NET degradation attenuates ricin-induced acute lung injury and protects mice from ARDS

Neutrophils are critical first responders of the innate immune system, rapidly recruited to sites of infection or sterile injury. Upon activation by pathogen- or damage-associated molecular patterns, neutrophils initiate antimicrobial responses, including cytokine release, phagocytosis, and the formation of neutrophil extracellular traps (NETs). While NETosis plays a protective role, excessive NET formation can exacerbate inflammation and tissue damage. Pulmonary exposure to ricin, a potent toxin derived from Ricinus communis, results in acute lung injury characterized by neutrophil infiltration, cytokine production, vascular leakage, and pulmonary edema. This study investigated the contribution of NETosis to ricin-induced lung pathology and explored the therapeutic potential of targeting NETosis with a long acting recombinant DNase I (PRX-119) to attenuate lung injury.

CD1 outbreed mice were pulmonary exposed to ricin, and bronchoalveolar lavage fluid (BALF) and lung tissues were collected at various time points post-exposure. NETosis was assessed by immunofluorescence and Western blot analysis of markers, including peptidyl arginine deiminase 4 (PAD4), citrullinated histone H3 (citH3) and myeloperoxidase (MPO). Therapeutic intervention included administration of a NET-degrading DNase agent in combination with an anti-ricin antibody. Cell-free DNA levels, NETosis markers, neutrophil infiltration, lung histopathology, vascular permeability and the expression of pro- and anti-inflammatory mediators were evaluated. Weight loss and survival were also monitored and compared between anti-ricin monotherapy and combined anti-ricin and plant-produced human recombinant long acting (LA) DNase I (PRX-119), a novel NET degradation therapy.

Ricin exposure led to elevated pulmonary levels of PAD4, citH3 and MPO, accompanied by extensive NET formation in both BALF and lung tissue. Mice receiving combined therapy with a newly developed DNase I - based agent (PRX-119) and an anti-ricin antibody treatment exhibited significantly improved survival and reduced weight loss compared to antibody monotherapy. The combined treatment not only significantly reduced NETosis markers, but also improved lung histopathology, reduced vascular leakage and pulmonary edema and altered the levels of proteins involved with pro- or anti-inflammatory effects, Dkk-1, CD93 and Periostin.

These findings demonstrate for the first time that NETosis plays a significant pathological role in ricin-induced lung injury. Moreover, they underscore the therapeutic potential of combining advanced NET-degrading agents, specifically PRX-119, an advanced DNAse I under development, with toxin-neutralizing antibodies as a promising strategy to reduce acute lung damage and enhance clinical outcomes.