Transcriptome sequencing and experiments reveal the effect of formyl peptide receptor 2 on liver homeostasis

Abstract

Background: Formyl peptide receptor 2 (Fpr2) is an important receptor in host resistance to bacterial infections. In previous studies, we found that the liver of Fpr2^-/- mice is the most severely damaged target organ in bloodstream infections, although the reason for this is unclear.

Aim: To investigate the role of Fpr2 in liver homeostasis and host resistance to bacterial infections.

Methods: Transcriptome sequencing was performed on the livers of Fpr2^-/- and wild-type (WT) mice. Differentially expressed genes (DEGs) were identified in the Fpr2^-/- and WT mice, and the biological functions of DEGs were analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) en-richment analysis. Quantitative real time-polymerase chain reaction (qRT-PCR) and western blot (WB) analyses were used to further validate the expression levels of differential genes. Cell counting kit-8 assay was employed to investigate cell survival. The cell cycle detection kit was used to measure the distribution of cell cycles. The Luminex assay was used to analyze cytokine levels in the liver. The serum biochemical indices and the number of neutrophils in the liver were measured, and hepatic histopathological analysis was performed.

Results: Compared with the WT group, 445 DEGs, including 325 upregulated genes and 120 downregulated genes, were identified in the liver of Fpr2^-/- mice. The enrichment analysis using GO and KEGG showed that these DEGs were mainly related to cell cycle. The qRT-PCR analysis confirmed that several key genes (CycA, CycB1, Cdc20, Cdc25c, and Cdk1) involved in the cell cycle had significant changes. The WB analysis confirmed a decrease in the expression of CDK1 protein. WRW4 (an antagonist of Fpr2) could inhibit the proliferation of HepG2 cells in a concentration dependent manner, with an increase in the number of cells in the G0/G1 phase, and a decrease in the number of cells in the S phase. Serum alanine aminotransferase levels increased in Fpr2^-/- mice. The Luminex assay measurements showed that interleukin (IL)-10 and chemokine (C-X-C motif) ligand (CXCL)-1 levels were significantly reduced in the liver of Fpr2^-/- mice. There was no difference in the number of neutrophils, serum C-reactive protein levels, and liver pathology between WT and Fpr2^-/- mice.

Conclusion: Fpr2 participates in the regulation of cell cycle and cell proliferation, and affects the expression of IL-10 and CXCL-1, thus playing an important protective role in maintaining liver homeostasis.

Keywords: Cdk1; Cell cycle; Cell proliferation; Differentially expressed genes; Formyl peptide receptor 2; RNA-sequencing.

Figure 1

RNA-sequencing analysis of distributions of all differentially expressed genes. A: Volcano map of the distributions of all differentially expressed genes in formyl peptide receptor 2 (Fpr2)^-/- and wild-type (WT) mice (n = 4). Red, green, and blue colors respectively represent upregulated genes, downregulated genes, and genes with no difference in expression; B: Venn diagram of gene counts expressed in the Fpr2^-/- and WT mice (n = 4). 9578 genes show identical expression between Fpr2^-/- and WT mice. 541 and 371 specific genes are expressed in Fpr2^-/- and WT mice, respectively. Fpr2: Formyl peptide receptor 2; WT: Wild-type.

Figure 2

Gene Ontology pathway enrichment analysis of differentially expressed genes. The bar graph shows the top 10 (red for biological processes, green for cellular component and blue for molecular function) enriched Gene Ontology pathways. A: Gene Ontology (GO) pathway enrichment analysis of all differentially expressed genes (DEGs); B: GO pathway enrichment analysis of upregulated DEGs; C: GO pathway enrichment analysis of downregulated DEGs. BP: Biological process; CC: Cellular component; MF: Molecular function.

Figure 3

Significant enrichment of Kyoto Encyclopedia of Genes and Genomes pathways with differentially expressed genes. The size of the bubble indicates the enrichment score, and the color indicates the significance of the enrichment.

Figure 4

The effect of formyl peptide receptor 2 deletion on cell cycle. A: Quantitative real time-polymerase chain reaction (qRT-PCR) validates the expression of key cell cycle genes regulated by formyl peptide receptor 2 (Fpr2), including CycA, CycB1, Cdc20, Cdc25c, and Mps1 (n = 6). P values are indicated on the graphs; B: qRT-PCR and western blot analyses are used to validate the changes in expression of CDK1; C: The effect of different concentrations of WRW4 on the proliferation activity of HepG2 cells detected by CCK-8 (n = 5); D: The cell cycle distribution of HepG2 cells treated with WRW4 (1 μM) and dimethyl sulfoxide determined by flow cytometry (n = 3). Data are shown as mean ± SD. ^aP < 0.0001 vs the 0 μM group; ^bP < 0.05 vs the dimethyl sulfoxide group; NS: No significant; Fpr2: Formyl peptide receptor 2; WT: Wild-type; DMSO: Dimethyl sulfoxide.

Figure 5

Liver function test in serum of wild-type and formyl peptide receptor 2^-/- mice (n = 4). The values are expressed as g/L for albumin levels, and U/L for alanine transaminase, lactate dehydrogenase, aspartate transaminase, and alkaline phosphatase levels. Data are expressed as mean ± SD. ^aP < 0.05, ^bP < 0.01 vs the wild-type group; NS: No significant; ALB: Albumin; ALP: Alkaline phosphatase; ALT: Alanine transaminase; AST: Aspartate transaminase; LDH: Lactate dehydrogenase; Fpr2: Formyl peptide receptor 2; WT: Wild-type.

Figure 6

Luminex assay analyses cytokines/chemokines concentration in liver of wild-type and formyl peptide receptor 2^-/- mice (n = 3). Mean concentration values are shown as normalized to tissue weight. Data are expressed as mean ± SD. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001 vs the wild-type group; NS: No significant; CXCL: Chemokine (C-X-C motif) ligand; IFN-γ: Interferon gamma; IL: Interleukin; MCP: Monocyte chemoattractant protein; MIP: Macrophage inflammatory protein; TNF: Tumor necrosis factor; Fpr2: Formyl peptide receptor 2; WT: Wild-type.

Figure 7

Evaluation of liver inflammation status. A: Serum C-reactive protein level in wild-type (WT) and formyl peptide receptor 2 (Fpr2)^-/- mice (n = 4); B: Flow cytometry analysis of the number of neutrophils (% of Ly6G⁺ cells) in the liver of WT and Fpr2^-/- mice (n = 3); C: Representative images of hematoxylin-eosin staining of liver tissue sections of Fpr2^-/- and WT mice. The image magnification is 200 × and 400 ×. NS: No significant difference. Fpr2: Formyl peptide receptor 2; WT: Wild-type; CRP: C-reactive protein.