Targeting transitioning lung monocytes/macrophages as treatment strategies in lung disease related to environmental exposures

Abstract

Background: Environmental/occupational exposures cause significant lung diseases. Agricultural organic dust extracts (ODE) and bacterial component lipopolysaccharide (LPS) induce recruited, transitioning murine lung monocytes/macrophages, yet their cellular role remains unclear.

Methods: CCR2 RFP⁺ mice were intratracheally instilled with high concentration ODE (25%), LPS (10 μg), or gram-positive peptidoglycan (PGN, 100 μg) for monocyte/macrophage cell-trafficking studies. CCR2 knockout (KO) mice and administration of intravenous clodronate liposomes strategies were employed to reduce circulating monocytes available for lung recruitment following LPS exposure. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected. Pro-inflammatory and/or pro-fibrotic cytokines, chemokines, and lung extracellular matrix mediators were quantitated by ELISA. Infiltrating lung cells including monocyte/macrophage subpopulations, neutrophils, and lymphocytes were characterized by flow cytometry. Lung histopathology, collagen content, vimentin, and post-translational protein citrullination and malondialdehyde acetaldehyde (MAA) modification were quantitated. Parametric statistical tests (one-way ANOVA, Tukey'smultiple comparison) and nonparametric statistical (Kruskal-Wallis, Dunn's multiple comparison) tests were used following Shapiro-Wilk testing for normality.

Results: Intratracheal instillation of ODE, LPS, or PGN robustly induced the recruitment of inflammatory CCR2⁺ CD11c^intCD11b^hi monocytes/macrophages and both CCR2⁺ and CCR2^- CD11c^-CD11b^hi monocytes at 48 h. There were also increases in CCR2⁺ CD4⁺ and CD8⁺ T cells and NK cells. Despite reductions in LPS-induced lung infiltrating CD11c^intCD11b^hi cells (54% reduction), CCR2 knockout (KO) mice were not protected against LPS-induced inflammatory and pro-fibrotic consequences. Instead, compensatory increases in lung neutrophils and CCL2 and CCL7 release occurred. In contrast, the depletion of circulating monocytes through the administration of intravenous clodronate (vs. vehicle) liposomes 24 h prior to LPS exposure reduced LPS-induced infiltrating CD11c^intCD11b^hi monocyte-macrophage subpopulation by 59% without compensatory changes in other cell populations. Clodronate liposome pre-treatment significantly reduced LPS-induced IL-6 (66% reduction), matrix metalloproteinases (MMP)-3 (36%), MMP-8 (57%), tissue inhibitor of metalloproteinases (61%), fibronectin (38%), collagen content (22%), and vimentin (40%). LPS-induced lung protein citrullination and MAA modification, post-translational modifications implicated in lung disease, were reduced (39% and 48%) with clodronate vs. vehicle liposome.

Conclusion: Highly concentrated environmental/occupational exposures induced the recruitment of CCR2⁺ and CCR2^- transitioning monocyte-macrophage and monocyte subpopulations and targeting peripheral monocytes may reduce the adverse lung consequences resulting from exposures to LPS-enriched inhalants.

Keywords: Endotoxin; Immunology; Inflammation; Lung disease; Monocytes; Organic dust.

Fig. 1

Inhalation of organic dust extract (ODE), lipopolysaccharide (LPS), and peptidoglycan (PGN) induce lung CCR2⁺ monocyte-macrophages (Mɸ) and monocytes. CCR2^RFP/+ mice were exposed once to ODE (25%), LPS (10 μg), PGN (100 μg), or saline control and euthanized at 48 h. Scatter plots with bars depict mean with SD delineating cells as total (gray), CCR2⁺ (green), and CCR2⁻ (red). A Total lung cells enumerated. B Representative contour plot of the four monocyte (mono)-Mɸ subpopulations across groups based upon CD11c and CD11b expression after removal of neutrophils gated from live CD45⁺ cells after excluding debris and doublets. C RFP ± staining by exposure group and subpopulation. D CD11c⁺CD11b^lo alveolar (Alv) Mɸ, CD11c⁺CD11b^hi activated (Act) Mɸ, CD11c^intCD11b^hi mono-Mɸ, and CD11c⁻CD11b.^hi monocytes determined by multiplying lung cell % population by total lung cells enumerated from lung sample. Statistical analyses were performed with Kruskal–Wallis with Dunn’s test for multiple comparisons (#p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001) vs. respective saline. N = 19 (saline), 9 (ODE), 8 (LPS), 5 (PGN)

Fig. 2

Ly6C expression of monocyte-macrophage (Mɸ) and monocyte subpopulations following organic dust extract (ODE), lipopolysaccharide (LPS), and peptidoglycan (PGN) exposure. C57BL/6 mice were exposed once to ODE (25%), LPS (10 μg), PGN (100 μg), or saline control and euthanized at 48 h. Scatter plots with bars depict mean with SD delineating cells as CCR2 + (green) and CCR2- (red). Expression of Ly6C by percent (A) and mean fluorescence intensity (MFI) (B) across CD11cintCD11bhi monocyte-Mɸ and CD11c-CD11b + monocyte subpopulations as determined by flow cytometry. Statistical analyses were performed with Kruskal–Wallis with Dunn’s test for multiple comparisons (#p < 0.05 vs. respective saline) and (*p < 0.05 denoted by line with brackets denoting difference between same inhalant exposure by CCR2 RFP positive vs. negative). N = 15 (saline), 9 (ODE), 4 (LPS), 5 (PGN)

Fig. 3

Organic dust extract (ODE) and lipopolysaccharide (LPS) inhalation induce lung infiltration of CCR2 + NK and T cells. C57BL/6 mice were exposed once to ODE (25%), LPS (10 μg), PGN (100 μg), or saline control and euthanized at 48 h. Scatter plots with bars depict mean with SD delineating cells as total (gray), CCR2 + (green), and CCR2- (red). CD11c-Ly6G + neutrophils, CD19 + B cells, CD3-NK1.1 + NK cells, CD3 + CD4 + T cells, and CD3 + CD8 + T cell infiltrates determined by flow cytometry on live CD45 + cells after exclusion of debris and doublets with lung cell % populations multiplied by total lung cells enumerated from lung sample. Gating strategy depicted in Supplemental Fig. 1. Statistical analyses were performed with Kruskal–Wallis with Dunn’s test for multiple comparisons (#p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001) vs. respective saline. N = 19 (saline), 9 (ODE), 8 (LPS), 5 (PGN)

Fig. 4

LPS-induced transitioning CD11c^intCD11b^hi monocytes/macrophages are reduced with systemic delivery of clodronate liposomes. Mice were pre-treated with vehicle (Veh) or clodronate (Clod) liposomes 24 h prior to a one-time treatment with LPS (10 μg) or saline (Sal) control and euthanized at 48 h. Scatter plot with bars depicting mean with SD. A Total lung cells enumerated. B Representative contour plot of the four monocyte (mono)-Mɸ subpopulations across groups based upon CD11c and CD11b expression after removal of neutrophils gated from live CD45⁺ cells after excluding debris and doublets. C Number of CD11c⁺CD11b^lo alveolar (Alv) Mɸ, CD11c⁺CD11b^hi activated (Act) Mɸ, CD11c^intCD11b^hi mono-Mɸ, and CD11c⁻CD11b.^hi monocytes determined by multiplying lung cell % population by total lung cells enumerated from lung sample. (#p < 0.05 vs. respective saline) and (*p < 0.05 denoted by line with brackets denoting difference between groups). N = 8 (Veh + Sal), 8 (Clod + Sal), 8 (Veh + LPS), 9 (Clod + LPS)

Fig. 5

Effects of systemic clodronate liposome delivery with LPS-induced lung inflammation, collagen, and infiltrating CCR2⁺ cells. Mice were pre-treated with vehicle (Veh) or clodronate (Clod) liposomes 24 h prior to a one-time treatment with LPS (10 μg) or saline (Sal) control and euthanized at 48 h. A Representative images from treatment groups stained by H&E, trichome, and CCR2 (red) with DAPI nuclei staining (blue) by confocal microscopy. Scatter plots with bars depict mean with SD of semi-quantitative lung inflammatory score (B) and integrated density of collagen (C), and CCR2 (D) quantified per each mouse. Statistical analyses were performed with Kruskal–Wallis with Dunn’s multiple comparison (inflammatory scores) and ANOVA with Tukey’s multiple comparison (collagen content and CCR2) (#p < 0.05 vs. respective saline) and (*p < 0.05 denoted by line with brackets denoting difference between groups). N = 8 (Veh + Sal), 8 (Clod + Sal), 8 (Veh + LPS), 9 (Clod + LPS). Line scale denotes 100 μm

Fig. 6

LPS-induced lung pro-fibrotic and inflammatory mediators modulated following systemic delivery of clodronate liposomes. Mice were pre-treated with vehicle (Veh) or clodronate (Clod) liposomes 24 h prior to a one-time treatment with LPS (10 μg) or saline (Sal) control and euthanized at 48 h. Scatter plots with bars depict mean with SD of protein levels of matrix metalloproteinase (MMP)-3, MMP-8, metalloproteinase inhibitor (TIMP-1), transforming growth factor (TGF)-β, IL-6, and neutrophil chemokine CXCL1 of lung homogenate. Statistical analyses were performed with ANOVA and Tukey’s multiple comparison test with significance (#p < 0.05 vs. respective saline) and (*p < 0.05 denoted by line with brackets denoting difference between groups) with % reduction noted. N = 8 (Veh + Sal), 8 (Clod + Sal), 8 (Veh + LPS), 9 (Clod + LPS)

Fig. 7

LPS-induced lung CIT and MAA autoantigens and vimentin expression decrease with systemic clodronate liposome delivery. Mice were pre-treated with vehicle (Veh) or clodronate (Clod) liposomes 24 h prior to a one-time treatment with LPS (10 μg) or saline (Sal) control and euthanized at 48 h. A Representative confocal microscopy images of lung tissue from treatment groups stained for citrulline (CIT, green) and malondialdehyde-acetaldehyde (MAA, red) modified proteins and vimentin (teal). Line scale denotes 70 μm. B Scatter plots with bars depict mean with SD of integrated density of CIT- and MAA-modified proteins and vimentin quantified per each mouse. Statistical analyses were performed with ANOVA with Tukey’s for multiple comparisons (#p < 0.05 vs. respective saline) and (*p < 0.05 denoted by line with brackets denoting difference between groups). N = 5 (Veh + Sal), 7 (Clod + Sal), 8 (Veh + LPS), 9 (Clod + LPS)