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

Abstract

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.

Keywords: BTB06584; CD61; IF1; LPS; acute lung inflammation; dexamethasone; ozone; propranolol.

Figure 1

Effect of combined ozone and LPS exposure in mice: (A) Experiment design of the study showing treatments in 6 different groups with N=3 in each group. The first group represents control group with 0.001% DMSO administered intraperitoneally (IP) 30 minutes before housing mice in cage where they breathed room air for 2 h. After 2 h, mice were lightly anesthetized with IP ketamine/xylazine and instilled with 50 μl sterile saline. The second group represents treatment group with 0.001% DMSO administered intraperitoneally (IP) 30 minutes before housing mice in custom cage where they breathed 0.005 ppm ozone for 2 h. After 2 h, mice were lightly anesthetized with IP ketamine/xylazine and instilled with 50 μg/50μl bacterial lipopolysaccharide (LPS) from E. coli strain 055:B5. The third to sixth groups represent treatment group with immune-modulators (1 mg/kg anti-CD61 antibody (clone 2C9.G2), 1 mg/kg BTB06584, 1 mg/kg propranolol and 0.1 mg/kg dexamethasone) administered intraperitoneally (IP) 30 minutes before housing mice in custom cage where they breathed 0.005 ppm ozone for 2 h. After 2 h, mice were lightly anesthetized with IP ketamine/xylazine and instilled with 50 μg/50μl bacterial lipopolysaccharide (LPS) from E. coli strain 055:B5. All mice were euthanized at 24 h time-point to collect peripheral blood, vascular perfusate, bronchoalveolar lavage (BAL), right lung for biochemical and chemokine analysis and left lung fixed in-situ with 4% paraformaldeyde, for cryosectioning and H&E histology. (B) Percent BAL cell viability assessed with trypan blue exclusion, across the 6 groups, ANOVA p=0.0012, (C) BAL total leukocyte counts (TLC) expressed as 10⁶ cells, ANOVA p<0.0001, (D) Lung vascular perfusate TLC expressed as 10⁶ cells, Kruskal-Wallis p=0.0615, (E) Peripheral blood TLC expressed as 10⁶ cells, ANOVA p=0.0201. (F) BAL cell immune-fluorescent staining of control, and the 4 treatment groups, for nucleus with DAPI shown in blue (a, b, c, d, e, f), NK1.1 shown in green (a’, b’, c’, d’, e’, f’), Gr1 shown in red (a’’, b’’, c’’, d’’, e’’, f’’), CX3CR1 shown in fuschia (a’’’, b’’’, c’’’, d’’’, e’’’, f’’’). The merged panels are shown in the sub-parts a’’’’, b’’’’, c’’’’, d’’’’, e’’’’, f’’’’, scale = 50μm. (G) Lung myeloperoxidase (MPO) biochemical quantification across groups expressed as U/mg of lung tissue, Kruskal-Wallis p=0.0404, (H) Lung eosinophil peroxidase (EPX) ELISA based quantification across groups expressed as μg/μg of lung tissue, ANOVA p=0.0013, (I) BAL protein content across groups expressed as μg/ml, ANOVA p<0.0001. Significance level was set at p<0.05. * represents p<0.05, ** p<0.01, *** p<0.001, and **** p<0.0001.

Figure 2

Chemokine, molecular and histology. (A) Bronchoalveolar lavage (BAL) fluid 33-plex chemokine analysis expressed as pg/ml in heat map, (B) Lung vascular perfusate 33-plex chemokine analysis expressed as pg/ml in heat map and (C) BAL/vascular perfusate ratio of 33 chemokines across the six groups: control, and 5 treatment groups namely vehicle, CD61 blocking (anti-CD61) antibody, BTB06584, propranolol and dexamethasone treatments. For magnetic bead-based immunoassay (Bio-rad Laboratories Ltd., CA, US), the following chemokines were analyzed: CXCL13 (B-lymphocyte chemoattractant), CCL27 (IL-11 R-alpha-locus chemokine (ILC)), CXCL5 (epithelial-derived neutrophil-activating peptide 78 (ENA-78)), CCL-11 (eotaxin-1), eotaxin-2 (CCL-24), CX3CL1 (fractalkine), GM-CSF (CSF-2), CCL1, IFNγ (interferon gamma), IL-10 (interleukin-10), IL-16 (interleukin-16), IL-1β (interleukin-1 beta), IL-2 (interleukin-2), IL-4 (interleukin-4), IL-6 (interleukin-6), CXCL-10 (interferon gamma-induced protein 10 (IP-10)), CXCL11 (Interferon-gamma-inducible protein 9 (IP-9)), KC (keratinocyte chemoattractant), MCP-1 (monocyte chemoattractant protein-1), MCP-3 (monocyte chemoattractant protein-3), MCP-5 (monocyte chemoattractant protein-5), MDC (macrophage-derived chemokine (CCL22)), MIP-1α (macrophage inflammatory protein-1 alpha), MIP-1β (macrophage inflammatory protein-1 beta), MIP-2 (macrophage inflammatory protein-2), MIP-3α (macrophage inflammatory protein-3 alpha), MIP-3β (macrophage inflammatory protein-3 beta), RANTES (regulated on activation, normal T cell expressed and secreted (CCL5)), CXCL-16, CXCL-12/SDF-1alpha (stromal cell-derived factor 1), TARC (thymus and activation regulated chemokine (TARC)), TECK (Thymus-Expressed Chemokine (CCL25)) and TNFα (tumor necrosis factor alpha) (D) BAL cell immune-fluorescent staining of control, and 4 treatment groups, for ATPase inhibitory factor 1 (IF1) shown in blue (a, b, c, d, e, f), Ki-67 shown in green (a’, b’, c’, d’, e’, f’), CD61 shown in red (a’’, b’’, c’’, d’’, e’’, f’’), Angiostatin shown in fuschia (a’’’, b’’’, c’’’, d’’’, e’’’, f’’’). The merged panels are shown in the sub-parts a’’’’, b’’’’, c’’’’, d’’’’, e’’’’, f’’’’, scale = 50μm. (E) Lung cryosection H&E across groups i.e. control (a), and 5 treatment groups namely vehicle (a’), CD61 blocking (anti-CD61) antibody (a’’), BTB06584 (a’’’), propranolol (a’’’’) and dexamethasone (a’’’’’) treatments. Scale bar = 100 μm, Br=bronchus, open arrows indicate bronchiolar epithelial damage, closed arrows indicate polymorphonuclear cells majority of which are eosinophilic in appearance, * represents clusters of polymorphonuclear cells. Note the dual color i.e. dark purple and eosinophilic presentation of lungs after ozone and LPS treatment in images from a’ to a’’’’’.