Control of systemic inflammation through early nitric oxide supplementation with nitric oxide releasing nanoparticles

Abstract

Amelioration of immune overactivity during sepsis is key to restoring hemodynamics, microvascular blood flow, and tissue oxygenation, and in preventing multi-organ dysfunction syndrome. The systemic inflammatory response syndrome that results from sepsis ultimately leads to degradation of the endothelial glycocalyx and subsequently increased vascular leakage. Current fluid resuscitation techniques only transiently improve outcomes in sepsis, and can cause edema. Nitric oxide (NO) treatment for sepsis has shown promise in the past, but implementation is difficult due to the challenges associated with delivery and the transient nature of NO. To address this, we tested the anti-inflammatory efficacy of sustained delivery of exogenous NO using i.v. infused NO releasing nanoparticles (NO-np). The impact of NO-np on microhemodynamics and immune response in a lipopolysaccharide (LPS) induced endotoxemia mouse model was evaluated. NO-np treatment significantly attenuated the pro-inflammatory response by promoting M2 macrophage repolarization, which reduced the presence of pro-inflammatory cytokines in the serum and slowed vascular extravasation. Combined, this resulted in significantly improved microvascular blood flow and 72-h survival of animals treated with NO-np. The results from this study suggest that sustained supplementation of endogenous NO ameliorates and may prevent the morbidities of acute systemic inflammatory conditions. Given that endothelial dysfunction is a common denominator in many acute inflammatory conditions, it is likely that NO enhancement strategies may be useful for the treatment of sepsis and other acute inflammatory insults that trigger severe systemic pro-inflammatory responses and often result in a cytokine storm, as seen in COVID-19.

Keywords: Cytokine storm; Inflammation; Nitric oxide; Sepsis; Septic shock; Vascular permeability.

Graphical abstract

Fig. 1

Central hemodynamics of mice before (BL) and after dosage with LPS and nanoparticles. (a) Mean arterial pressure significantly decreased 6 h after LPS dosage. (b) NO-np treatment normalized changes in HR following LPS dosage. N = 8/group. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 between groups; †p < 0.05 vs BL.

Fig. 2

Arteriolar microhemodynamics of mice after dosage with LPS, relative to baseline. Microhemodynamic aberrations appear more rapidly than changes in systemic hemodynamics. (a) LPS dosage results in vasodilation systemically, independent of treatment. (b) Flow decreases following LPS treatment, but NO-np treatment partially prevents this decrease in flow. N = 22 vessels/group. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 between groups; †p < 0.05 vs BL.

Fig. 3

Changes in functional capillary density (FCD) and vascular permeability after LPS dosage. (a) Treatment with NO-np increased FCD significantly compared to Control-np, thus improving capillary flow and microvascular O₂ transport; N = 8/group. (b) NO-np attenuated the increased permeability compared to animals that received Control-np. I₀ and I_i are the extravascular and intravascular intensity of FITC, respectively. (c) Representative image of the changes in vascular permeability. N = 12/group. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 between groups; †p < 0.05 vs BL or 0.5 h; ‡p < 0.05 vs 2 h.

Fig. 4

Survival curve over 72 h of animals treated with Control and NO-nps after LPS dosage. NO-np significantly improved 72-h survival, with only one animal dying over the observation period. Significance was measured via Log-Rank test. N = 9 & 12 at time = 0 for Control-np and NO-np, respectively.

Fig. 5

Fluorescence-activated cell sorting (FACS) of peritoneal macrophages from LPS-dosed animals after 72 h reveals that macrophages from Control-np had a primarily M1-like (inflammatory) phenotype, with M1-like macrophages representing 72% of all macrophages for Control-np, but only 16% of all macrophages for NO-np (a), but significantly more macrophages from animals treated with NO-np had an M2-like (anti-inflammatory) phenotype than animals treated with control-np, with M2-like macrophages representing 18% of macrophages from animals treated with Control-np, but 33% of macrophages from animals treated with NO-np (b). (a) n = 650 macrophages/group, (b) n = 585 macrophages/group.

Fig. 6

Cytokine profile of mice treated with NO-np and Control-np after LPS dosage after 24 and 48 h. At 24 h, anti-inflammatory cytokines (IL-10, TGFβ) were significantly higher in animals treated with NO-np compared to Control-np, but inflammatory cytokines showed an opposite trend. Data are presented as mean ± SD. Data between groups and between time points were analyzed with Welch's t-tests with Bonferroni correction. N = 4/group at 24 h & N = 2/group at 48 h for animals dosed with LPS; N = 3 for Sham at both time points. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 between groups; †p < 0.05 vs 24 h post-LPS.