Activation of JUN in fibroblasts promotes pro-fibrotic programme and modulates protective immunity

Introduction

Pulmonary fibrosis is a serious lung disease characterised by the spontaneous buildup of scar tissue in the absence of infectious or autoimmune diseases. It has a poor prognosis, with a 50% three-year survival rate, and currently, lung transplantation is the only curative treatment option. Despite the identification of genetic factors such as telomerase mutations, the exact cause of the disease remains unknown.

Fibroblasts and Macrophages in Pulmonary Fibrosis

• Fibroblasts are central to the fibrotic response, but comprehensive single-cell data on this heterogeneous population in pulmonary fibrosis is still lacking. Many fibroblast markers only identify small subsets of fibroblasts.
• Monocytes and macrophages are believed to play critical roles in injury and repair due to their involvement in innate immunity. Macrophage heterogeneity has also been implicated in fibrosis progression.

Study Objective and Methodology

In this study, the authors used single-cell mass cytometry and immunostaining to identify the specific immune mechanisms that promote fibrosis. They also proposed a therapeutic approach that could complement conventional anti-fibrotic drugs for the treatment of pulmonary fibrosis.

Key Findings

• Fibroblast frequency was five-fold higher in fibrotic lungs (80%) compared to normal lungs (15%). Principal component analysis revealed both an increased percentage and phenotypic differences in fibroblasts in fibrotic lungs compared to control lung fibroblasts.
  A distinct subset of fibroblasts specific to fibrotic lungs was identified, expressing high levels of CD47 and podoplanin.
• Immunofluorescent staining detected co-expression of CD47 with fibroblast-specific protein-1 (FSP1) and PD-L1 with smooth muscle actin (αSMA) in fibroblasts, indicating the upregulation of immune checkpoint proteins in the fibrotic lung.
• High-dimensional mass cytometry demonstrated co-activation of phospho JUN and AKT in patients with pulmonary fibrosis.
• The ratio of interstitial macrophages to alveolar macrophages differed significantly between fibrotic and normal lungs, suggesting a distinct composition of macrophages during fibrosis. Moreover, increased regulatory and exhausted T cells in human lungs suggest an immunosuppressive microenvironment in lung fibrosis.
• Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), doxycycline-inducible overexpression, and CRISPR-editing loss-of-function experiments indicated that JUN acts as an enhancer selector, modulating DNA accessibility in fibroblasts and potentially influencing profibrotic TGFbeta and Stat3 signalling pathways.
• Activation of JUN in lung fibroblasts promoted profibrotic programs and modulated protective immunity in a bleomycin-induced model of lung fibrosis. Conversely, immune checkpoint treatment prevents lung fibrosis.
• Blocking IL-6, CD47, and PD-L1 revealed two distinct mechanisms by which fibrosis was reversed in the same animal model, suggesting JUN signalling as a potential target for therapeutic intervention in pulmonary fibrosis.

Conclusion

Pulmonary fibrosis is a progressive and often fatal lung disease with limited curative treatments and a poor understanding of its underlying mechanisms. This study identifies several pathways that may contribute to disease progression. These pathways have already been well characterised in other medical indications, suggesting the possibility of therapeutic interventions. The significant data presented in this study could pave the way for novel drug development and combination therapies critical in halting the progression of this devastating disease.

Services

Epistem is a GCLP-accredited laboratory specialising in providing biomarker, target discovery and personalised medicine information. We offer microarray, qPCR and NGS services (including the 10X genomics platform for single-cell sequencing) for gene expression, whole-genome, and epigenetic analysis in all species. Our team can help you with your liver, kidney and lung fibrosis-related enquiries.

The Histology and Immunohistochemistry team at Epistem have a wealth of experience in developing IHC protocols for a range of biological targets. Our deep understanding of IHC methodology has allowed us to develop and troubleshoot hundreds of working protocols for both automated (Ventana Discovery Ultra) and manual applications. We offer a bespoke service which can be suited to your project. We can work readily with FFPE, frozen tissues, with chromogenic and fluorescent detection systems as well as RNAScope methodologies.