Abstract
Background/Aim: Patients with pneumonia after prolonged neutropenia are at increased risk for acute respiratory distress syndrome (ARDS). The key molecule of endothelial barrier breakdown in sepsis is lipopolysaccharide (LPS), which is a component of the outer membrane of gram-negative bacterial cell walls. Maintaining increased cyclic adenosine monophosphate (cAMP) levels in endothelial cells is effective in preventing endothelial dysfunction and microvascular permeability. The aim of this study was to elucidate whether roflumilast, a phosphodiesterase-4 (PDE-4) inhibitor, is effective in LPS-induced acute lung injury (ALI) during neutropenia recovery in a murine model. Materials and Methods: To induce neutropenia, all mice were administered intraperitoneal cyclophosphamide. On day 2 after neutropenia, mice were administered LPS by intra-tracheal instillation. In the prevention group, roflumilast was given orally on day 0, when neutropenia was induced. In the treatment group, roflumilast was administered orally 1 hour after LPS injection. Results: Roflumilast attenuated histopathological changes associated with LPS-induced lung injury. The accumulation of neutrophils and the concentrations of inflammatory cytokines IL-1β, TNF-α, and IL-6 in bronchoalveolar lavage fluids were inhibited effectively by roflumilast. Also, MMP-9 and TGF-β expression was attenuated in the roflumilast group. Conclusion: Roflumilast significantly attenuated LPS-induced ALI during neutropenia recovery.
According to the Berlin definition, acute respiratory distress syndrome (ARDS) is defined as acute respiratory failure not fully explained by heart failure or fluid overload with bilateral opacities in chest imaging within one week of new or worsening respiratory symptoms (1). Mortality is approximately 40% for patients with mild to severe ARDS (2). To date, there is still no definite treatment for patients with ARDS. Currently approved treatments include lung-protective mechanical ventilation, prone positioning, and supportive interventions. Among various pharmacologic medications, neuromuscular blockers and corticosteroids could reduce hospitalization and mortality rate (3, 4).
Patients with pneumonia after prolonged neutropenia are at increased risk for ARDS (5). Neutropenia recovery has been shown to be associated with deterioration of pre-existing lung injury and respiratory condition (6). Patients undergoing chemotherapeutic treatment may more frequently encounter this dangerous situation in neutropenia recovery.
Increased permeability of alveolar endothelial and epithelial barriers and uncontrolled activation of leukocytes, platelets, and coagulation pathways are the main pathophysiologic changes in acute lung injury (ALI) and ARDS (7). Lungs damaged by chemotherapy and infection may be sensitive to the influx of neutrophils (8).
Maintaining increased cyclic adenosine monophosphate (cAMP) levels in endothelial cells is effective for preventing endothelial dysfunction and microvascular permeability (9). Intracellular levels of these cyclic nucleotides are regulated by phosphodiesterases (PDEs). PDE-4 inhibition reduces the release of pro-inflammatory mediators from inflammatory cells, including neutrophils, lymphocytes, monocytes, macrophages, and eosinophils (10). It also represses structural lung cells, including epithelial cells, airway smooth muscle cells, and fibroblasts, from releasing pro-inflammatory mediators. In addition to their anti-inflammatory effect, PDE-4 inhibitors also have anti-remodelling properties by suppressing the epithelial-to-mesenchymal transition (EMT) (11). Additionally, PDE-4 inhibitors diminish airway hyperresponsiveness (12). Roflumilast, a selective PDE-4 inhibitor, is clinically used for patients with severe chronic obstructive pulmonary disease (COPD) with chronic bronchitis and a history of exacerbations (13).
The key molecule of endothelial barrier breakdown in sepsis is lipopolysaccharide (LPS), which is a component of the outer membrane of gram-negative bacterial cell walls. In our previous studies, we demonstrated that pravastatin, tyrosine kinase inhibitors, and neutrophil elastase attenuated LPS-induced ALI during neutropenia (14-16).
The aim of this study was to elucidate whether roflumilast, a PDE-4 inhibitor, is effective in LPS-induced ALI during neutropenia recovery in a murine model.
Materials and Methods
Animals. Five-week-old female ICR mice (n=5 per group; Orient Bio Experimental Animal Center, Kyoungki, Republic of Korea) were used. The animals were allocated to one of the following four groups: 1) control, 2) cyclophosphamide+LPS, 3) cyclophosphamide+ LPS+roflumilast (prevention group), 4) cyclophosphamide+LPS+ roflumilast (treatment group).
Neutropenia was induced by intraperitoneal injection of cyclophosphamide as shown in our previous studies (14-16). Control mice were injected with an equal volume of saline, instead of LPS. Two days after neutropenia, mice were administered LPS by intra-tracheal instillation to induce acute lung injury. In the prevention group, roflumilast was given orally on day 0, when neutropenia was induced. In the treatment group, roflumilast was administered orally 1 h after LPS administration on day 2 (roflumilast 1.0 mg/kg, suspended in equivalent amounts of polyethylene glycol 400+4% methylcellulose solution).
The experiments were approved by the Animal Subjects Committee (approval number: UJBCRL202001) of The Catholic University of Korea.
Morphological analysis. After sacrifice, lung tissues were inflated, fixed with 4% paraformaldehyde for 24 h in phosphate-buffered saline (PBS), and embedded in paraffin wax. Sections were cut at 4 μm thickness using a microtome and stained with hematoxylin and eosin using standard techniques for histological changes.
Bronchoalveolar lavage (BAL). The trachea was cannulated with silicone tube with a 22-gauge needle. The left lung was subjected to bronchoalveolar lavage (BAL), and fluid was withdrawn after washing the lung with 0.8 ml sterile PBS. Total cell counts in BAL fluid were calculated using a hemocytometer. The BAL fluid was processed using cytospin (5 min at 70×g) and stained with Diff-Quick (Sysmax, Tokyo, Japan). The percentages of macrophages, eosinophils, lymphocytes, and neutrophils in the BAL fluid were obtained by counting 500 leukocytes on randomly selected portions under light microscopy.
Sample preparation for myeloperoxidase (MPO) assays. MPO concentration in BAL fluid was determined using the mouse MPO kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. BAL fluid was centrifuged at 500×g for 10 min at 4°C, and the supernatant discarded. The leukocyte pellet was suspended again in extraction buffer.
Enzyme-linked immunosorbent assay (ELISA). The concentrations of IL-1β, tumor necrosis factor (TNF)-α, and IL-6 in BAL fluids were analyzed using an ELISA kit (R&D Systems). The protocol was performed according to the manufacturer’s instructions.
Nuclear and cytoplasmic protein extraction. Separated lung tissues promptly frozen in liquid nitrogen were disrupted using a TissueLyser II (QIAGEN, Hilden, Germany), containing a mixture of protease inhibitors for 1 min, after holding on ice for 10 min. After centrifugation at 4°C and 3,000 rpm for 10 min, the supernatants containing cytoplasmic proteins were discarded and preserved at −70°C. After incubation for 30 min on ice in a cold high-salt buffer, nuclear protein isolation was executed. After centrifugation at 4°C and 14,000 rpm for 25 min, supernatants containing the nuclear proteins were discarded and preserved at −70°C.
Western blotting. Protein samples were isolated by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk (Difco/Becton, Dickinson and Company, Atlanta, GA, USA) for 2 h at room temperature, followed by incubation overnight with primary antibody at 4°C. Next, the membrane was triple-washed with Tris-buffered saline containing 0.1% Tween 20 (TBST) for 30 min and incubated with the secondary antibody (horseradish peroxidase-conjugated IgG in 5% skim milk, 1:1,000) for 2 h at room temperature. The target proteins were obtained by an ECL western blotting analysis system (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Transforming growth factor beta (TGF-β) expression was measured.
Statistical analyses. The statistical significance of differences between groups was evaluated using analysis of variance (ANOVA) followed by Dunnett’s multiple range test. All data are expressed as mean±SD (standard deviations), and a p-value of <0.05 was considered to suggest statistical significance. SPSS version 15.0 software (SPSS Inc., Chicago, IL, USA) was used for the statistical analyses.
Results
Effect of roflumilast on histopathology. Compared with the control group (Figure 1A), acute alveolar damage and acute inflammation were observed in the cyclophosphamide+LPS group (Figure 1B). Roflumilast alleviated histopathological changes associated with LPS-induced lung injury in the prevention and treatment group, as shown in Figure 1C and D.
Effect of roflumilast on histopathology. A) Control group, B) Cyclophosphamide+lipopolysaccharide (LPS) group, C) Prevention group; Cyclophosphamide+LPS+roflumilast (given on day 0, when neutropenia was induced), D) Treatment group; Cyclophosphamide+LPS+roflumilast (given on day 2, after LPS administration).
Effects of roflumilast on inflammatory cells, inflammatory cytokines, and MPO. As shown in Table I, after intratracheal administration of LPS, the number of total cells and neutrophils in BAL fluid was increased. Roflumilast prevention and treatment significantly reduced both total cells and neutrophils in BAL fluid.
Effect of roflumilast on total and neutrophil cell counts (105/ml) in BAL fluid.
The concentrations of inflammatory cytokines, such as IL-1β, TNF-α, IL-6, and MPO in BAL fluid were dramatically increased in the cyclophosphamide+LPS group. Concentrations of inflammatory cytokines including IL-1β, TNF-α, and IL-6 were inhibited effectively by roflumilast (Figure 2). Although MPO concentrations were also reduced in the roflumilast prevention and treatment group, the difference was not statistically significant.
Effect of roflumilast on inflammatory cytokines. Concentrations of inflammatory cytokines including IL-1β, TNF-α, and IL-6 were significantly reduced by roflumilast. *p<0.05 for Cyclophosphamide+LPS versus Prevention or Treatment group.
Effects of roflumilast on MerTK, TLR-4, and MMP-9 expression. Expression levels of MerTK, TLR4, and MMP-9 genes were investigated by real-time RT-PCR. MMP-9 gene expression was increased by LPS administration, and significantly reduced in the roflumilast prevention group (Figure 3A). MerTK mRNA expression was decreased after LPS challenge and tended to increase in the roflumilast prevention and treatment groups; however, these increases were not significant. In contrast, there was no significant difference in TLR4 expression among the experimental groups.
Effect of roflumilast on the expression of MMP-9 and TGF-β. Expression of MMP-9 and TGF-β significantly decreased by roflumilast. *p<0.05 for Cyclophosphamide+LPS versus Prevention or Treatment group.
Effects of roflumilast on I-κB, ICAM, and TGF-β the expression. TGF-β expression increased after LPS administration, and significantly decreased in the roflumilast treatment group (Figure 3B). I-κB expression decreased after LPS administration during neutropenia recovery, whereas roflumilast administration increased I-κB expression, but not significantly. ICAM expression was increased in the cyclophosphamide+LPS group. Prevention or treatment with roflumilast decreased ICAM expression, but these decreases were not significant.
Discussion
According to a previous study, one-third of cancer patients who recovered from neutropenia experienced ARDS (5). These patients are prone to bacterial pneumonia or invasive fungal infections, especially when neutropenia is prolonged (17). Neutropenia recovery is associated with a high risk of deteriorating oxygenation and abnormal lung microvascular permeability (18).
Neutrophils not only play an important role in immune defense against infections, but also cause tissue damage, leading to inflammation (19). Especially, granulocyte colony-stimulating factor (G-CSF), which is used to treat patients with neutropenia, could enhance neutrophil functions (mobility, chemotaxis, and oxidant production) (8). However, there are no reliable therapeutic agents for patients with pneumonia during neutropenia recovery. Thus, it is important to elucidate the pathophysiology of ALI during neutropenia recovery.
In this study, we demonstrated that roflumilast significantly attenuated LPS-induced ALI during neutropenia recovery. Alveolar macrophages are the primary cells in the BAL fluid of patients with ARDS during neutropenia recovery. These alveolar macrophages are known to produce proinflammatory mediators, resulting in ARDS (5). Additionally, infiltration of activated neutrophils into the lung is a key process in the ALI inflammatory response (20). Both neutrophil recruitment and activation are required to cause ALI (21). In various ALI models, migration of polymorphonuclear leukocytes (PMN) into various compartments of the lung has been extensively investigated (22).
Cyclic AMP (cAMP) plays various roles in regulating intracellular responses, including anti-inflammatory effects. PDEs are enzymes that hydrolyze cyclic nucleotides. PDE4 inhibition results in increased cellular cAMP and reduces the release of proinflammatory cytokines from inflammatory and immune cells, including neutrophils, lymphocytes, eosinophils, monocytes, macrophages, as well as structural lung cells (epithelial cells, airway smooth muscle cells and fibroblasts) (10). Increased cAMP levels in endothelial cells prevent increased endothelial permeability and reduce pulmonary edema (9).
According to studies on the impact of PDE4-inhibitors on PMN migration, roflumilast significantly reduced PMN chemoattractants, such as IL-6, TNF-α, CXCL1, and CXCL 2/3 (23). Consequently, roflumilast is indicated for severe COPD patients with chronic bronchitis and a history of exacerbations (13). Additionally, Cortijo et al. demonstrated that roflumilast alleviated bleomycin-induced lung injury (24). Consistent with previous studies, the present study showed that inflammatory cytokines, such as IL-1β, TNF-α, and IL-6 were reduced in both the roflumilast prevention and treatment groups.
In ALI/ARDS pathogenesis, injury to the alveolar epithelial and endothelial capillary membranes and degradation of the basement membrane are present. Matrix metalloproteinase (MMPs) can degrade the extracellular matrix including the basement membrane, which can yield a variety of bioactive molecules (25). MMP-9 is thought to facilitate lung inflammation by degrading basement membranes, resulting in neutrophils that migrate into the lung tissue and airways (26). Previous studies showed that PDE-4 inhibition repressed the release of pro-inflammatory mediators, such as MMP-9, MPO, and reactive oxygen species (ROS) (27-29).
The results of this study correspond well with those from earlier experimental studies. Also, in this study, MMP-9 levels were significantly reduced in the roflumilast prevention group.
TGF-β has been shown to have pleiotropic effects that regulate various biological processes including development, tissue regeneration, immune responses, and tumorigenesis (30). In ALI, TGF-β directly increases alveolar epithelial permeability, leading to pulmonary edema (31). The results of this study showed that roflumilast reduced TGF-β.
Study limitations. We could not show a significant effect of roflumilast on MPO, MerTK, TLR-4, I-κB, or ICAM. Further investigation is needed to clarify the effect of roflumilast on various ALI/ARDS mediators. Also, future studies with larger sample sizes are required for greater statistical power.
Conclusion
In conclusion, roflumilast showed a protective role in treating LPS-induced ALI during neutropenia in a mouse model. This suggests that roflumilast could be a promising agent for treating ALI during neutropenia recovery.
Footnotes
Authors’ Contributions
KYK: Conception and design, data analysis, and interpretation; YAK: collection of data, administrative support, and provision of study materials, HSJ: collection of data, administrative support, and provision of study materials, JWK: conception, design, and interpretation.
Conflicts of Interest
The Authors wish to acknowledge the financial support of The Catholic University of Korea Uijeongbu St. Mary’s Hospital Clinical Research Laboratory Foundation during the program year of 2021.
- Received January 10, 2024.
- Revision received February 11, 2024.
- Accepted February 14, 2024.
- Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
