Pharmacological Targeting of BMP6-SMAD Mediated Hepcidin Expression Does Not Improve the Outcome of Systemic Infections With Intra-Or Extracellular Gram-Negative Bacteria in Mice

Alexander Hoffmann,Philipp Grubwieser,Markus Seifert,Anna-Maria Mitterstiller,David Haschka,Laura Von Raffay,Manuel Grander,Manfred Nairz,Maura Poli,Lara Valente De Souza,Günter Weiss

doi:10.3389/fcimb.2021.705087

Abstract

IntroductionHepcidin is the systemic master regulator of iron metabolism as it degrades the cellular iron exporter ferroportin. In bacterial infections, hepcidin is upregulated to limit circulating iron for pathogens, thereby increasing iron retention in macrophages. This mechanism withholds iron from extracellular bacteria but could be of disadvantage in infections with intracellular bacteria. We aimed to understand the role of hepcidin in infections with intra- or extracellular bacteria using different hepcidin inhibitors.MethodsFor the experiments LDN-193189 and oversulfated heparins were used, which interact with the BMP6-SMAD pathway thereby inhibiting hepcidin expression. We infected male C57BL/6N mice with either the intracellular bacterium Salmonella Typhimurium or the extracellular bacterium Escherichia coli and treated these mice with the different hepcidin inhibitors.ResultsBoth inhibitors effectively reduced hepcidin levels in vitro under steady state conditions and upon stimulation with the inflammatory signals interleukin-6 or lipopolysaccharide. The inhibitors also reduced hepcidin levels and increased circulating iron concentration in uninfected mice. However, both compounds failed to decrease liver- and circulating hepcidin levels in infected mice and did not affect ferroportin expression in the spleen or impact on serum iron levels. Accordingly, both BMP-SMAD signaling inhibitors did not influence bacterial numbers in different organs in the course of E.coli or S.Tm sepsis.ConclusionThese data indicate that targeting the BMP receptor or the BMP-SMAD pathway is not sufficient to suppress hepcidin expression in the course of infection with both intra- or extracellular bacteria. This suggests that upon pharmacological inhibition of the central SMAD-BMP pathways during infection, other signaling cascades are compensatorily induced to ensure sufficient hepcidin formation and iron restriction to circulating microbes.

Highlights

Hepcidin is the systemic master regulator of iron metabolism as it degrades the cellular iron exporter ferroportin
Macrophages are in the center of cellular iron disturbances in the course of inflammation as they can acquire iron via multiple pathways including transferrin receptor mediated iron uptake, molecular iron incorporation via divalent metal transporter 1 (DMT1), and the solute carrier family 39 member 14 (Zip14) (Hentze et al, 2010), via hemopexin or haptoglobin receptors (Hvidberg et al, 2005; Schaer et al, 2006), or via ingestion of senescent or damaged erythrocytes (Nairz et al, 2017)
To determine if LDN-193189 (LDN) or over-sulfated heparins can inhibit Hamp expression in vitro, we started experiments with murine FL83B cells, which are immortalized hepatocytes (Breslow et al, 1973).When unstimulated cells were treated with either LDN or osH for 6h, we observed a significant reduction of Hamp expression as determined by qualitative real time polymerase chain reaction (qPCR) with LDN being more potent than osH at the dosages used (Figure 1A)

Summary

Introduction

Hepcidin is the systemic master regulator of iron metabolism as it degrades the cellular iron exporter ferroportin. Macrophages are in the center of cellular iron disturbances in the course of inflammation as they can acquire iron via multiple pathways including transferrin receptor mediated iron uptake, molecular iron incorporation via divalent metal transporter 1 (DMT1), and the solute carrier family 39 (zinc transporter) member 14 (Zip14) (Hentze et al, 2010), via hemopexin or haptoglobin receptors (Hvidberg et al, 2005; Schaer et al, 2006), or via ingestion of senescent or damaged erythrocytes (Nairz et al, 2017) These pathways are differently affected by cytokines or bacterial products thereby causing increased iron incorporation into macrophages (Byrd and Horwitz, 1993; Weiss et al, 1997; Mulero and Brock, 1999; Ludwiczek et al, 2003). This protein is regulated by cytokines, radicals, or bacterial products which can determine the amount of iron being

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