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

CD1 outbred mice (females weighing 27–32 g) were purchased from Charles River Laboratories (England) and were kept in an animal husbandry facility for 4–8 days prior to commencement of the experiments. The animals were kept under a 12-hour light regime and had free access to food and water. The experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Israel Institute for Biological Research (protocols M-16-2023, M-12-24).

The use of ricin in the current study and purification of ricin from castor beans were conducted under the safety and environmental regulations of the Israel Institute for Biological Research, in compliance with the Israeli law.

Crude ricin was prepared from seeds of endemic Ricinus communis as previously described (Gal et al. 2014). Shortly, seeds were homogenized in a blender (Waring, Torrington, CT) in 5% acetic acid (Merck, Darmastadt, Germany)/PBS (Biological Industries, Beth-Haemek, Israel). The homogenate was centrifuged, and the clarified supernatant containing the toxin was subjected to ammonium sulfate (Merck, Darmastadt, Germany) precipitation (60% saturation). The precipitate was dissolved in PBS and dialyzed extensively against the same buffer. The toxin preparation appeared on a Coomassie blue (Bio-Rad, Rishon Le Zion, Israel)-stained nonreducing 10%polyacrylamide gel (ThermoFisher Scientific, Carlsbad, CA) as two major bands of molecular mass of ~ 65 kDa (ricin toxin, ~ 80%) and 120 kDa (Ricinus communis agglutinin, ~ 20%). Protein concentration was determined as 2.86 mg/ml by 280 nm absorption (Nanodrop 2000; ThermoFisher Scientific).

PRX-119 is a PEGylated plant-produced long-acting recombinant human DNase I. PRX-119 is produced in two steps, the non-PEGylated DNase I is produced using the Protalix Biotherapeutics plant cell-based expression system ProCellEx^® followed by PEGylation with 5 kDa PEG-aldehyde. This technology is covered under patents WO 2013/114,374 and WO 2022/074656.

Mice were anesthetized by intraperitoneal injection of 0.2 ml of ketamine (1.9 mg/mouse, Vetoquinol, Lure, France) and xylazine (0.19 mg/mouse, Eurovet Animal Health, AD Bladel, The Netherlands). Crude ricin was administered intranasally at a dose of 9.6 µg/kg body weight (2LD₅₀), at a volume of 25 µl per nostril.

Anti-ricin antibody treatment was administered by single intravenous administration (100 µl), 24 h after exposure to ricin. The antibody was purified from hyperimmune plasma of horses vaccinated with ricin (RR003, a purified F(ab)₂ antibody fragment, with a titer of 1716 Neutralizing Israeli Units) (Falach et al. 2018).

PRX-119 was administered intraperitoneally 5 mg/kg at a volume of 100 µl. Treatment commenced 24 h after exposure to ricin and was administered once a day for 14 days.

BALF was collected by instillation of 1 ml PBS at room temperature via a tracheal cannula, and centrifuged at 240 g at 4 °C for 5 min. Supernatants were collected and stored at − 20 °C until further use. Interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF), keratinocyte chemoattractant (KC), macrophage inflammatory protein-2 (MIP-2), Interleukin-1β (IL-1β), monocyte chemoattractant protein (MCP-1), vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNF-α) were quantified using ELISA kits (R&D Systems, USA), following the manufacturer’s instructions. Tailor-made Luminex mouse discovery assay was performed for measurement of the following mediators: C1qR1/CD93, CCL11/Eotaxin, CCL22/MDC, Chitinase 3-like 1/YKL-40, CXCL16, HGF, IL-1α/IL-1F1, IL-10, IL-13, MMP-9, Osteopontin, Periostin/OSF-2, Resistin, S100A9, Serpin E1/PAI-1, Thrombospondin-4, TIMP-1, Dkk-1, EMMPRIN/CD147 and LIX (R&D Systems, Biotest, USA).

Protein concentration in BALF was determined using Bradford reagent. Volume of 20 µl of BALF from each sample were loaded and separated on NuPage 4–12% Bis-Tris SDS-PAGE Gel (Invitrogen, NP0335BOX) and transferred to an iBlot Transfer Stack Nitrocellulose PVDF membrane (Invitrogen, IB01001). The membranes were blocked for one hour in room temperature in TBST solution (0.15 M NaCl, 0.05% Tween-20, 0.01 M Tris-HCl pH 8) containing 5% Skim Milk powder (Difco Skim Milk, BD 232100). The incubation with rabbit primary antibody against each of the tested proteins (anti-citH3, ab5103; anti-PAD4, ab214810, anti-MPO, ab208670, Abcam) was performed for 16 h at 4 °C with gentle agitation. After washing with TBST, the membranes were incubated for one hour at room temperature with goat anti-rabbit IRDye 680RD secondary antibody (Li-COR, 926−68071) diluted 1:10,000 in TBST solution with 5% skim milk powder. After washing, the bands were visualized using ODYSSEY CLx (LI-COR) imager. The intensity of each band was quantified using ImageJ software. Quantitation of citH3 in BALF was also performed using ELISA kit for citrullinated histone H3 (Cayman Chemicals, 501620, clone 11D3).

Cell-free DNA quantification was performed using Quant-iT™ PicoGreen™ dsDNA (Invitrogen) kit in accordance with manufacturer’s instructions. Briefly, a calibration curve was prepared using quantities from 0 to 1000 ng/ml DNA in x10 intervals. Then, 100 µl volumes of BALF samples were transferred into black 96 well plates (Greiner) and an equal volume of PicoGreen dsDNA reagent was added to each well. Fluorescent reads of the DNA were measured in a Microplate reader (Gen 5 3.04), excitation and emission at 485 nm and 538 nm, respectively.

One day before staining, round glass coverslips were inserted into 24 well plates and coated with 400 µl poly-L-lysine (ScienCell #0413) at a concentration of 10 µg/ml for one hour in at 37 °C. Subsequently, the wells were washed with double distilled water (DDW) and left to dry in air. BALF obtained from naïve or intoxicated animals was centrifuged and the cell pellet was separated and resuspended in an Opti-MEM medium containing 2% heat-inactivated Fetal Bovine Serum (FBS, Sigma-Aldrich). The resuspended cells were loaded on the coverslips for one hour at 37 °C to allow them to adhere. The cells were fixed with 4% paraformaldehyde for 15 min at room temperature and then dried. Next, the cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min at room temperature and blocked with 5% BSA in PBS for 40 min at room temperature. Cells were stained with primary antibody (anti-citH3, ab5103; or anti-MPO, ab208670, Abcam) resuspended in TBS containing 5% BSA and incubated over night at 4 °C. The next day, the cells were washed twice with TBS, incubated with anti-rabbit Alexa Fluor 594 secondary antibody (Invitrogen) for one hour at room temperature, and washed twice with TBS. For staining of extracellular cell free DNA, the cells were incubated with Sytox Green (Invitrogen) diluted 1:25,000 in PBS for 10 min at room temperature. The dye was then washed using TBS and nuclear staining was performed using Prolong^® Gold antifade reagent containing DAPI (Molecular probes).

Lungs were harvested, cut into small pieces and digested by incubation with 4 mg/ml Collagenase D (Roche, Mannheim, Germany) in PBS Ca² Mg² (Biological Industries, Beit Haemek, Israel) for 2 h at 37 °C. The tissue was then meshed through a 70 μm cell strainer. Neutrophils were isolated using a Histopaque gradient. Briefly, 3 ml Histopaque-1119 (Sigma) was inserted into 15 ml tubes and on top of it, 3 ml Histopaque-1077 (Sigma) was carefully layered. Above the gradient, 6 ml of lung cells suspended in PBS containing 5% BSA were cautiously added. The tubes were centrifuged at 700 g for 30 min at room temperature without brake and the neutrophils which resided in the interface between the gradient layers, were carefully collected. Neutrophils were isolated by negative selection kit (Miltenyi Biotec, 130-097-658). The isolated cells were counted and resuspended in Opti-MEM medium containing 2% heat-inactivated FBS. Then, 1–2 × 10⁶ cells were loaded on poly-L-Lysine coated glass coverslips in a 24 well plate. Subsequent preparations and NETs staining were performed as described above with respect to staining BALF neutrophils.

To visualize NETosis in lung tissue, lungs were collected and fixed in 4% buffered formaldehyde in PBS pH 7.2–7.4 (Bio Lab, Israel) for 2 weeks. Sections of 5 μm were prepared after paraffin embedding and using a microtome (RM 2255, Leica, Germany). The sections were deparaffinized and antigens were exposed by incubation with Target Retrieval Solution (DAKO) at 95 °C for 30 min. After blocking with 5% BSA in PBS, the slides were incubated with the primary antibodies rabbit anti-citH3 (ab5103, 1:200), and rat anti-Ly6B (ab53457, 1:200) (Abcam) over night at 4 °C. Subsequently, the slides were incubated for 1 h at room temperature with fluorescent secondary antibodies, Alexa Fluor 594-coupled goat anti-rat IgG (Abcam, ab150160, 1:100) and Atto550 goat anti-rabbit IgG (Rockland, 611-154-122s, 1:200). Nuclear staining was performed with Prolong^® Gold antifade reagent containing DAPI (Thermo Fisher Scientific). Image acquisition was performed using LSM 710 confocal microscope (Zeiss, Germany) with the following lasers argon multiline (458/488/514 nm), diode 405 nm, DPSS 561 nm and helium-neon 633 nm (objective lens LD C-Apochromat 40×/1.1 W Korr M27, image depth 8-bit, zoom 0.6, filters 410–521, 502–629, 570–609, 604–733 nm for DAPI, Sytox Green, citH3 and Ly6B, respectively). Analysis of the mean fluorescence intensity (MFI) of the staining was acquired using Zen 2008 (Zeiss, Germany).

To examine the lung morphology, tissue sections were stained with Hematoxylin and Eosin (H&E). The stained sections were photographed using 3D HISTECH Panoramic Midi II slide viewer (Budapest, Hungary), and analysis was performed using Case Viewer 2.4.0 software (Budapest, Hungary). Semi-quantitative assessment of the histological injury score in the lungs of mice was performed according to established published criteria: inflammatory cells, hyaline membrane, thickening of alveolar wall, enhanced injury, vessel congestion, and atelectasis (Silva et al. 2022). These parameters provided a score of 0 to 8, where 0 means “no phenomenon” and 8 means a severe phenomenon. Lungs were obtained from 5 mice in each treatment group. The score for each parameter was calculated as the average of five different fields. The percentage of the histological injury score for each animal was determined using the following equation: (sum of scores for the six evaluated parameters/48) × 100, where 48 is the maximum possible score.

Lung permeability was determined by the Evans Blue dye (EBD) extravasation method as follows: 7.5 mg/ml EBD (Sigma-Aldrich) was injected intravenously at a dose of 50 mg/kg, and allowed to circulate for 1 h. Mice were then anesthetized, and the lungs were perfused by cutting the left atrium and flushing with 5 ml PBS through the right ventricle. The lungs were removed and EBD was extracted by incubation of the tissues in 0.5 ml of formamide (Sigma-Aldrich) at 60 °C for 24 h. EBD optical density in the supernatant was measured at 620 nm in a Spectramax ABS Plus (Molecular Devices) spectrophotometer.

All statistical analyses were conducted with GraphPad Prism software (version 5.01, GraphPad Software Inc., La Jolla, CA, USA, 2007). Data were first tested for normality using the Shapiro–Wilk test. Simple comparisons were performed using the unpaired two-tailed Student’s t-test. For multiple comparisons, one-way or two-way analysis of variance (ANOVA) tests followed by Tukey’s or Bonferroni multiple comparisons test were applied, respectively. Survival data were analyzed using Kaplan–Meier survival curves, and differences between groups were assessed with the Log-rank (Mantel–Cox) test. Data are presented as means ± SEM. Differences were considered significant at p < 0.05.

While NETosis originally evolved to eliminate pathogens, it also occurs during sterile inflammation, where excessive neutrophil activation can cause tissue damage (Kovtun et al. 2018). In the lungs, NETs compromise the alveolar-capillary barrier, increasing permeability and leading to hemorrhages (Narasaraju et al. 2011). Following pulmonary exposure to ricin, extensive neutrophil infiltration has been observed constituting nearly half of all lung cells at certain time points post-exposure (Sapoznikov et al. 2015, 2019). To determine whether these pulmonary neutrophils undergo NETosis, we intranasally exposed mice to ricin and collected BALF at 24, 48, and 72 h post‑exposure. The presence and levels of PAD4, citH3, and MPO which are the hallmarks of NETosis, were assessed in BALF by Western blot analysis. Results revealed a significant increase in PAD4 levels in BALF at 24 h post-exposure, reaching peak levels at 48 h, followed by a decline at 72 h (Fig. 1A, B). CitH3 levels peaked at 48 h and remained high at 72 h (Fig. 1C, D), consistent with ELISA measurements of citH3 (Fig. 1E). Similar kinetics were observed for MPO (Fig. 1F, G). The presence of PAD4, citrullinated histones and extracellular MPO in BALF indicates that NETosis occurred in the lungs following exposure to ricin.

In addition, the level of cell-free DNA was quantified at different time-points following ricin exposure using Sytox Green dye. This dye intercalates into DNA that is accessible outside of intact cells, such as that released during NETosis or from dead/dying cells. The analysis showed that of cell-free DNA levels were already elevated 24 h post-ricin exposure and continued to raise at 48 and 72 h (Fig. 1H). Importantly, Sytox Green does not distinguish the source of extracellular DNA, which may originate from NETs, as well as from apoptotic or necrotic cells. The latter are abundant in the lung following ricin intoxication, owing to direct ribosome inactivation and inhibition of protein synthesis that ultimately leads to cell death (Wilhelmsen and Pitt 1996; Sapoznikov et al. 2015). Therefore, we combined Sytox Green (SG) staining with NET-specific markers to more precisely determine the contribution of NETs to extracellular DNA. To directly visualize NET formation in situ, BALF samples were analyzed for extracellular DNA and citH3 staining. Control samples displayed intact alveolar macrophages and neutrophils, whereas in BALF samples from exposed mice, only neutrophils were observed (Fig. 2A) due to rapid alveolar macrophage depletion following intoxication (Sapoznikov et al. 2015). At 24 h post-exposure, citH3-positive staining was detected, but characteristic NET structures of extracellular DNA were not yet visible. By 48 and 72 h, thread-like extracellular structures positive for citH3, extracellular DNA staining and extending from neutrophils were observed, indicating widespread NET formation (Fig. 2A). MPO staining revealed that the enzyme was released from neutrophils at 24–72 h post-exposure, but its distribution was not restricted to NETs, sometimes appearing as dispersed punctate staining near neutrophils (Fig. 2C, Ricin 48 h) rather than being associated with chromatin networks (Fig. 2D, Ricin 72 h). Therefore, we selected a combination of citH3 and extracellular DNA staining as markers of NETs throughout this study.

Since BALF neutrophils represent only a subgroup of total lung neutrophils, we next examined NETosis in neutrophils isolated directly from lung tissue. Neutrophils were enriched using density gradient centrifugation and isolated by magnetic sorting at 48 h post-exposure, the timepoint of peak NETosis, as was shown in Fig. 1. Isolated neutrophils were stained for extracellular DNA and citH3, revealing that lung-resident neutrophils underwent NETosis similar to BALF neutrophils, with characteristic extracellular chromatin networks (Fig. 3A, B). To ensure that the observed NETosis was not an artifact resulting from neutrophil activation during the isolation process, we quantified NETosis (extracellular DNA filaments positive for citH3) and compared the number of NET-forming neutrophils in both control- and ricin-intoxicated mice 48 h post-exposure. Quantification of NETosis confirmed significantly higher rates in neutrophils from exposed mice compared to controls (Fig. 3C).

To confirm NETosis within lung tissue in vivo, mice were exposed to ricin, and lungs were harvested at 24, 48, and 72 h post-exposure for immunofluorescent analysis. This approach allowed us to visualize the presence and localization of key NETosis markers within the lung parenchyma over time. Lung sections were stained for neutrophils (Ly6B), extracellular DNA, and citH3. Control lungs contained few neutrophils (Fig. 4A, C). At 24 h post-exposure, neutrophil recruitment was evident, but NETosis had not yet occurred (Fig. 4A, C, D, E). By 48 h, numerous neutrophils exhibited colocalized extracellular DNA and citH3 staining, indicative of NETosis (Fig. 4A, B, C, D, E). Neutrophils infiltrating the lungs through blood vessels could also be observed, already undergoing NETosis during the process of migration (Fig. 4A, B). At 72 h, NETosis remained extensive, but was slightly reduced compared to 48 h (Fig. 4A, D, E). These findings align with the Western blot results (Fig. 1).

Given the extensive NETosis observed in the lungs of mice that were exposed to ricin, we hypothesized that extracellular DNA release during this process contributes to lung inflammation and edema, exacerbating respiratory distress. Therefore, we evaluated whether treatment with a NET-degrading DNase I could alleviate lung pathology. For this purpose, we selected PRX-119, a PEGylated recombinant human DNase I that exhibits extended half-life in the circulation and has previously shown efficacy in sepsis mouse model of NET-related disease (Sharma et al. 2025). We employed the following experimental protocol to test the treatment: mice were intranasally intoxicated with ricin and treated at 24 h post-exposure with either PRX-119, anti-ricin antibody, or both (Fig. 5A). In our therapeutic approach, anti-ricin antibody treatment is always administered as a first-line intervention that prevents toxin binding to cells, thereby neutralizing its catalytic activity and downstream effects. Our previous studies have demonstrated that anti-ricin antibodies are essential for achieving partial survival in ricin-intoxicated animals. Notably, adjunctive therapies alone show limited or no efficacy in the absence of anti-ricin treatment. Therefore, additional targeted interventions are administered in combination with anti-ricin antibodies to enhance therapeutic outcomes (Gal et al. 2014, 2016).

At different time points post-exposure we quantified NETosis markers and NET degradation, and evaluated lung pathology (Fig. 5A). First, we examined the effect of the treatment on extracellular DNA content in the lungs. BALF was collected 72 h post-exposure from intranasally ricin-intoxicated mice, as well as from intoxicated mice treated with an anti-ricin antibody, PRX-119 as a standalone treatment, or a combination of anti-ricin antibody and PRX-119. The results indicated that in contrast to antibody treatment alone, which did not affect the high levels of extracellular DNA, PRX-119 treatment alone significantly reduced extracellular DNA levels in the lungs. Similarly, the combined treatment with the antibody and PRX-119 led to a significant decrease in extracellular DNA concentration (Fig. 5B). It is important to note that despite the significant reduction in extracellular DNA levels following PRX-119 treatment, high cell-free DNA levels remained in the lungs even 72 h post-exposure. Therefore, we measured DNA levels at later time points, 96- and 120-hours post-exposure. These measurements revealed that extracellular DNA levels continued to decline significantly following the combined treatment, compared to treatment with the anti-toxin antibody alone (Fig. 5C).

Next, the protein levels of key components involved in the NETosis process were analyzed by Western Blot to assess their trends following treatment. We observed a reduction in citH3 levels following antibody treatment alone, which was further enhanced following the combined therapy, reaching near-baseline levels (Fig. 6A, B). This reduction pattern was confirmed by ELISA for citH3 (Fig. 6C). PAD4 levels were also reduced following treatment with anti-ricin antibody alone, as well as in combination with PRX-119 treatment (Fig. 6D, E). These results demonstrate that the combination therapy, comprising an anti-toxin antibody and PRX-119, which degrades extracellular DNA, significantly reduced NETosis, nearly abolishing the process.

To further monitor the effect of the above-mentioned treatments on NETosis, we analyzed lung sections 48 h post-exposure by immunofluorescence. Extensive NETosis in ricin exposed untreated mice was evident, characterized by a high number of neutrophils positive for extracellular DNA and citH3. At the same time point, a significant reduction in NETosis was observed following antibody treatment alone. In agreement with the effects on NETosis markers, an almost complete resolution of NETosis was detected in the combination treatment group (Fig. 7A, B, C, D, E).

To evaluate the impact of treatment on lung pathology, histological analysis was performed on lung tissue sections from intoxicated and treated mice. Histological examination using hematoxylin and eosin (H&E) staining of the lungs from intoxicated mice revealed severely edematous, non-aerated lungs, heavily infiltrated with inflammatory cells and displaying increased tissue density. Focusing on perivascular regions, we observed significant neutrophil infiltration, marked expansion of these areas, and the presence of proteinaceous exudates. Pulmonary blood vessels were congested with red blood cells, surrounded by perivascular and peribronchiolar edema, and showed evidence of cellular debris indicative of cell death. Within the alveoli, prominent neutrophilic infiltration, cell death and edema were evident. The lungs exhibited extensive vascular leakage and fibrin accumulation within the alveolar airspaces. These pathological changes contrasted sharply with the normal, well-aerated lung structure observed in non-intoxicated control mice, which displayed an intact bronchiolar and alveolar architecture, contained alveolar macrophages and was devoid of neutrophil infiltration (Fig. 8A). It was observed that following antibody treatment alone, the lungs remained damaged, with poor aeration, inflammatory cell infiltration, and edematous foci (Fig. 8A). In contrast, lungs from mice that received combination therapy were better aerated, with only minimal inflammatory foci, well-preserved alveolar architecture, and very limited perivascular infiltration, appearing more similar to those of control mice (Fig. 8A). A semi-quantitative assessment of the lung histological injury score revealed that the score in the combination treatment group was significantly lower than that of the anti-ricin monotherapy group and that both treatment groups scored significantly lower than the intoxicated untreated group (42 ± 14%, 59 ± 9%, 65 ± 7% for combination treatment, monotherapy and ricin exposed untreated groups, respectively) (Fig. 8B). Increased permeability of the alveolar-capillary barrier and the influx of protein-rich fluid into the interstitium and alveolar space are hallmark features of pulmonary ricin intoxication. These processes lead to the development of pulmonary edema, respiratory failure, and ultimately, death (Gal et al. 2017). Previously, we had demonstrated that antibody treatment administered 24 h post-exposure to ricin reduced lung permeability (Sapoznikov et al. 2019). Here, we assessed whether the combined PRX-119 and antibody treatment could further mitigate the breakdown of the alveolar-capillary barrier compared to antibody treatment alone. To that end, lung permeability was assessed using the EBD Extravasation Assay. The results showed that while the increased permeability observed at 72 h post-exposure was partially reduced following antibody treatment, the combined treatment significantly improved barrier integrity compared to antibody treatment alone (Fig. 8C). These findings demonstrate that the combined treatment effectively mitigates the morphological and histological lung damage following pulmonary ricin exposure and significantly reduces the disruption of the alveolar-capillary barrier.

To evaluate the impact of PRX-119 treatment on the process of lung recovery and the reduction of inflammatory markers following ricin exposure, we examined changes in various pro- and anti-inflammatory cytokines. Cytokine levels were measured in the BALF of mice exposed to ricin and treated with either PRX-119 or anti-ricin antibody alone, or a combination of both therapies. We found that IL-6, the main pro-inflammatory cytokine which is elevated after ricin exposure (Gal et al. 2014, 2016), remained unaffected by PRX-119 treatment. However, antibody treatment against ricin significantly reduced its level. In agreement with this finding, the combined treatment did not provide any additional benefit in lowering this pro-inflammatory cytokine (Supplementary Fig. 1 A). Similarly, pro-inflammatory cytokines responsible for neutrophil recruitment to the lung tissue, such as G-CSF and KC (CXCL1), which were markedly elevated post-ricin intoxication, were significantly reduced following antibody treatment, with no further reduction observed with the combined treatment (Supplementary Fig. 1B, C). Other cytokines, including MIP-2 (CXCL2), IL-1β, MCP-1 (CCL2), VEGF, and TNF-α, which contribute to neutrophil and monocyte recruitment, increased endothelial permeability, and impaired alveolar fluid clearance, were moderately elevated after ricin exposure. PRX-119 alone had no impact on these cytokines. Unexpectedly, antibody monotherapy was found to increase the level of these cytokines in BALF, an effect which was not mitigated by the combined therapy (Supplementary Fig. 1D, E, F, G, H).

Since no significant differences were found in the levels of tested cytokines between antibody-only treatment and combined therapy, we sought to identify additional analytes that could characterize PRX-119 impact on lung recovery. For this purpose, we assembled a panel of factors involved in various pro- and anti-inflammatory processes that influence lung epithelial and endothelial cell repair, restoration of vascular leakage, reduction of pulmonary edema, fibrosis-related proteins, and markers of lung remodeling (Supplementary Fig. 2). This multi-analyte panel was assessed by Multiplex ELISA which was applied on BALF samples from three groups: ricin-exposed untreated mice, ricin-exposed mice treated with an anti-ricin antibody, and ricin-exposed mice treated with the combination of anti-ricin antibody and PRX-119 (Supplementary Fig. 3). Several analytes were identified as significantly altered following combination treatment. The protein Dkk-1 was elevated following ricin exposure, with even higher levels detected in mice that received antibody-only treatment. However, combination therapy significantly reduced Dkk-1 levels (Fig. 9A). Another protein, CD93 was found to be significantly lower in mice treated with combination therapy, whereas no substantial change was observed in the antibody-only treatment group compared to untreated ricin-exposed mice (Fig. 9B). Similarly, we found markedly elevated levels of Periostin in ricin-exposed mice, which were not significantly reduced by antibody monotherapy. In contrast, the combination therapy substantially decreased periostin levels (Fig. 9C). Altogether, these findings show that combination therapy reduced the levels of Dkk-1, CD93, and Periostin.

Having demonstrated that combined treatment with an anti-ricin antibody and PRX-119 inhibited NETosis, significantly mitigated lung damage and reduced the disruption of the alveolar-capillary barrier following pulmonary ricin exposure, we next evaluated its clinical effect in ricin-intoxicated mice. Mice were treated at 24 h post-exposure to ricin with either PRX-119, anti-ricin antibody, or with combination treatment. Weight loss and survival were monitored for 14 days. Mice treated with PRX-119 alone exhibited weight loss similar to ricin-intoxicated untreated mice and the animals in both groups succumbed to intoxication by day 4–6 (Fig. 10A, B). Animals receiving antibody treatment alone lost ~ 30% of body weight before beginning of recovery at around day 9. Interestingly, combination treatment further reduced morbidity as reflected by limited weight loss (20%) and earlier recovery beginning around day 6 (Fig. 10A). While PRX-119 alone did not improve survival, combination treatment significantly increased survival to 73%, in comparison to 40% survival following antibody treatment alone (Fig. 10B).

The present study was conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) Israeli law and was approved by the Ethics Committee of Animal Experiments of the Israel Institute for Biological Research (project identification codes M-16-2023, M-12-24).

Not applicable.

The authors declare no competing interests.

No datasets were generated or analysed during the current study.