TLR4 counteracts BVRA signaling in human leukocytes via differential regulation of AMPK, mTORC1 and mTORC2

Zhiyong Zhang,Louis F Amorosa,Mohan Nair,Beatrice Haimovich,Anna Petrova,Susette Coyle,Marie Macor,Leonard Y Lee

doi:10.1038/s41598-019-43347-8

Zhiyong Zhang, Louis F Amorosa + Show 6 more

Open Access

https://doi.org/10.1038/s41598-019-43347-8

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Abstract

TLR4 is implicated in diseases associated with chronic low-grade inflammation, yet homeostatic signaling mechanisms that prevent and/or are affected by chronic TLR4 activation are largely uncharacterized. We recently reported that LPS/TLR4 activates in human leukocytes signaling intermediates (SI), abbreviated TLR4-SI, which include mTORC1-specific effectors and targets, and that leukocytes of patients with T2D or after cardiopulmonary bypass (CPB) expressed similar SI. Extending these previous findings, here we show that TLR4-SI expression post-CPB was associated with low serum bilirubin and reduced preoperative expression of biliverdin reductase A (BVRA), the enzyme that converts biliverdin to bilirubin, in patient’s leukocytes. Biliverdin inhibited TLR4 signaling in leukocytes and triggered phosphorylation of mTORC2-specific targets, including Akt, PKCζ, AMPKα-LKB1-TSC1/2, and their association with BVRA. Torin, PP242, and a PKCζ inhibitory peptide, but not rapamycin, prevented these biliverdin-induced responses and TLR4 inhibition. In contrast, LPS/TLR4 triggered decreases in BVRA, AMPKα and PKCζ expression, and an increase in haptoglobin, a heme binding protein, in leukocytes in vivo and in vitro, indicating that activated TLR4 may suppress biliverdin/BVRA signaling. Significantly, compared to non-diabetics, BVRA and PKCζ expression was low and haptoglobin was high in T2D patients leukocytes. Sustained TLR4 activation may deregulate homeostatic anti-inflammatory BVRA/mTORC2 signaling and thereby contribute to chronic inflammatory diseases.

Highlights

Toll-like receptor 4 (TLR4) is implicated in morbidities associated with chronic low-grade inflammation, including insulin resistance and type 2 diabetes (T2D)[1,2]
Analyses of cardiopulmonary bypass (CPB) patient leukocytes and leukocytes exposed to LPS in vitro and in vivo uncovered an additional TLR4 regulatory mechanism that is controlled by biliverdin and biliverdin reductase A (BVRA)
We found that biliverdin/BVRA inhibits TLR4 signaling using multiple signaling intermediates (SI), including PKCζ, LKB1, AMPK, TSC1/2 and Akt

Summary

Introduction

Toll-like receptor 4 (TLR4) is implicated in morbidities associated with chronic low-grade inflammation, including insulin resistance and type 2 diabetes (T2D)[1,2]. Whereas LPS doses above 0.5 ng/kg elicited inflammatory responses that included IL-6 and TNF-α production and broad changes in leukocyte’s mRNA and signaling intermediates (SI) expression[7], doses below 0.5 ng/kg triggered only changes in leukocyte’s SI expression[8]. These observations suggested the hypothesis that the pattern of SI detected in leukocytes challenged with low LPS doses may be clinically relevant and serve as a tool for interrogating whether and how chronic low-grade TLR4 activation may deregulate homeostatic signaling mechanisms that prevent unwarranted TLR4 activation. We propose that in T2D patient’s leukocytes this loop is deregulated and that the signals are skewed in favor of TLR4

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