Circulating Microvesicle-Associated Inducible Nitric Oxide Synthase Is a Novel Therapeutic Target to Treat Sepsis: Current Status and Future Considerations

Abstract

To determine whether mitigating the harmful effects of circulating microvesicle-associated inducible nitric oxide (MV-A iNOS) in vivo increases the survival of challenged mice in three different mouse models of sepsis, the ability of anti-MV-A iNOS monoclonal antibodies (mAbs) to rescue challenged mice was assessed using three different mouse models of sepsis. The vivarium of a research laboratory Balb/c mice were challenged with an LD₈₀ dose of either lipopolysaccharide (LPS/endotoxin), TNFα, or MV-A iNOS and then treated at various times after the challenge with saline as control or with an anti-MV-A iNOS mAb as a potential immunotherapeutic to treat sepsis. Each group of mice was checked daily for survivors, and Kaplan-Meier survival curves were constructed. Five different murine anti-MV-A iNOS mAbs from our panel of 24 murine anti-MV-A iNOS mAbs were found to rescue some of the challenged mice. All five murine mAbs were used to genetically engineer humanized anti-MV-A iNOS mAbs by inserting the murine complementarity-determining regions (CDRs) into a human IgG_1,kappa scaffold and expressing the humanized mAbs in CHO cells. Three humanized anti-MV-A iNOS mAbs were effective at rescuing mice from sepsis in three different animal models of sepsis. The effectiveness of the treatment was both time- and dose-dependent. Humanized anti-MV-A iNOS rHJ mAb could rescue up to 80% of the challenged animals if administered early and at a high dose. Our conclusions are that MV-A iNOS is a novel therapeutic target to treat sepsis; anti-MV-A iNOS mAbs can mitigate the harmful effects of MV-A iNOS; the neutralizing mAb's efficacy is both time- and dose-dependent; and a specifically targeted immunotherapeutic for MV-A iNOS could potentially save tens of thousands of lives annually and could result in improved antibiotic stewardship.

Keywords: iNOS; inducible nitric oxide synthase; microvesicles; nanoparticles; sepsis; therapeutic target.

Figure 1

These are the combined data from five different experiments in which we tested all our murine anti-iNOS mAbs. Mice were dosed with LPS at 2 mg/kg at time zero and that was followed with a dose of MV-A iNOS at 4 h. At this dose of MV-A iNOS, 88% (22 of 25 died) of the challenged animals died of sepsis. As Lane #2 shows, if saline is injected at 4 h instead of MV-A iNOS, then more than 95% (24 of 25 lived) of the animals lived because a very low dose of LPS was used to prime the animals. Many of the murine anti-iNOS mAbs in our panel [1] did not rescue mice from death by sepsis since they did not neutralize the lethal effects of MV-A iNOS in vivo even though they all bound to iNOS. However, intervention with the anti-iNOS mAbs illustrated in Lanes # 3–7 did have a beneficial effect since they rescued some of the challenged mice from death by sepsis by stopping the sepsis cascade in these animals. The anti-iNOS mAbs depicted in Lanes # 3–7 were selected for continued investigation as potential candidate therapeutics to treat the sepsis pathology. The mouse monoclonal antibody secreting hybridoma cell lines were used to construct recombinant humanized anti-MV-A iNOS mAbs. Humanized anti-MV-A iNOS J mAb (rHJ mAb) is our lead immunotherapeutic candidate to treat sepsis.

Figure 2

These Kaplan–Meier survival curves demonstrate intervention with both the low dose (125 ng/gm body weight) and the high dose (1.25 µg/gm body weight) of rHJ mAb was effective at rescuing the mice from death by sepsis as compared with the saline control group. The earlier rHJ mAb was administered and the higher the dose given, the more effective it was. Similar results were also obtained when an LD₈₀ dose of TNFα or an LD₈₀ dose of MV-A iNOS was given IV as the challenge.

Figure 3

Published data by our team [6,7,8,33] demonstrate that what triggers sepsis is aberrant apoptosis that leads to secondary necrosis of cells induced to produce iNOS, to the release into the circulatory system of microvesicle-associated iNOS, and ultimately to the life-threatening sepsis cascade. This is a new pathophysiological pathway that was recently discovered and confirmed for the sepsis pathology. The left-hand side of the illustration depicts a cell that has been induced by the inflammatory cytokine storm to make iNOS. The red dashed line indicates that macrophages (Mø) do not recognize this cell as apoptotic. Thus, this induced apoptotic cell is not properly scavenged by a macrophage and instead undergoes secondary necrosis, which is depicted in the right-hand side of this illustration. The secondarily necrotic, induced cells swell, burst, and release their cellular contents into the circulatory system, including iNOS-containing microvesicles (depicted as extracellular green balls).

Figure 4

This illustration shows the causative role that microvesicle-associated iNOS (MV-A iNOS), shown as green balls, plays in sepsis. Once the MV-A iNOS is delivered as cargo contained in circulating microvesicles to receiver cells at distal sites in the body, the active iNOS enzyme is internalized into the receiver cells, where it produces toxic quantities of nitric oxide. This kills the receiver cell, which produces microperforations in barrier structures that then leak, and it also damages the myocardium of the heart, which leads to hemodynamic collapse and death.