Characterization of species-specific genes regulated by E2-2 in human plasmacytoid dendritic cells - PubMed (original) (raw)

Characterization of species-specific genes regulated by E2-2 in human plasmacytoid dendritic cells

Menglan Cheng et al. Sci Rep. 2015.

Abstract

Dendritic cells (DCs) are sentinels of the immune system and comprise two distinct subsets: conventional DCs (cDCs) and plasmacytoid DCs (pDCs). Human pDCs are distinguished from mouse pDCs phenotypically and functionally. Basic helix-loop-helix protein E2-2 is defined as an essential transcription factor for mouse pDC development, cell fate maintenance and gene programe. It is unknown whether E2-2 regulation contributes to this species-specific difference. Here we investigated the function of E2-2 in human pDCs and screened human-specific genes regulated by E2-2. Reduced E2-2 expression in human pDC cell line GEN2.2 resulted in diminished IFN-α production in response to CpG but elevated antigen presentation capacity. Gene expression profiling showed that E2-2 silence down-regulated pDC signature genes but up-regulated cDC signature genes. Thirty human-specific genes regulated by E2-2 knockdown were identified. Among these genes, we confirmed that expression of Siglec-6 was inhibited by E2-2. Further more, Siglec-6 was expressed at a higher level on a human pDC subset with drastically lower expression of E2-2. Collectively, these results highlight that E2-2 modulates pDC function in a species-specific manner, which may provide insights for pDC development and functions.

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Figures

Figure 1

Figure 1. E2-2 expression in GEN2.2 is silenced by shRNAs.

(A) E2-2 expression in GEN2.2 cells, purified human pDCs and PBMCs (n = 5) was measured by real-time PCR. (B) Immunobloting analysis of E2-2 in lysates of 293T cells transfected with E2-2 overexpression plasmid and control shRNA or E2-2-specific shRNA. GFP was used as a loading control. (C) E2-2 expression in GEN2.2 cells transduced with or without control shRNA or E2-2-specific shRNA was measured by real-time PCR.. * indicated p < 0.05. All the gels were run under the same experimental conditions as detailed in the Methods section, and full-length blots were cropped for final display.

Figure 2

Figure 2. Decreased IFNα and IL-6 production and higher T cell proliferation by E2-2 knockdown GEN2.2 cells.

(A) and (B) GEN2.2 cells were incubated with CpG B for 20 h. Different concentrations (0 ~ 1 μM) of CpG B stimulated cell supernantant was analyzed (right panel) and 0.2 μM was shown (left panel). (A) IFN-α in supernatants of E2-2 knockdown and control GEN2.2 cells was measured by ELISA. *** indicated p < 0.001. (B) IL-6 in supernatants of E2-2 knockdown and control GEN2.2 cells was measured by ELISA. ** indicated p < 0.01. (C) T cell priming capacity of E2-2 knockdown GEN2.2 cells. GEN2.2 cells transduced with or without control shRNA or E2-2-specific shRNA were co-cultured with CD4+CD45RA+ T cells, which were purified and labeled with CFSE. 7 days later, cells were analyzed for CFSE levels by flow cytometry. Numbers indicated percentage of CFSElow(proliferated) cells. Data are representative of at least three independent experiments.

Figure 3

Figure 3. Gene program regulated by E2-2 in human pDCs.

(A) Pairwise comparison of expression profile for E2-2 knockdown and control GEN2.2 cells. The scatter plot represents normalized log intensities of individual probes, with the probes increased or decreased >2 fold in GEN2.2 cells with E2-2-specific shRNA (E2-2i) and control shRNA (Ctrl) indicated in red and green, respectively. Some probes are highlighted in blue. (B) Expression of CLEC4C, GZMB, TCL1B and Siglec-6 in GEN2.2 cells with or without control shRNA or E2-2-specific shRNA. *, ** and *** indicated p < 0.05, p < 0.01 and p < 0.001.

Figure 4

Figure 4. Siglec-6 and E2-2 expression on human pDC subsets.

(A) Flow cytometry analysis of Siglec-6 expression on human pDCs from lineage negative PBMCs. Primary pDCs were marked as HLA-DR-CD11c-CD123+ and divided into two subsets according to Siglec-6 expression. (B) Siglec-6 and E2-2 expression in Siglec-6high or Siglec-6low pDC subsets. ** indicated p < 0.01. Data are representative of at least three donors.

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