All‑trans retinoic acid promotes macrophage phagocytosis and decreases inflammation via inhibiting CD14/TLR4 in acute lung injury

Abstract

Acute lung injury (ALI) is a common clinical emergency and all‑trans retinoic acid (ATRA) can alleviate organ injury. Therefore, the present study investigated the role of ATRA in ALI. Lipopolysaccharide (LPS)‑induced ALI rats were treated with ATRA and the arterial partial pressure of oxygen (PaO₂), lung wet/dry weight (W/D) ratio and protein content in the bronchial alveolar lavage fluid (BALF) were measured to evaluate the effect of ATRA on ALI rats. Alveolar macrophages were isolated from the BALF. The phagocytic function of macrophages was detected using the chicken erythrocyte phagocytosis method and flow cytometry. The viability of macrophages was measured using a Cell Counting Kit‑8 assay, and apoptosis was analyzed using a TUNEL assay and flow cytometry. The expression levels of Toll‑like receptor 4 (TLR4) and cluster of differentiation (CD)14 on the macrophage membrane were detected by immunofluorescence staining. The protein levels of TLR4, CD14, phosphorylated (p)‑65, p65, p‑IκBα and IκBα were analyzed using western blotting. The concentrations of IL‑6, IL‑1β and macrophage inflammatory protein‑2 in the plasma of rats were detected by ELISA. Macrophages were treated with IAXO‑102 (TLR4 inhibitor) to verify the involvement of CD14/TLR4 in the effect of ATRA on ALI. ATRA provided protection against LPS‑induced ALI, as evidenced by the increased PaO2 and reduced lung W/D ratio and protein content in the BALF. ATRA enhanced macrophage phagocytosis and viability and reduced apoptosis and inflammation in ALI rats. Mechanically, ATRA inhibited CD14 and TLR4 expression and NF‑κB pathway activation. ATRA enhanced macrophage phagocytosis and reduced inflammation by inhibiting the CD14/TLR4‑NF‑κB pathway in LPS‑induced ALI. In summary, ATRA inactivated the NF‑κB pathway by inhibiting the expression of CD14/TLR4 receptor in the alveolar macrophages of rats, thus enhancing the phagocytic function of macrophages in ALI rats, improving the activity of macrophages, inhibiting apoptosis, reducing the levels of inflammatory factors, and consequently playing a protective role in ALI model rats. This study may offer novel insights for the clinical management of ALI.

Keywords: Toll‑like receptor 4; acute lung injury; all‑trans retinoic acid; cluster of differentiation 14; inflammation; macrophage; phagocytosis.

Figure 1.

ATRA possesses protective effects on LPS-induced ALI rats. (A) Protein content in BALF detected using a kit. (B) PaO₂ in arterial blood detected using a blood gas analyzer. (C) Lung W/D ratio. (D) Lung tissue injury observed with hematoxylin and eosin staining. Magnification, ×200. (E) Lung injury score assessed with high score indicative of severe injury. N=10 in each group. A total of three independent experiments were repeatedly conducted and the data are expressed as mean ± standard deviation. Data analyzed using one-way analysis of variance, followed by Tukey's multiple comparisons test. *P<0.05, **P<0.01 vs. control group; ^#P<0.05, ^##P<0.01 vs. ALI group. ATRA, all-trans retinoic acid; LPS, lipopolysaccharide; ALI, acute lung injury; BALF, bronchial alveolar lavage fluid; PaO₂, arterial partial pressure of oxygen; W/D, wet/dry weight.

Figure 2.

ATRA enhances macrophage phagocytosis in ALI rats. (A) MPO activity of alveolar macrophages was measured. (B) Phagocytic rate and (C) phagocytic index of macrophages were detected using the chicken erythrocyte phagocytosis method. (D) MFI value of macrophages and (E) the percentage of phagocytic positive cells were detected using flow cytometry. The cell experiments were repeated three times independently. Data are expressed as mean ± standard deviation and analyzed using one-way analysis of variance, followed by Tukey's multiple comparisons test. **P<0.01 vs. control group; ^##P<0.01 vs. ALI group. ATRA, all-trans retinoic acid; ALI, acute lung injury; MPO, myeloperoxidase; MFI, mean fluorescence intensity.

Figure 3.

ATRA increases macrophage viability and inhibited apoptosis and decreases inflammatory cytokine levels in ALI rats. (A) CCK-8 assay was used to detect the viability of macrophages in ALI rats. (B) TUNEL and (C) flow cytometry were used to detect the apoptosis of macrophages in ALI rats. (D-F) ELISA kits were used to detect the levels of inflammatory cytokines (D) IL-6, (E) IL-1β and (F) MIP-2 in plasma of rats in each group. The cell experiments were repeated three times independently. Data are expressed as mean ± standard deviation and analyzed using one-way analysis of variance, followed by Tukey's multiple comparisons test. **P<0.01 vs. control group; ^##P<0.01 vs. ALI group. ATRA, all-trans retinoic acid; ALI, acute lung injury; MIP-2, macrophage inflammatory protein-2.

Figure 4.

ATRA inhibits CD14/TLR4 upregulation and the downstream NF-κB pathway activation in macrophages in LPS-induced ALI rats. (A) CD14 and TLR4 expressions on macrophage membrane are detected using immunofluorescence. (B) Key pathways in LPS signal transduction through the Kyoto Encyclopedia of Genes and Genomes database (https://www.kegg.jp/). (C) mRNA expressions of TLR4, CD14, P65 and IκBα are detected using reverse transcription quantitative PCR. (D) Protein levels of TLR4, CD14, p-P65, P65, p-IκBα and IκBα are detected using western blotting. The cell experiments were repeated three times independently. Data are expressed as mean ± standard deviation and analyzed using one-way or two-way analysis of variance, followed by Tukey's multiple comparisons test. *P<0.05, **P<0.01 vs. control group; ^#P<0.05, ^##P<0.01 vs. ALI group. ATRA, all-trans retinoic acid; CD, cluster of differentiation; TLR, Toll-like receptor; LPS, lipopolysaccharide; ALI, acute lung injury; p-, phosphorylated.

Figure 5.

ATRA enhances macrophage phagocytosis and reduces inflammatory cytokine levels via inhibiting CD14/TLR4 expressions in LPS-induced ALI rats. (A) mRNA expressions of TLR4, CD14, P65 and IκBα are detected using reverse transcription quantitative PCR. (B) Western blotting was used to detect protein levels of TLR4, p-P65, P65, p-IκBα and IκBα. (C) MPO activity in macrophages was measured using a kit. (D) MFI value of macrophages was measured using flow cytometry. (E) Percentage of macrophage phagocytic positive cells was determined using flow cytometry. (F) Levels of inflammatory cytokines (IL-6, IL-1β and MIP-2) in rat plasma were detected using ELISA kits. The cell experiments were repeated three times independently. Data were expressed as mean ± standard deviation and analyzed using one-way or two-way analysis of variance, followed by Tukey's multiple comparisons test. *P<0.05, **P<0.01 vs. the ALI + PBS group; ^#P<0.05, ^##P<0.01 vs. the ATRA + IAXO-102 group. ATRA, all-trans retinoic acid; CD, cluster of differentiation; TLR, Toll-like receptor; LPS, lipopolysaccharide; ALI, acute lung injury; p-, phosphorylated; MPO, myeloperoxidase; MFI, mean fluorescence intensity; MIP-2, macrophage inflammatory protein-2.