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.

Keywords: acute phase response; cell activation; cytokines; infection; inflammation; nitric oxide; protease.

Figure 1

S-NO-hAAT–activated macrophages kill intracellular Salmonella typhi. THP-1 cells (0.5 × 10⁶ per well) were pre- (A) or post- (B) treated with equimolar (27.5 μM) S-NO-hAAT, hAAT, and GSNO. The cells were also infected with live Salmonella typhi either prior or after treatment. CT, cells without treatment. In order to eliminate extracellular bacteria, the cells were washed and incubated with gentamicin, as detailed in the methods section. Remaining live bacteria were determined in cell lysates by counting CFU on blood agar, and exhibited logarithmic scale. (A,B) are representative results of two independent experiments (n = 3) for every condition. Mean ± SD, ^*p < 0.05 and ^****p < 0.0001.

Figure 2

Unlike unmodified hAAT, S-NO-hAAT increases inflammatory responses. Cytokine levels were determined in the supernatants of peritoneal macrophages (0.5 × 10⁶ per well) 48 h post-treatment with equimolar (27.5 μM) S-NO-hAAT, hAAT, GSNO and nitrosylation buffer (CT). (A) 10 ng/ml LPS stimulation 1 h post-treatment (n = 4). (B) S-NO-hAAT, hAAT, and GSNO treatment without added stimulus (n = 4). (C) mRNA relative levels in peritoneal macrophages (0.25 × 10⁶ per well, n = 6 from two independent experiments) 6 h post-treatment with equimolar (27.5 μM) S-NO-hAAT, hAAT, GSNO and nitrosylation buffer (CT). (D) Relative mRNA levels post S-NO-hAAT treatment at indicated time intervals. All data are presented as mean ± SD, ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001.

Figure 3

S-NO-hAAT activates MAPK signaling pathways. Peritoneal macrophages lysate (1 × 10⁷ per well) after incubation with 27.5 μM S-NO-hAAT for indicated time periods. (A) Kinase array, performed once for each time interval. Graph, densitometry analysis, mean ± SD. Below, representative assay blots. (B) Representative Western blot analysis of MAPK signaling proteins. (C) mRNA transcript levels of IL-1β and CXCL-1 of naïve (gray) or 1 h S-NO-hAAT treated (black) peritoneal macrophages (0.25 × 10⁶ per well) in the presence of signaling inhibitors or DMSO (–). mRNA transcript levels normalized to GAPDH (n = 3). Data are presented as mean ± SD. ns, non-significant, ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001.

Figure 4

Transnitrosylation-dependent activity of S-NO-hAAT. (A) Amount of remaining nitrosylated proteins in supernatant following S-NO-hAAT introduction to naïve (solid) and NEM-pretreated (dashed) peritoneal macrophages (1 × 10⁶ per well, n = 3). (B) DTNB or DMSO (vehicle)-pretreated peritoneal macrophages (0.25 × 10⁶ per well, n = 3) were examined for mRNA levels without S-NO-hAAT (gray) or 1 h after introducing it (black). Data are presented as mean ± SD, ^**p < 0.01, ^***p < 0.001, and ^****p < 0.0001.

Figure 5

hAAT duality: proposed mechanism. It is suggested that hAAT acts in two different manners according to its S-nitrosylation state. In an infected, inflamed, nitric oxide-rich site, hAAT is nitrosylated and can reduce the bacterial load by acting as an inflammatory trigger for immune cells. However, in the periphery, nitric oxide levels are low and hAAT is presumed to maintain its unmodified anti-inflammatory and tissue-protective activity profile.

Figure 6

Post-translational modifications in hAAT: pro-inflammatory outcomes. As an acute phase protein that rises during inflammation, hAAT may turn pro-inflammatory in an infected site as a result of S-nitrosylation (Cys232, pink), oxidation (Met351 and Met358, green), or proteolytic cleavage followed by release of its 36 amino-acids carboxyl terminal (C-36, red).