Mary Beth Humphrey

Cutting Edge: Inhibition of TLR and FcR Responses in Macrophages by Triggering Receptor Expressed on Myeloid Cells (TREM)-2 and DAP12

DAP12 is an ITAM-containing adapter that associates with receptors in myeloid and NK cells. DAP12-associated receptors can give activation signals leading to cytokine production; however, in some situations, DAP12 inhibits cytokine production stimulated through TLRs and FcRs. Here we show that Triggering Receptor Expressed on Myeloid cells (TREM)-2 is responsible for the DAP12-mediated inhibition in mouse macrophages. A chimeric receptor composed of the extracellular domain of TREM-2 and the cytoplasmic domain of DAP12 inhibited the TLR- and FcR-induced TNF production of DAP12-deficient macrophages, whereas a TREM-1 chimera did not. In wild-type macrophages, TREM-2 knockdown increased TLR-induced TNF production. A TREM-2 Fc fusion protein bound to macrophages, indicating that macrophages express a TREM-2 ligand. Thus, the interaction of TREM-2 and its ligand results in an inhibitory signal that can reduce the inflammatory response.

Role of ITAM‐containing adapter proteins and their receptors in the immune system and bone

Summary:  The immunoreceptor tyrosine‐based activation motif (ITAM) is a highly conserved region in the cytoplasmic domain of signaling chains and receptors and is a critical mediator of intracellular signals. ITAM‐mediated signals depend on the Syk or ζ‐associated protein of 70 kDa tyrosine kinases, and ITAM signaling is required for the differentiation and function of B and T cells in adaptive immunity. ITAM‐dependent receptors also regulate the function of innate immune cells, including natural killer cells, and myeloid‐derived cells such as macrophages, neutrophils, dendritic cells, and mast cells. Myeloid lineage cells also include osteoclasts (OCLs), the cells required for bone resorption, and recent studies show a critical role for the ITAM‐containing adapter proteins DAP12 and the FcRγ chain (Fcɛ receptor I γ chain) in OCL differentiation. Mice deficient in both the DAP12 and FcRγ ITAM‐bearing adapters are significantly osteopetrotic with a severe defect in OCL differentiation, demonstrating the requirement for ITAM signals in bone and further implicating this pathway in the development of highly specialized cell functions in hematopoietic cells. Regulation of osteoclastogenesis by ITAM‐dependent receptors suggests that OCLs, similar to related myeloid cells, are tightly controlled by arrays of receptors that allow them to sense and respond to their local microenvironment like other innate immune cells.

Association of a functional variant downstream of TNFAIP3 with systemic lupus erythematosus

Systemic lupus erythematosus (SLE, MIM152700) is an autoimmune disease characterized by self-reactive antibodies resulting in systemic inflammation and organ failure. TNFAIP3, encoding the ubiquitin-modifying enzyme A20, is an established susceptibility locus for SLE. By fine mapping and genomic re-sequencing in ethnically diverse populations, we fully characterized the TNFAIP3 risk haplotype and identified a TT>A polymorphic dinucleotide (deletion T followed by a T to A transversion) associated with SLE in subjects of European (P = 1.58 × 10−8, odds ratio = 1.70) and Korean (P = 8.33 × 10−10, odds ratio = 2.54) ancestry. This variant, located in a region of high conservation and regulatory potential, bound a nuclear protein complex composed of NF-κB subunits with reduced avidity. Further, compared with the non-risk haplotype, the haplotype carrying this variant resulted in reduced TNFAIP3 mRNA and A20 protein expression. These results establish this TT>A variant as the most likely functional polymorphism responsible for the association between TNFAIP3 and SLE.

Chronic Exposure to a TLR Ligand Injures Hematopoietic Stem Cells

Hematopoietic stem cells (HSC) can be harmed by disease, chemotherapy, radiation, and normal aging. We show in this study that damage also occurs in mice repeatedly treated with very low doses of LPS. Overall health of the animals was good, and there were relatively minor changes in marrow hematopoietic progenitors. However, HSC were unable to maintain quiescence, and transplantation revealed them to be myeloid skewed. Moreover, HSC from treated mice were not sustained in serial transplants and produced lymphoid progenitors with low levels of the E47 transcription factor. This phenomenon was previously seen in normal aging. Screening identified mAbs that resolve HSC subsets, and relative proportions of these HSC changed with age and/or chronic LPS treatment. For example, minor CD150HiCD48− populations lacking CD86 or CD18 expanded. Simultaneous loss of CD150Lo/−CD48− HSC and gain of the normally rare subsets, in parallel with diminished transplantation potential, would be consistent with age- or TLR-related injury. In contrast, HSC in old mice differed from those in LPS-treated animals with respect to VCAM-1 or CD41 expression and lacked proliferation abnormalities. HSC can be exposed to endogenous and pathogen-derived TLR ligands during persistent low-grade infections. This stimulation might contribute in part to HSC senescence and ultimately compromise immunity.

Osteoporosis in inflammatory bowel disease.

Osteoporosis commonly afflicts patients with inflammatory bowel disease, and many factors link the 2 states together. A literature review was conducted about the pathophysiology of osteoporosis in relation to inflammatory bowel disease. Screening guidelines for osteoporosis in general as well as those directed at patients with inflammatory bowel disease are reviewed, as are currently available treatment options. The purpose of this article is to increase physician awareness about osteopenia and osteoporosis occurring in patients with inflammatory bowel disease and to provide basic, clinically relevant information about the pathophysiology and guidelines to help them treat these patients in a cost-effective manner.

TREM2, a DAP12-associated receptor, regulates osteoclast differentiation and function

Deficiency of the signaling adapter protein DAP12 or its associated receptor TREM2 is associated with abnormal OC development in humans. Here we examine the role of TREM2 in mouse OC development and function, including migration and resorption in vitro. These results provide new evidence that TREM2 regulates OC function independent of its effects on multinucleated OC differentiation.

TREM2- and DAP12-Dependent Activation of PI3K Requires DAP10 and Is Inhibited by SHIP1

The inositol phosphatase SHIP1 binds to a receptor-adaptor complex on osteoclasts to prevent recruitment of PI3K and inhibit receptor signaling. Keeping Osteoclasts in Check Bone resorption is mediated by cells known as osteoclasts, which develop from myeloid precursor cells. Differentiation and activation of osteoclasts depend on the signaling of receptors whose responses are mediated by the adaptor protein DAP12. One such receptor is TREM2, which drives osteoclastogenesis; deficiencies in either DAP12 or TREM2 result in similar phenotypes. TREM2- and DAP12-dependent signaling also inhibits some Toll-like receptor (TLR)–dependent responses in macrophages, another myeloid cell type. Thus, the TREM2-DAP12 signaling axis may be both stimulatory and inhibitory; however, little is known about how DAP12 functions in these different contexts. Peng et al. found that ligation of TREM2 with a cross-linking antibody triggered DAP12-dependent osteoclastogenesis that required the recruitment of phosphatidylinositol 3-kinase (PI3K) to the TREM2-DAP12 signaling complex. This signaling pathway was inhibited by the Src homology 2 (SH2) domain–dependent recruitment to TREM2-DAP12 of the inositol phosphatase SHIP1, which prevented PI3K from binding to DAP12. In addition, signaling downstream of other receptors on osteoclasts differentially recruited SHIP1 to DAP12. The authors thus propose that the stimulatory or inhibitory nature of TREM2-DAP12 signaling is regulated by SHIP1. The activation and fusion of macrophages and of osteoclasts require the adaptor molecule DNAX-activating protein of 12 kD (DAP12), which contains immunoreceptor tyrosine-based activation motifs (ITAMs). TREM2 (triggering receptor expressed on myeloid cells–2) is the main DAP12-associated receptor in osteoclasts and, similar to DAP12 deficiency, loss of TREM2 in humans leads to Nasu-Hakola disease, which is characterized by bone cysts and dementia. Furthermore, in vitro experiments have shown that deficiency in DAP12 or TREM2 leads to impaired osteoclast development and the formation of mononuclear osteoclasts. Here, we demonstrate that the ligation of TREM2 activated phosphatidylinositol 3-kinase (PI3K), extracellular signal–regulated kinase 1 (ERK1) and ERK2, and the guanine nucleotide exchange factor Vav3; induced the mobilization of intracellular calcium (Ca2+) and the reorganization of actin; and prevented apoptosis. The signaling adaptor molecule DAP10 played a key role in the TREM2- and DAP12-dependent recruitment of PI3K to the signaling complex. Src homology 2 (SH2) domain–containing inositol phosphatase-1 (SHIP1) inhibited TREM2- and DAP12-induced signaling by binding to DAP12 in an SH2 domain–dependent manner and preventing the recruitment of PI3K to DAP12. These results demonstrate a previously uncharacterized interaction of SHIP1 with DAP12 that functionally limits TREM2- and DAP12-dependent signaling and identify a mechanism through which SHIP1 regulates key ITAM-containing receptors by directly blocking the binding and activation of PI3K.

The signaling adapter protein DAP12 regulates multinucleation during osteoclast development

Deficiency of the signaling adapter protein DAP12 is associated with bony abnormalities in both mice and humans. We identify specific DAP12‐associated receptors expressed by osteoclasts and examine function of DAP12 in murine osteoclasts in vivo and in vitro. These data show a new role for DAP12 signaling in regulating formation of multinucleated osteoclasts.

CMRF-35-like molecule-1, a novel mouse myeloid receptor, can inhibit osteoclast formation

By homology to triggering receptor expressed by myeloid cells-2, we screened the mouse expressed sequence tag database and isolated a new single Ig domain receptor, which we have expressed and characterized. The receptor is most similar in sequence to the human CMRF-35 receptor, and thus we have named it CMRF-35-like molecule (CLM)-1. By screening the mouse genome, we determined that CLM-1 was part of a multigene family located on a small segment of mouse chromosome 11. Each contains a single Ig domain, and they are expressed mainly in cells of the myeloid lineage. CLM-1 contains multiple cytoplasmic tyrosine residues, including two that lie in consensus immunoreceptor tyrosine-based inhibitory motifs, and we demonstrate that CLM-1 can associate with Src-homology 2 containing phosphatase-1. Expression of CLM-1 mRNA is down-regulated by treatment with receptor activator of NF-κB ligand (RANKL), a cytokine that drives osteoclast formation. Furthermore, expression of CLM-1 in the osteoclastogenic cell line RAW (RAW.CLM-1) prevents osteoclastogenesis induced by RANKL and TGF-β. RAW.CLM-1 cells fail to multinucleate and do not up-regulate calcitonin receptor, but they express tartrate-resistant acid phosphatase, cathepsin K, and β3 integrin, suggesting that osteoclastogenesis is blocked at a late-intermediate stage. Thus, we define a new family of myeloid receptors, and demonstrate that the first member of this family, CLM-1, is an inhibitory receptor, able to block osteoclastogenesis.

Genome-Wide DNA Methylation Study Identifies Significant Epigenomic Changes in Osteoarthritic Cartilage

To perform a genome‐wide DNA methylation study to identify DNA methylation changes in osteoarthritic (OA) cartilage tissue.