Transcriptional analysis of human peripheral blood mononuclear cells stimulated by Mycobacterium tuberculosis antigen

Front Cell Infect Microbiol. 2023 Sep 25:13:1255905. doi: 10.3389/fcimb.2023.1255905. eCollection 2023.

Abstract

Background: Mycobacterium tuberculosis antigen (Mtb-Ag) is a polypeptide component with a molecular weight of 10-14 kDa that is obtained from the supernatant of the H37Ra strain after heat treatment. It stimulates the activation and proliferation of γδT cells in the blood to produce an immune response against tuberculosis. Mtb-Ag is therefore crucial for classifying and detecting the central genes and key pathways involved in TB initiation and progression.

Methods: In this study, we performed high-throughput RNA sequencing of peripheral blood mononuclear cells (PBMC) from Mtb-Ag-stimulated and control samples to identify differentially expressed genes and used them for gene ontology (GO) and a Kyoto Encyclopedia of Genomes (KEGG) enrichment analysis. Meanwhile, we used PPI protein interaction network and Cytoscape analysis to identify key genes and qRT-PCR to verify differential gene expression. Single-gene enrichment analysis (GSEA) was used further to elucidate the potential biological functions of key genes. Analysis of immune cell infiltration and correlation of key genes with immune cells after Mtb-Ag-stimulated using R language.

Results: We identified 597 differentially expressed genes in Mtb-Ag stimulated PBMCs. KEGG and GSEA enrichment analyzed the cellular pathways related to immune function, and DEGs were found to be primarily involved in the TNF signaling pathway, the IL-17 signaling pathway, the JAK-STAT signaling pathway, cytokine-cytokine receptor interactions, and the NF-κB signaling pathway. Wayne analysis using GSEA, KEGG, and the protein-protein interaction (PPI) network showed that 34 genes, including PTGS2, IL-1β, IL-6, TNF and IFN-γ et al., were co-expressed in the five pathways and all were up-regulated by Mtb-Ag stimulation. Twenty-four DEGs were identified using qRT-PCR, including fourteen up-regulated genes (SERPINB7, IL20, IFNG, CSF2, PTGS2, TNF-α, IL36G, IL6, IL10, IL1A, CXCL1, CXCL8, IL4, and CXCL3) and ten down-regulated genes (RTN1, CSF1R CD14, C5AR1, CXCL16, PLXNB2, OLIG1, EEPD1, ENG, and CCR1). These findings were consistent with the RNA-Seq results.

Conclusion: The transcriptomic features associated with Mtb-Ag provide the scientific basis for exploring the intracellular immune mechanisms against Mtb. However, more studies on these DEGs in pathways associated with Mtb-Ag stimulation are needed to elucidate the underlying pathologic mechanisms of Mtb-Ag during Mtb infection.

Keywords: Mycobacterium tuberculosis; Mycobacterium tuberculosis antigen (Mtb-Ag); RNA sequencing; peripheral blood mononuclear cells; signaling pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Computational Biology / methods
  • Cyclooxygenase 2
  • Cytokines / metabolism
  • Gene Expression Profiling / methods
  • Humans
  • Leukocytes, Mononuclear
  • Mycobacterium tuberculosis*
  • Serpins*
  • Tuberculosis*

Substances

  • Cyclooxygenase 2
  • Cytokines
  • SERPINB7 protein, human
  • Serpins

Grants and funding

This work was supported by the Anhui Provincial Natural Science Foundation (1908085MH252, 2008085QH405), the Anhui Province Key Laboratory of Immunology in Chronic Diseases (KLICD-2022-Z3), the Anhui Province Key Laboratory of Clinical and Preclinical Research in Respiratory Disease (HX2022Z02), the 512 Talent Cultivation Plan of Bengbu Medical College (by51201309), and the Postgraduate Scientific Research Innovation Program of Bengbu Medical College (Byycx22013, Byycx23078).