Modulation of low-dose ozone and LPS exposed acute mouse lung inflammation by IF1 mediated ATP hydrolysis inhibitor, BTB06584

Ozone and bacterial lipopolysaccharide (LPS) are common air pollutants that are related to high hospital admissions due to airway hyperreactivity and increased susceptibility to infections, especially in children, older population and individuals with underlying conditions. We modeled acute lung inflammation (ALI) by exposing 6-8 week old male mice to 0.005 ppm ozone for 2 h followed by 50 μg of intranasal LPS. We compared the immunomodulatory effects of single dose pre-treatment with CD61 blocking antibody (clone 2C9.G2), ATPase inhibitor BTB06584 against propranolol as the immune-stimulant and dexamethasone as the immune-suppressant in the ALI model. Ozone and LPS exposure induced lung neutrophil and eosinophil recruitment as measured by respective peroxidase (MPO and EPX) assays, systemic leukopenia, increased levels of lung vascular neutrophil regulatory chemokines such as CXCL5, SDF-1, CXCL13 and a decrease in immune-regulatory chemokines such as BAL IL-10 and CCL27. While CD61 blocking antibody and BTB06584 produced maximum increase in BAL leukocyte counts, protein content and BAL chemokines, these treatments induced moderate increase in lung MPO and EPX content. CD61 blocking antibody induced maximal BAL cell death, a markedly punctate distribution of NK1.1, CX3CR1, CD61. BTB06584 preserved BAL cell viability with cytosolic and membrane distribution of Gr1 and CX3CR1. Propranolol attenuated BAL protein, protected against BAL cell death, induced polarized distribution of NK1.1, CX3CR1 and CD61 but presented with high lung EPX. Dexamethasone induced sparse cell membrane distribution of CX3CR1 and CD61 on BAL cells and displayed very low lung MPO and EPX levels despite highest levels of BAL chemokines. Our study unravels ATPase inhibitor IF1 as a novel drug target for lung injury.


Introduction
Lung inflammation is a common feature of many respiratory diseases like Acute Respiratory Distress Syndrome (ARDS) (1), asthma (2), Chronic Obstructive Pulmonary Disease (COPD) (3), and cystic fibrosis (4). It constitutes immune and neuro-humoral responses (5) from the vasculature and surrounding tissue in response to an infectious or other harmful stimulus. ARDS is a devastating complication of severe sepsis, from which patients have high mortality. Advances in treatment modalities including lung protective ventilation, prone positioning, use of neuromuscular blockade, and extracorporeal membrane oxygenation, have improved the outcome over recent decades; nevertheless, the mortality rate still remains high (6). Sepsis, ischemia reperfusion injury (as in stroke), hemodialysis, bacterial infections and multiple organ failure are marked by significant pulmonary neutrophil sequestration and concomitant neutropenia. This in turn, leads to immune suppression, which is another major cause of mortality (7,8). Neutrophils are observed in lungs at post-mortem in ARDS patients and their numbers correlate with increasing severity of ARDS (9). The activated neutrophils mount a robust immune response; however, they also damage the blood-air barrier and cause lung injury (10,11). The ultimate goal is to develop novel drug targets for lung inflammatory diseases without compromising the innate immune defense and the alveolar barrier.
Exposure to large concentrations (>1 ppm) of ozone for 6 h induces severe lung inflammation owing to its potent toxic effects (12,13). We have recently shown that ambient ozone (from 0.005 to 0.5 ppm for 2 h) also induces instant cell death, marked by accumulation of vascular neutrophil and platelet aggregates albeit without inducing an increase in BAL protein or cell counts. We have also shown that low dose single ozone exposure of 0.05 ppm for 2 h impairs cell redox functions including the suppression and perturbation of innate immune response to bacterial lipopolysaccharide (14,15). The dual burden of cell death and reduced immune capacity not only accumulates necrotic debris but also impairs the alveolar barrier and pathogen clearance (16,17).
There are a few reports that point towards protection offered by propranolol in models of stroke, ozone-induced lung injury (5) and it's atypical use in asthma patients refractory to long acting betaadrenergic agonists (LABAs), largely owing to downregulation of adrenergic agonists (18). In a mouse stroke model lung neutrophil sequestration and concomitant lymphopenia in peripheral blood, thymus and spleen have been shown to solely respond to beta adrenergic blockade with propranolol (19). Glucocorticoids such as dexamethasone are one of the most powerful anti-inflammatory agents available clinically and work by inducing downregulation of the nuclear factor NFkB (20). Although this anti-inflammatory effect is attributed to inhibition of neutrophil recruitment it remains to be tested if glucocorticoids contribute to a delay in resolution of lung inflammation as the dynamics of these changes are not known. Moreover, glucocorticoids suppress the immune system, which is counter to the repair phase of inflammation. Thus, there is a need to develop novel immunomodulatory therapeutics which would not impair the innate immune response. CD61 (Integrin b 3 ) is widely expressed in lungs and is an integral regulator of cell adhesion and cell-surface signaling (21). CD61 induces cortical actin reorganization, thus stabilizing focal adhesions (22). We and others have shown that CD61 knock-out mice, blocking antibodies and peptides like RGD amplify vascular leak, neutrophil recruitment and worsen survival in acute lung injury and sepsis models (23,24). Thus, CD61 is an important regulator of the alveolar barrier.
The F1F0 ATP synthase comprises a mitochondrial proton channel called F o , and a soluble catalytic F1 portion. The F1 portion comprises ab heterodimers, which under favorable pH conditions (>6.5), synthesize ATP due to the proton motive force (25)(26)(27). Thus, ATP synthase is a chemo-mechanical protein (25). Pathophysiological stress (measured by a drop in mitochondrial potential) induced by ozone or LPS cause ectopic (surface) expression of the b subunit of ATP synthase (ATPb), reversing the cellular energy flow (15, 28) which results in ATP hydrolysis and transport of protons against the gradient. We have shown in vitro that ATPb is upregulated and surface-expressed during acute neutrophil polarization where it binds with exogenously administered angiostatin, endogenous CD61, prevents neutrophil pseudopod formation, stabilizes microtubules, inhibits mitochondrial activation and ROS production and induces neutrophil apoptosis (29). This suggests an essential role of ATP synthase in integrin b 3 mediated neutrophil activation. Our in vitro observations were substantiated by in vivo studies where angiostatin administered subcutaneously (not intravenously) protected against LPS-induced lung vascular leak (30). Lung microangiography using state-of-the-art synchrotron dual K-edge subtraction imaging confirmed lung structure preservation by angiostatin treatment (31). The in vitro and in vivo data on the role of CD61 and angiostatin in lung injury, underscores the complex biology of ATP synthase in activated neutrophils.
Binding of angiostatin to surface-associated ATPb has been reported by various research groups (32,33). In the mitochondria, an additional protein, ''inhibitor of F1'' (IF1) is available to act as a ratchet ensuring that under conditions in which the electron motive force is diminished (such as relatively high pH below the membrane) the g chain of the F1 subunit cannot rotate in the clockwise direction and thus cannot needlessly consume ATP resources. Overexpression of mutant IF1in cell-lines or in vivo induces glycolytic enzymes and ROS production and protects against apoptosis and oxidative cell death (34, 35). A specific ATPase inhibitor, BTB06584 shows cardioprotection in ischemia reperfusion injury via IF1 (36). Because the contribution of ATPb during lung inflammation is not well understood, we hypothesized that the ATPase inhibitor, BTB06584, regulates neutrophil recruitment and activation in a combined low-dose ozone and bacterial lipopolysaccharide (LPS) exposed or the "dual-hit" model of ALI.
Thus, the goals of our current study were to understand the inflammatory lung phenotypes in dual ozone and LPS exposed mice subjected to immune-modulation by CD61 blocking antibody, ATPase inhibitor BTB06584, propranolol or dexamethasone in murine ALI and to characterize the expression of the angiostatin and it's binding proteins such as IF1 and CD61 in lung inflammation.

Mice
The study design was approved by the University of Saskatchewan's Animal Research Ethics Board and adhered to the Canadian Council on Animal Care guidelines for humane animal use. Six-eight week old male C57BL/6J (Stock No. 000664) mice were procured from Jackson Labs (CA, US).

Reagents and chemicals
All chemicals utilized for the experiments were purchased from Sigma-Aldrich Chemicals (MO, US) unless otherwise indicated.

Ozone exposures
For ozone exposures, mice were continuously exposed in an induction box for the desired times. These mice had free access to food and water while housed in the custom induction box. Ozone (0.005 ± 0.02 ppm) was generated, at 3 litres/minute, from ultrahigh-purity air using a silent-arc discharge O 3 calibrator cum generator (2B Technologies, CO, USA). Constant chamber air temperature (72 ± 3°F) and relative humidity (50 ± 15%) were maintained. Ozone concentrations were measured using a real-time ozone monitor (2B Technologies, CO, USA).

Experiment design
Eighteen mice were randomized into six groups (one control, one vehicle+ozone+LPS and four immune-modulator pre-treated + ozone+LPS) with three mice per group. The control mice were pretreated, 30 minutes before ozone exposure, as per respective IP doses, with vehicle (0.001% DMSO) and then returned to their cage to room air (RA) for 2 h. Mice were then anaesthetized intraperitoneally (IP) with ketamine (95 mg/kg) and xylazine (4.8 mg/kg) and instilled intranasally with 50 ml of sterile saline. These mice are represented as the control group ( Figure 1A). For the treatment groups, mice were pre-treated 30 minutes before ozone exposure as per respective IP doses, with vehicle or the immunemodulator treatment groups which were as follows: 1 mg/kg anti-CD61 antibody (clone 2C9.G2), 1 mg/kg BTB06584, 1 mg/kg propranolol and 0.1 mg/kg dexamethasone. Mice from treatment groups were exposed to 0.005 ppm ozone for 2 h as explained above. Immediately after the 2 h ozone exposures, mice were anaesthetized intraperitoneally (IP) with ketamine (95 mg/kg) and xylazine (4.8 mg/kg) and instilled with 50 mg/50 ml LPS into the external nares.
At 24 h, the mice were anesthetized with IP ketamine (190 mg/ kg) and xylazine (9.6 mg/kg) and prepared for further maneuvers. The experimental design is summarized in Figure 1A. Peripheral blood was collected in heparinized syringe by cardiac puncture. Mice were then tracheostomized, a custom non-collapsible polyethylene cannula was placed just before the end of tracheal bifurcation. Broncho-alveolar lavage (BAL) was collected with three consecutive washes, each with 0.5 ml PBS (phosphate buffered saline). The descending thoracic aorta was snipped at the midthoracic region. After clearing residual blood, lung vascular perfusate (LVP) was collected by perfusion through the right ventricle with PBS (0.25 ml X 2). The right lung lobes were ligated at the tracheo-bronchiolar hilus. The left lung was perfused with 0.5 ml freshly prepared 4% paraformaldehyde from a 20 cm water column to enable in situ fixation for 10 minutes and later cryo-embedded. Right lung was resected and flash frozen in liquid nitrogen and then stored at -80°C for further analysis. The collected samples are shown in the schematic in Figure 1A. BAL, peripheral blood and vascular perfusate were cytospun for immunefluorescence with one slide stained for NK1.1, Gr1 and CX3CR1, and the other slide stained for ATPase IF1, Ki-67, CD61 and angiostatin. BAL supernatant was utilized for total leukocyte counts (TLC), protein, and 33-plex chemokine quantification. Right lung homogenates were prepared for MPO, EPX, quantifications. Vascular perfusate was quantified for TLC, 33plex chemokine panel quantification. Peripheral blood samples were also analyzed for TLC, left lung cryosections were stained with hematoxylin and eosin (H&E) for histology.
End-points a) Total (TLC) leukocyte counts: Blood, BAL and lung vascular perfusate samples were centrifuged for 10 min at 3000 rpm. The supernatants were flash frozen and stored at -80°C until further analysis. The cells were reconstituted in PBS. TLC was performed by counting BAL, blood or lung perfusate cells on a hemocytometer. Trypan blue dye exclusion was utilized to quantify BAL cell viability under light microscopy. Blood TLC is presented as x10 6 cells per mL. BAL and lung perfusate TLC are expressed in x10 3 and x10 6 cells per collection, respectively. Acetic acid (2%) was added to lyse RBCs, in a 1:10 ratio for blood TLC and 1:2 ratio for lung vascular perfusate TLC. Cells (not more than 1X10 6 per slide) were then centrifuged (Shandon cytospin, Thermoscientific, US) to prepare cyotspins.
d) BAL protein analysis: In order to quantify ozone induced edema, i.e., we measured total protein content in the collected BAL fluid. Supernatant fractions were analyzed for their total protein concentration using a standard colorimetric assay (Pierce 660 nm protein assay, Thermoscientific, IL, US).

Statistical analysis
Results are expressed as mean ± SEM (N=3 per group). Effects of different treatments were analyzed by one way ANOVA or its nonparametric equivalent Kruskal-Wallis test followed by Tukey's or Dunn's multiple comparisons. A p value of <5% was considered significant. For multiplex chemokine analysis, one way ANOVA p values were adjusted for false discover rate by Benjamini-Hochberg correction. All analysis were done in GraphPad Prism 9 (Boston, MA, USA) Software, LLC.

BAL, perfusate and peripheral blood innate immune cell immune-fluorescence
Nuclear morphology, Gr1, CX3CR1 and NK1.1 were used to characterize BAL, lung vascular perfusate and peripheral blood cells. Majority of BAL cells were mononuclear and Gr1 positive but a few were NK1.1, Gr1 and CX3CR1 triple positive in the vehicle+ozone+LPS group ( Figure 1F Figures 1a''', 2a'''). Isotype control staining provided evidence that the observed staining pattern in anuclear fragments was not non-specific. We observed 5-6 fold lower staining in the isotype controls for mouse IgG2ak, rat IgG2ak and rabbit IgG (Supplementary Figure 3). Propranolol led to slightly few Gr1, NK1.1 and CX3CR1 triple positive cells ( Figure 1F

Lung myeloperoxidase (MPO) and eosinophil peroxidase (EPX)
To ascertain the number of neutrophils or eosinophils left after BAL, MPO and EPX were quantified in lung homogenates. To our surprise, we observed highest MPO in vehicle+ozone+LPS (81.00 ± 4.43 U/mg, p<0.05) but significance was only achieved against anti-CD61 antibody (6.61 ± 1.67 U/mg) treated group ( Figure 1G).

BAL ATPIF1, Ki-67, CD61 and angiostatin immune-fluorescence
In order to look at the expression of focal adhesions, magakaryocyte and platelet expressing CD61, IF1, antiproliferation marker angiostatin and the proliferation factor Ki-67, we sought to image these proteins in BAL cytospins. We observed higher IF1 expression in vehicle+ozone+LPS treated BAL cells compared to control group ( Figure 2D-a). Although we could not ascertain their distribution in the nuclear compartment, the distribution pattern of IF1 suggested its nuclear expression. Intracellular CD61 and angiostatin expression was also diffuse in BAL cells in control ( Figure 2D-a'', a''') and vehicle+ozone+LPS treated groups ( Figure 2D-b'', b'''). There was a predominant Ki-67 expression in vehicle+ozone+LPS group indicating the stimulation of cell proliferation in response to these exposures ( Figure 2D-b'). Numerous small CD61 and angiostatin positive entities were also observed in the BAL of vehicle+ozone+LPS treated cytospins ( Figure 2D-b'', b'''). The anti-CD61 antibody treated BAL cells displayed moderate staining for IF1, CD61, angiostatin and Ki-67 ( Figure 2D

Lung H&E staining
Lung cryosections revealed wide-spread alveolar and bronchiolar damage, hemorrhage, vascular leukocyte recruitment in vehicle ( Figure 2E-a'), CD61 blocking antibody ( Figure 2E-a'') and dexamethasone treatment groups ( Figure 2E-a'''''). Open arrows indicate bronchiolar epithelial damage, closed arrows indicate polymorphonuclear cells majority of which are eosinophilic in appearance, * represents clusters of polymorphonuclear cells noted especially in dexamethasone treated lung section ( Figure 2E-a'''''). Note the dual color i.e. dark purple and eosinophilic presentation of lungs after ozone and LPS treatment in images from Figure 2E-a' to a'''''.

Discussion
We have recently studied the effects of ozone alone in a time (up to 24 h) and dose (from 0.005 to 0.5 ppm) dependent manner (14). In addition, we have also studied the effects of LPS alone (30), and ozone+LPS (15) up to 72 h. We have shown that low dose ozone (0.05 ppm) does not induce increase in BAL total protein content. One of the reasons for lack of change in BAL protein could be due to protein degradation and wide-spread cell death as we do observe the cardinal signs of phlogistic cell death, extracellular DNA in BAL, vascular perfusate, and peripheral blood. We observed neutrophils, neutrophil and eosinophil chemokines in the vasculature, MPO in the lung tissue and large monocytic CD11b bright cells in BAL. The main reason for choosing the current ozone and LPS model at 24 h is based on these observations where lung inflammation is suppressed and delayed by 0.05 ppm ozone and 50 mg LPS; BAL protein peaked at 36 h, BAL cell counts peaked at 72 h unlike the BAL counts seen in ozone alone (peak at 6 h) or LPS alone (peak at 9 h). As very low doses of ozone (0.005 ppm) also compromise the alveolar barrier, induce BAL cell death and LPS is a strong neutrophil and innate immune response inducer, we wanted to assess the effects of novel immune interventions on innate immune response induced by the combined exposure to ozone and LPS. The current model utilizes 0.005 ppm ozone followed by LPS exposure resulting in a suppressed immune response while inducing lung MPO and EPX release which are direct indicators of neutrophil and eosinophil accumulation and degranulation (37). We further investigated the effects of immune stimulator propranolol and immune-suppressant dexamethasone which were compared to ATPase inhibitor, BTB06584 and CD61 blocking antibody. We have compiled our results in a comparative table (Table 1). We found that BTB06584 protects against cell death, induces robust lung neutrophil recruitment but does not induce massive eosinophil or neutrophil peroxidase release when compared to vehicle treated group.
Nuclear morphology, Gr1, CX3CR1 and NK1.1 fluorescence were used to characterize the BAL, lung vascular perfusate and peripheral blood cells. BAL cell counts and immune-fluorescent images showed large numbers of Gr1 and CX3CR1 double positive polymorphonuclear cells in the CD61 blocking antibody, BTB06584, propranolol and dexamethasone treatment groups compared to control or vehicle (ozone+LPS) treatment groups. Importantly, we observed debris, anuclear CX3CR1 and CD61 positive cells which could likely be platelets (38) or cell fragments called as microparticles (39). BAL cells from control, BTB06584, propranolol and dexamethasone treatment groups showed protection against cell death rather than signs of proliferation as visualized in the reduced Ki-67 staining in the immune-modulator treated groups compared to vehicle treatment group. Vehicle as well as immune-modulator treated groups showed leukopenia compared to control group. Only the CD61 blocking antibody, BTB06584 and dexamethasone treated mice showed high BAL protein compared to control, vehicle and propranolol treatment groups. This increase in BAL protein could be a result of either enhanced vascular permeability or higher amount of protein secreted in the BAL, which is a contentious hypothesis to be tested in carefully planned experiments. The vascular, and not the BAL, compartment of vehicle treated group showed predominance of neutrophil chemokine CXCL5, SDF-1a and the B cell chemokine, CXCL13, as indicated by the BAL to vascular perfusate chemokine ratios. These results are in agreement with our earlier study where the chemokine signature was similar after a single 0.05 ppm ozone+LPS exposure (15). All the treatment groups showed decrease in levels of regulatory BAL chemokines, namely IL-10 and CCL27. CD61 blocking antibody, BTB06584 and dexamethasone treatments induced significant increase in BAL chemokines namely RANTES, CXCL5, MIP-1a, MIP-1b, MIP-2, MIP-3a, IL-16, CXCL13, MCP-1, MCP-3, with KC, MDC and TARC being specific to CD61 blocking antibody treatment, eotaxin-2, IL-6 The symbols + to ++++ stand for the graded magnitude of the observed parameter in each group with + being the least and ++++ being the maximum. The symbol ↑ stands for an increase in the parameter described.
being specific to BTB06584 and IL-6, CXCL16 specific to dexamethasone treatment. Propranolol treatment led to increase in levels of BAL CXCL5, MIP-1a, CXCL13 and TARC. IL-16, KC, CXCL5, CXCL12 (i.e. SDF-1a), MIP-2 are important neutrophil chemokines (40-43). CXCL13 is a lymphoid expressed chemokine that induces B cell migration and is also a regulator of neutrophil migration (44). Other MIP isoforms are chemotactic for monocytes and lymphocytes. Eotaxin-2, RANTES and MCP-1 are known to mediate bronchial hyperresponsiveness, recruitment of mononuclear cells, eosinophil, platelet activation and allergic inflammation (45). Macrophage derived TARC and MDC are chemotactic not only for Th2 cells but also epithelial cells (46). The control, CD61 blocking antibody and dexamethasone treatment groups showed near baseline levels of MPO and EPX. Despite the non-significant increase in BAL leukocytes, the lungs of vehicle treated group had the maximum amount of myeloperoxidase (MPO). The lungs of vehicle treated group also had the maximum amount of eosinophil peroxidase (EPX) which was moderately increased in propranolol and BTB06584 treatment groups. Interestingly, we observed numerous polymorphonuclear cells in lung sections, majority of which were eosinophilic in appearance. Dexamethasone treated lung showed clusters of eosinophilic cells. The data from H&E histology and peroxidase assays suggests that the polymorphonuclear neutrophils and eosinophils are more adherent after ozone and LPS treatment, suggesting their activation. Taken together, our results indicate that both neutrophils and eosinophils orchestrate lung inflammation after single exposure to just 0.005 ppm ozone and 50 mg of intranasal LPS. The lungs present with widespread alveolar and bronchiolar damage, which was markedly exaggerated after pre-treatment with single IP dose of CD61 blocking antibody. The immunomodulators tested in our study induced significant protein and chemokine content in BAL as well as perfusate but BTB06584, propranolol and dexamethasone protected against excessive cell death, which might afford some degree of protection from ozone's toxic effects. Although our study was designed to analyze the effects of different treatments on acute lung inflammation parameters at 24 h time-point, it would be interesting to follow these parameters at later time-points and assess the late phase of inflammation and resolution.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The animal study was reviewed and approved by University Animal Care Committee (UACC).

Author contributions
GA secured funding (NSERC-DG), designed, executed the study and analyzed the results. PS performed downstream assays on lung homogenates. All authors contributed to the article and approved the submitted version.