Mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury

Abstract

Bone marrow-derived stromal cells (BMSCs) protect against acute lung injury (ALI). To determine the role of BMSC mitochondria in this protection, we airway-instilled mice first with lipopolysaccharide (LPS) and then with either mouse BMSCs (mBMSCs) or human BMSCs (hBMSCs). Live optical studies revealed that the mBMSCs formed connexin 43 (Cx43)-containing gap junctional channels (GJCs) with the alveolar epithelia in these mice, releasing mitochondria-containing microvesicles that the epithelia engulfed. The presence of BMSC-derived mitochondria in the epithelia was evident optically, as well as by the presence of human mitochondrial DNA in mouse lungs instilled with hBMSCs. The mitochondrial transfer resulted in increased alveolar ATP concentrations. LPS-induced ALI, as indicated by alveolar leukocytosis and protein leak, inhibition of surfactant secretion and high mortality, was markedly abrogated by the instillation of wild-type mBMSCs but not of mutant, GJC-incompetent mBMSCs or mBMSCs with dysfunctional mitochondria. This is the first evidence, to our knowledge, that BMSCs protect against ALI by restituting alveolar bioenergetics through Cx43-dependent alveolar attachment and mitochondrial transfer.

Figure 1. mBMSCs in live alveoli

Alv, alveolus. Green, calcein green. Red, mBMSCs with mitochondrial DsRed (a, b), Hoechst 33342 (c), fluorescence-tagged streptavidin (e), or calcein red (f). Mut, mBMSCs expressing mutant Cx43. (a) Images of mBMSCs (arrowheads) and alveolar septa (arrow). Line, 30 µm. Cartoons illustrate mBMSC migration to the imaged plane (blue rectangle). Pl, pleura. (b) Alveolar mBMSCs imaged at a fixed focal plane. Data are for 100 alveoli from 6 lungs for each group. * p < 0.05 versus LPS. (c) Two-photon images (optical slice thickness 0.6 µm) at focal planes 4 (top panel) and 1 µm (bottom panel) deep to the visceral pleura. Replicated in 6 lungs for each group. Line, 30 µm. (d) Quantification of lung mBMSCs by flow cytometry. n = 4 lungs for each bar; * p < 0.05 versus 0 h, †p < 0.05 versus bar on immediate left. (e) mBMSCs in thoracic lymph nodes. n = 6 lymph nodes for each bar; * p < 0.05 versus PBS. (f) Images show alveoli in an LPS lung (first image). Line, 20 µm. Magnification of the rectangle (second image) shows an mBMSC (arrow) in an alveolus. Line, 10 µm. Higher magnification (third image) shows green fluorescence in the mBMSC (arrow). Green channel renditions (fourth to sixth image) show responses to photobleaching in the area within the dotted line. Line, 3 µm. (g) Group data show FRAP responses in mBMSCs. n = 15 cells each bar, * p < 0.05 versus PBS. α-GA, α-glycyrrhetinic acid; Gap26, Cx43 inhibiting peptide.

Figure 2. Cx43 expression in mBMSCs and alveoli

(a) Images show Cx43 immunofluorescence (red) in live alveoli (Alv). Group data are for 100 alveoli from 3 lungs in each group. * p < 0.05 versus PBS. Line, 20 µm. (b) Image shows a DsRed-expressing mBMSC (red) and Cx43 immunofluorescence (green) in an alveolus. Tracings are line-scan analyses of the alveolar wall (dotted line). The data were replicated four times. Line, 10 µm. (c) Gels show mRNA (left) and protein (middle) expressions for Cx43, and densitometry for the immunoblots (right). AT2, alveolar type 2 cells. mRNA data replicated three times. n = 4 gels each bar, * p < 0.05 versus PBS. (d) Gels show PCR-amplified DNA sequences for different connexins in primary isolates of alveolar mixed cells (AC), AT2 cells and mBMSCs. Replicated four times.

Figure 3. Responses of alveolus-attached mBMSCs 4–8 h after LPS

(a) Image of an LPS lung (left) shows a mBMSC (green) in an alveolus (red). Alveolar margins are marked (dotted line). Line, 10 µm. High magnification of the selected region (rectangle) shows the mBMSC in green rendition in the second panel. CG, calcein green. M, microvesicles. N, nanotube. Line, 5 µm. Right panels show a microvesicle (arrows) at different time points in green (calcein green) and red (DsRed) renditions. Line, 3 µm. (b) Group data for LPS lungs. n = 40 mBMSCs from 5 lungs in each bar. mutBMSC, mBMSCs expressing mutant Cx43. (c) Flow cytometry of supernatants derived from homogenates of lungs isolated at the indicated time points after mBMSC instillation. n = 4 lungs for each bar, * p < 0.05 versus 0 h. (d) Images of an mBMSC (arrows) stained with Ca²⁺ dye, fluo-4, in a live alveolus (red). Line, 10 µm. (e, f) Group data are time course of fluo-4 responses in mBMSCs (e) and the effects of the indicated treatments (f). n = 9 mBMSCs from 3 lungs for each bar, * p < 0.05 versus PBS. WT, wild-type; Gap26, Cx43 inhibiting peptide; BAPTA-AM, Ca²⁺ chelator. (g) Images show a mBMSC (arrow) lying adjacent to the alveolar epithelium (green). The cartoon represents the imaging data at two time points. Mitochondria are brown. Line, 5 µm. (h) Group data for epithelial engulfment. n = 7 imaged fields from 3 lungs in each bar, * p < 0.05 versus left bar.

Figure 4. Mitochondrial transfer from BMSCs 24 h after LPS

(a) Image of an LPS lung shows AT2 cells (green) and instilled mBMSCs (arrowheads) expressing DsRed (red). Alv, alveoli. Line, 10 µm. (b) Green, red and merged renditions of a single AT2 cell show fluorescence of surfactant protein B (SPB) (upper panel) and DsRed (middle panel). Images were obtained at the indicated levels. The cartoon depicts the optical planes in the AT2 cell. The integrated image projection along the depth (Y-Z) axis, analyzed at the cross-point of the dotted lines in merge image, shows the intracellular location of DsRed fluorescence (bracket). Line, 2 µm. Replicated in 5 lungs (20 cells). (c) Gels show amplified DNA sequences from isolated AT2 cells. hCO1, human cytochrome oxidase 1; hCO2, human cytochrome oxidase 2; hBMSC, human BMSCs; mBMSC, mouse BMSCs; HEK, human embryonic kidney cells; mGapdh: mouse glyceraldehydes 3-phosphate dehydrogenase. Repeated 4 times. (d) Group data are flow cytometry determinations. WT, wild-type mBMSCs; mutBMSC, mBMSCs expressing mutant Cx43. n = 4 lungs for each bar, * p < 0.05 versus 0 h.

Figure 5. Effect of mBMSCs on alveolar bioenergetics

(a) Group data are colorimetric determinations in lung homogenates. WT, wild-type mBMSCs; RISP, Rieske iron-sulfur protein; siRISP, mBMSCs treated with siRNA against RISP; scRISP, mBMSCs treated with scrambled siRNA; mut, mBMSCs expressing a mutant Cx43; 3T3, mouse 3T3 fibrocytes. n = 4 lungs for each bar, * p < 0.05 versus PBS. (b) Images show immunofluorescence (upper panel) and GFP fluorescence (lower panel) of the ATP probe, Perceval. A633, Alexa fluor 633. Line, 20 µm. (c) Group data for ATP fluorescence. n = 3 imaged field for each bar, * p < 0.05 versus PBS. (d) Image (left) obtained 24 h after LPS, shows alveolar fluorescence of Perceval (green) and mBMSC mitochondria (red, arrows). Line, 20 µm. Magnified images of the selected region (rectangle in left) show an alveolus (Alv) in green, red and merge renditions. DsRed fluorescence is indicated (arrowheads). Line, 10 µm. (e) Yellow pseudocolor shows changes in alveolar GFP fluorescence (curved arrows). We superimposed the red channel rendition of DsRed mitochondria on the alveolar image. The tracings are line-scan analyses. Line, 10 µm. (f) Group data are changes in Perceval fluorescence. n = 5 areas in 3 lungs for each group, * p < 0.05 versus LPS alone.

Figure 6. Effect of mBMSCs on injury outcomes

(a-b) Inflation (arrow)-induced surfactant secretion for single experiments (a) and the group (b). WT, wild type mBMSCs; siRISP, mBMSCs containing siRNA against RISP; scRISP, mBMSCs containing scrambled siRNA; mut, mBMSCs expressing mutant Cx43; 3T3, mouse 3T3 fibrocytes. n = 12 cells in 4 lungs for each bar, * p < 0.05 versus LPS alone. (c) Group data for determinations in brochoalveolar lavage (BAL). n = 4 lungs for each bar, * p < 0.05 versus PBS. (d) Kaplan-Meier plots for mouse survival after instillation of LPS (10 mg kg⁻¹) followed 4 h later by mBMSCs. Cx43 KD, mBMSCs treated with siRNA against Cx43. n = 15 mice for each bar, * p < 0.05 versus LPS.