Furin inhibition protects against acute lung injury in a mouse model of Pseudomonas aeruginosa infection

Abstract

Pseudomonas aeruginosa (PA) is responsible for significant morbidity and mortality, particularly in patients with chronic lung diseases such as chronic obstructive pulmonary disease, bronchiectasis, cystic fibrosis, as well as hospital-acquired pneumonia and ventilator-associated pneumonia. The rise of antibiotic-resistant PA strains necessitates alternative treatment strategies. Among the different toxins secreted by PA, Exotoxin-A (Exo-A) becomes cytotoxic when cleaved by furin. This study investigates the therapeutic potential of furin inhibitor BOS-318 in mitigating acute lung injury induced by Exo-A and PA infection. Furin inhibition significantly improved survival rates and reduced lung injury in mouse pneumonia models using Exo-A and PA103. Additionally, BOS-318 accelerated bacterial clearance in vivo, and increased phagocytosis by alveolar macrophages. Bulk RNA-sequencing done on whole lung homogenate at 6 hours revealed an immune profile with decreased natural killer cell signaling in the BOS-318-treated group, possibly because of a decrease in natural killer recruitment observed at 24 hours, suggesting a role of furin in shaping the immune response. Moreover, administration of BOS-318 as a therapeutic strategy results in a protection of the lung epithelium. Overall, our findings demonstrate that furin inhibition protects against PA-induced acute lung injury and hastens bacterial clearance. These results are the first to characterize furin inhibition in animal models and supports its potential use as an adjunctive therapeutic strategy for treating PA infections.

Clinical Relevance.

Clinical Relevance Furin inhibition with BOS-318 offers a promising adjunctive therapy for Pseudomonas aeruginosa infections, which are a major cause of morbidity and mortality in patients with chronic lung diseases and hospital-acquired pneumonia. This study demonstrates that BOS-318 improves survival, reduces lung injury, and accelerates bacterial clearance in murine models, highlighting its potential to address antibiotic resistance and enhance immune-mediated pathogen eradication. These findings support further clinical evaluation of furin inhibitors as a novel strategy to protect lung function and improve outcomes in high-risk patient populations.

Introduction

Pseudomonas aeruginosa (PA), an opportunistic Gram-negative bacterial pathogen, is a common cause of morbidity and mortality in hospital-acquired pneumonia and ventilator-associated pneumonia, immunocompromised patients, and those with structural lung disease such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF).^1,2 Despite recent therapeutic advances, PA infection remains a significant challenge in CF. The prevalence of PA in adults with CF was estimated in 2021 at 34.2% and is associated with significant morbidity such that, on first colonization, extensive antibiotic courses are used in an attempt at eradication.^3,4 PA leads to increased lung function decline, pulmonary exacerbations, and mortality from terminal respiratory failure due to inflammation and airway damage.⁵ PA’s ability to form biofilms complicates treatment by hindering antibiotic penetration, reducing drug efficacy, and promoting antibiotic resistance. Despite effective CFTR modulator therapies, clonal lineages persist in patients with CF, particularly in those with advanced lung disease. Therefore, new therapies that assist eradication of PA and decrease PA-associated lung inflammation are needed in CF, and these advances could also greatly benefit COPD, hospital-acquired pneumonia, and ventilator-associated pneumonia patient populations.⁶ PA employs both intrinsic and acquired resistance mechanisms, making successful eradication increasingly challenging.⁶ Through the secretion of virulence factors, PA causes cellular damage, impairs the immune response, and enhances its own survival.⁷ Among these factors, exotoxin-A (Exo-A) is one of the most prevalent virulence factors in resistant clinical isolates of PA and mediates virulence by inhibiting protein synthesis in host cells as well as delivering effector proteins directly into host cells, disrupting cellular processes and contributing to infection.⁸ As a result, higher titers of immunoglobulin G antibody to Exo-A are associated with mortality in patients with CF.⁹ After being secreted by PA, Exo-A is internalized and cleaved by a proprotein convertase subtilisin/kexin 3 (PCSK3), also known as furin. Its toxic effect arises from its ability to inactivate cytosolic elongation factor 2, thereby inhibiting protein biosynthesis leading to cell death.^10-12 Exo-A has been shown to impair natural killer (NK) cell cytotoxicity functions.¹³ On the other hand, another virulence factor secreted by PA but not cleaved by furin, Exo-T has been shown to increase numbers of NK cells in the lung tissue during PA-induced pneumonia.¹⁴ There is no evidence that Exo-T contains a furin cleavage site, nor does Exo-T require cleavage for activation.¹⁵ BOS-318 has been reported to be an exceptionally potent and highly selective furin inhibitor, which prevents Exo-A-induced cell toxicity in human primary epithelial cells.¹⁶

For the first time, we report that BOS-318 modulates Exo-A and PA103-induced lung injury in animal models. Remarkably, inhibiting furin in our mouse models increased survival and reduced lung injury. In the live bacteria model, bacterial clearance was accelerated, facilitated by enhanced alveolar macrophage phagocytosis. Interestingly, furin inhibition also modulated NK infiltration in the lung tissue. Together, our findings support furin inhibition as a therapeutic strategy to treat PA infection.

Methods

Care and use of mice

Mice were housed under pathogen-free conditions at the University of California San Francisco Laboratory Animal Research Center and all experiments conformed to ethical principles and guidelines approved by University of California San Francisco Institutional Animal Care and Use Committee. Additional information provided in the online supplement.

Bacterial cultures

PA103 was kept at -80 °C and streaked onto plates overnight, after which 2 single colonies were selected to inoculate the subculture and promote purity using a previously published protocol.¹⁷ Additional information is provided in the online supplement. Lung injury models: A 200-µL solution of 100 µL PBS + 100 µL of 10% DMSO/90% corn oil (v/v) (veh) or 100 µL PBS + 100 µL BOS-318 (HY-147140, MCE) at 10 mM resuspended in vehicle-injected intraperitoneally (i.p.) 1 hour before oropharyngeal administration (o.a.) according to previously published method.¹⁸ Mice were then anesthetized with isoflurane 5%, then a 50-µL solution of 40 µL PBS + 5 µL DMSO or BOS-318 resuspended at 8.74 mM in DMSO + 5 µL Exo-A (0.25 µg) (Sigma P0184) or 5 µL PA103 was delivered via o.a. Following this o.a. challenge, additional i.p. treatments of PBS + vehicle or PBS + BOS-318 were given at 24, 48, and 72 hours, and mice were sacrificed at 96 hours in the nonlethal experiments. The treatment groups in the Exo-A model in Figure 1 were¹ PBS + vehicle i.p. and Exo-A o.a.² BOS-318 i.p. and Exo-A + BOS-318 o.a. In Supplemental Figure 1A, the groups were¹ PBS + vehicle i.p. and PBS + vehicle o.a. and² PBS + vehicle i.p. and BOS-318 o.a.

Figure 1.

Furin inhibition prevents exotoxin-A-induced acute lung injury in vivo. (A) Schematic of model, in which mice received intraperitoneal (i.p.) PBS + vehicle or PBS + BOS-318 (5 mM final concentration) 1 hour before oropharyngeal administration (o.a.) of 0.25 µg of Exo-A + PBS or 0.25 µg of Exo-A + BOS-318 (0.874 mM final concentration) followed by i.p. treatment with PBS + vehicle or PBS + BOS-318 (5 mM final) at 24, 48, and 72 hours. (B, C) Temperature and weight loss. **P < .01, ****P < .0001 with a 2-way analysis of variance with post hoc Tukey’s multiple comparisons test. (D) Survival curve. n = 9 mice per group, **P = .0025 with a log-rank test. (E) Bronchoalveolar lavage (BAL) protein concentration.***P = .0001 with unpaired t-test (n = 3–7 per group).

In the PA103 model used in Figures 2–6, the groups are¹ PBS + veh i.p. and PA103 + veh o.a. and² BOS-318 in veh i.p. and PA103 + BOS-318. All mice in the BOS-318 arms received BOS-318 both i.p. and o.a. In the nonlethal model, animals received 1 × 10⁵ colony-forming units (CFU) of PA103 o.a. In the survival experiment, animals received 2 × 10⁵ CFU of PA103 o.a. In the curative model used in Supplemental Figure 2, animals received 6.6 × 10⁴ CFU of PA103 o.a. in a total volume of 30 µL then received 1 hour later an i.p. injection of BOS-318 or vehicle as previously described, and an injection of 40 µL of DMSO or BOS-318 using the same concentration as previously described. Mice were euthanized at different time points to collect either lungs or bronchoalveolar lavage (BAL). BAL was used to count CFUs, analyzed by flow or stored for further analysis (see the online supplement).

Figure 2.

Furin inhibition decreases Pseudomonas aeruginosa-induced lung injury and mortality. (A) schematic depicting experimental procedure. Mice were injected intraperitoneally (i.p.) with 200 µL furin inhibitor BOS-318 5 mM final or vehicle 1 hour prior oropharyngeal aspiration (o.a.) with 2 × 10⁵ CFU of PA103 for survival experiment (B-D) or 1 × 10⁵ CFU (E–H) for animals aiming to be sacrificed in 45 µL with 5 µL of BOS-318 at 0.874 mM final or DMSO as a control. (B, C) Temperature and percentage of weight loss. *P < .05, **P < .01, ***P < .001, ****P < .0001. Mixed-effect analysis with a Tukey’s multiple comparisons test. (D) Survival curve (n = 10 mice per group), **P < .01 with a log-rank test. (E-G) Bronchoalveolar lavage protein and albumin concentration at 6, 24, and 72 hours after infection. *P = .05 with unpaired t-test (n = 7–8 per group). (H) Representative microphotograph of mouse left lung 24 hours after infection by hematoxylin and eosin. Scale 2.5 mm (left) and 200 µm (right). N = 2–6/group.

Figure 3.

Furin inhibitor reduces Pseudomonas aeruginosa-associated cytotoxicity. (A) Lungs from mice were collected 24 hours after infection and stained for TUNEL. Scale 600 µm. (B) Percentage of TUNEL-positive areas calculated in the left lobe using high-resolution photomicrographs. n = 2–6 mice/group. *P < .05 using unpaired t-test. (C–E) G6PD enzyme activity assayed in bronchoalveolar lavage (BAL) realized at 6, 24, and 72 hours after infection. n = 5–10 mice/group. *P < .05, ***P < .0001 using unpaired t-test.

Figure 4.

Furin inhibition improves bacterial clearance and neutrophilic inflammation. (A–C) BAL cytokine (MIP-2, MCP-1, KC) expression and neutrophil-elastase-DNA levels at 6, 24, and 72 hours after infection. (D–I) BAL analysis of CD45+, alveolar macrophages, and neutrophils numbers or percentage determined by flow cytometry using counting beads. n = 5–8 mice/group. (J–L) Neutrophil extracellular traps secretion in BAL. n = 4–10 mice/group. *P < .05, **P < .01, **P < .001, ****P < .0001.

Figure 5.

Furin inhibition improves bacterial clearance and reduces neutrophil infiltration. (A) Mouse lungs were collected at 2, 6, or 24 hours after infection and CFUs were counted (1 point per mouse). *P < .05 with unpaired t-test (n = 7–8 mice per group). (B, C) Mouse-isolated alveolar macrophages incubated with DMSO or BOS-318 for 2 hours, then media was replaced by pHrodo for 1 hour, 30 minutes, before recording phagocytosis by flow cytometry. ***P < .001 with paired t-test (n = 7 mice).

Figure 6.

Transcriptomic analysis of PA-infected lungs reveals a downregulation of natural killer cell-related genes. Bulk RNA_seq was performed on whole lungs 6 hours after infection. n = 4 mice/group. (A) Volcano plot displaying genes with FDR <0.05. (B) KEGG mouse pathways with DFR <0.01. (C) Heatmap displaying genes with FDR <0.01. (D, E) Lungs from mice were collected 24 hours after infection and stained for NK1.1. Scale 100 µm; n = 2–6 mice/group. **P < .01.

Histology

(see the online supplement).

Bulk-RNAseq

(see the online supplement).

Statistical analyses

Comparisons between 2 groups were done with relevant paired or unpaired t-test or Mann–Whitney U test when data were not normally distributed. Additional information provided in the online supplement.

Study approval

All animal experiments were approved by the Institutional Animal Care and Use Committee at University of California San Francisco.

Data availability

Bulk RNA_seq data and raw data have been provided as a supporting data file.

Results

Administration of a furin inhibitor protects mice from exotoxin-A lung injury

Previously, Douglas et al. demonstrated that a new furin inhibitor, BOS-318, prevents exotoxin-A induced cytotoxicity in explanted human bronchial epithelial cells,¹⁶ yet whether BOS-318 affects baseline inflammation in vivo is unknown. Here, we investigated whether furin inhibition prevents exotoxin-A induced lung injury in exogenous toxin and live bacterial mouse models of lung injury. To characterize safety, BOS-318 or vehicle (DMSO) was administered by o.a. at day 0. Another treatment was done by i.p. 1 hour before o.a. and every 24 hours until 72 hours, and mice were monitored and sacrificed at 96 hours (Supplemental Figure 1A). No differences were observed in total immune cell population, neutrophils and monocytes counts in the BAL between groups (Supplemental Figure 1B-D). Furin inhibition has been shown to prevent cleavage of newly expressed α and γ subunits of ENaC in vitro²¹ potentially affecting its activity, and sodium concentration in the respiratory tract. Here, the sodium concentration in the BAL remained unchanged after BOS-318 treatment (Supplemental Figure 1E). Therefore, BOS-318 does not affect sodium homeostasis or induce lung inflammation.

We then tested the inhibitor using an Exo-A-induced lung injury model. As described previously, mice were pretreated with BOS-318 or vehicle i.p. then administered Exo-A + BOS-318 or vehicle by o.a. (Figure 1A). Body temperature was significantly decreased in the vehicle group at 48 hours after Exo-A, whereas mice treated with the furin inhibitor remained relatively stable. Both groups had lower body temperatures, but the BOS-318-treated mice were significantly less hypothermic (Figure 1B). Mice treated with the vehicle also lost more weight than the BOS-318-treated group after 48 and 72 hours (Figure 1C). Moreover, mortality at 96 hours in the vehicle group was significantly greater with only 12.5% survivorship in the vehicle group compared to 78% in the treatment arm (Figure 1D). Taken together, BOS-318 conveyed a survival advantage in the Exo-A mouse model. To test for damage to the alveolar-capillary barrier, we measured total protein in the BAL, which was higher in the control group, suggesting a protective role of BOS-318 in Exo-A induced lung damage and permeability (Figure 1E). Overall, these results demonstrate that BOS-318, reduces Exo-A induced lung injury and mortality in mice.

Administration of a furin inhibitor decreases PA-induced lung injury and mortality

Exo-A is one of several extracellular toxins secreted by PA, which can induce leukopenia, acidosis, circulatory collapse, liver necrosis, pulmonary edema, hemorrhage, and acute tubular necrosis of the kidneys. Thus, we pursued a live bacterial model to evaluate the effects of BOS-318 against the full spectrum of PA-induced virulence. The Centers for Disease Control and Prevention has declared that Pseudomonas strain PA103 is a major concern in health care settings because of its ability to cause outbreaks and its high level of antibiotic resistance. Moreover, PA103, produces significantly higher levels of Exo-A than the prototypic strain PAO1.²² To assess the therapeutic potential of furin inhibition, we administered the furin inhibitor BOS-318 1 hour after intratracheal infection with 6.6 × 10⁴ CFU PA103 (Supplemental Figure 2A). BOS-318 treatment significantly reduced protein levels in BAL fluid compared to the vehicle group, indicating decreased lung permeability (Supplemental Figure 2B). However, therapeutic administration of BOS-318 did not affect bacterial clearance (Supplemental Figure 2C). Although the percentage of neutrophils remained statistically unchanged, total CD45⁺immune cell and neutrophil counts in BAL were significantly lower in the BOS-318-treated group (Supplemental Figure 2D, E). We compared then the curative effect of furin inhibition to its preventive effect. Mice were treated i.p. before infection with BOS-318 or vehicle (Figure 2A). In the survival experiment, mice were challenged with o.a. of 2 × 10⁵ CFU PA103. In the BOS-318-treated group, body temperature was significantly higher as early as 4 hours after PA103 injection (Figure 2B). Body weight was lower in the control group at 10 hours compared to the BOS-318-treated group (Figure 2C). All mice from the control group died within 48 hours, whereas the BOS-318 group exhibited 40% survival (Figure 2D). Next, we studied furin inhibition in a nonlethal infection model to characterize long-term effects of immune responses and tissue damage due to PA. Mice were infected with 1 × 10⁵ CFU. BAL protein levels were elevated as soon as 6 hours after infection and remained elevated at 72 hours (Figure 2E-G). The BOS-318-treated group had lower protein levels in BAL compared to the vehicle group at all times, indicative of reduced lung permeability. Similar differences were seen in BAL albumin content at 24 hours, suggesting that furin inhibition protected against vascular leak. At 24 hours, PA103 infection led to a dense inflammatory infiltrate characterized by alveolar wall edema and neutrophilic infiltration into the lung parenchyma, which was significantly reduced in the BOS-318 group (Figure 2H).

Furin inhibitor reduces PA-associated cytotoxicity

Furin is known to activate Exo-A, enhancing PA-induced cell death in the lung. Furin inhibition using BOS-318 has been shown to be effective in vitro to prevent PA-related toxin Exo-A induced cell toxicity.¹⁶ To investigate whether furin inhibition can rescue the lung epithelium from PA103 associated toxicity in vivo, we used TUNEL staining detecting DNA fragmentation to label cell death. Furin inhibition led to decreased TUNEL staining at 24 hours (P = .0135) (Figure 3A, B). As a second method to assess cell death in lung airspaces, we measured glucose-6-phosphate dehydrogenase (G6PD) activity in BAL. Damaged cells release G6PD, a cytosolic enzyme leaking from cells when plasma membrane integrity is compromised.²³ G6PD activity in BAL was significantly decreased in the BOS-318 group at 24 hours then 72 hours (Figure 3C-E), which correlates with albumin infiltration in the airspaces (Figure 2E-G). Overall, those results indicate that furin inhibition ameliorates PA-induced cell death and protects the lung epithelium.

Furin inhibition improves bacterial clearance and neutrophilic inflammation

The lung epithelium provides the first line of defense against respiratory pathogens by releasing inflammatory mediators that precipitate immune responses. We demonstrated that furin inhibition improves PA-associated cell death, which we postulated occurs by modulating inflammatory responses. We characterized major chemokines in the BAL in our mouse models. The murine chemokines KC (CXCL1) and MIP-2 (CXCL2) are the major chemoattractants responsible for neutrophil recruitment (Figure 4A-C). MIP-2 was significantly (and KC trending to be) less secreted in the BOS-318 group as early as 6 hours. Another chemokine, CCL2, also called monocyte chemoattractant protein-1 (MCP-1) was also decreased at 6 hours (Figure 4A-C), which suggests that BOS-318 reduces proinflammatory mediator secretion following PA103 infection. To explore the effects on immune cell recruitment to the lung, BAL samples were analyzed by flow cytometry. CD45+ cells in BAL did not differ at 6 hours between groups; however, BOS-318 treatment led to lower total white blood cell counts, which were predominantly neutrophilic at 24 and 72 hours (Figure 4D-F). In bacterial pneumonia, neutrophils drive a pro-inflammatory process called NETosis, or neutrophil extracellular trap (NET) formation. This web-like structure induces tissue damage and is targeted by therapies such as recombinant human DNase, or Pulmozyme, in human CF patients.^24,25 BAL NETs were significantly lower in the BOS-318 group at 24 and 72 hours compared to control (Figure 4J-L), mirroring the decreased neutrophil counts. Resident immune cells in the lung, such as alveolar macrophages (AMs), are responsible for pathogen detection and phagocytosis. AM counts was higher in the BOS-318 group in early time points (6 and 24 hours) and were more abundant at 24 and 72 hours (Figure 4G-I).

As immune cells are paramount for bacterial clearance, we explored the effect of BOS-318 treatment on bacterial load. CFU counts in whole lungs at 2 hours showed that the bacterial burden was unchanged between our 2 groups, indicating that the inhibitor does not directly affect very early bacteria growth or survival (Figure 5A). As early as 6 hours p.i., PA burden was significantly decreased in the BOS-318 group, this effect persisted at 24 hours. Interestingly, the decrease of bacterial burden at 6 hours after infection does not coincide with increased neutrophil counts in the BAL. However, more alveolar macrophages are present in the BOS-318 group. Alveolar macrophages have been described to contribute to PA clearance²⁶; hence, we explored the effect of furin-inhibition on alveolar macrophages phagocytosis. To do so, mouse alveolar macrophages were pre-treated with BOS-318 before exposure to Escherichia coli bioparticles conjugated with pHrodo. The fluorescence of the pHrodo dye dramatically increases as pH decreases from neutral to acidic in phagolysosomes. Hence, phagocytosis is detected by fluorescence detection using flow cytometry (Figure 5B). BOS-318 pretreated alveolar macrophages displayed increased phagocytosis capabilities (Figure 5C). This effect was specific to alveolar macrophages as furin inhibition did not modulate neutrophil phagocytosis activity (data not shown). These results suggest that furin inhibition accelerated bacterial clearance by increasing alveolar macrophage phagocytosis activity.

Transcriptomic analysis of PA-infected lungs reveals a downregulation of natural killer cell-related genes

Furin has pleiotropic effects by processing a wide range of precursor proteins into their active forms, influencing various biological processes. To identify pathways or molecular targets modulated by furin during early PA infection, we performed bulk RNA-seq on mouse lung samples at 6 hours after infection (Figure 5A-C). Few genes were upregulated in the BOS-318 group (Figure 5A), but no distinct pathways were highlighted using gene ontology enrichment analysis. Interestingly, the most significantly downregulated pathway in the BOS-318-treated group highlights a NK cell population signature and thus can be interpreted by a lower accumulation of NK cells in the lung tissue (Figure 5B). NK-related top genes downregulated in the BOS-318 group are shown in the volcano plot and the heatmap (Figure 5A, C), including granzymes A, B, Natural Killer Cell Granule Protein 7 (NKG7), killer cell lectin-like receptor subfamily A member 7 (klra7), or perforin. To decipher whether furin inhibition modulates actual signaling molecules or cell number, we then tested for NK cells in the lung tissue. We found that the influx of NK cells at 24 hours after infection was reversed by furin-inhibition. Our data show that furin inhibition limits NK cells infiltration in the lung following PA infection.

Discussion

In this study, we provide the first characterization of furin inhibition using selective BOS-318 in animal models, advancing an approach to modulate the host-pathogen immune response in bacterial pneumonia. Our major findings include: (1) BOS-318 attenuates Exo-A-induced lung permeability and mortality; (2) BOS-318 improves survival by reducing lung inflammation and tissue damage, evidenced by decreased protein leakage in BAL, decreased neutrophilic infiltration and NETosis, and a preservation of alveolar macrophages; (3) BOS-318 enhances phagocytosis by alveolar macrophages in vitro and bacterial clearance in vivo. We also showed a preventive effect of BOS-318 on NK cell infiltration. Together, these observations support that furin inhibition is an effective therapeutic approach to mitigate PA-mediated pulmonary damage.

The cytotoxic domain of Exo-A has been harnessed in immunotherapy approaches, where it is fused to tumor-related antigens, in order to penetrate cancer cell membranes and inhibit protein synthesis,²⁷ highlighting its potent biological activity. Indeed, targeting this toxin secreted by PA could dampen its virulence and reduce the severity of acute infections and chronic inflammation. Several non-specific furin inhibitors have been developed, including Hexa-D-arginine, which show protection in a Exo-A mouse model, however, the lack of specificity and potency of the inhibitors have resulted in these agents stalling in the preclinical pipeline.^28-30 The IC50 of BOS-318 is reported at 1.9 nM, comparable to Furin I, a previously described inhibitor with an IC50 of 1.3 nM. In Ref 14, Douglas et al. state that BOS-318 is specific to furin within the PCSK family, yet furin shares structural homology with the PCSK5/6/7 active sites. This implies that BOS-318 could act by a similar mechanism on other PCSK. However, the authors characterize the crystal structure of BOS-318 bound to furin, in which BOS-318 binds to an alternate site independent of the catalytic active site, accounting for the high selectivity and slow dissociation of BOS-318. Our findings are in concordance with previous work by Douglas et al.,¹⁶ which demonstrated that BOS-318 potently and selectively inhibited furin-mediated cleavage of Exo-A in vitro, thereby preventing its cytotoxic effects. By extending these observations to live bacteria animal models, our study advances the therapeutic effects of furin inhibition in a pathologic relevant context. Interestingly, Amirmozafari et al. observed in human subjects that expression of ToxA, an exotoxin-A related gene, correlates with antibiotic resistance.¹³ One compelling, unanswered question is whether BOS-318 administered synergistically with antibiotics would be effective in treating PA. If so, furin inhibition could potentiate current antibiotic susceptibility and thereby reduce antibiotic-resistance in bacteria.³¹

CF and COPD share a phenotype of chronic neutrophilic lung inflammation, punctuated by acute bacterial infections, giving them an unusual “acute-on-chronic” inflammatory phenotype, which stands out from other lung diseases.³² Both diseases result in structural damage in which PA colonization and infection increase mortality, yet this mechanism is not well understood. Definitive bacterial clearance from the lungs is key to preventing the unrelenting inflammation that leads to lung injury. Our data are the first to show that furin inhibition promotes bacterial clearance in animal models, which we propose is due to decreased neutrophilic infiltration and preservation of alveolar macrophages, resulting in enhanced phagocytic clearance of bacteria. The density of alveolar macrophages has been shown to correlate with bacterial lung clearance.³³ It has been reported that alveolar macrophages are essential for the rapid clearance of many pulmonary pathogens including PA,³⁴ and a future direction may be to examine the effects of BOS-318 on clearance of chronic PA lung infections, which has many key clinical implications Great effort is taken to eradicate PA in pediatric CF patients after initial colonization, which requires treatment for months with 2 or more intravenous antibiotics. Post-lung transplant patients colonized with PA in the sinuses undergo lifelong nebulized antibiotics to keep these pathogens at bay and out of the transplanted organs. Finally, while CFTR modulators improve mucociliary clearance and reduce inflammation, these therapies do not eradicate PA.³⁵

On the contrary, depletion of alveolar macrophages has been shown to increase pulmonary neutrophil infiltration, tissue damage, and sepsis.³⁶ Interestingly, we observed a downregulation of Cyp1a1 RNA in the lungs of BOS-318-treated mice. When overexpressed in macrophages, this enzyme can impair phagocytosis of bacteria and increase mortality in an E. coli model of intraperitoneal sepsis.³⁷ Pharmacologic Cyp1a1 inhibition improved survival and bacteria clearance of mice in sepsis.³⁷ Cyp1a1 activation may also suppress the cytotoxic effects of NK cells, particularly in anti-tumor immunity.³⁸ Three main hurdles in developing Cyp1a1 inhibitors has been selectivity and off-target effects, drug-drug interactions due to the common CYP pathway for metabolism, and membrane permeability which are key to reach intracellular targets. BOS-318 is highly selective, membrane-permeable, and may allow for impacting the downstream Cyp1a1 pathway and bypassing these obstacles. Thus, furin inhibition could augment bacterial clearance by increasing AM phagocytosis and modulating Cyp1a1 expression, although more studies are needed to understand the mechanisms in which furin inhibition refines alveolar macrophages phagocytosis and NK cell cytotoxicity.

In our model, furin inhibition strongly decreased chemoattractant MCP-1 concentration in BAL, which corresponds with attenuated neutrophil and NK cell recruitment. It has been observed that MCP-1 promotes NETosis in neutrophils in vitro.³⁹ NK cell recruitment has been reported to be dependent on MCP-1 and critical for clearance of lung pathogens, such as Aspergillus.⁴⁰ As reported by Eleman et al., NK cells could be helpful or harmful in PA infection,⁴¹ and more studies are needed to characterize these immune pathways. In a pneumo-toxin mouse model, Exo-A-induced NK cell recruitment is reported to induce liver damage.⁴² In COPD, NK cells may drive lung tissue destruction through cytotoxicity against lung epithelial cells.^43,44 In a pulmonary ischemia-reperfusion model, Calabrese et al. observed an increase of NK trafficking from peripheral reservoirs into the lung tissue, and antibody-mediated NK cell depletion improved ischemia-reperfusion model-induced acute lung injury.⁴⁵ NK cell depletion has also been shown to increase neutrophil recruitment in the lungs and mortality without impacting bacterial burden, suggesting a role for NK cells in modulating inflammation beyond bactericidality.⁴⁶ Our work suggests that furin inhibition could reduce neutrophil and NK cell infiltration as well as their associated damages by enhancing alveolar macrophage-mediated bacterial clearance. More work is needed to fully determine the importance of furin in NK cell biology.

Although our results are promising, several limitations need to be acknowledged. First, our study relied on murine models of Exo-A and PA-mediated acute lung injury, which may not fully recapitulate the acute-on-chronic, complex nature of infections observed in patients with CF or COPD. However, Douglas et al. reported that BOS-318 enhanced hydration and mucociliary transport in human CF epithelial cells.^16,47 Our mouse models were in male wild-type mice, which were chosen to minimize previously described sex-related variability in CF mouse models of PA infection.⁴⁸ Future studies are needed to characterize these gender differences. In addition, mouse models do not perfectly capture the structural human CF lung disease due to a lack of submucosal glands. Extension of these studies into other animal models such as CF pigs or ferrets are potential a future directions. Second, in our acute bacterial infection model, furin inhibition was administered either before and concurrently with infection or 1 hour after infection as a curative strategy, and future studies could attempt a rescue approach after bacterial pneumonia is established. Translating these results into chronic infection models will be critical to understand the long-term benefits and safety of furin inhibition such as CF.^49,50 Our hope, suggested by the therapeutic strategy results (Supplemental Figure 2), is that BOS-318 could offer a therapeutic option in patients with chronic PA lung infection as well as acute PA lung infection, which commonly trigger CF and COPD exacerbations. Finally, although BOS-318 is highly specific for furin, furin cleaves numerous substrates, which raises the question of specificity. Even if furin inhibition is safe and promotes resolution of PA acute lung infection, some studies suggest that furin inhibition could disrupt embryogenesis and homeostasis.^51,52 Thus, future studies employing lineage-specific furin deficiency models (eg, alveolar macrophages, NK, epithelial cells) may help clarify individual immune cell contributions during bacterial infection. Attention should be paid also in other cell types that could be important during acute and/or chronic infection such as endothelial cells. Indeed, silencing furin in mice resulted in reduced processing of pro-inflammatory and pro-fibrotic cytokine precursors endothelin-1 and TGF-β1.⁵³ Moreover, furin inhibition using previously described Hexa-D-arginine attenuated TGF‐β1‐induced fibroblast overproliferation and transmigration, and reduced pulmonary fibrosis in mouse model, suggesting that BOS-318 could also be used to treat airway fibrosis and remodeling like in COPD, CF and also idiopathic pulmonary fibrosis.³⁰

In summary, our study provides compelling evidence that furin inhibition using BOS-318 not only protects against Exotoxin A-mediated lung injury but also modulates key inflammatory pathways to enhance bacterial clearance. These findings pave the way for the use of protease inhibitors as new therapeutic strategies in the treatment of PA lung infections.

Author contributions

O.B. designed and conducted experiments, analyzed the data, and wrote the manuscript. M.K. performed experiments and analyzed the data, M.R.L. provided resources and edited the manuscript, M.M. performed experiments, analyzed the data, provided intellectual input and edited the manuscript. M.A.Y. provided intellectual input, provided resources and materials and edited the manuscript.

Supplementary material

Supplementary material is available at American Journal of Respiratory Cell and Molecular Biology online.

Conflicts of interest

The authors have declared that no conflict of interest exists. Please see the ICMJE disclosure forms, which have been provided as supplementary material.

Funding

We thank the UCSF Parnassus Flow CoLab, RRID: SCR_018206 and DRC Center Grant NIH P30 DK063720 for using their LSRII flow cytometer “Kermit,” and the UCSF Genomic CoLab for the bulk RNA_seq run and analysis. This work was supported by the UCSF Physician Scientist Scholars Program (5014-138401-2015664-45), the Cystic Fibrosis Foundation LeRoy Matthews Award (YU18L0) and NIAID grant RO1 AI160167.

Data availability

This article has a data supplement, which is accessible at the Supplements tab.