Nitric Oxide-Releasing Nanoparticles Prevent Propionibacterium acnes-Induced Inflammation by Both Clearing the Organism and Inhibiting Microbial Stimulation of the Innate Immune Response

Abstract

Propionibacterium acnes induction of IL-1 cytokines through the NLRP3 (NLR, nucleotide oligomerization domain-like receptor) inflammasome was recently highlighted as a dominant etiological factor for acne vulgaris. Therefore, therapeutics targeting both the stimulus and the cascade would be ideal. Nitric oxide (NO), a potent biological messenger, has documented broad-spectrum antimicrobial and immunomodulatory properties. To harness these characteristics to target acne, we used an established nanotechnology capable of generating/releasing NO over time (NO-np). P. acnes was found to be highly sensitive to all concentrations of NO-np tested, although human keratinocyte, monocyte, and embryonic zebra fish assays revealed no cytotoxicity. NO-np significantly suppressed IL-1β, tumor necrosis factor-α (TNF-α), IL-8, and IL-6 from human monocytes, and IL-8 and IL-6 from human keratinocytes, respectively. Importantly, silencing of NLRP3 expression by small interfering RNA did not limit NO-np inhibition of IL-1 β secretion from monocytes, and neither TNF-α nor IL-6 secretion, nor inhibition by NO-np was found to be dependent on this pathway. The observed mechanism by which NO-np impacts IL-1β secretion was through inhibition of caspase-1 and IL-1β gene expression. Together, these data suggest that NO-np can effectively prevent P. acnes-induced inflammation by both clearing the organism and inhibiting microbial stimulation of the innate immune response.

Figure 1. Nanoparticles Characterization

(a) Scanning electron microscopy revealed distinct spherical nanoparticles with irregular surface structure indicative of the porous matrix lattice (scale bar = 100 nm). (b) Using Dynamic light scattering (DLS), NO-np revealed an average hydrodynamic radius of 108.47 nm based on 40 acquisition attempts. The standard deviation was 14.2 nm (3.3%), proving that NO-np are homogenous in size.

Figure 2. Antimicrobial effects of Nitric Oxide releasing nanoparticles

(a) Susceptibility of P. acnes isolates to varying concentrations of empty nanoparticles (np) and NO-np (0.625, 1.25, 2.5 and 5 mg/ml), was investigated by real-time Bioscreen analysis and (b) colony form unit assay determination (mean CFU per ml). Experiments were repeated in triplicate and performed at least thrice on separate days. Asterisks denote p value significance calculated by unpaired two-tailed t test analysis. (*, p < 0.05; **, p < 0.01).

Figure 3. NO-np significantly inhibits P. acnes induced pro-inflammatory cytokines in PMBC and keratinocytes

P. acnes stimulated PBMCs expression of IL-1β (a), TNF-α (b), IL-6 (c), and IL-8 (e), and HaCaT cell expression of IL-6 (d) and IL-8 (f) with and without various concentrations of NO-np. NO-np at 2.5 mg/ml was used as a control. IL-1β and TNF-α were undetectable in the HaCaT supernatant via ELISA (data not shown)). The data is presented as the mean of triplicate wells ± SD and is representative of three individual experiments. (*, p < 0.05; **, p < 0.01).

Figure 4. NO-np inhibits PBMC IL-1β secretion by directly down-regulating IL-1β gene and Caspase-1 gene expression via the inflammasome pathway

Cells were infected by P. acnes at MOI 0.5 with and without NO-np and with scramble siRNA, siNLRP3 or siNLRP1, and IL-1β secretion in culture supernatants was tabulated via ELISA (NO-np 2.5 mg/ml) (a). The expressions of Caspase-1, IL-1β, NLRP3, and ASC were measured by real-time PCR (b-e). Data are shown as the mean of triplicate wells ± SD and is representative of three individual experiments. (*, p < 0.05; **, p < 0.01).

Figure 5. NO-np down-regulates P. acnes induced Caspase-1 protein expression and activity in PBMC cells

Peripheral blood mononuclear cells (PBMC) were isolated from whole blood of normal healthy donors. Transfected cells were plated into 6–well cell culture plates, cultured at 37°C overnight, and co-cultured with or without 2.5 mg/mL control-np and 2.5 mg/ml NO-nanoparticle for an hour. Cells were then infected by P. acnes at MOI 0.5 for 24 hours. Cells were lysed, and caspase-1 protein expression was measured with western blot (a) and activity assay (b). a) Western blot using anti-caspase-1: Cells alone (Lane 1); P. acnes (Lane 2); Control np (Lane 3); NO-np (Lane 4); P. acnes in the presence of NO-np (lane 5); P. acnes in the presence of Control np (lane 6). b) Caspase-1 activity assay: cells were normalized as percentage of control cells. NO-np significantly decreases PBMC caspase-1 protein activity in the presence of P. acnes compared to control-np. (*, p < 0.05)

Figure 6. Nitric Oxide releasing Nanoparticles are non-toxic

NO-np at various concentrations (1.25, 2.5 and 5 mg/ml, respectively) were incubated with HaCaT cells (a) or PBMCs (b). Cells were collected and evaluated for viability using MTT assay. (c) Representative images of zebrafish embryos at 120 hpf: control (top) and exposed to NO-np (bottom). No significant differences were observed in larval morphology or behavioral endpoints (p > 0.05 for each endpoint evaluated, Fisher's Exact test). Error bars denote SEM.