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

ABSTRACT: The pathogenesis of many inflammatory diseases is associated with the uncontrolled activation of nuclear factor kappa B (NF-?B) in macrophages. Previous studies have shown that in various cell types, heat shock protein 70 (Hsp70) plays a crucial role in controlling NF-?B 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-1? and tumor necrosis factor-? (TNF-?) and levels of phosphorylated NF-?B 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-? and IL-6, as well as NF-?B 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-?B activation and pro-inflammatory cytokine expression. Collectively, our study reveals that Hsp70 may regulate inflammatory responses through NF-?B activation and cytokine expression in canine macrophages.

SUBMITTER: Lyu Q

PROVIDER: S-EPMC7555705 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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

Lyu Qingkang Q Wawrzyniuk Magdalena M Rutten Victor P M G VPMG van Eden Willem W Sijts Alice J A M AJAM Broere Femke F

International journal of molecular sciences 20200904 18

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 underlyin ...[more]

PMID: 32899721

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Project description:BackgroundNeuroinflammation is the response of the central nervous system to events that interfere with tissue homeostasis and represents a common denominator in virtually all neurological diseases. Activation of microglia, the principal immune effector cells of the brain, contributes to neuronal injury by release of neurotoxic products. Toll-like receptor 4 (TLR4), expressed on the surface of microglia, plays an important role in mediating lipopolysaccharide (LPS)-induced microglia activation and inflammatory responses. We have previously shown that curcumin and some of its analogues harboring an α,β-unsaturated 1,3-diketone moiety, able to coordinate the magnesium ion, can interfere with LPS-mediated TLR4-myeloid differentiation protein-2 (MD-2) signaling. Fluoroquinolone (FQ) antibiotics are compounds that contain a keto-carbonyl group that binds divalent ions, including magnesium. In addition to their antimicrobial activity, FQs are endowed with immunomodulatory properties, but the mechanism underlying their anti-inflammatory activity remains to be defined. The aim of the current study was to elucidate the molecular mechanism of these compounds in the TLR4/NF-κB inflammatory signaling pathway.MethodsThe putative binding mode of five FQs [ciprofloxacin (CPFX), levofloxacin (LVFX), moxifloxacin, ofloxacin, and delafloxacin] to TLR4-MD-2 was determined using molecular docking simulations. The effect of CPFX and LVFX on LPS-induced release of IL-1β and TNF-α and NF-κB activation was investigated in primary microglia by ELISA and fluorescence staining. The interaction of CPFX and LVFX with TLR4-MD-2 complex was assessed by immunoprecipitation followed by Western blotting using Ba/F3 cells.ResultsCPFX and LVFX bound to the hydrophobic region of the MD-2 pocket and inhibited LPS-induced secretion of pro-inflammatory cytokines and activation of NF-κB in primary microglia. Furthermore, these FQs diminished the binding of LPS to TLR4-MD-2 complex and decreased the resulting TLR4-MD-2 dimerization in Ba/F3 cells.ConclusionsThese results provide new insight into the mechanism of the anti-inflammatory activity of CPFX and LVFX, which involves, at least in part, the activation of TLR4/NF-κB signaling pathway. Our findings might facilitate the development of new molecules directed at the TLR4-MD-2 complex, a potential key target for controlling neuroinflammation.

Dataset Information

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

Publications

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

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