TLR5 agonist entolimod reduces the adverse toxicity of TNF while preserving its antitumor effects.

Gary J Haderski,Christopher P Johnson,Venkatesh Natarajan,Ilia M Toshkov,Geraldine M Paszkiewicz,Lyudmila G Burdelya,Bojidar M Kandar,Craig M Brackett,Andrei V Gudkov,Anatoli S Gleiberman,Partha Mukhopadhyay

doi:10.1371/journal.pone.0227940

Abstract

Tumor necrosis factor alpha (TNF) is capable of inducing regression of solid tumors. However, TNF released in response to Toll-like receptor 4 (TLR4) activation by bacterial lipopolysaccharide (LPS) is the key mediator of cytokine storm and septic shock that can cause severe tissue damage limiting anticancer applications of this cytokine. In our previous studies, we demonstrated that activation of another Toll-like receptor, TLR5, could protect from tissue damage caused by a variety of stresses including radiation, chemotherapy, Fas-activating antibody and ischemia-reperfusion. In this study, we tested whether entolimod could counteract TNF-induced toxicity in mouse models. We found that entolimod pretreatment effectively protects livers and lungs from LPS- and TNF-induced toxicity and prevents mortality caused by combining either of these agents with the sensitizer, D-galactosamine. While LPS and TNF induced significant activation of apoptotic caspase 3/7, lipid tissue peroxidation and serum ALT accumulation in mice without entolimod treatment, these indicators of toxicity were reduced by entolimod pretreatment to the levels of untreated control mice. Entolimod was effective when injected 0.5-48 hours prior to, but not when injected simultaneously or after LPS or TNF. Using chimeric mice with hematopoiesis differing in its TLR5 status from the rest of tissues, we showed that this protective activity was dependent on TLR5 expression by non-hematopoietic cells. Gene expression analysis identified multiple genes upregulated by entolimod in the liver and cultured hepatocytes as possible mediators of its protective activity. Entolimod did not interfere with the antitumor activity of TNF in mouse hepatocellular and colorectal tumor models. These results support further development of TLR5 agonists to increase tissue resistance to cytotoxic cytokines, reduce the risk of septic shock and enable safe systemic application of TNF as an anticancer therapy.

Highlights

During infection, stimulation of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) from the outer membrane of gram-negative bacteria induces hematopoietic cells, macrophages, to express a variety of pro-inflammatory cytokines [1]
The toxic side effects of anticancer radiation therapy (RT) and chemotherapy have been found to be caused in part by activation of TLR4 signaling by endogenous protein HMGB1 released by activated immunocytes and tumor cells under stress conditions [4, 6, 7]
In hepatocellular carcinoma (HCC) (BNL and Hepa 1–6) tumor models, we showed that entolimod prevented mouse mortality caused by TNF combined with D-galactosamine (D-GalN) sensitization but did not diminish tumor growth suppression

Summary

Introduction

Stimulation of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) from the outer membrane of gram-negative bacteria induces hematopoietic cells, macrophages, to express a variety of pro-inflammatory cytokines [1]. LPS activation of the proinflammatory NF-кB and IFN signaling pathways in TLR4-expressing cells promotes strong production of cytotoxic cytokines such as TNF-α (TNF), IL-8, IL-6, IL-1β, IL-1, IL-12 and IFN-γ and release of reactive oxygen and nitrogen species [2, 3]. Dysregulation of the inflammatory response can lead to excessive and self-amplifying systemic cytokine release due to autocrine feedback mechanisms. This “cytokine storm” can lead to a dangerous sepsis-like syndrome in humans involving widespread inflammation, tissue damage and organ failure [2, 5]. TLR4 signaling has been reported to be involved in the hepatic immune response after RT and induction of the cytotoxic cytokines responsible for acute and chronic liver injury following various stresses [4]

Objectives

Methods

Results

Conclusion