Leucine Reconstitutes Phagocytosis-Induced Cell Death in E. coli-Infected Neonatal Monocytes-Effects on Energy Metabolism and mTOR Signaling.

Stephan Dreschers,Thorsten W Orlikowsky,Kim Ohl,Julia Möllmann,Klaus Tenbrock

doi:10.3390/ijms22084271

Stephan Dreschers, Thorsten W Orlikowsky + Show 3 more

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https://doi.org/10.3390/ijms22084271

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Abstract

MΦ differentiate from circulating monocytes (Mo). The reduced ability of neonatal Mo to undergo apoptosis after E. coli infection (phagocytosis-induced cell death (PICD)) could contribute to sustained inflammatory processes. The objective of our study was to investigate whether immune metabolism in Mo can be modified to gain access to pro-apoptotic signaling. To this end, we supplemented Mo from neonates and from adults with the branched amino acid leucine. In neonatal Mo, we observed increased energy production via oxidative phosphorylation (Oxphos) after E. coli infection via Seahorse assay. Leucine did not change phagocytic properties. In neonatal Mo, we detected temporal activation of the AKT and mTOR pathways, accompanied with subsequent activation of downstream targets S6 Kinase (S6K) and S6. FACS analyses showed that once mTOR activation was terminated, the level of anti-apoptotic BCL-2 family proteins (BCL-2; BCL-XL) decreased. Release of cytochrome C and cleavage of caspase-3 indicated involvement of the intrinsic apoptotic pathway. Concomitantly, the PICD of neonatal Mo was initiated, as detected by hypodiploid DNA. This process was sensitive to rapamycin and metformin, suggesting a functional link between AKT, mTOR and the control of intrinsic apoptotic signaling. These features were unique to neonatal Mo and could not be observed in adult Mo. Supplementation with leucine therefore could be beneficial to reduce sustained inflammation in septic neonates.

Highlights

Sepsis-induced mortality and morbidity peaks during infancy and diagnostic and therapeutic efforts have been made, a recent statement of the WHO designates neonatal sepsis as a main threat for the future, since 9–20% of sepsis-suffering newborns still die [1]
We previously demonstrated that monocyte-derived neonatal macrophages (MΦ) are defective in their metabolic repertoire, pointing to a potentially pivotal role of mTOR signaling [4]
We examined in vitro whether (i) E. coli infection and leucine supplementation alter the energy metabolism in CBMo and PBMo exceedingly and (ii) whether the signaling induced by leucine and infection affects the AKT/mTOR checkpoint with comparable reactions in CBMo and PBMo

Summary

Introduction

Sepsis-induced mortality and morbidity peaks during infancy and diagnostic and therapeutic efforts have been made, a recent statement of the WHO designates neonatal sepsis as a main threat for the future, since 9–20% of sepsis-suffering newborns still die [1]. The stressful transition from immune tolerance of the intrauterine surroundings to the situation after birth, with molecules initiating defensive immune responses, supports a state of sustained inflammation [2]. The mTORC1 complex measures the nutrient state of a cell and is involved in TLR-dependent, TRIF-mediated signaling, facilitating anti-inflammatory responses [5,6]. This signaling pathway is thought to establish a tolerogenic state in alarmin (S100)-exposed neonatal Mo [7]. The first protein complex is strongly inhibited by rapamycin, whereas the latter is frequently reported to be rapamycin resistant [8]

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