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. 2019 Aug;44(2):617-629.
doi: 10.3892/ijmm.2019.4208. Epub 2019 May 23.

Classical Dendritic Cells Regulate Acute Lung Inflammation and Injury in Mice With Lipopolysaccharide‑induced Acute Respiratory Distress Syndrome

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Free PMC article

Classical Dendritic Cells Regulate Acute Lung Inflammation and Injury in Mice With Lipopolysaccharide‑induced Acute Respiratory Distress Syndrome

Lang Li et al. Int J Mol Med. .
Free PMC article

Abstract

Classical dendritic cells (cDCs) are involved in the pathogenesis of inflammatory lung diseases; however, their contributions in acute respiratory distress syndrome (ARDS), which is pathophysiologically inflammatory, remain unknown. The present study aimed to explore the regulatory effects of pulmonary cDCs on acute lung inflammation and injury in lipopolysaccharide (LPS)‑induced ARDS. Fms‑like tyrosine kinase 3‑ligand (FLT3L) and lestaurtinib, a specific activator and an inhibitor of FLT3 signaling respectively, were used separately for the pretreatment of C57BL/6 mice for 5 consecutive days. ARDS was induced by intratracheal injection of LPS, and mice were sacrificed 6 and 24 h later. Flow cytometry was used to measure the aggregation and maturation of pulmonary cDCs. The ratio of lung wet weight to body weight (LWW/BW) and histopathological analyses were assessed to evaluate lung edema and lung injury. Tumor necrosis factor‑α and interleukin (IL)‑6 levels were measured by ELISA to evaluate acute lung inflammation. The levels of interferon‑γ, IL‑1β, IL‑4 and IL‑10, and the expression of the transcription factors T‑box‑expressed‑in‑T‑cells (T‑bet) and GATA binding protein 3, were quantified by ELISA, RT‑qPCR and western blotting to evaluate the balance of the Th1/Th2 response. Myeloperoxidase (MPO) activity was measured to evaluate neutrophil infiltration. The results demonstrated that the aggregation and maturation of pulmonary cDCs reached a peak at 6 h after LPS challenge, followed by a significant decrease at 24 h. FLT3L pretreatment further stimulated the aggregation and maturation of pulmonary cDCs, resulting in elevated lung MPO activity and increased T‑bet expression, which in turn led to aggravated LWW/BW, acute lung inflammation and injury. However, lestaurtinib pretreatment inhibited the aggregation and maturation of pulmonary cDCs, decreased lung MPO activity and T‑bet expression, and eventually improved LWW/BW, acute lung inflammation and injury. The present results suggested that pulmonary cDCs regulated acute lung inflammation and injury in LPS‑induced ARDS through the modulation of neutrophil infiltration and balance of the Th1/Th2 response.

Figures

Figure 1
Figure 1
Regulation of FLT3 signaling in lung tissue by FLT3L and lestaurtinib. mRNA and protein expression levels of Akt, ERK1/2 and STAT5 were measured by reverse transcription-quantitative PCR and western blotting, respectively, to confirm regulation of FLT3 signaling by FLT3L and lestaurtinib. (A) mRNA expression of Akt. (B) mRNA expression of ERK1/2. (C) mRNA expression of STAT5. β-actin was used as a reference gene. (D) Western blot analysis of Akt, ERK1/2 and STAT5 and their phosphorylated forms. (E) Ratio of p-Akt to total Akt levels. (F) Ratio of p-ERK to total ERK levels. (G) Ratio of p-STAT5 to total STAT5 levels. β-actin was used as a reference protein. Data are presented as the mean ± standard deviation (n=3). *P<0.05 vs. Control; #P<0.05 vs. ARDS; &P<0.05 vs. FLT3L+ARDS; and $P<0.05 vs. lestaurtinib+ARDS. FLT3, Fms-like tyrosine kinase 3; FLT3L, Fms-like tyrosine kinase 3-ligand; p-, phosphorylated; ARDS, acute respiratory distress syndrome.
Figure 2
Figure 2
Aggregation and maturation of pulmonary cDCs peaks at 6 h post-LPS challenge. The aggregation and maturation of pulmonary cDCs were measured by flow cytometry. (A) Representative dot plots and (B) quantification showing the percentage of cDCs (CD11c+CD11b+) among total lung cells in the control and ARDS groups at 6 and 24 h post-LPS challenge. (C) Representative histograms and (D) quantification showing the expression of MHC II in pulmonary cDCs in the control and ARDS groups at 6 and 24 h post-LPS challenge. (E) Representative histograms and (F) quantification showing the expression of CD80 in pulmonary cDCs in the control and ARDS groups at 6 and 24 h post-LPS challenge. Results are presented as the mean ± standard deviation of values obtained from six mice per group at each time point. Gray histograms represent the isotype control. *P<0.05 vs. 6 h-Control; #P<0.05 vs. 6 h-ARDS; and &P<0.05 vs. 24 h-Control. cDCs, classical dendritic cells; LPS, lipopolysaccharide; ARDS, acute respiratory distress syndrome; MHC II, major histocompatibility complex class II.
Figure 3
Figure 3
Effect of FLT3 signaling on the aggregation and maturation of pulmonary cDCs. The aggregation and maturation of pulmonary cDCs were measured by flow cytometry at 6 h post-LPS challenge. (A) Representative dot plots and (B) quantification showing the percentage of cDCs (CD11c+CD11b+) among total lung cells in the experimental groups. (C) Representative histograms and (D) quantification showing the expression of MHC II in pulmonary cDCs in the experimental groups. (E) Representative histograms and (F) quantification showing the expression of CD80 in pulmonary cDCs in the experimental groups. Data are presented as the mean ± standard deviation (n=6). *P<0.05 vs. Control; #P<0.05 vs. ARDS; &P<0.05 vs. FLT3L+ARDS; and $P<0.05 vs. lestaurtinib+ARDS. FLT3, Fms-like tyrosine kinase 3; cDCs, classical dendritic cells; LPS, lipopolysaccharide; MHC II, major histocompatibility complex class II; ARDS, acute respiratory distress syndrome; FLT3L, Fms-like tyrosine kinase 3-ligand.
Figure 4
Figure 4
Effect of cDC manipulation on acute lung inflammation in ARDS. (A) Levels of TNF-α and (B) IL-6 were measured using ELISA to evaluate acute lung inflammation at 6 h post-LPS challenge. Data are presented as the mean ± standard deviation (n=6). *P<0.05 vs. Control; #P<0.05 vs. ARDS; &P<0.05 vs. FLT3L+ARDS; and $P<0.05 vs. lestaurtinib+ARDS. cDC, classical dendritic cell; ARDS, acute respiratory distress syndrome; TNF, tumor necrosis factor; IL, interleukin; LPS, lipopolysaccharide; FLT3L, Fms-like tyrosine kinase 3-ligand.
Figure 5
Figure 5
Effect of cDC manipulation on lung edema and lung injury. LWW/BW and the lung injury score were measured to assess lung edema and lung injury at 6 and 24 h post-LPS challenge, respectively. (A) Hematoxylin and eosin staining (magnification, ×200) of lung tissues at 6 h and (B) at 24 h post-LPS challenge. (C) Lung injury score at 6 h and (D) at 24 h post-LPS challenge. (E) LWW/BW at 6 h and (F) at 24 h post-LPS challenge. Lung injury score is expressed as arbitrary mean units. Data are presented as the mean ± standard deviation (n=6). *P<0.05 vs. Control; #P<0.05 vs. ARDS; &P<0.05 vs. FLT3L+ARDS; and $P<0.05 vs. lestaurtinib+ARDS. cDC, classical dendritic cell; LWW/BW, lung wet weight to body weight; LPS, lipopolysaccharide; ARDS, acute respiratory distress syndrome; FLT3L, Fms-like tyrosine kinase 3-ligand.
Figure 6
Figure 6
Effect of cDC manipulation on neutrophil infiltration. MPO activity was measured by chromometry to assess neutrophil infiltration (A) at 6 h and (B) at 24 h post-LPS challenge. MPO activity is expressed as units per gram of sample. Data are presented as the mean ± standard deviation (n=6). *P<0.05 vs. Control; #P<0.05 vs. ARDS; &P<0.05 vs. FLT3L+ARDS; $P<0.05 vs. lestaurtinib+ARDS. cDC, classical dendritic cell; MPO, myeloperoxidase; LPS, lipopolysaccharide; ARDS, acute respiratory distress syndrome; FLT3L, Fms-like tyrosine kinase 3-ligand.
Figure 7
Figure 7
Effect of cDC manipulation on the balance of the Th1/Th2 response. The mRNA and protein expression levels of T-bet and GATA-3 were measured by reverse transcription-quantitative PCR and western blotting to evaluate the balance of the Th1/Th2 response. (A) mRNA expression of T-bet and (B) GATA-3. β-actin was used as a reference gene. (C) Representative blots from western blotting analysis of T-bet and GATA-3 protein expression. (D) Protein expression levels of T-bet and (E) GATA-3. β-actin was used as a reference protein. Data are presented as the mean ± standard deviation (n=3). *P<0.05 vs. Control; #P<0.05 vs. ARDS; &P<0.05 vs. FLT3L+ARDS; and $P<0.05 vs. lestaurtinib+ARDS. cDC, classical dendritic cell; Th, T helper cell; T-bet, T-box-expressed-in-T-cells; GATA-3, GATA binding protein 3; ARDS, acute respiratory distress syndrome; FLT3L, Fms-like tyrosine kinase 3-ligand.
Figure 8
Figure 8
Effect of cDC manipulation on Th1/Th2 cytokine production. (A) Levels of IFN-γ, (B) IL-1β, (C) IL-4 and (D) IL-10 were measured in the lungs by ELISA at 24 h post-LPS challenge, in order to evaluate the Th1/Th2 cytokine production. Data are presented as the mean ± standard deviation (n=6). *P<0.05 vs. Control; #P<0.05 vs. ARDS; &P<0.05 vs. FLT3L+ARDS; and $P<0.05 vs. lestaurtinib+ARDS. cDC, classical dendritic cell; Th, T helper cell; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; ARDS, acute respiratory distress syndrome; FLT3L, Fms-like tyrosine kinase 3-ligand.
Figure 9
Figure 9
FLT3L expression in normal and ARDS lung tissues. FLT3L protein expression levels in normal and ARDS lung tissues were evaluated by western blotting at 6 h post-LPS challenge. Data are presented as the mean ± standard deviation (n=3). *P<0.05 vs. Control. FLT3L, Fms-like tyrosine kinase 3-ligand; ARDS, acute respiratory distress syndrome.

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