Innate Immune Training Initiates Efferocytosis to Protect against Lung Injury

Abstract

Innate immune training involves myelopoiesis, dynamic gene modulation, and functional reprogramming of myeloid cells in response to secondary heterologous challenges. The present study evaluates whether systemic innate immune training can protect tissues from local injury. Systemic pretreatment of mice with β-glucan, a trained immunity agonist, reduces the mortality rate of mice with bleomycin-induced lung injury and fibrosis, as well as decreasing collagen deposition in the lungs. β-Glucan pretreatment induces neutrophil accumulation in the lungs and enhances efferocytosis. Training of mice with β-glucan results in histone modification in both alveolar macrophages (AMs) and neighboring lung epithelial cells. Training also increases the production of RvD1 and soluble mediators by AMs and efferocytes. Efferocytosis increases trained immunity in AMs by stimulating RvD1 release, thus inducing SIRT1 expression in neighboring lung epithelial cells. Elevated epithelial SIRT1 expression is associated with decreased epithelial cell apoptosis after lung injury, attenuating tissue damage. Further, neutrophil depletion dampens the effects of β-glucan on macrophage accumulation, epigenetic modification in lung macrophages, epithelial SIRT1 expression, and injury-mediated fibrosis in the lung. These findings provide mechanistic insights into innate immune training and clues to the potential ability of centrally trained immunity to protect peripheral organs against injury-mediated disorders.

Keywords: alveolar macrophages; efferocytosis; inflammation; lung injury; trained immunity.

Figure 1

β‐Glucan‐induced trained immunity protects against lung injury‐induced fibrosis. a) Schematic diagram of in vivo training preceded by lung injury. Mice were intraperitoneally administered β‐glucan, followed 7 d later by intratracheal instillation of bleomycin, and monitored for 21 d. b) Survival of PBS‐ or β‐glucan‐trained mice with bleomycin‐induced pulmonary fibrosis. PBS‐PBS n = 12 mice, β‐Glu‐PBS; PBS‐Bleo; β‐Glu‐Bleo n = 18 mice per group. ***p < 0.0001 by log‐rank test. c) Visualization of collagen deposits in lung sections by sirius red staining. Collagen (red) and muscle fiber (yellow). Scale bar = 100 µm. d) Hydroxyproline content of lung tissues 21 d after bleomycin instillation. n = 6–12 mice per group. **p < 0.01; ***p < 0.001 by one‐way ANOVA.

Figure 2

Systemic β‐glucan pretreatment led to accumulation of myeloid cells in the lungs in the absence of tissue injury, increasing apoptotic neutrophils. a,b) Infiltrating and accumulating immune cells in the BALF of untrained and trained mice. Numbers of a) neutrophils (CD11b⁺Gr1⁺) and b) macrophages (F4/80⁺CD45⁺) in BALF 10 d after intraperitoneal injection of PBS (untrained) or β‐glucan (trained). n = 6 mice per group. c) Numbers of monocytes (F4/80⁻CD11c^highSiglecF⁻), monocyte‐derived AMs (Mo‐AM; F4/80⁺CD11c^highSiglecF⁻), and tissue‐resident AMs (TR‐AM; F4/80⁺CD11c^lowSiglecF⁺) in the BALF of untrained and trained mice 10 d after intraperitoneal injection of PBS or β‐glucan. n = 6 mice per group. d) Fold change in numbers of apoptotic neutrophils (AnnexinV⁺Gr1⁺) in the BALF of trained mice 0, 3, 7, 10, 14, 17, 21, and 28 d after intraperitoneal injection of β‐glucan. n = 4 mice per group. e) Effect of neutrophil depletion through treatment of mice with anti‐Ly6G mAb on bleomycin‐induced pulmonary fibrosis. Representative images showing collagen type I at 14 dpbi in the lung sections of untrained (PBS) and trained (β‐glucan) mice (left panels). Scale bar = 50 µm. Quantification of the mean fluorescence intensity of collagen (right panels). n = 3–4 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001; by two‐sided paired a,b) Student's t‐tests or c–e) one‐way ANOVA.

Figure 3

Immune training with β‐glucan accelerates efferocytosis of AMs. a) Phagocytosis of phosphatidylserine‐coated beads by untrained or in vitro‐trained bone marrow‐derived macrophages incubated in the absence (M0‐type) or presence of LPS/IFN‐γ (M1‐type) or IL‐4 (M2‐type). The numbers of cells phagocytosing fluorescent beads were measured by flow cytometry depending on relative fluorescence intensity (low, medium, or high, as indicated in extended data Figure 4). n = 5 mice per group. b) Representative confocal microscopy images showing the phagocytic capability of M2 type macrophages cultured as in (a), with phagocytosis quantified. Scale bar = 100 µm. n = 3 independent cultures per group. c) Relative expression of the Il4ra and Il13ra genes in AMs (sorted as SiglecF⁺Gr1⁻) 7 d after intraperitoneal injection of β‐glucan. n = 5 mice per group. d,e) Ex vivo efferocytosis of d) apoptotic neutrophils and e) apoptotic epithelial cells by AMs (SiglecF⁺Gr1⁻) isolated from untrained (PBS) and β‐glucan‐trained mice 7 d after β‐glucan pretreatment. n = 5 mice per group. f) Relative count of apoptotic cells (left) and total cells (right) in the BALF in the trained mice with lung injury after treatment with a specific efferocytosis blocker, BMS794833. n = 6 mice per group. g) Effect of blocking efferocytosis through treatment of mice with BMS794833 on collagen type I production in the lungs of trained mice with lung injury. Representative images of collagen at 14 dpbi in the lung sections of untrained (PBS) and trained (β‐glucan) mice. Scale bar = 50 µm. h) Quantification of the mean fluorescence intensity of collagen (g). n = 3–4 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001; by a–f) t‐tests or h) one‐way ANOVA.

Figure 4

Immune training with β‐glucan increases the transcription of genes involved with phagocytosis and induces epigenetic modification in lung macrophages. a) Relative expression of genes associated with phagocytosis (Pparg, Lxra, Abca1, and Stab1) in AMs (SiglecF⁺Gr1⁻) isolated from untrained and trained mice 7 d after β‐glucan treatment. n = 5 mice per group. b) Relative expression of Alox15 in whole lungs isolated from untrained (n = 5) and β‐glucan (n = 6) mice 14 d after β‐glucan treatment. c,d) Global modification of the histones c) H3K27ac and d) H3K4me3 in lung macrophages (F4/80⁺ cells‐sorted) from untrained and trained mice 7 d after PBS or β‐glucan treatment. n = 5–6 mice per group. e) ChIP‐qPCR analysis of H3K4me3 enrichment at the Alox15 and IL‐1b locus in lung macrophages (F4/80⁺ cells‐sorted) isolated from untrained and trained mice 7 d after PBS or β‐glucan treatment. One dot represents a pool of lung macrophages isolated from six untrained or trained mice. f–h) Effect of neutrophil depletion through treatment of mice with anti‐Ly6G mAb on the number of macrophages and epigenetic modification in lung macrophages of mice with bleomycin‐induced pulmonary fibrosis. Representative images of H3K4me3 in F4/80⁺ cells at 14 dpbi in the lung sections of untrained (PBS) and trained (β‐glucan) mice (f). Scale bar = 20 µm. Percentages of g) macrophages (F4/80⁺ cells) and h) H3K4me3⁺F4/80⁺ cells in the lung sections (right panel). n = 3‐4 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001 by a–e) t‐tests or g,h) one‐way ANOVA.

Figure 5

β‐Glucan‐enhanced efferocytosis promotes production of resolvin D1 and reduces tissue damage following lung injury. a–c) Levels of RvD1 in the conditioned media of untrained and a) in vivo‐trained AMs and b,c) efferocytes. Levels of RvD1 in the conditioned media of b) AMs engulfing apoptotic neutrophils and c) AMs engulfing apoptotic lung epithelial cells. n = 4–6 per group. d) Effect of blocked efferocytosis on the level of RvD1 in the culture of AMs engulfing apoptotic neutrophils. n = 6 per group. e) Levels of RvD1 in the BALF of untrained (PBS) and trained (β‐glucan) mice 10 d after PBS (sham) or bleomycin instillation. n = 5 mice per group. f) Representative images showing apoptotic cells (TUNEL) and epithelial cells (E‐cadherin) in the lung sections of untrained (PBS) and trained (β‐glucan) mice 7 d after PBS (sham) or bleomycin instillation. Scale bar = 50 µm. g) Quantification of fluorescence intensity as shown in (e). n = 5 mice per group. h) Number of cells in the bronchoalveolar lavage fluid (BALF) of untrained and trained mice following PBS or bleomycin instillation. n = 6 mice per group, except for n = 5 mice for PBS‐Bleo on day 21. i) Levels of the lung injury marker, glycocalyx, in the lungs of untrained and trained mice 7 and 14 d after bleomycin instillation. n = 6 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001 by a–c) t‐tests, d,e,g,i) one‐way ANOVA, and h) two‐way ANOVA.

Figure 6

β‐Glucan‐trained immunity induces epithelial SIRT1 expression in the lungs, which requires neutrophil accumulation and macrophage efferocytosis. a) Epithelial SIRT1 protein in the lungs of untrained and trained mice before and after injury. The lungs were isolated 7 d after PBS or bleomycin instillation. Scale bar = 100 µm. b) Quantification of SIRT1⁺ E‐cadherin⁺‐doubly positive cells as in (a). n = 6 mice per group. c) Effect of neutrophil depletion through treatment of mice with anti‐Ly6G mAb on epithelial SIRT1 protein levels in the lungs of trained mice with lung injury. Representative images of epithelial SIRT1 at 14 dpbi in the lung sections of untrained (PBS) and trained (β‐glucan) mice (left panels). Scale bar = 100 µm. d) Quantification of SIRT1⁺ E‐cadherin⁺‐doubly positive cells as in (c). n = 5 mice per group. e) Effect of blocked efferocytosis through treatment of mice with BMS794833 on the expression of epithelial SIRT1 protein in the lungs of trained mice with lung injury. Representative images of epithelial SIRT1 at 14 dpbi in the lung sections of untrained (PBS) and trained (β‐glucan) mice. Scale bar = 50 µm. f) Quantification of SIRT1⁺ E‐cadherin⁺‐doubly positive cells as in (e). n = 3 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001; by one‐way ANOVA.

Figure 7

β‐Glucan‐trained macrophages induce epithelial SIRT1 expression, reducing epithelial cell apoptosis upon application of genotoxic and oxidative cellular stress. a) Relative Sirt1 expression in lung epithelial cells cocultured in transwell with AMs isolated from untrained and in vivo‐trained mice. Epithelial cells were isolated from the lungs of normal untrained mice. n = 6 per group. b Relative Sirt1 expression in lung epithelial cells incubated with the conditioned media of efferocytes. Efferocytes were obtained from coculture of AMs from untrained or trained mice with apoptotic neutrophils. Epithelial cells were isolated from the lungs of normal untrained mice. n = 6 per group. c) Effect of knockdown of the RvD1 receptor, Fpr2, on Sirt1 expression in lung epithelial cells cocultured in transwell plates with AMs isolated from untrained or trained mice. n = 6 per group. d,e) Global histone modification (H3K27ac and H3K9ac) in lung epithelial cells (EpCAM⁺ cells) from untrained and trained mice 7 d after β‐glucan pretreatment. n = 6 per group. f–h) Apoptosis of lung epithelial cells following bleomycin treatment in vitro. Prior to bleomycin treatment, epithelial cells were incubated with conditioned media of untrained or f) in vivo‐trained AMs or g) efferocytes. Epithelial cells were isolated from the lungs of normal untrained mice. Efferocytes were obtained from coculture of AMs from untrained or trained mice with apoptotic neutrophils. Negative control, none; Positive control, bleomycin but no conditioned media. n = 5 per group. h Apoptosis of SIRT1‐knockdown lung epithelial cells following bleomycin treatment in vitro. Epithelial cells isolated from the lungs of normal untrained mice were incubated with conditioned media of in vivo‐trained AMs. n = 5 per group. *p < 0.05; **p < 0.01; ***p < 0.001; by a,b,d‐f) t‐tests or c, g,h) one‐way ANOVA

Figure 8

Systemic β‐glucan‐trained immunity promotes production of proresolving and anti‐inflammatory lipid mediators in the lungs. a,b) Levels of endogenous lipid mediators in lung tissues collected a) 7 and b) 14 d after PBS or bleomycin instillation into untrained and trained mice. Lipidomics were performed with the lungs of six mice per group, but some missing data are not shown because the corresponding lipid mediators were not detected. *p < 0.05; **p < 0.01; ***p < 0.001 by t‐tests.

Figure 9

Schematic diagram illustrating the mechanism of systemic β‐glucan‐trained immunity to attenuate lung injury. Created with BioRender.com.