Abstract Rationale Chronic infections plague patients with lung diseases. Mesenchymal stem cells (MSCs) are anti-inflammatory and antimicrobial paracrine signaling vehicles that have potential for treating pulmonary infections. We have demonstrated the safety of MSCs in patients with cystic fibrosis (NCT02866721) and preclinical antimicrobial potency against Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus, Streptococcus pneumonia, and a couple of non-tuberculous mycobacterium (NTM) species. The MSC therapeutic conundrum, which has prevented FDA approval, stems from their exquisite functional diversity and milieu sensitivity. We hypothesized that MSCs can be strategically optimized with antimicrobial-inducing effectors, thereby promoting a targeted MSC molecular signature to treat lung infections. Methods Bone marrow-derived MSCs (n = 12 donors) were analyzed for their antimicrobial potency against P. aeruginosa and NTMs, quantifying pathogen survival and growth behavior. C57BL/6J wild-type mice were infected with 106 colony-forming units (CFUs) of P. aeruginosa or the NTMs with MSCs (106/100ul saline) delivered through the retro-orbital sinus at 24 hours, followed by thermal imaging for clinical score, culminating at day 7 with euthanasia to analyze lung infection and inflammation. MSCs’ molecular analysis was done by RNAseq, focusing on antimicrobial peptides: cathelicidin, lipocalin, hepcidin, human beta-defensins, heme oxygenase 1, CCL20, surfactant protein A and D, all validated by RT-PCR. Multiparametric statistics were utilized to identify the MSC molecular signature, which correlated with in vitro and in vivo antimicrobial potency. MSC inducing effectors (proprietary) were utilized to recapitulate the MSC antimicrobial potency. MSC transduction with an AMP expression vector was pursued for targeted antimicrobial delivery. Statistics included Quality by Design® for effector selection and ANOVA multiparametric analysis for functional correlation using GraphPad® Prism 9. Results The MSCs had diverse and pathogen-specific antimicrobial potency against P. aeruginosa and the NTMs. MSCs were analyzed for AMP gene expression, secretome phenotype, and functional specificity. MSC-derived LL-37 (p = 0.05) defined antimicrobial activity against P. aeruginosa, with heme oxygenase 1, CCL20, and IL-6 correlating with anti-NTMs activity (p ≤ 0.05). MSC-derived SP-A provided antimicrobial activity against both bacteria (p ≤ 0.05). MSCs’ ideal AMP signatures were assessed in vitro and in vivo for antimicrobial potency, demonstrating functional consistency defined by molecular signature (p 0.05). Utilizing effectors, MSCs were induced to successfully enhance antimicrobial potency in vitro (p 0.05) and in vivo (p 0.05). Targeted MSC AMP delivery by transduction also demonstrated efficient management of infection. Conclusions The MSC antimicrobial AMP molecular signatures can be utilized to predict functional potency. Effectors or target AMP delivery can be used to promote efficient MSC antimicrobial therapeutic delivery. This abstract is funded by: The Marcus Foundation, Cytsitic Fibrosis Foundation, David and Virginia Baldwin Fund, CWRU CTSC Annual Award
Bonfield et al. (Fri,) studied this question.
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