Polarization of Low-Grade Inflammatory Monocytes Through TRAM-Mediated Up-Regulation of Keap1 by Super-Low Dose Endotoxin.

Allison Rahtes,Liwu Li

doi:10.3389/fimmu.2020.01478

Abstract

Subclinical endotoxemia [low levels of bacterial endotoxin (LPS) in the blood stream] has been correlated with chronic inflammatory diseases, with less-understood mechanisms. We have previously shown that chronic exposure to super low doses of LPS polarizes monocytes/macrophages to a pro-inflammatory state characterized by up-regulation of pro-inflammatory regulators such as p62 and simultaneous down-regulation of anti-inflammatory/resolving regulators such as Nrf2. Building upon this observation, here we show that chronic exposure to super-low doses of LPS leads to accumulation of the Nrf2-inhibitory protein Keap1 in murine monocytes. This is accompanied by increases of p62 and MLKL, consistent with a disruption of autolysosome function in polarized monocytes challenged by super-low dose LPS. Monocytes subjected to persistent super-low dose LPS challenge also accumulate higher levels of IKKβ. As a consequence, SLD-LPS challenge leads to an inflammatory monocyte state represented by higher expression of the inflammatory marker Ly6C as well as lower expression of the anti-inflammatory marker CD200R. Further analysis revealed that Keap1 levels are significantly enriched in the Ly6Chi pro-inflammatory monocyte population. Finally, we show that the TLR4 signaling adaptor TRAM is essential for these effects. Together our study provides novel insight into signaling mechanisms behind low-grade inflammatory monocyte polarization unique to chronic super-low dose LPS exposure.

Highlights

Emerging studies reveal dynamic programming processes of innate leukocytes by varying dosages of bacterial endotoxin Lipopolysaccharide (LPS) [1,2,3]
To further explore the molecular mechanism behind this phenomenon we examined the effects of chronic Subclinical low-dose LPS (SLD-LPS) exposure on Nrf2’s regulatory protein Keap1 using our previously developed chronic exposure 5-days bonemarrow-derived monocytes (BMDMs) model, which mimics the establishment of a non-resolving low-grade inflammatory state [20]
We observed that cells treated with chronic SLD-LPS (100 pg/mL) had the opposite effect, exhibiting a significant increase in Keap1 expression as compared to cells treated with PBS or high-dose (1 μg/mL) LPS (Figure 1A) To further determine whether this novel Keap1 accumulation was due to effects at the protein or transcript level, we performed qRT-PCR to assess the effects of chronic SLD-LPS on keap1 transcription and observed that SLD-LPS lead to a significant increase in keap1 mRNA levels, which was observed in high-dose LPS treated cells (Figure 1B)

Summary

Introduction

Emerging studies reveal dynamic programming processes of innate leukocytes by varying dosages of bacterial endotoxin Lipopolysaccharide (LPS) [1,2,3]. Monocytes/macrophages persistently challenged with subclinical low-dose LPS (100 pg−1 ng/mL) are programmed into a low-grade inflammatory state characterized by sustained expression of pro-inflammatory mediators [4]. Monocytes/macrophages challenged with prolonged high doses of LPS have shown increased compensatory resolving processes including autolysomemediated degradation of p62 and IKKβ [8] as well as the upregulation of anti-oxidative responses through the induction of Nrf2 [9]. SLD-LPS treatment reduces the activity of the anti-inflammatory mediator Nrf, which may collectively contribute to the establishment of non-resolving low-grade inflammatory state [11]. Cells challenged with high dose LPS exhibit reduced Keap levels, potentially through lysosome-mediated degradation, which correlates with elevated Nrf and anti-inflammatory compensation [13]. Given the knowledge gap regarding the effects of SLD-LPS, our current study will focus on examining the effects of SLD-LPS on Keap regulation in primary monocytes

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