Characteristics Of Host Responses To Inhaled Pseudomonas Aeruginosa And Biotoxins Based On Transcriptomics

The lungs are an important part of the human respiratory system and are often exposed to foreign substances due to their connection with the outside.Foreign substances invading the lungs may damage the lung cells and make them unable to work properly.Foreign substances come from a variety of sources,including bacteria,viruses,toxins,dust,etc.After invading the lungs through the respiratory tract,the invasion of foreign substances sensed by the lungs and an inflammatory response will occur.Usually,the delicate balance between pro-inflammatory and anti-inflammatory processes of lung immunity is crucial for maintaining the integrity of the lungs.Once the lung immune balance is broken,it can lead to two situations:one is that the anti-inflammatory ability is stronger than the pro-inflammatory ability,which will lead to the inability of the lungs to effectively clear pathogens,resulting in serious infection.The second is that the pro-inflammatory ability is stronger than the anti-inflammatory ability,which can clear the pathogens,but the inflammation cannot be controlled,leading to lung injury or even death.In the study of the mechanism of lung injury,high-throughput sequencing technology based on transcriptomics is used to rapidly,accurately and comprehensively detect gene expression levels,providing a powerful tool for a deeper understanding of the complex immune response mechanisms involved in lung injury.Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen and one of the most common causative agents of nosocomial infections in intensive care units,capable of inducing acute lung infections.In-depth investigation of the mechanisms underlying P.aeruginosa-induced pneumonia is necessary to identify alternative therapeutic strategies,as antibiotic treatment may be ineffective due to resistance.The cellular and molecular mechanisms involved in bacterial clearance,inflammation initiation,and resolution in the host following P.aeruginosa lung infection remain unclear.In this study,single-cell sequencing technology（sc RNA-seq）was used to characterize the intrinsic responses of lung cells during acute infection with P.aeruginosa in mice.Our results showed that neutrophils rapidly infiltrate the lungs in large numbers after P.aeruginosa infection and exhibit enhanced bactericidal ability,making them the most important cell type involved in bacterial clearance.Fibroblasts are activated and transformed into inflammatory fibroblasts,which participate in immune cell recruitment and inflammation amplification.Among the subpopulations of fibroblasts,Col13a1⁺matrix fibroblasts,rather than Col14a1⁺matrix fibroblasts,are likely the primary source of inflammatory fibroblasts.In the inflammation resolution stage,non-immune cells such as type II alveolar epithelial cells exhibit high expression of the circadian transcription factor Dbp and its downstream target gene Per3,which may be involved in inflammation resolution and tissue repair.Notably,early in infection,type II alveolar epithelial cells perceive bacteria and secrete CCL-20 by upregulating the NF-κB signaling pathway,potentially recruiting CCR6⁺ILC3s.ILC3s secrete IL-17at damaged type II alveolar epithelial cells to recruit neutrophils and secrete IL-22 to maintain the integrity of type II alveolar epithelial cells and stimulate the secretion of antimicrobial peptides to kill bacteria.This study deepens our understanding of the dynamic cellular responses of lung cell types during P.aeruginosa-induced acute lung infection.These findings provide potential therapeutic strategies for non-antibiotic resistant P.aeruginosa infections.LPS is one of the main constituents of the cell wall in Gram-negative bacteria.SEB is produced by the Gram-positive opportunistic pathogen,Staphylococcus aureus.Emerging evidence suggests that intraperitoneal injection of LPS combined with low-dose aerosolized SEB exposure can cause severe lung injury and even death,while SEB or LPS alone cause neither mortality nor severe pulmonary symptoms in mice.However,pulmonary effects from exposure to aerosolized SEB potentiated by LPS have not been evaluated.This study investigates the global transcriptome profile of lung tissue in mice after exposure to aerosolized SEB potentiated by LPS or LPS alone.A mouse model of intratracheal exposure to LPS-potentiated aerosolized SEB is established and described through histological examination.Transcriptome analysis revealed LPS-potentiated aerosolized SEB affected mouse lungs within 72 h post-SEB inhalation,gradually causing lung injury starting from 24 h post-inhalation.Hub genes leading to lung injury at 48 h post-inhalation have been identified.We speculated that under LPS potentiation,low-dose SEB produces a super-antigenic response in the lungs of mice,activating T lymphocytes and exacerbating the degree of lung injury.To our knowledge,this study represents investigate pulmonary transcriptional responses of LPS-potentiated aerosolized low-dose SEB exposure.This research helps to elucidate the molecular mechanisms underlying the process by which the two bacterial components combine was combined to produce lung damage and provides an insight into potential treatments for alleviating inflammation of the lung when co-infection is present.RT is a 60–65 k Da protein extracted from the seeds of the castor plant（Ricinus communis）,a widespread plant in tropical regions that is readily available in the global market.Specific therapeutics are not available for acute lung injury（ALI）induced by RT.Inhibiting the host immune response in the course of pulmonary ricinosis is hypothesized to be of benefit and can be achieved by impairing granulocyte-macrophage colony-stimulating factor（GM-CSF）signaling,thereby reducing the pro-inflammatory response to exogenous foreign body invasion.However,it is unknown whether mice with impaired GM-CSF signaling can survive after RT inhalation.To test this,colony stimulating factor 2 receptor alpha（Csf2ra）knockout（KO）mice that lack GM-CSF signaling and wild-type（WT）mice models of intratracheal exposure to a lethal dose(2×LD₅₀)of RT were established.Survival was greater in Csf2ra KO mice21 days after RT inhalation compared with WT mice.Highly co-expressed genes that probably attenuated the pro-inflammatory response in the lung of Csf2ra KO mice were identified.Bioinformatics analysis revealed that transcriptome changes involved mostly inflammation-related genes after RT exposure in both Csf2ra KO mice and WT mice.However,the activity levels of pro-inflammatory pathways,such as the TNF signaling pathway and NF-κB signaling pathway,in Csf2ra KO mice were significantly decreased and the degree of neutrophil infiltration inhibited after RT-exposure relative to WT mice.RT-q PCR and flow cytometry validated results of RNA-Seq analysis.This work provides potential avenues for host-directed therapeutic applications that can mitigate the severity of ALI-induced by RT.