Double-stranded RNA analog and type I interferon regulate expression of Trem paired receptors in murine myeloid cells

Abstract

Background: Triggering receptors expressed on myeloid cells (Trem) proteins are a family of cell surface receptors used to control innate immune responses such as proinflammatory cytokine production in mice. Trem genes belong to a rapidly expanding family of receptors that include activating and inhibitory paired-isoforms.

Results: By comparative genomic analysis, we found that Trem4, Trem5 and Trem-like transcript-6 (Treml6) genes typically paired receptors. These paired Trem genes were murine-specific and originated from an immunoreceptor tyrosine-based inhibition motif (ITIM)-containing gene. Treml6 encoded ITIM, whereas Trem4 and Trem5 lacked the ITIM but possessed positively-charged residues to associate with DNAX activating protein of 12 kDa (DAP12). DAP12 was directly associated with Trem4 and Trem5, and DAP12 coupling was mandatory for their expression on the cell surface. In bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs), and splenic DC subsets, polyinosinic-polycytidylic acid (polyI:C) followed by type I interferon (IFN) production induced Trem4 and Treml6 whereas polyI:C or other TLR agonists failed to induce the expression of Trem5. PolyI:C induced Treml6 and Trem4 more efficiently in BMDMs than BMDCs. Treml6 was more potentially up-regulated in conventional DC (cDCs) and plasmacytoid DC (pDCs) than Trem4 in mice upon in vivo stimulation with polyI:C.

Discussion: Treml6-dependent inhibitory signal would be dominant in viral infection compared to resting state. Though no direct ligands of these Trem receptors have been determined, the results infer that a set of Trem receptors are up-regulated in response to viral RNA to regulate myeloid cell activation through modulation of DAP12-associated Trem4 and ITIM-containing Treml6.

Keywords: Dendritic cells; Evolution; Macrophages; Paired receptors; RNA sensors; Trem family; Type I interferon.

Fig. 1

Deduced amino acid sequences of murine paired Trems, Treml7 and Treml8. Murine paired Trems and other mammalian Treml7 and Treml8 are aligned. Boxs indicate potential N-linked glycosylation sites predicted by NetNGlyc program (http://www.cbs.dtu.dk/services/NetNGlyc/). ‘#’ represents the ITIM, defined as (I/V/L/S)-X-Y-X-X-(L/V). ‘+’ represents the lysine residue in the transmembrane region (TM), which is presumed to be involved in the interaction with DAP12. SP, signal peptide; Ig, immunoglobulin; CPR, cytoplasmic region. Black shaded area, 100–90 % identity; gray shaded area, 89–70 % identity; light gray shaded area, 69–50 % identity. Mm; mouse (Mus musculus), Oc; rabbit (Oryctolagus cuniculus), Cf; dog (Canis lupus familiaris), Ec; horse (Equus caballus)

Fig. 2

Comparative genome analysis of Trem genes in vertebrates. a Synteny block including Trem and their neighbor genes. The arrows represent the genes and their transcriptional orientations annotated in the Ensembl genome data for each species. Gray and black arrows indicate ITIM-containing Trem and other Trem genes, respectively. This figure is not drawn to scale. b Neighbor-joining tree of vertebrate Trem genes based on extracellular Ig domains. Numbers at the nodes represent the bootstrap confidence level in percentage. Only bootstrap values over 40 are indicated. Hs; human (Homo sapiens), Mm; mouse (Mus musculus), Oc; rabbit (Oryctolagus cuniculus), Cf; dog (Canis lupus familiaris), Ec; horse (Equus caballus), Bt; cow (Bos taurus), Ss; pig (Sus scrofa), Md; opossum (Monodelphis domestica), Gg; chicken (Gallus gallus), Xt; frog (Xenopus tropicalis). c Evolutionary model of murine paired Trem genes. Firstly, ancestral paired Trem gene and Treml7 rose from common ancestral gene that encored two ITIMs in its CPR. After brunching to murine linage, ancestral paired Trem gene containing ITIMs were duplicated, and the one of them evolved murine Treml6 gene. Another duplicated gene was translocated between Trem1 and Foxp4 genes and lost ITIMs. Finally, the common ancestor of Trem5 and Trem4 genes was duplicated, and evolved murine Trem5 and Trem4

Fig. 3

DAP12 binds Trem5 and Trem4 and facilitates Trem’s post-translational modification and cell-surface expression. a DAP12 is required for the post-translational modification of Trem5 and Trem4. Whole-cell lysates (WCL) prepared from HEK293FT cells transfected with the indicated combinations of HA-paired Trem proteins and/or FLAG-DAP12 were analyzed by immunoblotting with anti-FLAG or anti-HA Abs. b Association of DAP12 with paired Trem proteins. WCL prepared from HEK293FT cells transfected with the indicated combinations of HA-paired Trem proteins and FLAG-DAP12 were immunoprecipitated with anti-HA Ab and detected with anti-FLAG or anti-HA Abs. c DAP12 is required for cell surface expression of Trem5 and Trem4. Paired Trems were transfected into HEK293FT cells with or without DAP12. HA-paired Trem proteins on HEK293FT cells were analyzed using anti-HA mAb by FACS

Fig. 4

Expression of paired Trem genes in BMDCs. a Paired Trems expression after TLR agonist stimulations in BMDCs. Concentrations of stimulants used were as follows: Pam3CSK4, 5 μg/ml; polyI:C (pIC), 20 μg/ml; LPS, 0.1 μg/ml; Flagellin A (FlaA), 0.3 μg/ml; CpG, 2.5 μg/ml with DOTAP. b Type I IFN production in Wild-type, Ticam-1 ^−/− and Mavs ^−/− BMDCs. BMDCs prepared from each mouse line were stimulated with pIC (20 μg/ml). 24 h later, the mounts of IFNα and IFNβ in culture supernatants were measured by ELISA. c Treml6 and Trem4 mRNA expressions after pIC stimulation in KO mice-derived BMDCs. Wild-type, Ticam-1−/−, Mavs−/− and Ifnar1−/− derived BMDCs were stimulated by pIC (20 μg/ml) for 12 h. d Type I IFN signal is indispensable for Treml6 and Trem4 expression of BMDCs derived from Wild-type mice. BMDCs were stimulated with pIC (20 μg/ml) and anti-IFN receptor blocking antibody or isotype control (10 μg/ml) for 12 h. In these experiments, mRNA expressions of paired Trem genes were measured by qPCR and their expression levels were normalized to that of Gapdh mRNA. Data are representative of three independent experiments. The data shown are representative of at least two independent experiments. Data are means ± SD of three independent samples. *p < 0.05. **p < 0.01

Fig. 5

Expression of paired Trem genes in BMDMs. a. Dose-dependent expression of paired Trem genes in response to polyI:C stimulation in BMDMs. Day 7-harvested BMDMs (2 × 10⁵ cells /well) were incubated with polyI:C for 12 h, the concentration of polyI:C were 10 μg/ml, 20 μg/ml and 50 μg/ml. b Time-dependent expression of paired Trem genes in response to polyI:C stimulation in BMDMs. Day 7-harvested BMDMs (2 × 10⁵ cells /well) were incubated with polyI:C by the concentration of 20 μg/ml. The incubation times with polyI:C were 4, 8 and 12 h. Relative expression levels of Trem genes were evaluated by qPCR. The data shown are representative of at least two independent experiments. Data are means ± SD of three independent samples. *p < 0.05. **p < 0.01

Fig. 6

Expression of paired Trem genes in tissues and splenic DC subsets. a Tissue distributions of paired Trem transcripts in adult healthy mice. N.D., not detected. b Paired Trem expression after polyI:C stimulation in spleen. Wild-type mice were intraperitoneally injected with polyI:C (200 μg/ml). After 3 h, total RNA was isolated from spleen in each mouse. c Treml6 and Trem4 expression after polyI:C stimulation in splenic cDCs and pDCs. Wild-type mice were intraperitoneally injected with polyI:C (200 μg/ml). After 3 h, total RNA was isolated from FACS-sorted cDCs and pDCs. In these experiments, mRNA expressions of paired Trem genes were measured by qPCR and their expression levels were normalized to that of Gapdh mRNA. Data are representative of three independent experiments. The data shown are representative of at least two independent experiments. Data are means ± SD of three independent samples. *p < 0.05. **p < 0.01