Let-7b-5p loaded Mesenchymal Stromal Cell Extracellular Vesicles reduce Pseudomonas-biofilm formation and inflammation in CF Bronchial Epithelial Cells

Abstract

Cystic Fibrosis (CF) is a multiorgan disease caused by mutations in the 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, while let-7b-5p reduces biofilm formation and inflammation. In a preclinical CF mice model, we reported that let-7b-5p-loaded MSC EVs reduced P. aeruginosa burden, immune cells, and proinflammatory cytokines in the lungs. We hypothesize four complementary mechanisms for the observed in-vivo effects of the let-7b-5p loaded MSC EVs: antimicrobial activity, anti-inflammatory properties, inhibition of antibiotic-resistant P. aeruginosa biofilm formation in CF airways, and stimulation of anti-inflammatory macrophage behaviors. This study focused on the second and third mechanisms and demonstrates that MSC EVs engineered to contain let-7b-5p effectively blocked the formation of antibiotic-resistant P. aeruginosa biofilms on primary human bronchial epithelial cells (pHBECs) while also reducing P. aeruginosa-induced inflammation. This approach holds promise for improving outcomes for people with CF. Future work will focus on optimizing delivery strategies and expanding the clinical applicability of MSC EVs to target other CF-associated pathogens.

Keywords: Cystic Fibrosis; Human Bronchial Epithelial Cells; Inflammation; Mesenchymal Stromal Cell Extracellular Vesicles; Pseudomonas aeruginosa biofilms.

Figure 1:

A) ATCC MSC EVs were significantly better at reducing IL-6 secretion by CF-pHBEC compared to PACT MSC EVs (p = 0.0020). B) ATCC MSC EVs (p = 0.0004) were more potent than PACT MSC EVs (p = 0.0086) in reducing IL-8 secretion by CF-pHBEC. CF-pHBEC were exposed to MSC EVs for 6 hours and then apical and basolateral supernatants from CF-pHBEC were collected and analyzed by ELISA. The experiments included CF-pHBEC from two donors (ΔF508/ΔF508), each with 3 replicates (yielding 6 data points per treatment group). 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 < 0.01; ***p < 0.001; mean ± SEM depicted.

Figure 2:

P. aeruginosa (Pa) alone or in combination with PC, NC MSC EVs, or let-7b MSC EVs were not cytotoxic as compared to untreated CF-pHBEC 6 hours after exposure. 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). PC (process control) = unconditioned media passed though EV preparation process, NC = negative control miRNA.

Figure 3:

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. PC pre-treated P. aeruginosa significantly increased biofilm volume over 5h (p =5.02 × 10⁻⁷), whereas there was no significant increase in biofilm volumes of P. aeruginosa pre-treated with MSC EVs or let-7b MSC EVs. MSC EV pre-treated P. aeruginosa and let-7b MSC EV pre-treated P. aeruginosa have significantly lower (p = 0.000163 and 0.00762, respectively) biofilm volumes compared to PC pre-treated P. aeruginosa at 5h. There was no difference in the attachment of P. aeruginosa to pHBEC at 0 hours in all three treatments. 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) MSC EV pre-treated P. aeruginosa and let-7b MSC EV pre-treated P. aeruginosa have significantly lower (p = 0.023 and 0.005, respectively) biofilm CFU/mL compared to PC pre-treated P. aeruginosa at 5h. 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. 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 < 0.05, **p < 0.01; ***p < 0.001; PC (process control) = unconditioned media passed through the EV preparation process.

Figure 3:

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. PC pre-treated P. aeruginosa significantly increased biofilm volume over 5h (p =5.02 × 10⁻⁷), whereas there was no significant increase in biofilm volumes of P. aeruginosa pre-treated with MSC EVs or let-7b MSC EVs. MSC EV pre-treated P. aeruginosa and let-7b MSC EV pre-treated P. aeruginosa have significantly lower (p = 0.000163 and 0.00762, respectively) biofilm volumes compared to PC pre-treated P. aeruginosa at 5h. There was no difference in the attachment of P. aeruginosa to pHBEC at 0 hours in all three treatments. 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) MSC EV pre-treated P. aeruginosa and let-7b MSC EV pre-treated P. aeruginosa have significantly lower (p = 0.023 and 0.005, respectively) biofilm CFU/mL compared to PC pre-treated P. aeruginosa at 5h. 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. 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 < 0.05, **p < 0.01; ***p < 0.001; PC (process control) = unconditioned media passed through the EV preparation process.

Figure 4:

let-7b-5p MSC EVs significantly reduce P. aeruginosa stimulated: (A) IL-8 (p-value = 0.014) but not (B) IL-6 cytokine levels in CF-pHBEC at 6 hours. The fold changes of mRNA transcript levels of IL-8 (C) and IL-6 (D) are not significantly altered by the PC or MSC EV treatments. The 2^−ΔΔCt method was used to analyze relative gene expression, with UBC serving as the reference gene (the UBC ΔCT was unchanged by experimental treatment) and untreated cells as the control. Each colored line represents one CF donor across all treatment conditions. 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 < 0.05, **p < 0.01; PC (process control) = unconditioned media passed through EV preparation process, NC = negative control.

Figure 4:

let-7b-5p MSC EVs significantly reduce P. aeruginosa stimulated: (A) IL-8 (p-value = 0.014) but not (B) IL-6 cytokine levels in CF-pHBEC at 6 hours. The fold changes of mRNA transcript levels of IL-8 (C) and IL-6 (D) are not significantly altered by the PC or MSC EV treatments. The 2^−ΔΔCt method was used to analyze relative gene expression, with UBC serving as the reference gene (the UBC ΔCT was unchanged by experimental treatment) and untreated cells as the control. Each colored line represents one CF donor across all treatment conditions. 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 < 0.05, **p < 0.01; PC (process control) = unconditioned media passed through EV preparation process, NC = negative control.

Figure 5:

CF-pHBEC from three donors kill P. aeruginosa (Pa) after 6 hours. The CFUs fell from 3 × 10⁸ CFU/mL Inoculum) to < 4.4 ×10⁴ CFU/mL. 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 in the bacterial burden assay; n = 3 CF-pHBEC donors (ΔF508ΔF508); ***p < 0.001 comparing P. aeruginosa (Pa) inoculum to all four treated CF-pHBEC groups ± ETI; PC (process control), NC = negative control.