PRMT4 inhibitor TP-064 inhibits the pro-inflammatory macrophage lipopolysaccharide response in vitro and ex vivo and induces peritonitis-associated neutrophilia in vivo

Yiheng Zhang,Miriam De Boer,Ezra J Van Der Wel,Miranda Van Eck,Menno Hoekstra

doi:10.1016/j.bbadis.2021.166212

Abstract

Previous in vitro studies have shown that protein arginine N-methyltransferase 4 (PRMT4) is a co-activator for an array of cellular activities, including NF-κB-regulated pro-inflammatory responses. Here we investigated the effect of PRMT4 inhibitor TP-064 treatment on macrophage inflammation in vitro and in vivo.Exposure of RAW 264.7 monocyte/macrophages to TP-064 was associated with a significant decrease in the production of pro-inflammatory cytokines upon a lipopolysaccharide challenge. Similarly, thioglycollate-elicited peritoneal cells isolated from wildtype mice treated with TP-064 showed lowered mRNA expression levels and cytokine production of pro-inflammatory mediators interleukin (IL)-1β, IL-6, IL-12p40, and tumor necrosis factor-α in response to lipopolysaccharide exposure. However, TP-064-treated mice exhibited an ongoing pro-inflammatory peritonitis after 5 days of thioglycollate exposure, as evident from a shift in the peritoneal macrophage polarization state from an anti-inflammatory LY6ClowCD206hi to a pro-inflammatory LY6ChiCD206low phenotype. In addition, TP-064-treated mice accumulated (activated) neutrophils within the peritoneum as well as in the blood (7-fold higher; P < 0.001) and major organs such as kidney and liver, without apparent tissue toxicity. TP-064 treatment downregulated hepatic mRNA expression levels of the PRMT4 target genes glucose-6-phosphatase catalytic subunit (−50%, P < 0.05) and the cyclin-dependent kinases 2 (−50%, P < 0.05) and 4 (−30%, P < 0.05), suggesting a direct transcriptional effect of PRMT4 also in hepatocytes.In conclusion, we have shown that the PRMT4 inhibitor TP-064 induces peritonitis-associated neutrophilia in vivo and inhibits the pro-inflammatory macrophage lipopolysaccharide response in vitro and ex vivo. Our findings suggest that TP-064 can possibly be applied as therapy in NF-κB-based inflammatory diseases.

Highlights

Macrophages and their monocyte progenitors play an important role in host defense and inflammation, as well as the resolution of inflam mation and stimulation of repair, processes strongly influenced by their plasticity
Previous studies in fibroblasts have suggested that genetic protein arginine Nmethyltransferase 4 (PRMT4) deficiency is associated with a reduced protein expression of the macrophage inflammatory protein 2 (MIP-2) and interferon-γ-induced protein 10 (IP-10) after LPS exposure [8]
We found that the mRNA expression level of pro-inflammatory mediators, like CXC and CC motif ligand 1 (CXCL1; 10-fold, P < 0.001), CXC and CC motif ligand 2 (MIP-2; CXCL2; 7-fold, P < 0.05), CXC and CC motif ligand 10 (IP-10; CXCL10; 5-fold, P < 0.01), and C–C motif chemokine ligand 2 (CCL2; 5-fold, P < 0.001) were all significantly increased in the thioglycollate-elicited peritoneal macro phages isolated from TP-064-treated mice as compared to dimethyl sulfoxide (DMSO)-treated controls (Fig. 3A)

Summary

Introduction

Macrophages and their monocyte progenitors play an important role in host defense and inflammation, as well as the resolution of inflam mation and stimulation of repair, processes strongly influenced by their plasticity. Upon exposure to LPS, M1 macrophages are formed that promote inflammation, amongst others by stimulating the production of vast amounts of proinflammatory cytokines like interleukin-6 (IL-6), TNFα, IL-12, and IL1β [1,2]. These cytokines play a central role in host defense, but are on the other hand associated with chronic pathologies, including atherosclerosis, cancer, diabetes, and asthma [3,4,5].

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