Transcriptome Analysis of Bronchoalveolar Lavage Fluid From Children With Mycoplasma pneumoniae Pneumonia Reveals Natural Killer and T Cell-Proliferation Responses

Abstract

Background: Mycoplasma pneumoniae pneumonia (MPP) is one of the most common community-acquired pneumonia; this study is to explore the immune-pathogenesis of children MPP.

Methods: Next-generation transcriptome sequencing was performed on the bronchoalveolar lavage fluid cells from six children with MPP and three children with foreign body aspiration as control. Some of the results had been validated by quantitative real-time PCR in an expanded group of children.

Results: Results revealed 810 differentially expressed genes in MPP group comparing to control group, of which 412 genes including RLTPR, CARD11 and RASAL3 were upregulated. These upregulated genes were mainly enriched in mononuclear cell proliferation and signaling biological processes. Kyoto encyclopedia of genes and genomes pathway analysis revealed that hematopoietic cell linage pathway, natural killer cell-mediated cytotoxicity pathway, and T cell receptor signaling pathway were significantly upregulated in MPP children. In addition, significant alternative splicing events were found in GNLY and SLC11A1 genes, which may cause the differential expressions of these genes.

Conclusion: Our results suggest that NK and CD8+ T cells are over activated and proliferated in MPP children; the upregulated IFNγ, PRF1, GZMB, FASL, and GNLY may play important roles in the pathogenesis of children MPP.

Keywords: CD8+ T cells; Mycoplasma pneumoniae pneumonia; bronchoalveolar lavage fluid; children; interferon gamma; natural killer cells.

Figure 1

The radiological images and bronchoscopic images of the subjects enrolled in sequencing. (A1) shows radiological image of control1, emphysema (air trapping) in the left lung was observed; (A2) shows radiological image of control 3, foreign body (red arrow) blocked the right main bronchus; (A3) shows radiological image of Mycoplasma pneumoniae pneumonia (MPP) 1, patchy density shadows in the right lung were observed; (A4) shows radiological image of MPP3, atelectasis of the right middle lobe was found; (A5) shows radiological image of MPP4, which revealed large density shadows (consolidation) in the right middle lobe; (A6) shows radiological image of MPP6, multiple necrotizing sacs (red arrows) were observed in the right lung. The wall of the sacs was very thin, which was different from the lung abscess; (B1) is the bronchoscopic image of control 2, a peanut was observed in the right main bronchus, which blocked the airway; (B2) is the bronchoscopic image of MPP2, lines on mucosa (red arrow) were observed in the right upper lobe; (B3) is the bronchoscopic image of MPP5, which shows diffused mucosal nodules (red arrow) in distal part of the left main bronchus; (B4) is the bronchoscopic image of MPP6, which shows the erosion of mucosa and secretions in the bronchus; (B5) is the bronchoscopic image of MPP4, which shows sputum plugging; (B6) is the bronchoscopic image of MPP3, the red arrow shows the proliferation of fibrous tissue occluded the bronchus orifice completely.

Figure 2

Evaluation of each bronchoalveolar lavage fluid sample included in this study and differentially expressed genes between Mycoplasma pneumoniae pneumonia (MPP) group and control group. (A) The correlation coefficient heat map of MPP group and control group. Correlation matrix shows a high consistency of measurements within each group, R² ≥ 0.8 is needed for the up-coming analyzing. (B) Principal component analysis (PCA) plot of the sequencing samples. PCA is conducted to evaluate the clustering nature of the samples. The repeatability of the samples has been shown. Each point represents one sample, the red circles represent the samples in the MPP group, and the green triangles represent the samples in the control group. Percentages are contribution ratios. (C) Volcano plot of genes differentially expressed between MPP group and control group. Each point represents one gene that is detectable in both groups. The red points represent the significantly upregulated (Up-R) genes; the green points represent the significantly downregulated (Down-R) genes. (D) Cluster of 810 genes showing significantly regulated genes between MPP group and control group. All of the genes that are differentially expressed between MPP group and control group by adjusted p value < 0.05 have been selected.

Figure 3

Gene ontology (GO) analysis of the deferentially expressed genes between Mycoplasma pneumoniae pneumonia (MPP) group and control group. (A) Top 30 GO terms were enriched as biochemical processes (BP), cellular components (CCs), or molecular function (MF). The numbers of deferentially expressed genes between MPP and control in each category were compared. Red bars represent upregulated genes, and blue bars represent downregulated genes. (B) Directed acyclic graph of GO enrichment of upregulated genes, the square represents the top 10 GO terms based on adjusted p values; red squares or red circles represent higher degrees of enrichment.

Figure 4

Differential expression genes (DEGs) mapped to hematopoietic cell lineage Kyoto encyclopedia of genes and genomes pathway. (A) Model diagram shows hematopoietic cell lineage pathway, red arrows indicate upregulated gene expression. (B) Cluster analysis of DEGs that mapped to hematopoietic cell lineage pathway. (C) Quantitative real-time PCR validation of DEGs mapped to hematopoietic cell lineage pathway.

Figure 5

Differential expression genes (DEGs) mapped to natural killer (NK) cell-mediated cytotoxicity Kyoto encyclopedia of genes and genomes pathway. (A) Model diagram showing NK cell-mediated cytotoxicity pathway, red arrows indicate upregulated gene expression. (B) Cluster analysis of DEGs that mapped to NK cell-mediated cytotoxicity pathway. (C) Quantitative real-time PCR validation of DEGs mapped to NK cell-mediated cytotoxicity pathway.

Figure 6

Differential expression genes (DEGs) mapped to T cell receptor signaling pathway Kyoto encyclopedia of genes and genomes pathway. (A) Model diagram showing T cell receptor signaling pathway, red arrows indicate upregulated gene expression. (B) Cluster analysis of DEGs that mapped to T cell receptor signaling pathway. (C) Quantitative real-time PCR validation of DEGs mapped to T cell receptor signaling pathway.

Figure 7

Differential alternative splicing events. (A) Summary of the differential alternative splicing event analysis results. (B) Read distribution plot for granulysin (GNLY) with differential isoform expression due to retained intron between Mycoplasma pneumoniae pneumonia (MPP) group and control group. (C) Read distribution plot for solute carrier family 11 (SLC11A1) with differential isoform expression due to alternative 3′ splice sites (A3SS) between MPP group and control group.