Hsp70 and NF-kB Mediated Control of Innate Inflammatory Responses in a Canine Macrophage Cell Line.

Qingkang Lyu,Femke Broere,Willem Van Eden,Victor P M G Rutten,Alice J A M Sijts,Magdalena Wawrzyniuk

doi:10.3390/ijms21186464

Qingkang Lyu, Femke Broere + Show 4 more

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https://doi.org/10.3390/ijms21186464

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Abstract

The pathogenesis of many inflammatory diseases is associated with the uncontrolled activation of nuclear factor kappa B (NF-κB) in macrophages. Previous studies have shown that in various cell types, heat shock protein 70 (Hsp70) plays a crucial role in controlling NF-κB activity. So far, little is known about the role of Hsp70 in canine inflammatory processes. In this study we investigated the potential anti-inflammatory effects of Hsp70 in canine macrophages as well as the mechanisms underlying these effects. To this end, a canine macrophage cell line was stressed with arsenite, a chemical stressor, which upregulated Hsp70 expression as detected by flow cytometry and qPCR. A gene-edited version of this macrophage cell line lacking inducible Hsp70 was generated using CRISPR-Cas9 technology. To determine the effects of Hsp70 on macrophage inflammatory properties, arsenite-stressed wild-type and Hsp70 knockout macrophages were exposed to lipopolysaccharide (LPS), and the expression of the inflammatory cytokines IL-6, IL-1β and tumor necrosis factor-α (TNF-α) and levels of phosphorylated NF-κB were determined by qPCR and Western Blotting, respectively. Our results show that non-toxic concentrations of arsenite induced Hsp70 expression in canine macrophages; Hsp70 upregulation significantly inhibited the LPS-induced expression of the pro-inflammatory mediators TNF-α and IL-6, as well as NF-κB activation in canine macrophages. Furthermore, the gene editing of inducible Hsp70 by CRISPR-Cas9-mediated gene editing neutralized this inhibitory effect of cell stress on NF-κB activation and pro-inflammatory cytokine expression. Collectively, our study reveals that Hsp70 may regulate inflammatory responses through NF-κB activation and cytokine expression in canine macrophages.

Highlights

Macrophages constitute a major component of the innate immune response
The abnormal activation of NF-κB in macrophages leads to excessive pro-inflammatory cytokine expression, as can be seen in many inflammatory diseases, such as rheumatoid arthritis [11], neurodegenerative diseases [28], inflammatory bowel disease [29] and type I diabetes [30]
Previous tudies showed that heat shock protein 70 (Hsp70) was implicated in inflammatory responses by modulating NF-κB activity [31,32,33]

Summary

Introduction

Macrophages constitute a major component of the innate immune response. They are originally derived from myeloid progenitors [1] and ubiquitously distributed throughout the body tissues, including lung, liver, gut and brain, comprising the innate defense against pathogen invasion and tissue damage. Macrophage activation, which is associated with an inflammatory response, induces the production and secretion of a variety of inflammatory mediators, including interleukin-6 (IL-6), IL-1β, tumor necrosis factor-α (TNF-α) and nitric oxide (NO) [3,4]. The release of those inflammatory mediators tunes the immune response, facilitates the resolution of inflammation, and protects the organism from pathogen invasion. The modulation of those inflammatory mediators may have a potentially therapeutic effect on severe disease-associated inflammation

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