Leukocyte mono-immunoglobulin-like receptor 8 (LMIR8)/CLM-6 is an FcRγ-coupled receptor selectively expressed in mouse tissue plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) produce large amounts of type-I interferon (IFN) in response to viral infection or self nucleic acids. Leukocyte mono-immunoglobulin-like receptor 8 (LMIR8), also called CMRF-35-like molecule-6 (CLM-6), is a putative activating receptor among mouse LMIR/CLM/CD300 members; however, the expression and function of LMIR8 remain unclear. Here, we characterize mouse LMIR8 as a pDC receptor. Analysis of Flag-tagged LMIR8-transduced bone marrow (BM)-derived mast cells demonstrated that LMIR8 can transmit an activating signal by interacting with immunoreceptor tyrosine-based activating motif (ITAM)-containing FcRγ. Flow cytometric analysis using a specific antibody for LMIR8 showed that LMIR8 expression was restricted to mouse pDCs residing in BM, spleen, or lymph node. FcRγ deficiency dampened surface expression of LMIR8 in mouse pDCs. Notably, LMIR8 was detected only in pDCs, irrespective of TLR9 stimulation, suggesting that LMIR8 is a suitable marker for pDCs in mouse tissues; LMIR8 is weakly expressed in Flt3 ligand-induced BM-derived pDCs (BMpDCs). Crosslinking of transduced LMIR8 in BMpDCs with anti-LMIR8 antibody did not induce IFN-α production, but rather suppressed TLR9-mediated production of IFN-α. Taken together, these observations indicate that LMIR8 is an FcRγ-coupled receptor selectively expressed in mouse tissue pDCs, which might suppress pDC activation through the recognition of its ligands.

The CD300e molecule in mice is an immune-activating receptor.

CD300 molecules (CD300s) belong to paired activating and inhibitory receptor families, which mediate immune responses. Human CD300e (hCD300e) is expressed in monocytes and myeloid dendritic cells and transmits an immune-activating signal by interacting with DNAX-activating protein 12 (DAP12). However, the CD300e ortholog in mice (mCD300e) is poorly characterized. Here, we found that mCD300e is also an immune-activating receptor. We found that mCD300e engagement triggers cytokine production in mCD300e-transduced bone marrow-derived mast cells (BMMCs). Loss of DAP12 and another signaling protein, FcRγ, did not affect surface expression of transduced mCD300e, but abrogated mCD300e-mediated cytokine production in the BMMCs. Co-immunoprecipitation experiments revealed that mCD300e physically interacts with both FcRγ and DAP12, suggesting that mCD300e delivers an activating signal via these two proteins. Binding and reporter assays with the mCD300e extracellular domain identified sphingomyelin as a ligand of both mCD300e and hCD300e. Notably, the binding of sphingomyelin to mCD300e stimulated cytokine production in the transduced BMMCs in an FcRγ- and DAP12-dependent manner. Flow cytometric analysis with an mCD300e-specific Ab disclosed that mCD300e expression is highly restricted to CD115+Ly-6Clow/int peripheral blood monocytes, corresponding to CD14dim/+CD16+ human nonclassical and intermediate monocytes. Loss of FcRγ or DAP12 lowered the surface expression of endogenous mCD300e in the CD115+Ly-6Clow/int monocytes. Stimulation with sphingomyelin failed to activate the CD115+Ly-6Clow/int mouse monocytes, but induced hCD300e-mediated cytokine production in the CD14dimCD16+ human monocytes. Taken together, these observations indicate that mCD300e recognizes sphingomyelin and thereby regulates nonclassical and intermediate monocyte functions through FcRγ and DAP12.

Disrupting ceramide-CD300f interaction prevents septic peritonitis by stimulating neutrophil recruitment.

Sepsis is a serious clinical problem. Negative regulation of innate immunity is associated with sepsis progression, but the underlying mechanisms remains unclear. Here we show that the receptor CD300f promotes disease progression in sepsis. CD300f -/- mice were protected from death after cecal ligation and puncture (CLP), a murine model of septic peritonitis. CD300f was highly expressed in mast cells and recruited neutrophils in the peritoneal cavity. Analysis of mice (e.g., mast cell-deficient mice) receiving transplants of wild-type or CD300f -/- mast cells or neutrophils indicated that CD300f deficiency did not influence intrinsic migratory abilities of neutrophils, but enhanced neutrophil chemoattractant production (from mast cells and neutrophils) in the peritoneal cavity of CLP-operated mice, leading to robust accumulation of neutrophils which efficiently eliminated Escherichia coli. Ceramide-CD300f interaction suppressed the release of neutrophil chemoattractants from Escherichia coli-stimulated mast cells and neutrophils. Administration of the reagents that disrupted the ceramide-CD300f interaction prevented CLP-induced sepsis by stimulating neutrophil recruitment, whereas that of ceramide-containing vesicles aggravated sepsis. Extracellular concentrations of ceramides increased in the peritoneal cavity after CLP, suggesting a possible role of extracellular ceramides, CD300f ligands, in the negative-feedback suppression of innate immune responses. Thus, CD300f is an attractive target for the treatment of sepsis.

Ceramide-CD300f binding suppresses experimental colitis by inhibiting ATP-mediated mast cell activation.

OBJECTIVE: Extracellular ATP mediates mast cell-dependent intestinal inflammation via P2X7 purinoceptors. We have previously shown that CD300f (also called the leucocyte mono-immunoglobulin-like receptor 3 (LMIR3)) suppresses immunoglobulin E-dependent and mast cell-dependent allergic responses by binding to ceramide. The aim of the present study was to clarify the role of ceramide-LMIR3 interaction in the development of IBD. DESIGN: The dextran sodium sulfate (DSS)-induced colitis model was used in wild-type (WT), LMIR3(-/-), mast cell-deficient Kit(W-sh/W-sh), Kit(W-sh/W-sh)LMIR3(-/-) or Kit(W-sh/W-sh) mice engrafted with WT or LMIR3(-/-) bone marrow-derived mast cells (BMMCs). The severity of colitis was determined by clinical and histological criteria. Lamina propria cell populations were assessed by flow cytometry. Production of chemical mediators from lamina propria cells was measured by real-time reverse transcription PCR. Production of chemical mediators from ATP-stimulated BMMCs in the presence or absence of ceramide was measured by ELISA. The severity of DSS-induced colitis was assessed in mice given either an Fc fusion protein containing an extracellular domain of LMIR3, and anticeramide antibody, or ceramide liposomes. RESULTS: LMIR3 deficiency exacerbated DSS-induced colitis in mice. Kit(W-sh/W-sh) mice harbouring LMIR3(-/-) mast cells exhibited more severe colitis than those harbouring WT mast cells. Ceramide-LMIR3 interaction inhibited ATP-stimulated activation of BMMCs. DSS-induced colitis was aggravated by disrupting the ceramide-LMIR3 interaction, whereas it was suppressed by treating with ceramide liposomes. CONCLUSIONS: LMIR3-deficient colonic mast cells were pivotal in the exacerbation of DSS-induced colitis in LMIR3(-/-) mice. Ceramide liposomes attenuated DSS-induced colitis by inhibiting ATP-mediated activation of colonic mast cells through ceraimide-LMIR3 binding.

A novel cell-cycle-indicator, mVenus-p27K-, identifies quiescent cells and visualizes G0-G1 transition.

The quiescent (G0) phase of the cell cycle is the reversible phase from which the cells exit from the cell cycle. Due to the difficulty of defining the G0 phase, quiescent cells have not been well characterized. In this study, a fusion protein consisting of mVenus and a defective mutant of CDK inhibitor, p27 (p27K(-)) was shown to be able to identify and isolate a population of quiescent cells and to effectively visualize the G0 to G1 transition. By comparing the expression profiles of the G0 and G1 cells defined by mVenus-p27K(-), we have identified molecular features of quiescent cells. Quiescence is also an important feature of many types of stem cells, and mVenus-p27K(-)-transgenic mice enabled the detection of the quiescent cells with muscle stem cell markers in muscle in vivo. The mVenus-p27K(-) probe could be useful in investigating stem cells as well as quiescent cells.

Hes1 upregulation contributes to the development of FIP1L1-PDGRA-positive leukemia in blast crisis.

We have previously shown that elevated expression of Hairy enhancer of split 1 (Hes1) contributes to blast crisis transition in Bcr-Abl-positive chronic myelogenous leukemia. Here we investigate whether Hes1 is involved in the development of other myeloid neoplasms. Notably, Hes1 expression was elevated in only a few cases of 65 samples with different types of myeloid neoplasms. Interestingly, elevated expression of Hes1 was found in two of five samples of Fip1-like1 platelet-derived growth factor receptor-α (FIP1L1-PDGFA)-positive myeloid neoplasms associated with eosinophilia. Whereas FIP1L1-PDGFRα alone induced acute T-cell leukemia or myeloproliferative neoplasms in mouse bone marrow transplantation models, mice transplanted with bone marrow cells expressing both Hes1 and FIP1L1-PDGFRα developed acute leukemia characterized by an expansion of myeloid blasts and leukemic cells without eosinophilic granules. FIP1L1-PDGFRα conferred cytokine-independent growth to Hes1-transduced common myeloid progenitors, interleukin-3-dependent cells. Imatinib inhibited the growth of common myeloid progenitors expressing Hes1 with FIP1L1-PDGFRα, but not with imatinib-resistant FIP1L1-PDGFRα mutants harboring T674I or D842V. In contrast, ponatinib efficiently eradicated leukemic cells expressing Hes1 and the imatinib-resistant FLP1L1-PDGFRΑ mutant in vitro and in vivo. Thus, we have established mouse models of FIP1L1-PDGFRA-positive leukemia in myeloid blast crisis, which will help elucidate the pathogenesis of the disease and develop a new treatment for it.

Myelodysplastic syndromes are induced by histone methylation­altering ASXL1 mutations.

Recurrent mutations in the gene encoding additional sex combs-like 1 (ASXL1) are found in various hematologic malignancies and associated with poor prognosis. In particular, ASXL1 mutations are common in patients with hematologic malignancies associated with myelodysplasia, including myelodysplastic syndromes (MDSs), and chronic myelomonocytic leukemia. Although loss-of-function ASXL1 mutations promote myeloid transformation, a large subset of ASXL1 mutations is thought to result in stable truncation of ASXL1. Here we demonstrate that C-terminal­truncating Asxl1 mutations (ASXL1-MTs) inhibited myeloid differentiation and induced MDS-like disease in mice. ASXL1-MT mice displayed features of human-associated MDS, including multi-lineage myelodysplasia, pancytopenia, and occasional progression to overt leukemia. ASXL1-MT resulted in derepression of homeobox A9 (Hoxa9) and microRNA-125a (miR-125a) expression through inhibition of polycomb repressive complex 2­mediated (PRC2-mediated) methylation of histone H3K27. miR-125a reduced expression of C-type lectin domain family 5, member a (Clec5a), which is involved in myeloid differentiation. In addition, HOXA9 expression was high in MDS patients with ASXL1-MT, while CLEC5A expression was generally low. Thus, ASXL1-MT­induced MDS-like disease in mice is associated with derepression of Hoxa9 and miR-125a and with Clec5a dysregulation. Our data provide evidence for an axis of MDS pathogenesis that implicates both ASXL1 mutations and miR-125a as therapeutic targets in MDS.

Fyn is not essential for Bcr-Abl-induced leukemogenesis in mouse bone marrow transplantation models.

The Bcr-Abl oncogene causes human Philadelphia chromosome-positive (Ph(+)) leukemias, including B-cell acute lymphoblastic leukemia (B-ALL) and chronic myeloid leukemia (CML) with chronic phase (CML-CP) to blast crisis (CML-BC). Previous studies have demonstrated that Src family kinases are required for the induction of B-ALL, but not for CML, which is induced by Bcr-Abl in mice. In contrast, it has been reported that Fyn is up-regulated in human CML-BC compared with CML-CP, implicating Fyn in the blast crisis transition. Here, we aimed to delineate the exact role of Fyn in the induction/progression of Ph(+) leukemias. We found that Fyn is expressed in mouse hematopoietic cells at varying stages of development, including c-kit(+)Sca-1(+)Lin(-) cells. Notably, Fyn is highly expressed in some of human lymphomas, but not in human Ph(+) leukemias including CML-BC. In mouse bone marrow transplantation models, mice transplanted with wild-type or Fyn-deficient bone marrow cells transduced with Bcr-Abl showed no differences in the development of B-ALL or CML-like diseases. Similarly, Fyn deficiency failed to impact the development of myeloid CML-BC induced by Bcr-Abl and Hes1. Elevated expression of Fyn was not found in mouse samples of Bcr-Abl-mediated CML- and CML-BC-like diseases. Thus, Fyn is not required for the pathogenesis of Bcr-Abl-mediated leukemias.

Characterization of leukocyte mono-immunoglobulin-like receptor 7 (LMIR7)/CLM-3 as an activating receptor: its similarities to and differences from LMIR4/CLM-5.

Here we characterize leukocyte mono-Ig-like receptor 7 (LMIR7)/CLM-3 and compare it with an activating receptor, LMIR4/CLM-5, that is a counterpart of an inhibitory receptor LMIR3/CLM-1. LMIR7 shares high homology with LMIR4 in the amino acid sequences of its Ig-like and transmembrane domains. Flow cytometric analysis demonstrated that LMIR4 was predominantly expressed in neutrophils, whereas LMIR7 was highly expressed in mast cells and monocytes/macrophages. Importantly, LMIR7 engagement induced cytokine production in bone marrow-derived mast cells (BMMCs). Although FcRγ deficiency did not affect surface expression levels of LMIR7, it abolished LMIR7-mediated activation of BMMCs. Consistently we found significant interaction of LMIR7-FcRγ, albeit with lower affinity compared with that of LMIR4-FcRγ. Our results showed that LMIR7 transmits an activating signal through interaction with FcRγ. In addition, like LMIR4, LMIR7 synergizes with TLR4 in signaling. Analysis of several chimera receptors composed of LMIR4 and LMIR7 revealed these findings: 1) the transmembrane of LMIR7 with no charged residues maintained its surface expression at high levels in the absence of FcRγ; 2) the extracellular juxtamembrane region of LMIR7 had a negative effect on its surface expression levels; and 3) the strong interaction of LMIR4 with FcRγ depended on the extracellular juxtamembrane region as well as the transmembrane domain of LMIR4. Thus, LMIR7 shares similarities with LMIR4, although they are differentially regulated in their distribution, expression, and function.