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. 2021 Jul 7:12:692286.
doi: 10.3389/fimmu.2021.692286. eCollection 2021.

Butorphanol Promotes Macrophage Phenotypic Transition to Inhibit Inflammatory Lung Injury via κ Receptors

Guangxin Luan  1 , Fan Pan  1 , Lina Bu  1 , Kaixuan Wu  1 , Aizhong Wang  1 , Xiaotao Xu  1
Affiliations

Butorphanol Promotes Macrophage Phenotypic Transition to Inhibit Inflammatory Lung Injury via κ Receptors

Guangxin Luan et al. Front Immunol. .

Abstract

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is characterized by diffuse inflammation of the lung parenchyma and refractory hypoxemia. Butorphanol is commonly used clinically for perioperative pain relief, but whether butorphanol can regulate LPS-induced alveolar macrophage polarization is unclear. In this study, we observed that butorphanol markedly attenuated sepsis-induced lung tissue injury and mortality in mice. Moreover, butorphanol also decreased the expression of M1 phenotype markers (TNF-α, IL-6, IL-1β and iNOS) and enhanced the expression of M2 marker (CD206) in alveolar macrophages in the bronchoalveolar lavage fluid (BALF) of LPS-stimulated mice. Butorphanol administration reduced LPS-induced numbers of proinflammatory (M1) macrophages and increased numbers of anti-inflammatory (M2) macrophages in the lungs of mice. Furthermore, we found that butorphanol-mediated suppression of the LPS-induced increases in M1 phenotype marker expression (TNF-α, IL-6, IL-1β and iNOS) in bone marrow-derived macrophages (BMDMs), and this effect was reversed by κ-opioid receptor (KOR) antagonists. Moreover, butorphanol inhibited the interaction of TLR4 with MyD88 and further suppressed NF-κB and MAPKs activation. In addition, butorphanol prevented the Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF)-mediated IFN signaling pathway. These effects were ameliorated by KOR antagonists. Thus, butorphanol may promote macrophage polarization from a proinflammatory to an anti-inflammatory phenotype secondary to the inhibition of NF-κB, MAPKs, and the TRIF-mediated IFN signaling pathway through κ receptors.

Keywords: acute lung injury; butorphanol; inflammation; macrophage; κ receptor.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Graphical Abstract
Graphical Abstract
Graphical summary of the effects and mechanisms of butorphanol on sepsis-induced ALI. Upon binding of LPS to TLR4, macrophages are activated and accumulated at the site of infection where they are polarized into the M1 (a pro-inflammatory) and M2 (an anti-inflammatory) phenotypes. However, butorphanol may mitigate sepsis-induced ALI by promoting macrophage transition from M1 to M2 phenotype secondary to the inhibition of NF-κB and MAPKs, and the TRIF-mediated IFN signaling pathway through κ receptors.
Figure 1
Figure 1
Butorphanol ameliorated LPS-induced inflammatory lung injury. Mice were injected with LPS (10 mg·kg-1 i.p.) or butorphanol (8 mg·kg-1 i.p.) or butorphanol (4 mg·kg-1 i.p. or 8 mg·kg-1 i.p.) plus LPS (10 mg·kg-1 i.p.) and analyzed after 24 h. (A) Hematoxylin and eosin staining of lung sections. Scale bar: 50 μm. (B) Lung injury score. (C, D) Lungs obtained at 24 h after LPS challenge were used to determine the wet/dry weight ratio and MPO activity. (E) The total protein level in BALF was measured with a BCA assay. (F–H) The BALF expression levels of TNF-α, IL-6, and IL-1β were determined by ELISA. (I–M) The mRNA levels of TNF-α, IL-6, IL-1β, iNOS, and CD206 in CD11b+ alveolar macrophages in the BALF of mice were measured with qRT-PCR. (N) Mouse survival was monitored for 72 h after a lethal dose of LPS (20 mg·kg-1 i.p.) alone or in combination with butorphanol (4 mg·kg-1 i.p. or 8 mg·kg-1 i.p.) 30 min after LPS challenge. n = 10 per group. The results are the means ± SEM of ten independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post hoc test when comparing multiple independent groups and the log-rank test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 2
Figure 2
Butorphanol administration inhibited M1 macrophage polarization in LPS-induced lung injury. Mice were injected with LPS (10 mg·kg-1 i.p.) or butorphanol (8 mg·kg-1 i.p.) or butorphanol (4 mg·kg-1 i.p. or 8 mg·kg-1 i.p.) plus LPS (10 mg·kg-1 i.p.) and analyzed after 24 h. (A) Sections were stained with antibodies against F4/80 (red) and iNOS (green). Scale bar: 25 μm. (B) The image analysis results are presented as the percentage of iNOS-positive F4/80 cells. The results are the means ± SEM of ten independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post hoc test when comparing multiple independent groups. ****P < 0.0001.
Figure 3
Figure 3
Butorphanol administration promoted M2 macrophage polarization in LPS-induced lung injury. Mice were injected with LPS (10 mg·kg-1 i.p.) or butorphanol (8 mg·kg-1 i.p.) or butorphanol (4 mg·kg-1 i.p. or 8 mg·kg-1 i.p.) plus LPS (10 mg·kg-1 i.p.) and analyzed after 24 h. (A) Sections were stained with antibodies against F4/80 (red) and CD206 (green). Scale bar: 25 μm. (B) The image analysis results are presented as the percentage of CD206-positive F4/80 cells. The results are the means ± SEM of ten independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post hoc test when comparing multiple independent groups. ***P < 0.001, ****P < 0.0001.
Figure 4
Figure 4
Butorphanol inhibited M1 macrophage activation. Macrophages were collected and cultured from the bone marrow of mice and treated with different concentrations of butorphanol (0-8 μM) for 24 h. (A) CCK-8 analysis of cell viability. Cells were incubated with LPS (1 μg·mL-1) alone or butorphanol (4 μM or 8 μM) plus LPS (1 μg·mL-1) for 24 h. (B–D) TNF-α, IL-6, and IL-1β protein levels in the supernatant of macrophage cultures were determined by ELISA. (E–H) The mRNA expression of proinflammatory cytokines (TNF-α, IL-6, IL-1β, and iNOS) in cells was measured by qRT-PCR. (I) The levels of iNOS were measured by western blot analysis. (J) Densitometric analysis of iNOS levels in (I) was performed with normalization to β-tubulin. Cells were incubated with IFN-γ (100 ng·mL-1) alone or butorphanol (4 μM or 8 μM) plus IFN-γ (100 ng·mL-1) for 24 h. (K–N) The mRNA expression of proinflammatory cytokines (TNF-α, IL-6, IL-1β, and iNOS) in cells was measured by qRT-PCR. The results are the means ± SEM of six independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post hoc test when comparing multiple independent groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 5
Figure 5
Butorphanol promoted M2 macrophage polarization. Macrophages were collected and cultured from the bone marrow of mice and incubated with LPS (1 μg·mL-1) alone or butorphanol (4 μM or 8 μM) plus LPS (1 μg·mL-1) for 24 h. (A, B) The mRNA expression of M2 markers (CD206 and Arg-1) in cells was measured by qRT-PCR. (E) The protein levels of CD206 were measured by western blot analysis. (F) Densitometric analysis of CD206 expression in (E) was performed with normalization to β-tubulin. Macrophages were incubated with IL-4 (20 ng·mL-1) alone or butorphanol (4 μM or 8 μM) plus IL-4 (20 ng·mL-1) for 24 h. (C, D) The mRNA expression of M2 markers (CD206 and Arg-1) in cells was measured by qRT-PCR. (G) The protein levels of CD206 and Arg-1 were measured by western blot analysis. (H, I) Densitometric analysis of CD206 and Arg-1 levels in (G) was performed with normalization to β-tubulin. Macrophages were incubated with IL-4 (20 ng·mL-1) alone or butorphanol (4 μM or 8 μM) plus IL-4 (20 ng·mL-1) for 30 min. (J) The protein levels of phosphorylated STAT6 and total STAT6 were measured by western blot analysis. (K) Densitometric analysis of the phosphorylated STAT6 levels in (J) was performed with normalization to the respective total protein. The results are the means ± SEM of six independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post hoc test when comparing multiple independent groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Figure 6
Figure 6
Butorphanol regulated macrophage polarization through the KOR in LPS-induced BMDMs. Macrophages were collected and cultured from the bone marrow of mice and incubated with LPS (1 μg·mL-1) alone or butorphanol (8 μM) plus LPS (1 μg·mL-1), nor-BNI (KOR antagonist, 5 μM) plus butorphanol (8 μM) plus LPS (1 μg·mL-1), or naloxegol (MOR antagonist, 5 μM) plus butorphanol (8 μM) plus LPS (1 μg·mL-1) for 24 h. Cells were pretreated with nor-BNI or naloxegol for 30 min. (A, B) TNF-α and IL-6 protein levels in the supernatant of macrophage cultures were determined by ELISA. (C–F) The mRNA levels of TNF-α, IL-6, iNOS and CD206 in cells were measured by qRT-PCR. The results are the means ± SEM of six independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post hoc test when comparing multiple independent groups. *P < 0.05, **P < 0.01, ****P < 0.0001.
Figure 7
Figure 7
Butorphanol prevented the interaction of MyD88 with TLR4 and inhibited NF-κB activation through KOR in M1-polarized BMDMs. Macrophages were collected and cultured from the bone marrow of mice and incubated with LPS (1 μg·mL-1) alone, butorphanol (8 μM) plus LPS (1 μg·mL-1), or nor-BNI (KOR antagonist, 5 μM) plus butorphanol (8 μM) and LPS (1 μg·mL-1) for 15 min or 30 min. Cells were pretreated with nor-BNI for 30 min. (A) To observe the interaction of MyD88 with TLR4, cells were fixed, permeabilized, and stained, and images were obtained by confocal microscopy. Scale bar: 25 μm. (B) The levels of phosphorylated IKKβ and total IKKβ were measured by western blot analysis. (C) Densitometric analysis of the phosphorylated IKKβ levels in (B) was performed with normalization to the respective total protein. (D) The translocation of NF-κB in cells was examined by confocal microscopy. Scale bar: 25 μm. The results are the means ± SEM of six independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post hoc test when comparing multiple independent groups. ****P < 0.0001.
Figure 8
Figure 8
Butorphanol inhibited M1 phenotype macrophage polarization through the MAPK signaling pathway. Macrophages were collected and cultured from the bone marrow of mice and treated with LPS (1 μg·mL-1) alone or butorphanol (4 μM or 8 μM) plus LPS (1 μg·mL-1) for 30 min. (A) The levels of phosphorylated MAPKs and their respective total proteins were measured by western blot analysis. (B–D) Densitometric analysis of the phosphorylated MAPKs levels in (A) was performed with normalization to the respective total proteins. Macrophages were incubated with LPS (1 μg·mL-1) alone, butorphanol (8 μM) plus LPS (1 μg·mL-1), the p38 inhibitor SB203580 (10 µM) plus LPS (1 μg·mL-1), the ERK inhibitor PD98059 (10 µM) plus LPS (1 μg·mL-1), or the JNK inhibitor SP600125 (10 µM) plus LPS (1 μg·mL-1) for 24 h. (E, F) IL-6 and IL-1β protein levels in the supernatant of macrophage cultures were determined by ELISA. (G–I) The mRNA levels of IL-6, IL-1β, and iNOS in cells were measured by qRT-PCR. The results are the means ± SEM of six independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post hoc test when comparing multiple independent groups. *P < 0.05, ***P < 0.001, ****P < 0.0001.
Figure 9
Figure 9
Butorphanol prevented the phosphorylation of MAPKs through KOR in M1-polarized BMDMs. Macrophages were collected and cultured from the bone marrow of mice and incubated with LPS (1 μg·mL-1) alone, butorphanol (8 μM) plus LPS (1 μg·mL-1) or nor-BNI (KOR antagonist, 5 μM) plus butorphanol (8 μM) plus LPS (1 μg·mL-1) for 30 min. Cells were pretreated with nor-BNI for 30 min. (A) The levels of phosphorylated MAPKs and their respective total proteins were measured by western blot analysis. (B–D) Densitometric analysis of the phosphorylated MAPKs levels in (A) was performed with normalization to the respective total proteins. The results are the means ± SEM of six independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post hoc test when comparing multiple independent groups. ****P < 0.0001.
Figure 10
Figure 10
Butorphanol decreased LPS-induced the elevation of IRF7 and IFN-β through KOR in M1-polarized BMDMs. Macrophages were collected and cultured from the bone marrow of mice and incubated with LPS (1 μg·mL-1) alone, butorphanol (8 μM) plus LPS (1 μg·mL-1) or nor-BNI (KOR antagonist, 5 μM) plus butorphanol (8 μM) plus LPS (1 μg·mL-1) for 6 h. Cells were pretreated with nor-BNI for 30 min. (A–C) The mRNA levels of IRF3, IRF7, and IFN-β in cells were measured by qRT-PCR. The results are the means ± SEM of six independent experiments. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post hoc test when comparing multiple independent groups. *P < 0.05, **P < 0.01, ****P < 0.0001.
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