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Increased susceptibility of irradiated mice to Aspergillus fumigatus infection via NLRP3/GSDMD pathway in pulmonary bronchial epithelia.

ABSTRACT:

Background

Aspergillus fumigatus infection is difficult to diagnose clinically and can develop into invasive pulmonary aspergillosis, which has a high fatality rate. The incidence of Aspergillus fumigatus infection has increased die to widespread application of radiotherapy technology. However, knowledge regarding A. fumigatus infection following radiation exposure is limited, and the underlying mechanism remains unclear. In this study, we established a mouse model to explore the effect of radiation on A. fumigatus infection and the associated mechanisms.

Methods

In this study, a mouse model of A. fumigatus infection after radiation was established by irradiating with 5 Gy on the chest and instilling 5 × 10⁷/ml Aspergillus fumigatus conidia into trachea after 24 h to explore the effect and study its function and mechanism. Mice were compared among the following groups: normal controls (CON), radiation only (RA), infection only (Af), and radiation + infection (RA + Af). Staining analyses were used to detect infection and damage in lung tissues. Changes in protein and mRNA levels of pyroptosis-related molecules were assessed by western blot analysis and quantitative reverse transcription polymerase chain reaction, respectively. Protein concentrations in the serum and alveolar lavage fluid were also measured. An immunofluorescence colocalization analysis was performed to confirm that NLRP3 inflammasomes activated pyroptosis.

Results

Radiation destroyed the pulmonary epithelial barrier and significantly increased the pulmonary fungal burden of A. fumigatus. The active end of caspase-1 and gasdermin D (GSDMD) were highly expressed even after infection. Release of interleukin-18 (IL-18) and interleukin-1β (IL-1β) provided further evidence of pyroptosis. NLRP3 knockout inhibited pyroptosis, which effectively attenuated damage to the pulmonary epithelial barrier and reduced the burden of A. fumigatus.

Conclusions

Our findings indicated that the activation of NLRP3 inflammasomes following radiation exposure increased susceptibility to A. fumigatus infection. Due to pyroptosis in lung epithelial cells, it resulted in the destruction of the lung epithelial barrier and further damage to lung tissue. Moreover, we found that NLRP3 knockout effectively inhibited the pyroptosis and reducing susceptibility to A. fumigatus infection and further lung damage. Overall, our results suggest that NLRP3/GSDMD pathway mediated-pyroptosis in the lungs may be a key event in this process and provide new insights into the underlying mechanism of infection. Video abstract.

SUBMITTER: Wu DM

PROVIDER: S-EPMC9238178 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Aspergillus fumigatus (A. fumigatus) is a ubiquitous fungus which reproduces asexually by releasing abundant airborne conidia (spores), which are easily respirable. In allergic and immunocompromised individuals A. fumigatus can cause a wide spectrum of diseases, including allergic bronchopulmonary aspergillosis, aspergilloma and invasive aspergillosis. Previous studies have demonstrated that A. fumigatus conidia are internalized by macrophages and lung epithelial cells; however the exact transcriptional responses of airway epithelial cells to conidia are currently unknown. Thus, the aim of this study was to determine the transcriptomic response of the human bronchial epithelial cell line (16HBE14o-) following interaction with A. fumigatus conidia. We used fluorescence-activated cell sorting (FACS) to separate 16HBE14o- cells having bound and/or internalized A. fumigatus conidia expressing green fluorescent protein from cells without spores. Total RNA was then isolated and the transcriptome of 16HBE14o- cells was evaluated using Agilent Whole Human Genome microarrays.Immunofluorescent staining and nystatin protection assays demonstrated that 16HBE14o- cells internalized 30-50% of bound conidia within six hrs of co-incubation. After FAC-sorting of the same cell culture to separate cells associated with conidia from those without conidia, genome-wide analysis revealed a set of 889 genes showing differential expression in cells with conidia. Specifically, these 16HBE14o- cells had increased levels of transcripts from genes associated with repair and inflammatory processes (e.g., matrix metalloproteinases, chemokines, and glutathione S-transferase). In addition, the differentially expressed genes were significantly enriched for Gene Ontology terms including: chromatin assembly, G-protein-coupled receptor binding, chemokine activity, and glutathione metabolic process (up-regulated); cell cycle phase, mitosis, and intracellular organelle (down-regulated).We demonstrate a methodology using FACs for analyzing the transcriptome of infected and uninfected cells from the same cell population that will provide a framework for future characterization of the specific interactions between pathogens such as A. fumigatus with human cells derived from individuals with or without underlying disease susceptibility.