Extracellular vesicles from lung tissue drive bone marrow neutrophil recruitment in inflammation

Abstract

Extracellular vesicles (EVs) are single-membrane vesicles that play an essential role in long-range intercellular communications. EV investigation has been explored largely through cell-culture systems, but it remains unclear how physiological EVs exert homeostatic or pathological functions in vivo. Here, we report that lung EVs promote chemotaxis of neutrophils in bone marrow through delivery of double stranded DNA (dsDNA). We have identified and characterized EVs containing dsDNA collected from both human and murine lung tissues using newly developed approaches. Our analysis of EV proteomics together with single-cell RNA sequencing data reveals that type II alveolar epithelial cells are the main source of the lung EVs. Furthermore, we demonstrate that the lung EVs accumulate in bone marrow and enhance neutrophil recruitment under inflammation conditions. Moreover, lung EV-DNA stimulates neutrophils to release the chemokines CXCL1 and CXCL2 via DNA-TLR9 signalling. Our findings establish a molecular basis of lung EVs in enhancement of host immune response to bacterial infection and provide new insights into understanding of vesicle-mediated systematic communications.

Keywords: dsDNA; lung EVs; neutrophil chemotaxis.

FIGURE 1

High‐quality EVs are collected from human and murine lung tissues. (a) Representative TEM images of multiple extracellular vesicles and MVEs. Blue, green and red arrows indicated small EVs, large EVs and MVEs, respectively. Scale bars, 500 nm. (b) Schematic of EVs isolation from fresh human and murine lung tissues. (c) Percentage values of live and dead cells after digestion of human and murine lung tissues detected by flow cytometry (mean ± SEM; human samples n = 4, murine samples n = 5). (d) Immunoblotting analysis of protein extracts of the pellets from serial centrifugations of murine samples (P300 g, P3 kg, P10 kg, P110 indicated pellets of 300 × g, 3000 × g, 10,000 × g and 110,000 × g centrifugations, respectively). Immunoblotting was carried out using antibodies to GM130, Calnexin, TOM20, VDAC1, ALIX, CD9, TSG101, Flotillin‐1, β‐actin. (e) Immunoblotting analysis of protein extracts of the pellets from serial centrifugations of human samples. Immunoblotting was carried out using antibodies to GPRC5A, AGER, ALIX, CD9, CD81, β‐actin. (f) Representative TEM images of lung EVs. Scale bars, 200 nm. (g) Size distribution of lung EVs detected by NTA (mean ± SEM; n = 3 acquisitions/sample). (h) Schematic of EV purification by sucrose density centrifugation. Densities of ten fractions are shown on the far right. Shaded fractions represent enrichment of lung EVs. (i) Immunoblotting analysis of protein extracts of the ten fractions of human lung EVs. Immunoblotting was carried out using antibodies to GPRC5A, AGER, ALIX, CD9, CD81. (j) Immunoblotting analysis of protein extracts of the ten fractions of murine lung EVs. Immunoblotting was carried out using antibodies to ALIX, Flotillin‐1. (k) Numbers of particles in different fractions detected by NTA (mean ± SEM; n = 3 acquisitions/sample)

FIGURE 2

Protein profiling proves vesicle‐ and tissue‐specificity of lung EVs. (a) Pearson correlation analysis of the expression of proteins between lung cells and lung EVs from human samples. (b) A Venn diagram of the proteins overlapping between human and murine lung EVs. (c) Differential expressed proteins between lung cells and lung EVs were identified using R package “limma.” Adjusted P < 0.005 and log (fold change) ≥ 1 was considered as statistically significant. The top 30 higher expressed proteins in individual groups were illustrated by R package “pheatmap.” (d) Analysis of EV proteins in human, mouse and overlapping sets using GO terms related to cellular component. The graph shows the percentage of proteins identified by mass spectrometry that fall into the designated GO category relative to the total number of proteins in the category. Top 10 categories in overlapping sets are shown

FIGURE 3

Analysis of EV proteomics together with scRNA‐seq data assesses lung EV origin. (a) Schematic of the EV origin assessment by taking advantage of scRNA‐seq data and LC–MS/MS results. (b) Percentage of lung EV proteins identified to be “Cluster Markers,” “Non‐Cluster Markers” or “Not detected” in human and mouse LC–MS/MS results. (c,d) Percentage of the number of cells of each cluster in total cells (Cell%) and percentage of EVs from each cluster (t‐EV%) in human (c) and mouse (d) data, shown as classification of cell cluster. (e,f) Percentage of EVs from each single cell (s‐EV%) in human (e) and mouse (f) data, shown as classification of cell cluster (mean ± SEM; n = cell number of each cluster). (g,h) Log(t‐EV%/cell%) of each cell cluster in human (g) and mouse (h) data. (i) Quantification of particle and protein of EVs isolated from different cell lines including immune cell lines (human cell lines: Raji, K562, Jurkat) and non‐immune cell lines (human cell lines: HECV, H1299, HCC827, A549; mouse cell line: LLC). (j) GSEA was performed focusing on gene set of EVs release in four groups, including immune cells and non‐immune cells in both human and mouse lung scRNA‐seq data, ATII and B in mouse scRNA‐seq data, ATII and T in human scRNA‐seq data

FIGURE 4

Lung EVs contain dsDNA. (a) Representative images of negative stain TEM of low‐density fractions of lung EVs (F3, F4) and high‐density fractions of non‐vesicular components (F8, F9) after sucrose density centrifugation. NV, non‐vesicular components. Scale bar: 200 nm. (b) Quantification of DNA extracted from ten fractions by Qubit (mean ± SEM, n = 3). NV, non‐vesicular components. (c) Representative plots of lung EVs and non‐vesicular components staining with DiI from flow cytometry analysis. NC, negative control. NV, non‐vesicular components. (d) Representative plots of lung EVs staining with DAPI from flow cytometry analysis. (e) CNVs in chromosomes of both the EV‐DNA and cellular DNA derived from murine lung tissue. (f) A Venn diagram of all the CNVs overlapping between the cellular and EV‐DNA derived from murine lung tissues. (g) Quantification of DNA extracted from ten fractions treated with or without DNase I. Low‐density fractions (F1–F5) were collected as EVs and high‐density fractions (F6–F10) were collected as non‐vesicular components. NV, non‐vesicular components. (mean ± SEM, n = 3). (h) Flow cytometry analysis of DAPI positive lung EVs treated with DNase I or not (mean ± SEM, n = 3). Statistics by two‐tailed unpaired Student's t test (*, P < 0.05). (i) Representative images of DAPI positive lung EVs labelled with DiI. Scale bars, 500 nm

FIGURE 5

Lung EVs labelled with fluorescence are transferred into bone marrow. (a) Representative IVIS images of mice and their femurs 48 h post‐injection with DiR‐lung EVs or DiR‐liposomes as control. (b) Representative images of DiI positive cells in different organs from mice 48 h post‐injection with DiI‐liposomes or DiI‐lung EVs detected by flow cytometry. (c) Ratio of DiI positive cells in bone marrow detected by flow cytometry. Mice were injected with DiI labelled lung EVs in different routes 48 h before their bone marrow cells were harvested (mean ± SEM; n = 3). NC, negative control, without injection; i.n., intranasal instillation; i.p., intraperitoneal injection; i.v., tail intravenous injection. (d) Proportion of different cell types in DiI positive cells in bone marrow from mice injected with DiI labeled lung EVs. Neutrophils were gated with CD45⁺ CD11b⁺ Ly6G^high Ly6C^low and monocytes were gated with CD45⁺ CD11b⁺ Ly6G^low Ly6C^high. (e) Representative confocal image of DiI positive neutrophils isolated from mice injected with DiI labelled lung EVs. Scale bars, 20 μm. (f) System of ROSA26‐CAG‐LSL‐tdTOMATO mice infected by AAV‐GFP or AAV‐Cre through nasal instillation. (g) Ratio of cells with GFP or Tomato in bone marrows from mice treated with AAV‐GFP or AAV‐Cre through nasal instillation for 4 weeks (mean ± SEM; n  = 3). Statistics by two‐tailed unpaired Student's t test (****, P < 0.0001. ns, not significant, P > 0.05)

FIGURE 6

Lung EV‐DNA enhances chemotaxis of neutrophils. (a) Representative flow cytometry plots for neutrophils recruited in peritoneal cavity 3 h after intraperitoneal injection with thioglycolate. Mice were injected with gradient doses of lung EVs or equal volume of PBS 3 days before in a–c. (b) Absolute number of neutrophils recruited in peritoneal cavity 3 h after intraperitoneal injection with thioglycolate (mean ± SEM; n = 4). Statistics by two‐tailed unpaired Student's t test (*, P < 0.05. **, P < 0.01. ns, not significant, P > 0.05). (c) Proportion of neutrophils in CD45⁺ cells in peripheral blood 3 h after intraperitoneal injection with thioglycolate (mean ± SEM; n = 3–4). Statistics by two‐tailed unpaired Student's t test (ns, not significant, P > 0.05). (d) Representative flow cytometry plots for neutrophils recruited in peritoneal cavity 3 h after intraperitoneal injection with thioglycolate. Mice were injected with lung EVs, lung EVs pretreated with DNase I, non‐vesicular components with equal DNA content or equal volume of PBS 3 days before in d–f. NV, non‐vesicular components. (e) Absolute number of neutrophils recruited in peritoneal cavity 3 h after intraperitoneal injection with thioglycolate. (mean ± SEM; n = 3–4). Statistics by two‐tailed unpaired Student's t test (*, P < 0.05. **, P < 0.01. ns, not significant, P > 0.05). (f) Proportion of neutrophils in CD45⁺ cells in peripheral blood 3 h after intraperitoneal injection with thioglycolate (mean ± SEM; n = 3–4). Statistics by two‐tailed unpaired Student's t test (ns, not significant, P > 0.05). (g) Quantification of protein, RNA and particle concentration of lung EVs treated with or without DNase I (mean ± SEM, n = 3). Statistics by two‐tailed unpaired Student's t test (ns, not significant, P > 0.05). (h) Size distribution of lung EVs treated with or without DNase I detected by NTA (mean ± SEM; n = 3 acquisitions/sample). (i) Immunoblotting analysis of protein extracts of lung EVs treated with or without DNase I. Immunoblotting was carried out using antibodies to ALIX

FIGURE 7

Lung EV‐DNA triggers the release of neutrophil mobilizing chemokines through TLR9 signaling. (a) mRNA sequencing analysis of chemokine and cytokine levels in neutrophils treated with lung EVs or PBS as control for 18 h. (b) GSEA was performed focusing on neutrophil‐chemotaxis genes between neutrophils treated with lung EVs and neutrophils treated with PBS as control. (c) Representative cytokine array panels of supernatants taken from 18 h culture of neutrophils incubated with lung EVs, lung EVs pretreated with DNase I, or PBS as control. (d) Real‐time PCR analysis of Cxcl1 and Cxcl2 levels in murine neutrophils treated with lung EVs, lung EVs pretreated with DNase I or PBS as control for 18 h (mean ± SEM; n = 4). Statistics by two‐tailed unpaired Student's t test (*, P < 0.05. **, P < 0.01. ***, P < 0.001. ****, P < 0.0001). (e) Real‐time PCR analysis of Cxcl1 and Cxcl2 levels in neutrophils isolated from mice injected with lung EVs, lung EVs pretreated with DNase I or PBS as control (mean ± SEM; n = 4). Statistics by two‐tailed unpaired Student's t test (**, P < 0.01. ***, P < 0.001. ns, not significant, P > 0.05). (f) Lysates of HEK293T transfected with Flag‐tagged TLR9 were incubated in the presence or the absence of biotinylated lung EV‐DNA. The bound proteins were immunoprecipitated with streptavidin microbeads and blotted by an anti‐Flag antibody. (g) Immunoblotting analysis for TLR9‐Flag immunoprecipitated with biotinylated lung EV‐DNA in the absence or in the presence of 1 μg unbiotinylated lung EV‐DNA. (h–i) Real‐time PCR analysis of Cxcl1 and Cxcl2 levels in bone marrow neutrophils pretreated with TLR9 inhibitory oligodeoxynucleotide ODN2088 (h) and inhibitor E6446 (i) for 5 h prior to incubation with or without lung EVs (mean ± SEM; n = 4). Statistics by two‐way repeated‐measures ANOVA followed by the Bonferroni post test (**, P < 0.01. ***, P < 0.001. ****, P < 0.0001. ns, not significant, P > 0.05)

FIGURE 8

Lung EVs enhance the recruitment of neutrophils during Salmonella infection. (a) Quantification of dsDNA in lung EVs isolated from mice 12 h after injection with LPS (n = 3) or infection by S. typhimurium (n = 4). Statistics by two‐tailed unpaired Student's t test (**, P < 0.01, ***, P < 0.001). (b) Flow cytometry analysis of DNA positive lung EVs (DAPI⁺) isolated from mice injected with PBS and LPS (mean ± SEM, n = 3). Statistics by two‐tailed unpaired Student's t test (*, P < 0.05). (c) Representative flow cytometry plots for neutrophils recruited in peritoneal cavity 3 h post S. typhimurium infection. Mice were injected with lung EVs, lung EVs pretreated with DNase I, non‐vesicular components with equal DNA content or equal volume of PBS 3 days before. NV, non‐vesicular components. (d) Absolute number of neutrophils recruited in peritoneal cavity 3 h post S. typhimurium infection (mean ± SEM; n = 5). Statistics by two‐tailed unpaired Student's t test (**, P < 0.01. ns, not significant, P > 0.05). (e) Representative flow cytometry plots for neutrophils in peripheral blood 3 h post S. typhimurium infection. (f) Proportion of neutrophils in CD45⁺ cells in peripheral blood 3 h post S. typhimurium infection (mean ± SEM; n = 5). Statistics by two‐tailed unpaired Student's t test (*, P < 0.05. ***, P < 0.001. ns, not significant, P > 0.05). (g) Mouse survival curves post S. typhimurium infection. PBS treated mice and lung EV‐treated mice were inoculated intraperitoneally with 1 × 10⁷ CFU/mouse bacteria in g, h (n = 13). Statistics by Mantel‐Cox test (****, P < 0.0001). (h) Absolute quantification of S. typhimurium DNA in peripheral blood 12 h post infection were detected by Real‐time PCR (mean ± SEM; n = 6). Statistics by two‐tailed unpaired Student's t test (*, P < 0.05). (i) Mouse survival curves post S. typhimurium infection. DMSO treated mice and GW4869 treated mice were inoculated intraperitoneally with 1 × 10⁷ CFU/mouse bacteria in i, j (n = 15). Statistics by Mantel‐Cox test (***, P < 0.001). (j) Absolute quantification of S. typhimurium DNA in peripheral blood 12 h post infection were detected by Real‐time PCR (mean ± SEM; n = 6). Statistics by two‐tailed unpaired Student's t test (*, P < 0.05). (k) Schema depicting regulations of lung EVs on bone marrow neutrophils