Background
Some multiple immune receptors, such as signal regulatory protein (SIRP), CD200R, killer inhibitory receptor (KIR), lymphocyte antigen 49 (Ly49), CD300, have a highly homologous extracellular domain [
1‐
4]. These receptors are termed “paired receptors” that are generally defined by the following characteristics: 1. they have a high level of amino acid sequence identity within their extracellular domains, 2. they are encoded by different genes, but located in the same gene cluster, 3. they have both activating receptor associated by immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor, such as DNAX activating protein of 12 kDa (DAP12), and immunoreceptor tyrosine-based inhibition motif (ITIM)-containing inhibitory receptors [
4‐
6]. Their activating and inhibitory receptors recognize endogenous/exogenous components and often differ in their binding specificity. Paired receptors expressed on myeloid cells including dendritic cell (DC) subsets have been poorly understood in their functional features concerning innate-adaptive interplay, whereas paired NK receptors including Ly49, KIR, and Natural Killer group 2 (NKG2)/CD94, have been characterized to explain the “missing self” activity of NK cells [
3,
7]. Therefore, we attempted to investigate the role of uncharacterized paired receptors in myeloid cells. We first searched for genes of myeloid paired receptors in mammalian genome database and analyzed their expression profiling and immune-related function.
Triggering receptors expressed on myeloid cells (Trem) proteins are a family of cell surface receptors expressed broadly on myeloid cells [
8]. The receptors are a structurally-related protein family that generally contain a single immunoglobulin (Ig) domain in the extracellular region, and their gene cluster is located on mouse chromosome 17C and human chromosome 6p21.1 [
9,
10]. Inhibitory Trem proteins, containing cytoplasmic ITIM recruit protein tyrosine phosphatases such as Src homology 2 domain-containing phosphatase 1 (SHP-1) and SHP-2 [
11]. On the other hand, activating Trem proteins lack cytoplasmic signaling elements, but contain a charged residue in their transmembrane domain that facilitates the association with DAP12, which functions as docking sites for Syk tyrosine kinases that mediate PLCγ and PI3K activation [
12,
13].
Trem and Trem-like genes such as novel immune type receptors have also been reported in teleost [
14‐
16]. Human Trem1 is an activating receptor expressed at high levels on neutrophils and monocytes that infiltrate human tissues infected with bacteria [
17]. Recent studies have shown that Trem1 has a critical role in negative regulation of proinflammatory cytokine production in vivo [
18,
19]. Trem2 is also activating receptor expressed on DCs, osteoclasts and microglia and control not only immune function such as maturation of DCs but also osteoclastogenesis [
20,
21]. Functional DAP12-associated Trem3 is present in mice, but not primates [
22]. Treml4 binds to dead cells and accelerates antigen presentation and priming of CD4
+ and CD8
+ T cells in vivo [
23,
24]. Trem4 (also known as pDC-Trem) is involved in IFN-α production from Fms-related tyrosine kinase 3 (Flt3) ligand-induced plasmacytoid DCs (pDC), and TLR signals are essential for Trem4 expression [
25]. Nevertheless, other Trem and Treml receptors have been poorly characterized in mice, although they show myeloid cell-dominant expression [
8]. There is so far no report on the comprehensive analysis of the mouse Trem/Treml gene cluster, and we found paired receptor-like signatures in several Trem receptors on database search.
In this study, we focused on orphan and uncharacterized Trem genes, named murine Trem4 (deposited as pDC-Trem [
25]), Trem5 (known as 9830107B12Rik) and Treml6 (known as B430306N03Rik), and elucidated their evolutional stands in vertebrates. The ligands of these receptors remain undetermined, but we noticed that polyI:C signal dynamically up-regulated the Trem4 and Treml6. Although the extracellular immunoglobulin (Ig) domain of Trem4, Trem5 and Treml6 were closely related to each other, Treml6 encoded ITIM in its cytoplasmic region, whereas Trem4 and Trem5 lacked ITIM but had positively-charged residues to associate with DAP12. Together, these expression features indicate that these receptors are typical paired receptors, which modulate innate signal in myeloid cells like the case of Trem1 and Trem2 [
26]. Trem4 and Treml6 may be up-regulated on the cell-surface in myeloid cells by viral RNA to counteract or modulate presetting immune response.
Methods
Mice
All mice were backcrossed with C57BL/6 mice more than seven times before use.
Ifnar1
−/− mice were obtained from the Jackson Laboratory.
Ticam-1
−/− and
Mavs
−/− mice were generated in our laboratory [
27,
28]. All mice were maintained under specific pathogen-free conditions in the animal facility of the Hokkaido University Graduate School of Medicine. Female mice 8–12 weeks of age were used in all experiments, all of which were performed according to the guidelines issued by Hokkaido University Animal Care and Use Committee, who approved this study as ID number: 08–0243, “Analysis of immune modulation by toll-like receptors”. Mice were sacrificed according to the rule of animal euthanasia. Animal experiments protocols and guidelines were approved by the Animal Safety Center, Hokkaido University, Japan.
Cells
Bone marrow-derived dendritic cells (BMDCs) were generated by culturing bone-marrow cells for 6 days in RPMI 1640 (GIBCO Invitrogen GmbH, Karlsruhe, Germany) with 10 % heat-inactivated FBS containing GM-CSF (10 ng/ml, Peprotech, Rocky Hills, NJ, USA). Bone marrow-derived macrophages (BMDMs) were prepared by culturing bone-marrow cells in RPMI 1640 for 6 days with 10 % heat-inactivated FBS containing 30 % L929 supernatant. Concentrations of stimulants used were as follows: Pam3CSK4 (Roche, Indianapolis, IN, USA), 5 μg/ml; polyI:C (GE Healthcare Biosciences, Uppsala, Sweden), 20 μg/ml; LPS (Sigma–Aldrich, St. Louis, MO, USA), 0.1 μg/ml; Flagellin A (FlaA), 0.3 μg/ml; CpG (M362, Invivogen, San Diego, CA, USA), 2.5 μg/ml with DOTAP (Roche, Indianapolis, IN, USA). FlaA was synthesized in our previous study. Wild-type, Ticam-1
−/−, Mavs
−/− and Ifnar1
−/− BMDCs were stimulated with polyI:C (20 μg/ml). For blocking experiment of type I IFN receptor, anti-IFN receptor-neutralizing antibody (Biolegend, San Diego, CA, USA) or isotype control antibody (Biolegend, San Diego, CA, USA) were added at a final concentration of 10 μg/ml. HEK293FT cells were maintained in DMEM (GIBCO Invitrogen GmbH, Karlsruhe, Germany) supplemented with 10 % heat-inactivated FBS and antibiotics. For isolation of cDCs and pDCs, spleens were treated with 400 IU Mandle U/ml collagenase D (Roche, Indianapolis, IN, USA) at 37 °C for 25 min in HBSS (Sigma–Aldrich, St. Louis, MO, USA). EDTA was added, and the cell suspension was incubated for an additional 5 min at 37 °C. After removal of red blood cells with ammonium chloride–potassium lysis buffer, CD11c+ DCs were isolated using CD11c and PDCA-1 MACS beads (Miltenyi Biotec, Auburn, CA, USA). MACS-sorted DCs were stained with anti-B220–FITC (RA3-6B2, eBiosciences, San Diego, CA, USA) and anti-CD11c–APC (N418, Biolegend, San Diego, CA, USA) and CD11c+/B220− cDCs and CD11c+/B220+ pDCs were sorted by using FACS Aria II (BD Biosciences, San Jose, CA, USA). The purity of sorted cells was > 98 %.
Database analysis
A BLAST search of the vertebrate Trem genes was performed by the amino acid sequences of human and mouse Trem genes as queries using the Ensembl genome databases (
http://www.ensembl.org/index.html). The genome assembly versions of species used in this paper were as follows: GRCh37 of human, GRCm38 of mouse, OryCun2.0 of Rabbit, CanFam3.1 of Dog, EquCab2 of Horse, UMD3.1 of Cow, Sscrofa10.2 of Pig, BROADO5 of opossum, Galgal4 of chicken, JGI_4.2 of frog and Zv9 of zebrafish. Domain structures of Trem proteins were analyzed by SMART program (
http://smart.embl-heidelberg.de). A phylogenetic tree based on the amino acid sequences of Ig domain was constructed by the Neighbor-joining (NJ) method in the ClustalX version 2 program and the MEGA version 5 program [
29,
30]. The distance matrix was obtained by calculating p-distances for all pairs of sequences. Sites containing gaps were excluded from the analysis using the pairwise deletion option. The reliability of branching patterns was assessed by bootstrap analysis (1000 replications). Vertebrate Trem genes used for phylogenetic and comparative genomic analysis are listed in supplementary Table
1.
Table 1
Identity matrix comparison of Ig domains in murine Trem proteins
Trem5 | — | — | — |
Treml6 | 80.4 % | — | — |
Trem4 | 78.4 % | 88.7 % | — |
Trem1 | 27.3 % | 23.9 % | 26.4 % |
Trem2 | 23.9 % | 23.9 % | 24.8 % |
Trem3 | 22.2 % | 22.4 % | 23.1 % |
Treml1 | 32.7 % | 21.9 % | 24.3 % |
Treml2 | 24.8 % | 23.2 % | 25.7 % |
Treml4 | 34.0 % | 32.1 % | 34.0 % |
RNA isolation and cDNA synthesis
Total RNA from BMDCs, BMDMs, splenic DC subsets, and tissues was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), after which 0.1–1 mg RNA was reverse transcribed using a high-capacity cDNA transcription kit with an RNase inhibitor kit (Applied Biosystems Foster City, CA, USA), according to the manufacturer’s instructions.
Gene cloning and expression vectors
Murine Treml6/Trem4/DAP12 genes and Trem5 gene were amplified by KOD-plus DNA polymerase (Toyobo, Osaka, Japan) from polyI:C-stimulated BMDCs and unstimulated spleen cDNA, respectively. The primers for the Trem5 gene: 5′-GTGGTGGCTCCTGAATCC-3′ (forward) and 5′-CTCAGCTCCTCCTTTGACATAGACT-3′ (reverse), Treml6 gene: 5′-TGCCCAGCTTCTCTCTCATC-3′ (forward) and 5′-TAGGAAGTTGCCCCTCCACA-3′ (reverse), Trem4 gene: 5′-TGCCCAGCTTCTCTCTCATC-3′ (forward) and 5′-AGCGAACCCTGAGTAATACAGTCAG-3′ (reverse), Dap12 gene: 5′-GCCCCTGGACTGTGGTGT-3′ (forward) and 5′-GATCCCAGAGAGGGCTTGTT-3′ (reverse). The open reading frame (ORF) sequences of Trem5, Treml6, Trem4 and Dap12 genes were completely conformed to reported sequences of NM_001177896, NM_177083, AB372005 and NM_011662, respectively. These cDNA encoding the full-length ORF was cloned into a pEF-BOS multi-cloning site, and an HA or FLAG sequence was inserted behind signal peptide sequence of paired Trem and DAP12 genes, respectively [
31]. The plasmids were sequenced to confirm that there were no PCR errors.
Immunoblotting and immunoprecipitation
HEK293FT cells were transfected in 6-well plates with plasmids encoding FLAG-tagged DAP12 and/or HA-tagged paired Trem genes by using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The plasmid amounts were normalized by the addition of empty plasmid. At 24 h after transfection, cells were lysed with lysis buffer (20 mM Tris–HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 10 % glycerol, 1 % Nonidet P-40, 30 mM NaF, 5 mM Na3VO4, 20 mM iodoacetamide, 2 mM phenylmethylsulfonyl fluoride), and then proteins were immunoprecipitated with mouse anti-HA monoclonal antibody (Sigma–Aldrich, St. Louis, MO, USA). The lysates and precipitates were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with rabbit anti-HA polyclonal antibody (Sigma–Aldrich, St. Louis, MO, USA) or mouse anti-FLAG M2 monoclonal antibody (Sigma–Aldrich, St. Louis, MO, USA).
Flow cytometric analysis
At 24 h after transfection, transfected cells were incubated with FACS buffer (DPBS containing 0.1 % BSA and 0.1 % sodium azide) containing 10 % goat serum (Cedarlane, Ontario, Canada) on ice. After incubation, the cells were washed twice with FACS buffer and labeled with anti-HA polyclonal antibody and anti-FLAG M2 monoclonal antibody for 30 min on ice. After washing, anti-mouse IgG fluorescein (FITC) labeled secondary antibody (American Qualex, San Clemente, CA) and anti-rabbit IgG phycoerythrin (PE)-labeled secondary antibody (Biolegend, San Diego, CA, USA) was added and the cells were further incubated for 30 min on ice. Stained cells were then analyzed using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA).
ELISA
BMDCs (3–5 × 105) seeded on 24-well plates were collected and analyzed for cytokine levels with enzyme-linked immunosorbent assay (ELISA). ELISA kits for mouse IFN-α and IFN-β were purchased from PBL Biomedical Laboratories.
Quantitative real-time PCR
Quantitative real-time PCR (qPCR) was performed using the Step One Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The primers for Trem5 gene: 5′-ATTCAAGTGCTCCTGCCAAT-3′ (forward) and 5′-AATGGAGATCGGGTCCTGT-3′ (reverse), Treml6 gene: 5′-CAGCCCCGTCACTAACAGAA-3′ (forward) and 5′-CGAATGTGAGCAGTAGCAGAA-3′ (reverse), Trem4 gene: 5′-CCACTGTTGTTTCCTCTCCTGA-3′ (forward) and 5′-AGTCCACAGACCACGGGAAT-3′ (reverse), and Gapdh: 5′- GCCTGGAGAAACCTGCCA −3′ (forward) and 5′- CCCTCAGATGCCTGCTTCA −3′ (reverse).
Discussion
This report is the first to provide evidence that mouse has paired Trem receptors in myeloid cells and increases the expression levels in response to type I IFN. The term “paired receptor” is commonly used to describe families of membrane receptors that have similar extracellular regions but different TM and CPR. Trem4/Trem5 vs. Treml6 are typical paired receptors in the murine linage (Figs.
1 and
2b). PolyI:C stimulation triggers the mRNA expression of Trem4 and Treml6 via type I IFN, which is an output produced through the MDA5/MAVS pathway in BMDCs (Figs.
4b,
c,
d and
6c). All paired Trem genes are highly expressed in secondary lymphoid organs such as the spleen, which implies that the pair receptor effect coincides with an event of type I IFN induction. The Syk/SLP76-PKC or PI3K pathways are known to be activated downstream of their signal, where type I IFN also participates. Therefore, Trem4, Trem5 and Treml6 may participate in innate immune response in a myeloid-specific manner.
We also identified two novel ITIM-containing Trem genes, named as Treml7 in the dog and horse genomes and Treml8 in the rabbit and opossum genomes (Fig.
2a,
b). Interestingly, either of these genes are lacking in other mammalian genomes. This finding indicates that species-specific construction occurs in paired Trem receptors. Phylogenetic analysis shows that the murine paired Trem genes are most closely related to Treml7 genes, but not Treml8. Hence, murine paired Trem genes and Treml7 are likely to originate from a common ancestral gene. A possible evolution of murine paired Trem genes is summarized in Fig.
2c. Initially, the ancestral paired Trem gene and Treml7 arose from a common ancestral gene that encoded two ITIMs in its CPR. After branching off of the murine linage, the ancestral paired Trem gene containing ITIMs was duplicated, and the murine Treml6 gene evolved from one of the duplicated genes. Another duplicated gene was translocated between the Trem1 and Foxp4 genes and lost its ITIMs. Finally, the common ancestor of Trem4 and Trem5 genes was duplicated, and evolved into murine Trem4 and Trem5.
Notably, the above-mentioned model clearly shows that activating Trem4 and Trem5 are derived from the ancestral ITIM-containing Trem gene. In human, siglec-14 or human/murine NK receptors fall into the same family [
1,
37‐
39]. Barclay and Hatherley propose the “counterbalance theory” to explain the evolution of paired receptors [
6]. In this theory, the driving force in the evolution of paired receptors is that pathogens target the inhibitory receptors on immune cells and then activating receptors evolve from these inhibitory receptors [
6].
Although macrophages and DCs express Trem4, Trem5 and Treml6, natural ligands for these Trems have not been identified in either endogenous or microbial origin. How these receptors interact with NK cells, therefore, remains undetermined. Trem proteins other than Trem4/Treml6 are involved in inflammatory response [
26]. Trem1 is a pro-inflammatory mediator that amplifies endotoxin-induced shock in sepsis [
17,
40]. Trem1 constitutively associates with DAP12 and recognizes endogenous ligands, which is indispensable for induction of intracellular signals [
17,
41]. The Trem1 signal appears to act in concert with other pattern-recognition receptors (PRRs) including TLRs and modify the cytokine/chemokine profile in myeloid cells, but its physiological mechanism remains largely elusive. Trem2 also couples with DAP12, and make a crosstalk with TLR signaling [
42]. Trem2 essentially (but not simply) suppresses inflammatory responses, although their endogenous ligands are largely unknown [
26,
41,
42]. Trem1 and Trem2 appear differentially work in the context of inflammation and microbial environment.
Watarai et al. showed that the Trem4-DAP12 complex mediates positive signaling for type I IFN production in Flt3 ligand-induced pDCs [
25]. Here we find that direct association of DAP12 with Trem4 and Trem5 is required for their post-translational modification and cell surface expression (Fig.
3). Both receptors may lead to activation of Src-family kinases and subsequent phosphorylation of paired tyrosine residues in the ITAM of DAP12. On the other hand, activation of the MAVS pathway and type I IFN are required for surface-expression of Trem4. PolyI:C induce high surface-expression of Trem4 in the presence of DAP12, while polyI:C alone sustains high expression of Treml6 independent of DAP12. Notably, none of the TLR agonists tested participate in their expression. There is a gene-inducing program in DCs that evokes DC priming in response to internalization of viral RNA [
43], where DAP12-associated Trem4 induction would be involved. Since infected DCs usually induce cell death [
44], viral RNA is likely to be extrinsically taken up into DC/macrophages in the form of exosome or debris. What is responsible for Trem5 expression in combination with DAP12 is unknown. Trem5 expression might be controlled by other innate receptors such as C-type lectin receptors and NOD-like receptors.
BMDMs more efficently induce up-regulation of Trem4 and Treml6 in response to polyI:C compared to BMDCs (Fig.
4a vs. Fig.
5b). Trem-DAP12 complex may facilitate macrophage activation by enhancing phagocytosis and cytokine production. In fact, the Trem2-DAP12 axis plays a critical role in evoking phagocytosis in addition to production of inflammatory cytokines in macrophages and granulocytes in mice challenged with LPS [
45]. Taken together with our results that both Trem4 and Treml6 are expressed through type I IFN signaling in macrophages and DC subsets, Trem proteins may be engaged in fine-tuning of DC maturation and myeloid immune response via the DAP12 axis and IFN-inducing pathways. The repertoire of Trem receptors has been evolutionally selected depending on species and environment. In mouse, Trem proteins modify allergic states and host response to some infections [
46,
47], which will be etiologically connected with the profiles of the Trem expression.
Ethics approval
This research does not involve human materials. All animal research protocols for this work were reviewed and approved by the Animal Safety Center, Hokkaido University, Japan.
Availability of data and materials
The dataset supporting the conclusions of this article is included within the article.
Acknowledgements
Jun Kasamatsu was a Research Fellow of the Japan Society for the Promotion of Science. We thank Drs. H. Takaki, A. Maruyama, L. C. Ring, T. Suzuki, M. Tatematsu, M. Okamoto and Ms. Sato in our laboratory for their fruitful discussions. We also thank Ms. K. Matsumura (Tohoku University Graduate School of Medicine) for technical \ort.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Design of the research, JK, MM, TS; Performing experiments, JK, MD, KF; Data interpretation, JK, MD, MM, MK, KF, HO, TS; Supplying materials, MM, MA, HS, MK, HO, TS; Writing manuscript, JK, TS. All authors read and approved the final manuscript.