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

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.

Aging associated altered response to intracellular bacterial infections and its implication on the host

“…This surprising observation prompted us to examine changes in the expression levels of Ser ine P rotease In hibitors (SERPIN) A1, B1 and B9 in the liver tissue. SERPINs are mostly known to have a protective function during chronic inflammation and an increase in their levels are correlated with resolution of inflammation induced damage to the host (Law et al ., 2006; Choi et al ., 2019; Kaner et al ., 2019; Rieder et al ., 2019). Significant downregulation of SERPINs A1 and B9 were seen in infected compared to uninfected young mice (Fig 5B and 5C) possibly to potentiate the immune response towards clearing the bacterial burden.…”

Post-Translational Modifications of Circulating Alpha-1-Antitrypsin Protein

“…A recent study reported that S-nitrosylation facilitates the antibacterial activity of AAT by promoting its ability to activate innate immune cells [116]. Although the precise proportion of S-NO-AAT generated in vivo is not known, nor the ratio between AAT/S-NO-AAT, the authors of this latter study postulated that during infection, AAT becomes S-nitrosylated, and thus can assist in the reduction of the bacterial burden.…”

“…Protein S-nitrosylation involves covalent attachment of a nitric oxide (NO) group to cysteine residues via direct interactions between reactive nitrogen species and protein thiol residues, or via nitrosylation by S-nitrosoglutathione [115]. The only cysteine residue of AAT (Cys232) undergoes S-nitrosylation (S-NO-AAT) in the inflammatory milieu [116]. The reverse process, protein denitrosylation, is driven by two enzyme systems: glutathione/S-nitrosoglutathione reductase and thioredoxin/thioredoxin reductase [117].…”

“…The reverse process, protein denitrosylation, is driven by two enzyme systems: glutathione/S-nitrosoglutathione reductase and thioredoxin/thioredoxin reductase [117]. Denitrosylation can also be self-induced by the S-NO-protein, as observed in S-NO-AAT, via transfer of the NO molecule to cellular targets with free thiols [116,118].…”

“…The bacteriostatic effects of S-NO-AAT enabled via transnitrosylation was 20-to 3000-fold more effective than for other S-nitrosylated albumins and glutathione [118]. Moreover, S-NO-AAT appears to stimulate a significantly stronger anti-bacterial response in macrophages, compared to native AAT or S-nitrosoglutathione [116]. In addition, S-NO-AAT can prevent hepatocyte apoptosis in rats [118,119].…”