Inhibition of peptidyl arginine deiminase-4 ameliorated pulmonary fibrosis via modulating M1/M2 polarisation of macrophages

Abstract

Pulmonary fibrosis (PF) arises from dysregulated wound healing, leading to excessive extracellular matrix (ECM) deposition and impaired lung function. Macrophages exhibit high plasticity, polarizing to pro-inflammatory M1 during early inflammation and anti-inflammatory, fibrosis-inducing M2 during later stages of PF. Additionally, neutrophils and neutrophil extracellular traps (NETs) release mediated by peptidyl arginine deiminase (PAD-4), also play a key role in PF progression. PAD-4 inhibitor chloro-amidine (CLA) has shown anti-fibrotic effects in bleomycin (BLM) induced PF mouse model in our earlier study. Here, we have demonstrated that CLA also exhibited inhibition of macrophage polarisation in in-vitro in THP-1 monocytes and in-vivo in BLM induced PF. THP-1 monocytes were exposed to NETs isolated from phorbol 12-myristate-13-acetate (PMA) stimulated and PMA plus CLA treated differentiated HL-60 (dHL-60) cells. Monocytes exposed to stimulated NETs resulted in increased oxidative stress, disrupted mitochondrial membrane potential and increased M1 and M2 macrophage markers. These alterations were abrogated in THP-1 cells upon exposure to CLA treated NETs. Further, CLA treatment in BLM induced mice improved abnormal BALF, biochemical, and histological parameters in line with our previous findings. Additionally, CLA also reduced M1 and M2 markers time-dependently, as shown by immunofluorescence (IF), western blot, and RT-PCR analysis. CLA treatment led to decreased expression of PAD-4, M1-related pro-inflammatory cytokines and M2-related pro-fibrotic cytokines and mediators, as confirmed by western blot and ELISA analysis. Thus, it is established that inhibition of PAD-4 lead to mitigation of macrophage polarisation and a combined anti-fibrotic effect is achieved which can be explored further.