The Cell-Permeable Derivative of the Immunoregulatory Metabolite Itaconate, 4-Octyl Itaconate, Is Anti-Fibrotic in Systemic Sclerosis.

Sharadha Dayalan Naidu,Stefan Przyborski,Albena T Dinkova-Kostova,John Henderson,Steven O′Reilly,Richard Stratton

doi:10.3390/cells10082053

Abstract

Systemic sclerosis (SSc) is an autoimmune connective tissue disease that leads to skin fibrosis. Altered metabolism has recently been described in autoimmune diseases and SSc. Itaconate is a product of the Krebs cycle intermediate cis-aconitate and is an immunomodulator. This work examines the role of the cell-permeable derivative of itaconate, 4-octyl itaconate (4-OI), in SSc. SSc and healthy dermal fibroblasts were exposed to 4-OI. The levels of collagen Nrf2-target genes and pro-inflammatory cytokines interleukin 6 (IL-6) and monocyte chemotactic protein 1 (MCP-1) were determined. Levels of reactive oxygen species (ROS) as well as the gene expression of collagen and Cellular Communication Network Factor 2 (CCN2) were measured after transforming growth factor beta 1 (TGF-β1) stimulation in the presence or absence of 4-OI. Wild-type or Nrf2-knockout (Nrf2-KO) mouse embryonic fibroblasts (MEFs) were also treated with 4-OI to determine the role of Nrf2 in 4-OI-mediated effects. 4-OI reduced the levels of collagen in SSc dermal fibroblasts. Incubation with 4-OI led to activation of Nrf2 and its target genes heme oxygenase 1 (HO-1) and NAD(P)H quinone oxidoreductase 1 (NQO1). 4-OI activated antioxidant response element (ARE)-dependent gene expression, reduced inflammatory cytokine release and reduced TGF-β1-induced collagen and ROS production in dermal fibroblasts. The effects of 4-OI are dependent on Nrf2. The cell-permeable derivative of itaconate 4-OI is anti-fibrotic through upregulation of Nrf2 and could be a potential therapeutic option in an intractable disease.

Highlights

Systemic sclerosis (SSc) is an autoimmune idiopathic connective tissue disease that is characterised by vascular abnormalities, inflammation and fibrosis [1,2]
We recently showed that the cell-permeable itaconate derivative 4-octyl itaconate (4-OI) reduced collagen in dermal fibroblasts from diffuse SSc patients [13], and we sought to examine that further
To confirm that 4-OI induces antioxidant response element (ARE)-dependent transcription, we transfected dermal fibroblasts with an ARE-driven luciferase reporter, and we could see a significant increase in ARE-luciferase activity by 2-fold (p = 0.0001, Student’s t-test, n = 4) (Figure 1C)

Summary

Introduction

Systemic sclerosis (SSc) is an autoimmune idiopathic connective tissue disease that is characterised by vascular abnormalities, inflammation and fibrosis [1,2]. Several studies have suggested a role of the immune system in the activation of the fibroblasts to differentiate into myofibroblasts that secrete the excessive extracellular matrix that underpins the main pathology of the disease [1]. The activated fibroblasts secrete exuberant levels of extracellular matrix (ECM), become contractile and resistant to cell apoptosis, and this results in fibrosis [3]. Emerging data have suggested a role for immunometabolism in inflammatory diseases [4]. Numerous studies have suggested alterations in cellular metabolism can lead to inflammatory diseases, including, among others, rheumatoid arthritis [5]. A recently described metabolite derived from the Krebs cycle, which is generated by the enzyme aconitate decarboxylase 1 (ACOD1) ( known as IRG1), is enormously upregulated after TLR stimulation [6] in immune cells [7]. There are tiny amounts of itaconate, but upon exposure to lipopolysaccharide (LPS), this can be released in millimolar levels [8]

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