Lipocalin-2 promotes acute lung inflammation and oxidative stress by enhancing macrophage iron accumulation

Abstract

Lipocalin-2 (LCN2) is an acute-phase protein that regulates inflammatory responses to bacteria or lipopolysaccharide (LPS). Although the bacteriostatic role of LCN2 is well studied, the function of LCN2 in acute lung damage remains unclear. Here, LCN2 knockout (KO) mice were used to investigate the role of LCN2 in LPS-treated mice with or without recombinant LCN2 (rLCN2). In addition, we employed patients with pneumonia. RAW264.7 cells were given LCN2 inhibition or rLCN2 with or without iron chelator deferiprone. LCN2 KO mice had a higher survival rate than wild-type (WT) mice after LPS treatment. In addition to elevated LCN2 levels in serum and bronchoalveolar lavage fluid (BALF), LPS treatment also increased LCN2 protein in alveolar macrophage lysates of BALF. LCN2 deletion attenuated neutrophil and macrophage infiltration in the lungs of LPS-treated mice as well as serum and BALF interleukin-6 (IL-6). Circulating proinflammatory cytokines and LCN2-positive macrophages were prominently increased in the BALF of pneumonia patients. In addition to increase of iron-stained macrophages in pneumonia patients, increased iron-stained macrophages and oxidative stress in LPS-treated mice were inhibited by LCN2 deletion. In contrast, rLCN2 pretreatment aggravated lung inflammation and oxidative stress in LPS-treated WT mice and then resulted in higher mortality. In RAW264.7 cells, exogenous LCN2 treatment also increased inflammation and oxidative stress, whereas LCN2 knockdown markedly diminished these effects. Furthermore, deferiprone inhibited inflammation, oxidative stress, and phagocytosis in RAW264.7 cells with high LCN2 levels, as well as LPS-induced acute lung injury in WT and LCN2 KO mice. Thus, these findings suggest that LCN2 plays a key role in inflammation and oxidative stress following acute lung injury and that LCN2 is a potential therapeutic target for pneumonia or acute lung injury.

Keywords: Acute lung injury; Inflammation; Iron; Lipocalin-2; Macrophage; Oxidative stress.

Figure 1

LCN2 increases in the BALF and lung of LPS-treated mice. (A) Percent survival in LPS-treated WT and LCN2 KO mice for 8days. Mice (n = 10) were intratracheally given with 20 mg/kg LPS. (B) ELISA analysis of serum LCN2 protein levels (n = 3-8). (C) Western blot and quantification of supernatant (s) and alveolar macrophage lysates (c) LCN2 proteins in the BALF (n = 3-8). β-actin served as a loading control. (D) Western blot and quantification of LCN2 and its receptor 24p3R proteins in lung tissues (n = 5-8). β-actin served as a loading control. (E) Representative images of LCN2 immunofluorescence. Scale bar, 50 μm. LCN2-positive cells were counted and analyzed in three fields (100x100 μm²) for each slide (n = 6). Differences between four groups were evaluated using two-way ANOVA followed by Tukey's multiple comparisons test. *P < 0.05 versus WT saline. †P < 0.05 versus WT LPS. All data are presented as mean ± SEM.

Figure 2

LCN2 deletion attenuates lung oxidative stress and iron accumulation in LPS-treated mice. (A) Western blot and quantification of HO-1 and SOD-2 protein levels. β-actin served as a loading control. (n = 4-7). (B) Representative double immunofluorescence images of CD11b and 4-HNE in lung sections. Nuclei were stained with DAPI. Scale bar, 50 μm. (C) Co-localized CD11b and 4-HNE-positive cells were counted and analyzed in three fields (100x100 μm²) for each slide (n = 6). (D) Representative images of Perls Prussian blue staining in lung sections and BALF slides. Arrows indicate iron-stained alveolar macrophages. Scale bar, 50 μm. (E) In lung tissues and BALF slides (D), iron-stained macrophages were counted and analyzed in four fields (100x100 μm²) for each slide (n = 6). (F) Representative triple immunofluorescence images of F4/80, LCN2, and ferritin in lung sections. Scale bar, 50 μm. (G) Co-localized F4/80, LCN2, and ferritin-positive cells were counted and analyzed in three fields (100x100 μm²) for each slide (n = 6). Differences between four groups were evaluated using two-way ANOVA followed by Tukey's multiple comparisons test. *P < 0.05 versus WT saline. †P < 0.05 versus WT LPS. All data are presented as mean ± SEM.

Figure 3

Pretreatment with rLCN2 promotes acute lung inflammation in LPS-treated WT and LCN2 KO mice. (A) Percent survival following intratracheal instillation with 20 mg/kg LPS after rLCN2 pretreatment. (n = 10). (B) Experimental schematic of rLCN2 pretreatment in LPS-treated mice. (C-D) ELISA analyses of LCN2 concentrations in serum (C) and BALF (D). (n = 4-8). (E) Representative images of Ly6G and F4/80 immunoreactivity in lung sections of rLCN2+LPS-treated WT and LCN2 KO mice. Scale bar, 20 μm. (F) Ly6G- and F4/80-positive cells were counted and analyzed in two fields (300x300 μm²) for each slide (n = 6). (G) Western blot and quantification of LCN2, F4/80, and IL-6 proteins in lung tissues (n = 5-7). β-actin served as a loading control. Differences between two groups were evaluated using unpaired Student's t-tests. *P < 0.05 versus LPS-treated WT. †P < 0.05 versus rLCN2+LPS-treated WT. All data are presented as mean ± SEM.

Figure 4

Pretreatment with rLCN2 promotes lung oxidative stress and iron deposition in LPS-treated WT and LCN2 KO mice. (A) Western blotting and quantification of lung HO-1, 4-HNE, and SOD-2 protein levels in lung tissues. β-actin served as a loading control. (n = 5-7). (B) Representative images of Perls Prussian blue iron staining in lung sections and BALF slides. Arrows indicate iron-stained alveolar macrophages. Scale bar, 50 μm. (n = 4 mice). (C) In lung sections and BALF slides (B), iron-positive alveolar macrophages were counted and analyzed in four fields (100x100 μm²) for each slide (n = 6). (D) Quantification of total lung iron levels using an iron assay kit. (n = 6 mice). Differences between two groups were evaluated using unpaired Student's t-tests. *P < 0.05 versus LPS-treated WT. †P < 0.05 versus rLCN2+LPS-treated WT. All data are presented as mean ± SEM.

Figure 5

Effect of LCN2 deletion on proinflammatory cytokines in LPS-treated RAW264.7 cells. (A) Western blot analysis of cellular lysate LCN2, 24p3R, IL-6, TNF-α, iNOS, and media LCN2 in LPS-treated RAW264.7 cells. (B) Schematic drawing of LPS-treated RAW264.7 cells medium (LTM) treatment method in LTM-treated RAW264.7 cells. (C) Western blot analysis of cellular lysate LCN2, 24p3R, IL-6, TNF-α, iNOS, and media LCN2 in LTM-treated RAW264.7 cells. (D) Western blot analysis of cellular lysate LCN2, 24p3R, IL-6, TNF-α, iNOS, and media LCN2 in recombinant LCN2 (rLCN2)-treated RAW264.7 cells. (E-F) Western blot analysis (E) and quantification (F) of cellular lysate LCN2, 24p3R, IL-6, TNF-α, iNOS, HO-1, and media LCN2 in siLCN2+LPS-treated RAW264.7 cells from three independent experiment. β-actin served as a loading control. Differences between four groups were evaluated using two-way ANOVA followed by Tukey's multiple comparisons test. *P < 0.05 versus CTL. †P < 0.05 versus LPS. All data are presented as mean ± SEM.

Figure 6

Effects of iron chelator deferiprone on inflammation and oxidative stress in LTM or rLCN2-treated RAW264.7 cells. (A, C) Western blot analysis and quantification of cellular lysate LCN2, 24p3R, IL-6, TNF-α, iNOS, HO-1, SOD-2, 4-HNE, IRP1, ferritin, and media LCN2 in deferiprone (DFP)+ LTM-treated RAW264.7 cells from three independent experiment. β-actin served as a loading control. (B, D) Western blot analysis and quantification of cellular lysate LCN2, 24p3R, IL-6, TNF-α, iNOS, HO-1, SOD-2, 4-HNE, IRP1, ferritin, and media LCN2 in DFP+rLCN2-treated RAW264.7 cells from three independent experiment. β-actin served as a loading control. (E-F) Detection and quantification of superoxide by MitoSOX™ Red in DFP+LTM (E)- and DFP/rLCN2 (F)-treated RAW264.7 cells. DAPI was used for nuclear staining. Scale bar, 25 μm. Bar graphs indicate MitoSOX™ Red fluorescence intensity. Differences between four groups were evaluated using two-way ANOVA followed by Tukey's multiple comparisons test. *P < 0.05 versus CTL. †P < 0.05 versus LTM or rLCN2. All data are presented as mean ± SEM.

Figure 7

Effects of iron chelator deferiprone on phagocytosis in LTM or rLCN2-treated RAW264.7 cells. (A-B) Representative images obtained after 30 min incubation with zymosan red particle in deferiprone (DFP)+LTM (A)- and DFP+rLCN2 (B)-treated RAW264.7 cells. DAPI was used for nuclear staining. Scale bar, 25 μm. Bar graphs indicate the number of zymosan red particles. Differences between four groups were evaluated using two-way ANOVA followed by Tukey's multiple comparisons test. *P < 0.05 versus CTL. †P < 0.05 versus LTM or rLCN2. All data are presented as mean ± SEM.