Biology subjects

Barnaby, R.

Publications and source records attributed to Barnaby, R..

7 recordsLinked to original sources

bioRxiv2026

Polymicrobial extracellular vesicles reduce the innate immune response of human cystic fibrosis bronchial epithelial cells

Chronic antibiotic-resistant cystic fibrosis (CF) lung infections are the leading cause of death in adults with CF. Despite advances in highly effective modulator therapies, microbial communities persist in the CF lung. The pathogenesis of CF airway infections can be exacerbated by pathogens such as Pseudomonas aeruginosa, which communicates with primary human bronchial epithelial cells (pHBEC) by secreting bacterial extracellular vesicles (bEVs) that diffuse through mucus and deliver virulence factors, DNA, and RNA to pHBEC. However, most CF lung infections are polymicrobial in nature, and therefore, the contribution of polymicrobial bEVs remains to be determined. By using a polymicrobial culture model representing a pulmotype detected in [~]34% of lung infections in people with CF (pwCF), comprised of P. aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown in synthetic sputum medium under anoxia, we report that each bacterial genus in the polymicrobial community secretes bEVs containing proteins and RNAs predicted to promote the establishment of chronic infection by enhancing virulence, biofilm formation, and upregulating the stress response and pro-inflammatory pathways in pHBEC. This response is most pronounced in CF pHBEC. Elexacaftor/Tezacaftor/Ivacaftor (ETI), a highly effective modulator therapy, does not ameliorate the response or return it to WT levels. Bacterial EVs also inhibited ETI CFTR Cl- currents by CF pHBEC. These studies provide insight into why ETI does not eliminate polymicrobial lung infections and a hyperinflammatory lung environment in pwCF. IMPORTANCECystic fibrosis (CF) is a genetic disease characterized by chronic polymicrobial lung infections that, if untreated, are one of the primary causes of death in CF. Elexacaftor/Tezacaftor/Ivacaftor (ETI) has many positive clinical outcomes, but it does not eliminate chronic polymicrobial lung infections or inflammation. Using a new biologically relevant co-culture model, we have demonstrated that bacteria secrete vesicles (bEVs) that contain proteins and RNAs. We observed that these RNA-loaded bEVs are predicted to promote the pathogenesis of chronic CF lung infections by enhancing bacterial virulence and biofilm formation, as well as upregulating the pro-inflammatory response in lung cells. ETI does not ameliorate the response of lung cells to bEVs. Our research will facilitate the development of more effective approaches to eliminate infection and inflammation in CF and other lung diseases characterized by chronic polymicrobial infections and excessive inflammation.

microbiology↗

bioRxiv2025

Outer Membrane Vesicles Secreted by Bacteroides fragilis Inhibit CFTR Chloride Secretion by Human Colon Organoids

The goals of this study were to develop a model to study host pathogen interactions in primary human colon organoids and to test the hypothesis that Bacteroides fragilis toxin (BFT-2) secreted in outer membrane vesicles (OMVs) modulates mucosal immunity and CFTR Cl- secretion. Since Bacteroides species reside in mucus, OMVs are likely to represent a mechanism of communication between Bacteroides and the host. Two strains of Bacteroides were studied, Enterotoxigenic Bacteroides fragilis (ETBF), which produces BFT-2, and the non-toxigenic Bacteroides fragilis strain NCTC 9343 (NTBF) that does not produce BFT-2. We also utilized two additional strains of Bacteroides fragilis: one in which bft-2 was knocked out (ETBF {Delta}bft), and one that was engineered to contain bft-2 (NTBF+bft). We report that Bacteroides fragilis OMVs reduced CFTR Cl- secretion but had no effect on tight junction or cell adhesion proteins, transepithelial resistance (TER) or cytokine secretion by primary human colon organoids. NTBF OMVs containing BFT-2 were more effective in reducing CFTR Cl- secretion than NTBF lacking BFT-2. We conclude that OMVs secreted by Bacteroides can be an important mechanism of host pathogen interactions in the colon by reducing CFTR Cl- secretion.

microbiology↗

bioRxiv2025

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

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. NEW AND NOTEWORTHYThis is the first study demonstrating that let-7b-5p loaded Mesenchymal Stromal Cell Extracellular Vesicles (MSC EVs) block antibiotic-resistant P. aeruginosa biofilm formation and reduce inflammation in CF primary human bronchial epithelial cells. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/656674v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@3754b3org.highwire.dtl.DTLVardef@99dadcorg.highwire.dtl.DTLVardef@efc4b3org.highwire.dtl.DTLVardef@15aed48_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Extracellular Vesicles Derived from Mesenchymal Stromal Cells Reduce Pseudomonas aeruginosa Lung Infection and Inflammation in Mice

The World Health Organization and the U.S. Centers for Disease Control and Prevention have reported that antibiotic resistant infections with Pseudomonas aeruginosa present a significant health risk world-wide. In the genetic disease Cystic Fibrosis (CF), chronic antibiotic resistant Pseudomonas lung infections and persistent inflammation remain the leading causes of mortality. While highly effective modulator therapy (HEMT) dramatically improves lung function in CF, they fail to eradicate chronic infections or eliminate the associated hyperinflammatory state. Thus, there is an urgent need for innovative therapies that can simultaneously eliminate antibiotic resistant P. aeruginosa lung infection and the attendant hyperinflammatory lung environment. Mesenchymal stromal cell-derived extracellular particles (MSC EPs) represent a promising solution, offering potent anti-inflammatory and antimicrobial properties while being safe and non-toxic. This study demonstrates using a CF mouse model of infection that MSC EPs reduce acute P. aeruginosa lung infection and inflammation. As the first investigation of MSC EPs in CF mice, this research underscores the dual effects of MSC EPs; reducing inflammation and bacterial burden. These findings mark an important advancement in antimicrobial therapy, addressing the unmet need for reducing antibiotic resistant infections and hyperinflammation in CF as well as other diseases with chronic, antibiotic resistant P. aeruginosa infections.

microbiology↗

bioRxiv2024

Gene expression responses of CF airway epithelial cells exposed to elexacaftor/tezacaftor/ivacaftor (ETI) suggest benefits beyond improved CFTR channel function

The combination of elexacaftor/tezacaftor/ivacaftor (ETI, Trikafta) reverses the primary defect in Cystic Fibrosis (CF) by improving CFTR mediated Cl- and HCO3- secretion by airway epithelial cells (AEC), leading to improved lung function and less frequent exacerbations and hospitalizations. However, studies have shown that CFTR modulators like ivacaftor, a component of ETI, has numerous effects on CF cells beyond improved CFTR channel function. Because little is known about the effect of ETI on CF AEC gene expression we exposed primary human AEC to ETI for 48 hours and interrogated the transcriptome by RNA-seq and qPCR. ETI increased defensin gene expression (DEFB1) an observation consistent with reports of decreased bacterial burden in the lungs of people with CF (pwCF). ETI also decreased MMP10 and MMP12 gene expression, suggesting that ETI may reduce proteolytic induced lung destruction in CF. ETI also reduced the expression of the stress response gene heme oxygenase (HMOX1). qPCR analysis confirmed DEFB1, HMOX1, MMP10 and MMP12 gene expression results observed by RNA-seq. Gene pathway analysis revealed that ETI decreased inflammatory signaling, cellular proliferation and MHC Class II antigen presentation. Collectively, these findings suggest that the clinical observation that ETI reduces lung infections in pwCF is related in part to drug induced increases in DEFB1, and that ETI may reduce lung damage by reducing MMP10 and MMP12 gene expression, which is predicted to reduce matrix metalloprotease activity. Moreover, pathway analysis also identified several genes responsible for the ETI induced reduction in inflammation observed in people with CF. New and NoteworthyGene expression responses by CF AEC exposed to ETI suggest that in addition to improving CFTR channel function, ETI is likely to increase resistance to bacterial infection by increasing levels of beta defensin 1 (hBD-1). ETI may also reduce lung damage by suppressing MMP10, and reduce airway inflammation by repressing proinflammatory cytokine secretion by AEC cells.

pharmacology and toxicology↗

bioRxiv2024

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in Cystic Fibrosis

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa. The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lung, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection. New and noteworthyThe experiments in this report identify a novel mechanim whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet haves in OMVs secreted by P. aeruginiosa, which reduced the OMV-LPS induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF. Graphical abstractThe anti-inflammatory effect of tobramycin mediated by 5' tRNA-fMet halves secreted in P. aeruginosa OMVs. (A) P. aeruginosa colonizes the CF lungs and secrets OMVs. OMVs diffuse through the mucus layer overlying bronchial epithelial cells and induce IL-8 secretion, which recruits neutrophils that causes lung damage. (B) Tobramycin increases 5' tRNA-fMet halves in OMVs secreted by P. aeruginosa. 5' tRNA-fMet halves are delivered into host cells after OMVs fuse with lipid rafts in CF-HBEC and down-regulate protein expression of MAPK10, IKBKG, and EP300, which suppresses IL-8 secretion and neutrophils in the lungs. A reduction in neutrophils in CF BALF is predicted to improve lung function and decrease lung damage. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/578737v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@d9d5e7org.highwire.dtl.DTLVardef@16a5715org.highwire.dtl.DTLVardef@7b35ddorg.highwire.dtl.DTLVardef@10d4dc0_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2021

Poly (acetyl arginyl) glucosamine attenuates Pseudomonas aeruginosa in a rat lung infection model

Pseudomonas aeruginosa is a common opportunistic pathogen that can cause chronic infections in multiple disease states, including respiratory infections in patients with cystic fibrosis (CF) and non-CF bronchiectasis. Like many opportunists, P. aeruginosa forms multicellular biofilm communities that are widely thought to be an important determinant of bacterial persistence and resistance to antimicrobials and host immune effectors during chronic/recurrent infections. Poly (acetyl, arginyl) glucosamine (PAAG) is a glycopolymer which has antimicrobial activity against a broad range of bacterial species, and also has mucolytic activity which can normalize rheologic properties of cystic fibrosis mucus. In this study, we sought to evaluate the effect of PAAG on P. aeruginosa bacteria within biofilms in vitro, and in the context of experimental pulmonary infection in a rodent infection model. PAAG treatment caused significant bactericidal activity against P. aeruginosa biofilms, and a reduction in the total biomass of preformed P. aeruginosa biofilms on abiotic surfaces, as well as on the surface of immortalized cystic fibrosis human bronchial epithelial cells. Studies of membrane integrity indicated that PAAG causes changes to P. aeruginosa cell morphology and dysregulates membrane polarity. PAAG treatment reduced infection and consequent tissue inflammation in experimental P. aeruginosa rat infections. Based on these findings we conclude that PAAG represents a novel means to combat P. aeruginosa infection, which may warrant further evaluation as a therapeutic.

microbiology↗