofibroblast differentiation and secrete chemokines, notably CCL2 and
CCL18 that attract inflammatory cells, including monocyte derived macrophages. In recent work, we have defined
altered chromatin accessibility and thereby inferred altered transcription factor (TF) activity that regulate the profibrotic
macrophage phenotype. We hypothesize that basic helix-loop-helix (bHLH) TFs regulating profibrotic SPP1-expressing
macrophage chromatin structure mediate signals from IL-6 to alter profibrotic macrophage gene expression and
phenotype. Tocilizumab, an IL-6 receptor inhibitor, is an approved SSc therapy that blocks strikingly upregulated levels
of circulating CCL18 seen in SSc-ILD patients. Our previous scRNA-seq studies show that profibrotic macrophages are
the main cell type expressing and upregulating CCL18. Our recent unpublished multiome-seq studies show that the
proximal CCL18 promoter harbors a duplicated consensus motif for the bHLH family of TFs and infer strongly increased
TF binding to this motif in SSc-ILD profibrotic macrophages. Thus, IL-6 appears to activate macrophage CCL18
expression by promoting TF binding to this motif, providing a paradigm for understanding profibrotic macrophage
activation. We propose to define the specific bHLH transcription factor regulating the CCL18 promoter upon IL-6
stimulation. Further, we propose to define the upstream signaling leading to TF binding to the CCL18 enhancer by
inhibiting known IL-6 signaling mediators in SSc-ILD macrophages and IL-6 stimulated primary cultures from heathy
lungs.

Project Title: Investigating the role of fibroblast trained immunity in the pathogenesis of systemic sclerosis associated pulmonary fibrosis.

Project Summary: Fibroblasts shift between immunosuppressive and inflammatory states and may be persistently altered by inflammatory stimuli, in a process known as
trained immunity. Trained immunity is classically associated with innate/myeloid cells, interestingly, fibroblasts also exhibit memory-like responses.
Examples include transcriptional and epigenetic modifications, enhanced functional response to subsequent stimuli and metabolic alterations, leading
to negative pathological outcomes. Importantly, how trained immunity in lung fibroblasts influences lung fibrosis development and whether this
process holds therapeutic promise, has not been addressed. Crosstalk between immune cells and fibroblasts drives SSc pathogenesis. Immune-derived
cytokines activate fibroblasts, leading to fibrosis. The NFκB transcription factor cRel/REL orchestrates epigenetic and metabolic changes in immune cells,
implicating this molecule as a putative regulator of trained immunity. Our preliminary data show conditional myeloid cell deletion of cRel reduced
fibrosis in mouse models, suggesting cRel/REL signalling is crucial for fibroblast-immune cell interactions in lung fibrosis. We have shown REL is
constitutively active in SSc patient fibroblasts and is associated with a distinct transcriptional profile linked to fibrosis. Inhibiting REL with IT603 reduced
fibrotic markers in human dermal fibroblasts. Furthermore, SSc-ILD myofibroblasts have elevated REL levels and expression in the lung is localised in
immune cells and fibroblastic foci. Our preliminary findings indicate that trained immunity in lung fibroblasts, driven by cRel/REL, may contribute to lung
fibrosis, representing a potential therapeutic target. This project employs a multifaceted approach integrating transcriptomics, metabolomics, and
spatial analysis to unravel the role of fibroblast trained immunity in lung fibrosis, focusing on translating findings into potential treatments.

Project Title: To B or not to B: the Topoisomerase-1 B cell response and progression of interstitial lung disease in Systemic Sclerosis.
Project Summary: Systemic Sclerosis (SSc) is hallmarked by the presence of different anti-nuclear antibodies, each associated with distinct clinical phenotypes. Antitopoisomerase-1 antibody (ATA)+ SSc patients frequently develop interstitial lung disease (ILD), the leading cause of death in this population. We and others
have demonstrated that ATA of different isotypes associate with disease progression. Moreover, we observed that the frequency of circulating, ATA-IgGsecreting B cells correlate with the extend and severity of ILD. These findings, together with the first reports on the successes of CD19 CAR T-cell therapy in
SSc, indicate that the autoreactive B cell response drives disease activity and the development/progression of ILD. Here, we aim to understand the
contribution of the ATA B cell response to disease, focusing on the development and progression of ILD. Specifically, we will analyze patient ATA clonal
repertoires and their ability to differentiate patients with and without ILD using a novel, ATA-specific mass spectrometry-based IgG1 Fab profiling approach.
Furthermore, ATA-expressing B cells will be phenotypically and functionally analyzed in relation to patient ILD status using spectral flow cytometry. Together,
we expect to define (1) the molecular make-up of ATA and ATA-expressing B cell responses in relation to ILD development, (2) the potential of assessing (the
activity of) the ATA B cell response as biomarker for disease progression and ILD-risk stratification (to be substantiated in subsequent prospective studies),
and (3) possible novel targets rendering the ATA B cell response amenable for therapeutic intervention.