Mesenchymal stromal cell extracellular vesicles reduce Pseudomonas biofilm formation, and let-7b-5p loading confers additional anti-inflammatory effects

Abstract

Cystic fibrosis (CF) is a multiorgan disease caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, leading to chronic pulmonary infections and hyperinflammation. Among pathogens colonizing the CF lung, Pseudomonas aeruginosa is predominant, infecting over 50% of adults with CF, and becoming antibiotic-resistant over time. Current therapies for CF, while providing tremendous benefits, fail to eliminate persistent bacterial infections, chronic inflammation, and irreversible lung damage, necessitating novel therapeutic strategies. Our group engineered mesenchymal stromal cell-derived extracellular vesicles (MSC EVs) to carry the microRNA let-7b-5p as a dual anti-infective and anti-inflammatory treatment. MSC EVs are low-immunogenicity platforms with innate antimicrobial and immunomodulatory properties, whereas let-7b-5p reduces inflammation. This study demonstrates that MSC EVs effectively blocked the formation of antibiotic-resistant P. aeruginosa biofilms on primary human bronchial epithelial cells (pHBECs), and let-7b-5p loading into MSC EVs conferred additional anti-inflammatory effects by reducing P. aeruginosa-induced IL-8 secretion by pHBECs. This approach holds promise for improving outcomes for people with CF, and future work will focus on optimizing delivery strategies and expanding the clinical applicability of MSC EVs to target other CF-associated pathogens.NEW & NOTEWORTHY This is the first study demonstrating that mesenchymal stromal cell extracellular vesicles (MSC EVs) block antibiotic-resistant P. aeruginosa biofilm formation and that let-7b-5p-loaded MSC EVs reduce inflammation in CF primary human bronchial epithelial cells.

Keywords: Pseudomonas aeruginosa biofilms; cystic fibrosis; human bronchial epithelial cells; inflammation; mesenchymal stromal cell extracellular vesicles.

Figure 1:

CF-pHBECs were exposed to MSC EVs for 6 hours. Apical and basolateral supernatants were collected and analyzed by ELISA to measure (A) IL-6 and (B) IL-8 secretion. The experiments included CF-pHBEC from two donors (ΔF508/ΔF508), each with 3 replicates (yielding 6 data points per treatment group). The male donor is represented by blue squares, while the female donor is represented by orange circles. Linear mixed-effects models with the donor as a random effect were used to test the statistical significance between the control and EV groups. **p = 0.0020 (Figure A) **p = 0.0086 (Figure B) and; ***p = 0.0004; mean ± SD depicted.

Figure 1:

CF-pHBECs were exposed to MSC EVs for 6 hours. Apical and basolateral supernatants were collected and analyzed by ELISA to measure (A) IL-6 and (B) IL-8 secretion. The experiments included CF-pHBEC from two donors (ΔF508/ΔF508), each with 3 replicates (yielding 6 data points per treatment group). The male donor is represented by blue squares, while the female donor is represented by orange circles. Linear mixed-effects models with the donor as a random effect were used to test the statistical significance between the control and EV groups. **p = 0.0020 (Figure A) **p = 0.0086 (Figure B) and; ***p = 0.0004; mean ± SD depicted.

Figure 2.

(A-D) Transmission electron microscopy (TEM) of MSC-derived extracellular vesicles (EVs) and process control (PC) samples. (A) TEM image of MSC EVs at low magnification (x6000). (B) TEM image of MSC EVs at high magnification (x50000). (C) TEM image of PC at low magnification (x6000). (D) TEM image of PC at high magnification (x50000). Regions indicated by boxes in panels A and C are shown at higher magnification in panels B and D, respectively. Scale bars: 500 nm (A, C) and 100 nm (B, D). (E) Protein marker analysis of MSC EVs. Capillary western blot was performed for CD81 detection. ELISA was conducted to assess ALIX and albumin levels.

Figure 2.

(A-D) Transmission electron microscopy (TEM) of MSC-derived extracellular vesicles (EVs) and process control (PC) samples. (A) TEM image of MSC EVs at low magnification (x6000). (B) TEM image of MSC EVs at high magnification (x50000). (C) TEM image of PC at low magnification (x6000). (D) TEM image of PC at high magnification (x50000). Regions indicated by boxes in panels A and C are shown at higher magnification in panels B and D, respectively. Scale bars: 500 nm (A, C) and 100 nm (B, D). (E) Protein marker analysis of MSC EVs. Capillary western blot was performed for CD81 detection. ELISA was conducted to assess ALIX and albumin levels.

Figure 3:

CF-pHBECs were exposed for 6 hours to P. aeruginosa (Pa) alone or in combination with process control (PC), negative control (NC) MSC EVs, or let-7b MSC EVs. Cytotoxicity was compared to untreated (control) CF-pHBECs. Each colored line represents one CF donor across all treatment conditions. Linear mixed-effects models with donor as random effect were used to test for statistical significance between untreated and the treatment groups. n = 3 CF donors (ΔF508/ΔF508). Male donor is represented by squares, while female donors are represented by circles. PC (process control) = unconditioned media passed though EV preparation process, NC = negative control miRNA.

Figure 4:

CF-pHBEC from three donors were exposed to P. aeruginosa (Pa) alone or in combination with process control (PC), negative control (NC) MSC EVs, or let-7b MSC EVs for 6 hours, in the absence or presence of ETI pre-exposure.Total bacterial burden was assessed by CFU enumeration of apical and basolateral supernatants, as well as the cell lysates. Each colored line represents one CF donor across all treatment conditions. Male donor is represented by squares, while female donors are represented by circles. Linear mixed-effects models with donor as random effect were used to test for statistical significance in the bacterial burden assay; n = 3 CF-pHBEC donors (ΔF508ΔF508); ***p = 1 x 10⁻⁶; comparing P. aeruginosa (Pa) inoculum to all four treated CF-pHBEC groups ± ETI; PC (process control), NC = negative control.

Figure 5:

Representative images of maximum intensity projections from z-stacks of biotic biofilms of P. aeruginosa pre-treated with PC at (A) 0h and at B) 5h after the 1 hour of exposure to enable the bacteria to attach to pHBEC. Panels C and D are images of P. aeruginosa pre-treated with MSC EVs at 0h (C) and at 5h (D) on pHBEC monolayers. (E) Summary of data of biofilm volumes of P. aeruginosa exposed to PC, MSC EVs or let-7b-5p MSC EVs on pHBEC. Volume renderings of 3D Z-stacks from 1-5 random areas of each monolayer were generated using Keyence software following the manufacturer’s instructions. Each data point represents the P. aeruginosa biofilm volume on a monolayer from an individual pHBEC donor, with colored lines connecting measurements from the same donor at 0 and 5h. Each colored line represents a unique pHBEC donor, and lines of the same color are technical replicates (different imaged areas) of the same donor. (F) Biofilm CFU/mL were assessed by scraping the pHBECs, and plating the bacteria on LB agar at the end of the experiment. The lines connect CFUs formed on monolayers from each donor across the different pre-treatment conditions. Colored lines and symbols represent replicates of the same donor. The male donor is represented by squares, while female donors are represented by circles. Linear mixed-effects models with donor as random effect were used to test statistical significance, and for the biofilm volumes, interaction analyses between time and treatment (to determine if the treatment effect was modified by time) were also included in the model; n = 3 pHBEC donors; ***p = 0.000163 and 0.00762 (Figure E), **p-value = 0.023 and *** p-value = 0.005 (Figure F); PC (process control) = unconditioned media passed through the EV preparation process.

Figure 5:

Representative images of maximum intensity projections from z-stacks of biotic biofilms of P. aeruginosa pre-treated with PC at (A) 0h and at B) 5h after the 1 hour of exposure to enable the bacteria to attach to pHBEC. Panels C and D are images of P. aeruginosa pre-treated with MSC EVs at 0h (C) and at 5h (D) on pHBEC monolayers. (E) Summary of data of biofilm volumes of P. aeruginosa exposed to PC, MSC EVs or let-7b-5p MSC EVs on pHBEC. Volume renderings of 3D Z-stacks from 1-5 random areas of each monolayer were generated using Keyence software following the manufacturer’s instructions. Each data point represents the P. aeruginosa biofilm volume on a monolayer from an individual pHBEC donor, with colored lines connecting measurements from the same donor at 0 and 5h. Each colored line represents a unique pHBEC donor, and lines of the same color are technical replicates (different imaged areas) of the same donor. (F) Biofilm CFU/mL were assessed by scraping the pHBECs, and plating the bacteria on LB agar at the end of the experiment. The lines connect CFUs formed on monolayers from each donor across the different pre-treatment conditions. Colored lines and symbols represent replicates of the same donor. The male donor is represented by squares, while female donors are represented by circles. Linear mixed-effects models with donor as random effect were used to test statistical significance, and for the biofilm volumes, interaction analyses between time and treatment (to determine if the treatment effect was modified by time) were also included in the model; n = 3 pHBEC donors; ***p = 0.000163 and 0.00762 (Figure E), **p-value = 0.023 and *** p-value = 0.005 (Figure F); PC (process control) = unconditioned media passed through the EV preparation process.

Figure 5:

Representative images of maximum intensity projections from z-stacks of biotic biofilms of P. aeruginosa pre-treated with PC at (A) 0h and at B) 5h after the 1 hour of exposure to enable the bacteria to attach to pHBEC. Panels C and D are images of P. aeruginosa pre-treated with MSC EVs at 0h (C) and at 5h (D) on pHBEC monolayers. (E) Summary of data of biofilm volumes of P. aeruginosa exposed to PC, MSC EVs or let-7b-5p MSC EVs on pHBEC. Volume renderings of 3D Z-stacks from 1-5 random areas of each monolayer were generated using Keyence software following the manufacturer’s instructions. Each data point represents the P. aeruginosa biofilm volume on a monolayer from an individual pHBEC donor, with colored lines connecting measurements from the same donor at 0 and 5h. Each colored line represents a unique pHBEC donor, and lines of the same color are technical replicates (different imaged areas) of the same donor. (F) Biofilm CFU/mL were assessed by scraping the pHBECs, and plating the bacteria on LB agar at the end of the experiment. The lines connect CFUs formed on monolayers from each donor across the different pre-treatment conditions. Colored lines and symbols represent replicates of the same donor. The male donor is represented by squares, while female donors are represented by circles. Linear mixed-effects models with donor as random effect were used to test statistical significance, and for the biofilm volumes, interaction analyses between time and treatment (to determine if the treatment effect was modified by time) were also included in the model; n = 3 pHBEC donors; ***p = 0.000163 and 0.00762 (Figure E), **p-value = 0.023 and *** p-value = 0.005 (Figure F); PC (process control) = unconditioned media passed through the EV preparation process.

Figure 5:

Representative images of maximum intensity projections from z-stacks of biotic biofilms of P. aeruginosa pre-treated with PC at (A) 0h and at B) 5h after the 1 hour of exposure to enable the bacteria to attach to pHBEC. Panels C and D are images of P. aeruginosa pre-treated with MSC EVs at 0h (C) and at 5h (D) on pHBEC monolayers. (E) Summary of data of biofilm volumes of P. aeruginosa exposed to PC, MSC EVs or let-7b-5p MSC EVs on pHBEC. Volume renderings of 3D Z-stacks from 1-5 random areas of each monolayer were generated using Keyence software following the manufacturer’s instructions. Each data point represents the P. aeruginosa biofilm volume on a monolayer from an individual pHBEC donor, with colored lines connecting measurements from the same donor at 0 and 5h. Each colored line represents a unique pHBEC donor, and lines of the same color are technical replicates (different imaged areas) of the same donor. (F) Biofilm CFU/mL were assessed by scraping the pHBECs, and plating the bacteria on LB agar at the end of the experiment. The lines connect CFUs formed on monolayers from each donor across the different pre-treatment conditions. Colored lines and symbols represent replicates of the same donor. The male donor is represented by squares, while female donors are represented by circles. Linear mixed-effects models with donor as random effect were used to test statistical significance, and for the biofilm volumes, interaction analyses between time and treatment (to determine if the treatment effect was modified by time) were also included in the model; n = 3 pHBEC donors; ***p = 0.000163 and 0.00762 (Figure E), **p-value = 0.023 and *** p-value = 0.005 (Figure F); PC (process control) = unconditioned media passed through the EV preparation process.

Figure 6:

CF-pHBEC from three donors were exposed to P. aeruginosa (Pa) alone or in combination with process control (PC), negative control (NC) MSC EVs, or let-7b MSC EVs for 6 hours. Cytokine levels of (A) IL-8 and (B) IL-6 were measured by ELISA. Relative mRNA transcript levels of (C) IL-8 and (D) IL-6 were analyzed using the 2^−ΔΔCt method, with UBC serving as the reference gene (the UBC ΔCT was <1) compared to untreated cells as the control. Each colored line represents one CF donor across all treatment conditions. The male donor is represented by squares, while the female donors are represented by circles. Linear mixed-effects models with donor as a random effect were used to test for statistical significance. (E) Heatmap showing significant cell stress and inflammation DEGs (differentially expressed genes with (p < 0.05, ∣ log₂FC ∣ > 1 ) between let-7b-5p MSC EV treatment and P. aeruginosa treatment, and how they change in other conditions (untreated control, PC, NC MSC EV) relative to P. aeruginosa only. Red and blue represent the upregulation or downregulation of a gene, respectively, while white represents no change. FPR1, a predicted target of let-7b-5p is indicated with an arrow. (F) Heatmap showing the enrichment scores of the two KEGG pathways in PC, NC MSC EV and let-7b-5p exposures, all compared to P. aeruginosa. Red and blue represent upregulation or downregulation of a pathway, respectively. n = 3 CF-pHBEC donors (ΔF508/ΔF508); *p-value = 0.014 (Figure A); *p < 0.05, **p < 0.01 (Figure E; exact p-values in Supplementary Table 1); PC (process control) = unconditioned media passed through EV preparation process, NC = negative control.

Figure 6:

CF-pHBEC from three donors were exposed to P. aeruginosa (Pa) alone or in combination with process control (PC), negative control (NC) MSC EVs, or let-7b MSC EVs for 6 hours. Cytokine levels of (A) IL-8 and (B) IL-6 were measured by ELISA. Relative mRNA transcript levels of (C) IL-8 and (D) IL-6 were analyzed using the 2^−ΔΔCt method, with UBC serving as the reference gene (the UBC ΔCT was <1) compared to untreated cells as the control. Each colored line represents one CF donor across all treatment conditions. The male donor is represented by squares, while the female donors are represented by circles. Linear mixed-effects models with donor as a random effect were used to test for statistical significance. (E) Heatmap showing significant cell stress and inflammation DEGs (differentially expressed genes with (p < 0.05, ∣ log₂FC ∣ > 1 ) between let-7b-5p MSC EV treatment and P. aeruginosa treatment, and how they change in other conditions (untreated control, PC, NC MSC EV) relative to P. aeruginosa only. Red and blue represent the upregulation or downregulation of a gene, respectively, while white represents no change. FPR1, a predicted target of let-7b-5p is indicated with an arrow. (F) Heatmap showing the enrichment scores of the two KEGG pathways in PC, NC MSC EV and let-7b-5p exposures, all compared to P. aeruginosa. Red and blue represent upregulation or downregulation of a pathway, respectively. n = 3 CF-pHBEC donors (ΔF508/ΔF508); *p-value = 0.014 (Figure A); *p < 0.05, **p < 0.01 (Figure E; exact p-values in Supplementary Table 1); PC (process control) = unconditioned media passed through EV preparation process, NC = negative control.

Figure 6:

CF-pHBEC from three donors were exposed to P. aeruginosa (Pa) alone or in combination with process control (PC), negative control (NC) MSC EVs, or let-7b MSC EVs for 6 hours. Cytokine levels of (A) IL-8 and (B) IL-6 were measured by ELISA. Relative mRNA transcript levels of (C) IL-8 and (D) IL-6 were analyzed using the 2^−ΔΔCt method, with UBC serving as the reference gene (the UBC ΔCT was <1) compared to untreated cells as the control. Each colored line represents one CF donor across all treatment conditions. The male donor is represented by squares, while the female donors are represented by circles. Linear mixed-effects models with donor as a random effect were used to test for statistical significance. (E) Heatmap showing significant cell stress and inflammation DEGs (differentially expressed genes with (p < 0.05, ∣ log₂FC ∣ > 1 ) between let-7b-5p MSC EV treatment and P. aeruginosa treatment, and how they change in other conditions (untreated control, PC, NC MSC EV) relative to P. aeruginosa only. Red and blue represent the upregulation or downregulation of a gene, respectively, while white represents no change. FPR1, a predicted target of let-7b-5p is indicated with an arrow. (F) Heatmap showing the enrichment scores of the two KEGG pathways in PC, NC MSC EV and let-7b-5p exposures, all compared to P. aeruginosa. Red and blue represent upregulation or downregulation of a pathway, respectively. n = 3 CF-pHBEC donors (ΔF508/ΔF508); *p-value = 0.014 (Figure A); *p < 0.05, **p < 0.01 (Figure E; exact p-values in Supplementary Table 1); PC (process control) = unconditioned media passed through EV preparation process, NC = negative control.

Figure 6:

CF-pHBEC from three donors were exposed to P. aeruginosa (Pa) alone or in combination with process control (PC), negative control (NC) MSC EVs, or let-7b MSC EVs for 6 hours. Cytokine levels of (A) IL-8 and (B) IL-6 were measured by ELISA. Relative mRNA transcript levels of (C) IL-8 and (D) IL-6 were analyzed using the 2^−ΔΔCt method, with UBC serving as the reference gene (the UBC ΔCT was <1) compared to untreated cells as the control. Each colored line represents one CF donor across all treatment conditions. The male donor is represented by squares, while the female donors are represented by circles. Linear mixed-effects models with donor as a random effect were used to test for statistical significance. (E) Heatmap showing significant cell stress and inflammation DEGs (differentially expressed genes with (p < 0.05, ∣ log₂FC ∣ > 1 ) between let-7b-5p MSC EV treatment and P. aeruginosa treatment, and how they change in other conditions (untreated control, PC, NC MSC EV) relative to P. aeruginosa only. Red and blue represent the upregulation or downregulation of a gene, respectively, while white represents no change. FPR1, a predicted target of let-7b-5p is indicated with an arrow. (F) Heatmap showing the enrichment scores of the two KEGG pathways in PC, NC MSC EV and let-7b-5p exposures, all compared to P. aeruginosa. Red and blue represent upregulation or downregulation of a pathway, respectively. n = 3 CF-pHBEC donors (ΔF508/ΔF508); *p-value = 0.014 (Figure A); *p < 0.05, **p < 0.01 (Figure E; exact p-values in Supplementary Table 1); PC (process control) = unconditioned media passed through EV preparation process, NC = negative control.

Figure 6:

CF-pHBEC from three donors were exposed to P. aeruginosa (Pa) alone or in combination with process control (PC), negative control (NC) MSC EVs, or let-7b MSC EVs for 6 hours. Cytokine levels of (A) IL-8 and (B) IL-6 were measured by ELISA. Relative mRNA transcript levels of (C) IL-8 and (D) IL-6 were analyzed using the 2^−ΔΔCt method, with UBC serving as the reference gene (the UBC ΔCT was <1) compared to untreated cells as the control. Each colored line represents one CF donor across all treatment conditions. The male donor is represented by squares, while the female donors are represented by circles. Linear mixed-effects models with donor as a random effect were used to test for statistical significance. (E) Heatmap showing significant cell stress and inflammation DEGs (differentially expressed genes with (p < 0.05, ∣ log₂FC ∣ > 1 ) between let-7b-5p MSC EV treatment and P. aeruginosa treatment, and how they change in other conditions (untreated control, PC, NC MSC EV) relative to P. aeruginosa only. Red and blue represent the upregulation or downregulation of a gene, respectively, while white represents no change. FPR1, a predicted target of let-7b-5p is indicated with an arrow. (F) Heatmap showing the enrichment scores of the two KEGG pathways in PC, NC MSC EV and let-7b-5p exposures, all compared to P. aeruginosa. Red and blue represent upregulation or downregulation of a pathway, respectively. n = 3 CF-pHBEC donors (ΔF508/ΔF508); *p-value = 0.014 (Figure A); *p < 0.05, **p < 0.01 (Figure E; exact p-values in Supplementary Table 1); PC (process control) = unconditioned media passed through EV preparation process, NC = negative control.

Figure 6:

CF-pHBEC from three donors were exposed to P. aeruginosa (Pa) alone or in combination with process control (PC), negative control (NC) MSC EVs, or let-7b MSC EVs for 6 hours. Cytokine levels of (A) IL-8 and (B) IL-6 were measured by ELISA. Relative mRNA transcript levels of (C) IL-8 and (D) IL-6 were analyzed using the 2^−ΔΔCt method, with UBC serving as the reference gene (the UBC ΔCT was <1) compared to untreated cells as the control. Each colored line represents one CF donor across all treatment conditions. The male donor is represented by squares, while the female donors are represented by circles. Linear mixed-effects models with donor as a random effect were used to test for statistical significance. (E) Heatmap showing significant cell stress and inflammation DEGs (differentially expressed genes with (p < 0.05, ∣ log₂FC ∣ > 1 ) between let-7b-5p MSC EV treatment and P. aeruginosa treatment, and how they change in other conditions (untreated control, PC, NC MSC EV) relative to P. aeruginosa only. Red and blue represent the upregulation or downregulation of a gene, respectively, while white represents no change. FPR1, a predicted target of let-7b-5p is indicated with an arrow. (F) Heatmap showing the enrichment scores of the two KEGG pathways in PC, NC MSC EV and let-7b-5p exposures, all compared to P. aeruginosa. Red and blue represent upregulation or downregulation of a pathway, respectively. n = 3 CF-pHBEC donors (ΔF508/ΔF508); *p-value = 0.014 (Figure A); *p < 0.05, **p < 0.01 (Figure E; exact p-values in Supplementary Table 1); PC (process control) = unconditioned media passed through EV preparation process, NC = negative control.