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Time-Course Transcriptome Analysis of Lungs From Mice Infected With Hypervirulent Klebsiella pneumoniae via Aerosolize Intratracheal Inoculation

ABSTRACT: Time-Course Transcriptome Analysis of Lungs From Mice Infected With Hypervirulent Klebsiella pneumoniae via Aerosolize Intratracheal Inoculation

PROVIDER: PRJNA820708 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR18503129_1.fastq.gz	Fastqsanger.gz
	SRR18503129_2.fastq.gz	Fastqsanger.gz
	SRR18503130_1.fastq.gz	Fastqsanger.gz
	SRR18503130_2.fastq.gz	Fastqsanger.gz
	SRR18503131_1.fastq.gz	Fastqsanger.gz

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Project description:Purpose:Investigated transcriptome profile changes in the lungs of infected mice at different time points, to increase our understanding of hvKp pulmonary infection and to screen for genes or targets of research value. microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: Animals were randomly divided into four infection groups and one control group (five mice per group), and the infected mice were euthanized at 12, 24, 48 and 60 h post-infection (hpi), respectively, while the control group mice were euthanized immediately after delivery of PBS at the 0 h time point. The lungs of the mice were isolated and divided into two parts for subsequent histopathologic examination or total RNA extraction. Results: Using the control group (0 hpi) as a reference, 6247 DEGs were identified across four time points after hvKp infection. At 12 hpi, 939 upregulated DEGs and 439 downregulated DEGs were detected, and by 60 hpi, 2647 upregulated DEGs and 2806 downregulated DEGs were detected. Overall, the number of upregulated and downregulated DEGs increased over time and a total of 688 genes were upregulated and 240 genes downregulated in common for all time points. And 12 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.9548. To get a more holistic view, DEGs were clustered into nine clusters according to their temporal expression patterns.WGCNA was performed with the detected DEGs. The dynamic tree cutting algorithm in the WGCNA package was used to process the hierarchical clustering tree, and a total of 28 different modules were finally obtained. Conclusions: Our work both validates the results of previous studies on K. pneumoniae pulmonary infections and provides new additions and insights on hvKp pulmonary infections, which may contribute to the development of therapeutic approaches to reduce hvKp lung inflammation.

Project description:Hypervirulent Klebsiella pneumoniae (hvKp) can cause life-threatening community-acquired infections among healthy young individuals and is thus of concern for global dissemination. In this study, a mouse model of acute primary hvKp pneumonia was established via aerosolized intratracheal (i.t.) inoculation, laying the foundation for conducting extensive studies related to hvKp. Subsequently, a time-course transcriptional profile was created of the lungs from the mouse model at 0, 12, 24, 48 and 60 hours post-infection (hpi) using RNA Sequencing (RNA-Seq). RNA-Seq data were analyzed with the use of Mfuzz time clustering, weighted gene co-expression network analysis (WGCNA) and Immune Cell Abundance Identifier for mouse (ImmuCellAI-mouse). A gradual change in the transcriptional profile of the lungs was observed that reflected expected disease progression. At 12 hpi, genes related to acute phase inflammatory response increased in expression and lipid metabolism appeared to have a pro-inflammatory effect. At 24 hpi, exacerbation of inflammation was observed and active IFN-γ suggested that signaling promoted activation and recruitment of macrophages occurred. Genes related to maintaining the structural integrity of lung tissues showed a sustained decrease in expression after infection and the decrease was especially marked at 48 hpi. TNF, IL-17, MAPK and NF-kB signaling pathways may play key roles in the immunopathogenesis mechanism at all stages of infection. Natural killer (NK) cells consistently decreased in abundance after infection, which has rarely been reported in hvKp infection and could provide a new target for treatment. Genes Saa1 and Slpi were significantly upregulated during infection. Both Saa1, which is associated with lipopolysaccharide (LPS) that elicits host inflammatory response, and Slpi, which encodes an antimicrobial protein, have not previously been reported in hvKp infections and could be important targets for subsequent studies. To t our knowledge, this paper represents the first study to investigate the pulmonary transcriptional response to hvKp infection. The results provide new insights into the molecular mechanisms underlying the pathogenesis of hvKp pulmonary infection that can contribute to the development of therapies to reduce hvKp pneumonia.

Project description:Carbon black nanoparticles (CBNP) are the most widely produced nanomaterial and are thus a concern for occupational exposures. CBNPs are known to cause inflammation upon inhalation; however, detailed time course data to evaluate the changing nature of inflammatory responses have not been evaluated. In this study, we investigate the short- and longer-term effects of CBNP following a single intratracheal exposure. Female C57BL/6BomTac mice were exposed to CBNPs (printex 90) via a single intratracheal instillation of 162 µg/mouse alongside vehicle controls. Mice were examined 3 hours (hrs), and 1, 2, 3, 4, 5, 14, and 42 days (d) post-exposure. Global gene expression was assessed using DNA microarrays in parallel with analysis of pulmonary inflammation assessed as bronchoalveolar lavage (BAL) cell counts and DNA strand breaks assessed by the comet assay. Pulmonary inflammation in terms of increased neutrophil influx was obverved at all time points. Increased levels of DNA strand breaks were observed in BAL cells 3 hrs post exposure and in lung tissue 2-5 d post exposure. Approximately 2600 significant differentially expressed genes (±1.5 fold; p ≤0.05) across all of the time-points in the lungs of exposed mice were identified upon comparison to time-matched controls. Transcriptional alterations were enriched for genes involved in common respiratory infectious diseases. The perturbed biological processes and pathways were primarily related to immune-inflammatory responses and acute phase responses, consistent with the BAL inflammatory profiles. Gene and pathway analysis revealed a biphasic inflammatory response, with changes occurring as early as 3 hrs post-exposure and declining to base-levels by 3 d post-exposure, but with inflammatory pathways increasing in response again at 14 d and persisting to 42 d post-exposure. Other perturbed pathways included DNA repair and apoptosis, cell cycle regulation, and muscle contraction. Single CBNP exposure induced a biphasic inflammatory response that lasted until 42 d post-exposure. Such persistent inflammatory response raises concerns over the chronic effects of CBNP exposure since the administered dose corresponds to pulmonary deposition in mice during nine 8-hour working days at the current occupational exposure limit for carbon black.

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Time-Course Transcriptome Analysis of Lungs From Mice Infected With Hypervirulent Klebsiella pneumoniae via Aerosolize Intratracheal Inoculation

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