Attenuation of pulmonary ACE2 activity impairs inactivation of des-Arg9 bradykinin/BKB1R axis and facilitates LPS-induced neutrophil infiltration

Abstract

Angiotensin-converting enzyme 2 (ACE2) is a terminal carboxypeptidase with important functions in the renin-angiotensin system and plays a critical role in inflammatory lung diseases. ACE2 cleaves single-terminal residues from several bioactive peptides such as angiotensin II. However, few of its substrates in the respiratory tract have been identified, and the mechanism underlying the role of ACE2 in inflammatory lung disease has not been fully characterized. In an effort to identify biological targets of ACE2 in the lung, we tested its effects on des-Arg⁹ bradykinin (DABK) in airway epithelial cells on the basis of the hypothesis that DABK is a biological substrate of ACE2 in the lung and ACE2 plays an important role in the pathogenesis of acute lung inflammation partly through modulating DABK/bradykinin receptor B1 (BKB1R) axis signaling. We found that loss of ACE2 function in mouse lung in the setting of endotoxin inhalation led to activation of the DABK/BKB1R axis, release of proinflammatory chemokines such as C-X-C motif chemokine 5 (CXCL5), macrophage inflammatory protein-2 (MIP2), C-X-C motif chemokine 1 (KC), and TNF-α from airway epithelia, increased neutrophil infiltration, and exaggerated lung inflammation and injury. These results indicate that a reduction in pulmonary ACE2 activity contributes to the pathogenesis of lung inflammation, in part because of an impaired ability to inhibit DABK/BKB1R axis-mediated signaling, resulting in more prompt onset of neutrophil infiltration and more severe inflammation in the lung. Our study identifies a biological substrate of ACE2 within the airways, as well as a potential new therapeutic target for inflammatory diseases.

Keywords: C-X-C motif chemokine 5; angiotensin-converting enzyme 2; bradykinin receptor B1; endotoxin; lung inflammation.

Fig. 1.

des-Arg⁹ bradykinin (DABK) is a substrate of angiotensin I-converting enzyme 2 (ACE2) in human airway epithelia. Luciferase assays were used to assess bradykinin receptor B1 (BKB1R) activity under several conditions. A: BKB1R activity in HEK293 cells in the presence of DABK (across a range of doses) with or without ACE2 (n = 5). HEK293 cells were transfected with plasmids expressing activator protein-1 (AP-1) luciferase and human BKB1R (B1), with or without a human ACE2 (hACE2) construct. RLU, relative light units. B: BKB1R activity under the same transfection conditions as in A. Additional interventions included stimulation with DABK alone, DABK application in the presence of airway surface liquid (ASL) collected from air-liquid interface cultures of human airway epithelial cells (contains ACE2), or application of DABK, ASL, and ACE2 inhibitor DX600 (n = 6). C: BKB1R activity in air-liquid interface (ALI) cultures of human airway epithelia. The cultures were transduced with an adenovirus expressing the AP-1 luciferase reporter at multiplicity of infection = 1. DABK was applied apically, either alone or in the presence of ACE2-neutralizing antibody (α-ACE2) or isotope goat IgG (n = 6). D: BKB1R activity in cultured airway epithelia as described in C [DABK (ALI)], but ACE2 abundance was reduced by covering air-liquid interface cultures with media for 7 days (DABK subm; n = 5). All comparisons are undertaken between control (CTL) and treated groups, unless otherwise indicated in the figure. *P < 0.05, **P < 0.01.

Fig. 2.

Endotoxin inhalation reduces pulmonary angiotensin I-converting enzyme 2 (ACE2) activity and induces neutrophil infiltration into the lung. Mouse lung bronchoalveolar lavage fluid (BALF) measured for ACE2 enzymatic activity, plus or minus ACE2 inhibitor DX600 (A), bronchoalveolar lavage total neutrophil [polymorphonuclear neutrophils (PMN)] and macrophage (MAC) counts (B), and percentage of PMN and macrophages in BALF (C). RLU, relative light units. For each time point and group, n ≥ 5. *P < 0.05, **P < 0.01.

Fig. 3.

Endotoxin induces reduction of angiotensin I-converting enzyme 2 (ACE2) protein abundance in the lung. A: enzymatic activity of recombinant human ACE2 (rhACE2) protein alone (10 ng) or coincubated with 10 μg endotoxin at 37°C for 60 min was measured by fluorometric-based substrate assay (n = 5). N.S, not significant. B: ACE2 mRNA (mACE2) expression in mouse lung was detected by quantitative reverse transcriptase-PCR (qRT-PCR). Control (CTL) mouse lung or mouse lung exposed to LPS for various times as indicated was collected, and total RNA was extracted for qRT-PCR (n ≥ 5). C: ACE2 protein abundance in control mouse lungs or mouse lungs that received LPS through inhalation was measured by ELISA (n ≥ 5). D: ACE2 activity in mouse lungs repressed by LPS inhalation for 6 h was restored by Bay11-7082 (Bay11), an antagonist for NF-κB signaling (n ≥ 5). RLU, relative light units. E: representative micrograph of Western blot showing that Bay11-7082 partially blocked the reduction in ACE2 protein abundance in mouse lungs that inhaled LPS for 6 h. p-IKKα/β, phospho-IKKα/β. *P < 0.05, **P < 0.01.

Fig. 4.

Lack of pulmonary angiotensin I-converting enzyme 2 (ACE2) activity promotes neutrophil infiltration of the lung and exacerbates LPS-induced lung inflammation and injury. A: schematic depiction of treatment time lines. B: wild-type mice received the ACE2 inhibitor DX600 (1 mg/kg inhalation) both before and after LPS treatment via nasal instillation. Neutrophil infiltration of the lung at indicated times post-LPS inhalation was measured by polymorphonuclear neutrophil (PMN) counts in bronchoalveolar lavage fluid (BALF). CTL, control. C: ACE2 contents in mouse lung from the same experimental groups as in B are measured by ELISA. D: PMN numbers in BALF of wild-type (WT) and ACE2^−/− mice at an array of LPS exposure times were counted (n ≥ 5). E–G: inhibition of ACE2 activity exacerbates LPS-induced lung injury as manifested by representative images of hematoxylin and eosin staining (E), lung permeability (F), and edema (G). H–J: lack of active ACE2 enhances lung inflammation response to LPS stimulation as demonstrated by representative immunohistochemical images of inducible nitric oxide synthase (iNOS, cyan; 43) and cleaved caspase-3 (CC3, red) in the lung (H) and proinflammatory cytokine levels [C-X-C motif chemokine 1 (KC), I; and TNF-α, J] in BALF. K: impact of ACE2 inhibition on LPS-induced NF-κB activation illustrated by Western blot. W/D ratio, wet weight-to-dry weight ratio; DAPI, 4′,6-diamidino-2-phenylindole; p-IKKα/β, phospho-IKKα/β. In all experimental groups, n ≥ 5. Comparison is conducted as indicated in B–D and between the LPS-alone group and the LPS-plus-ACE2 inhibitor DX600 group (F–I). *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar = 50 μm.

Fig. 5.

Endotoxin-induced des-Arg⁹ bradykinin (DABK)/bradykinin receptor B1 (BKB1R) activation contributes to pulmonary neutrophil infiltration. A–C: expression of BKB1R in air-liquid interface primary airway epithelial cell cultures, controls (CTL) or treated with lipooligosaccharide-myeloid differentiation factor 2 (LOS:MD-2). A: human BKB1R (hBKB1R) mRNA expression as assessed by quantitative reverse transcriptase-PCR (qRT-PCR), in the absence (CTL) or presence of LOS:MD-2 stimulation (n = 6). B: BKB1R protein expression as assessed by immunoblot. Cells from three different donors were used per treatment group (CTL or LOS:MD-2 treatments). C: expression of BKB1R in well-differentiated airway epithelia minus or plus LOS:MD-2 treatment. Blue, To-pro-3 (nuclei); red, β-tubulin; green, BKB1R. D–F: neutrophil infiltration of the lung with or without BKB1R antagonist treatment. D: bronchoalveolar lavage (BAL) neutrophil abundance in LPS-treated mice in the presence of BKB1R antagonist [Leu⁸]-DABK. BKB1R inhibition reduced neutrophil infiltration 6 h post-LPS instillation. E: BAL polymorphonuclear neutrophil counts in wild-type (WT) and BKB1R^−/− mouse lungs exposed to LPS for 6 h. N.S, not significant. F: neutrophil abundance in mouse lungs treated with BKB1R antagonist and/or angiotensin I-converting enzyme 2 (ACE2) inhibitor. Treatment with BKB1R antagonist reduces accelerated neutrophil infiltration 3 h post-LPS challenge in mice pretreated with ACE2 inhibitor DX600. For each experimental group, n = 6–9. **P < 0.01, ***P < 0.001. Scale bar = 50 μm.

Fig. 6.

Effects of bradykinin receptor B1 (BKB1R) antagonist treatment on acute lung injury induced by LPS exposure. A: time lines outline the experimental protocol. B: inhibiting BKB1R improved outcomes of LPS-mediated reduction of body weight (n ≥ 5). C: inhibiting BKB1R alleviated LPS-induced neutrophil infiltration in mouse lung (n ≥ 5). D: blocking BKB1R partially restored LPS-mediated reduction of angiotensin I-converting enzyme 2 (ACE2) protein content in mouse lung. E: blockade of BKB1R reduces LPS-induced NF-κB activation as assessed by Western blot. As indicated in separated lanes, lane 1, control (CTL) mouse lung; lane 2, mouse lung exposed to LPS for 24 h; lane 3, mouse lung that inhaled BKB1R inhibitor for 24 h; and lane 4, mouse lung treated with BKB1R inhibitor before LPS inhalation. The panels at top show phospho-IKKα/β (p-IKKα/β) protein expression; the panels at bottom display housekeeping protein β-actin expression. F–H: inhibition of BKB1R mitigated LPS-induced lung injury as manifested by representative images of hematoxylin and eosin staining (F), reduced lung permeability (G), and lung edema (H). I–K: lack of BKB1R activation alleviated lung inflammation response to LPS inhalation as demonstrated by representative immunohistochemical images of inducible nitric oxide synthase (iNOS; I) and cleaved caspase-3 (CC3, red; J) in the lung and proinflammatory cytokine levels [C-X-C motif chemokine 1 (KC), K; and TNF-α, L] in bronchoalveolar lavage fluid (BALF). W/D ratio, wet weight-to-dry weight ratio; DAPI, 4′,6-diamidino-2-phenylindole. In all experimental groups, n ≥ 5. Comparison analysis is conducted among LPS vs. trial A (*), LPS vs. trial B (+), and LPS vs. trial C (#). *+# P < 0.05, ** ++ P < 0.01, *** +++ ### P < 0.001. Scale bar = 50 μm.

Fig. 7.

Endotoxin enhances airway epithelial C-X-C motif chemokine 5 (CXCL5) expression, and bradykinin receptor B1 (BKB1R) blockade attenuates the induction. A: LPS-induced neutrophil-recruiting chemokine production in bronchoalveolar lavage fluid (BALF), as assessed by ELISA. MIP2, macrophage inflammatory protein-2; KC, C-X-C motif chemokine 1. B: LPS-induced CXCL5 production in BALF is inhibited by BKB1R antagonist, 6 h post-LPS challenge. CTL, control. C: effects of CXCL5 depletion with a neutralizing antibody (α-CXCL5) on LPS-induced neutrophil infiltration 6 h post-LPS challenge. PMN, polymorphonuclear neutrophils, MAC, macrophages, N.S, not significant. D: inhibition of ACE2 activity in airway epithelia enhances CXCL5 production in response to des-Arg⁹ bradykinin (DABK) treatment. DABK was applied to air-liquid interface cultured human airway epithelial cells apically for 6 h to activate BKB1R. Airway surface liquid ACE2 activity was inhibited by DX600. In all experimental groups, n > 6. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 8.

Schematic of how attenuated pulmonary angiotensin I-converting enzyme 2 (ACE2) activity may influence endotoxin-induced des-Arg⁹ bradykinin (DABK)/B1 receptor signaling and neutrophil infiltration. ACE2 is expressed on the apical surface of well-differentiated ciliated epithelia. There it inhibits DABK/bradykinin receptor B1 (BKB1R) activation by inactivating DABK, cleaving a single amino acid residue from its carboxyl terminus. Following exposure to infectious or inflammatory stimuli, ACE2 activity is impaired, leaving the DABK/BKB1R axis more active. This promotes the production and release of chemokines such as C-X-C motif chemokine 5 (CXCL5) from airway epithelial cells. By binding to receptors such as C-X-C motif chemokine receptor 2 (CXCR2) on neutrophils, these chemokines recruit neutrophils from the bone marrow or other peripheral reservoirs to the lung. Exacerbated neutrophil infiltration of the lung contributes to the pathogenesis of acute lung inflammation.