S-Nitrosylation of α1-Antitrypsin Triggers Macrophages Toward Inflammatory Phenotype and Enhances Intra-Cellular Bacteria Elimination.

Ziv Kaner,Moran Benhar,Yossef Av-Gay,Peleg Rider,Eli C Lewis,Ronen Schuster,Rotem Engelman,David Greenberg

doi:10.3389/fimmu.2019.00590

Abstract

Background: Human α1-antitrypsin (hAAT) is a circulating anti-inflammatory serine-protease inhibitor that rises during acute phase responses. in vivo, hAAT reduces bacterial load, without directly inhibiting bacterial growth. In conditions of excess nitric-oxide (NO), hAAT undergoes S-nitrosylation (S-NO-hAAT) and gains antibacterial capacity. The impact of S-NO-hAAT on immune cells has yet to be explored.Aim: Study the effects of S-NO-hAAT on immune cells during bacterial infection.Methods: Clinical-grade hAAT was S-nitrosylated and then compared to unmodified hAAT, functionally, and structurally. Intracellular bacterial clearance by THP-1 macrophages was assessed using live Salmonella typhi. Murine peritoneal macrophages were examined, and signaling pathways were evaluated. S-NO-hAAT was also investigated after blocking free mambranal cysteine residues on cells.Results: S-NO-hAAT (27.5 uM) enhances intracellular bacteria elimination by immunocytes (up to 1-log reduction). S-NO-hAAT causes resting macrophages to exhibit a pro-inflammatory and antibacterial phenotype, including release of inflammatory cytokines and induction of inducible nitric oxide synthase (iNOS) and TLR2. These pro-inflammatory effects are dependent upon cell surface thiols and activation of MAPK pathways.Conclusions: hAAT duality appears to be context-specific, involving S-nitrosylation in a nitric oxide rich environment. Our results suggest that S-nitrosylation facilitates the antibacterial activity of hAAT by promoting its ability to activate innate immune cells. This pro-inflammatory effect may involve transferring of nitric oxide from S-NO-hAAT to a free cysteine residue on cellular targets.

Highlights

Human α1-antitrypsin is a 52 kDa glycoprotein that is mainly produced by hepatocytes, and circulates at steady-state levels of 1.05–1.64 mg/ml [1]
Macrophage bacterial killing activity was examined using PMA-primed THP-1 cells infected with Salmonella typhi after or prior to treatment with Human α1-antitrypsin (hAAT), SNO-hAAT, or GSNO (Figure 1)
Analysis of CFU counts obtained from lysed macrophages showed that antibacterial activity was enhanced by S-NO-hAAT

Summary

Introduction

Human α1-antitrypsin (hAAT) is a 52 kDa glycoprotein that is mainly produced by hepatocytes, and circulates at steady-state levels of 1.05–1.64 mg/ml [1]. During acute phase responses, such as during infection, circulating levels of hAAT rise more than 4-fold [2]. Innate immune cells display unique behaviors when exposed to physiologic concentrations of hAAT. During conditions of high hAAT levels, sera content displays an abrupt shift toward lower IL-1β, IL-6, TNFα and IL-8, and higher IL-10 levels [5, 8,9,10,11,12,13,14,15,16]. Human α1-antitrypsin (hAAT) is a circulating anti-inflammatory serine-protease inhibitor that rises during acute phase responses. The impact of S-NO-hAAT on immune cells has yet to be explored

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