Cutting edge: signal-regulatory protein beta 1 is a DAP12-associated activating receptor expressed in myeloid cells

Signal-regulatory proteins (SIRPs) are cell-surface glycoproteins expressed on myeloid and neural cells that have been shown to recruit SH2 domain-containing protein phosphatase 1 (SHP-1) and SHP-2 and to regulate receptor tyrosine kinase-coupled signaling. One SIRP of unknown function, designated SIRP beta 1, contains a short cytoplasmic domain that lacks sequence motifs capable of recruiting SHP-1 and SHP-2. Using a SIRP-specific mAb, we show that SIRP beta 1 is expressed in monocytes and dendritic cells and associates with the signal transduction molecule DAP12. SIRP beta 1/DAP12 complex formation was required for efficient cell-surface expression of SIRP beta 1. Stimulation of this complex induced tyrosine phosphorylation, mitogen-activated protein kinase activation, and cellular activation. Thus, SIRP beta 1 is a new DAP12-associated receptor involved in the activation of myeloid cells.     

Effects of aging on triggering receptor expressed on myeloid cells (TREM)-1-induced PMN functions

Triggering receptor expressed on myeloid cell-1 (TREM-1) is a recently described receptor that has many effects on polymorphonuclear neutrophil (PMN), as the engagement of this receptor on PMN can induce phagocytosis, respiratory burst and degranulation. We studied the effects of aging on TREM-1 engagement in human PMN. PMN from elderly were found to have impaired response following TREM-1 engagement. Notably they were not able to modulate the TREM-1-induced respiratory burst as PMN from young did. TREM-1 engagement could not reverse PMN survival following incubation with LPS or GM-CSF in the elderly whereas it did in the young. The phosphorylation of TREM-1 signal transduction molecules was altered with aging. Finally, TREM-1 engagement could not drive the recruitment of TREM-1 in the lipid-rafts of the elderly explaining in part the altered response. The observed alterations in TREM-1 response are possibly an important contributing factor in the higher incidence of sepsis-related deaths in the elderly population.     

Cutting edge: expression patterns of surface and soluble triggering receptor expressed on myeloid cells-1 in human endotoxemia

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a recently identified molecule involved in the amplification of inflammation. To determine the regulation of TREM-1, we studied TREM-1 expression and soluble TREM-1 plasma levels upon i.v. LPS challenge in healthy humans in vivo and in vitro. Granulocyte TREM-1 expression was high at baseline and immediately down-regulated upon LPS exposure along with an increase in soluble TREM-1. Monocytes displayed a gradual up-regulation of TREM-1 upon LPS in vivo and in vitro. In vitro studies extended these findings to highly purified lipoteichoic acid and Streptococcus pneumoniae. Nonbacterial TLR ligands such as polyinosine-polycytidylic acid and imidazoquinoline, as well as the TLR9 ligand CpG, did not impact TREM-1 expression. The LPS-induced alterations in TREM-1 surface expression were not a result of increased TNF-alpha or IL-10. Inhibitor studies disclosed a PI3K-dependent pathway in LPS-induced up-regulation of TREM-1 on monocytes, whereas MAPK played a limited role.     

Cutting edge: Toll-like receptor (TLR)2- and TLR4-mediated pathogen recognition in resistance to airborne infection with Mycobacterium tuberculosis

Innate resistance against Mycobacterium tuberculosis is thought to depend critically on engagement of pattern recognition receptors on macrophages. However, the relative contribution of these receptors for containing M. tuberculosis infection has remained unexplored in vivo. To address this issue, we infected mice defective in CD14, TLR2, or TLR4 with M. tuberculosis by aerosol. Following infection with 100 mycobacteria, either mutant strain was as resistant as congenic control mice. Granuloma formation, macrophage activation, and secretion of proinflammatory cytokines in response to low-dose aerosol infection were identical in mutant and control mice. However, high-dose aerosol challenge with 2000 CFU M. tuberculosis revealed TLR2-, but not TLR4-defective mice to be more susceptible than control mice. In conclusion, while TLR2 signaling contributes to innate resistance against M. tuberculosis in borderline situations, its function, and that of CD14 and TLR4, in initiating protective responses against naturally low-dose airborne infection is redundant.     

Lipopolysaccharide-induced up-regulation of triggering receptor expressed on myeloid cells-1 expression on macrophages is regulated by endogenous prostaglandin E2

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a recently identified cell surface molecule that is expressed by neutrophils and monocytes. TREM-1 expression is modulated by various ligands for TLRs in vitro and in vivo. However, the influence of PGE(2), a potential mediator of inflammation, on TREM-1 expression has not been elucidated. In this study, we examined the effects of PGE(2) on LPS-induced TREM-1 expression by resident murine peritoneal macrophages (RPM) and human PBMC. PGE(2) significantly induced murine TREM-1 (mTREM-1) expression by RPM. Up-regulation of TREM-1 expression was specific to PGE(2) among arachidonic acid metabolites, while ligands for chemoattractant receptor-homologous molecule expressed on Th2 cells and the thomboxane-like prostanoid receptor failed to induce mTREM-1 expression. PGE(2) also increased expression of the soluble form of TREM-1 by PBMC. LPS-induced TREM-1 expression was regulated by endogenous PGE(2) especially in late phase (>2 h after stimulation), because cyclooxygenase-1 and -2 inhibitors abolished this effect at that points. A synthetic EP4 agonist and 8-Br-cAMP also enhanced mTREM-1 expression by RPM. Furthermore, protein kinase A, PI3K, and p38 MAPK inhibitors prevented PGE(2)-induced mTREM-1 expression by RPM. Activation of TREM-1 expressed on PGE(2)-pretreated PBMC by an agonistic TREM-1 mAb significantly enhanced the production of IL-8 and TNF-alpha. These findings indicate that LPS-induced TREM-1 expression on macrophages is mediated, at least partly, by endogenous PGE(2) followed by EP4 and cAMP, protein kinase A, p38 MAPK, and PI3K-mediated signaling. Regulation of TREM-1 and the soluble form of TREM-1 expression by PGE(2) may modulate the inflammatory response to microbial pathogens.     

Cutting edge: functional interactions between toll-like receptor (TLR) 2 and TLR1 or TLR6 in response to phenol-soluble modulin

Toll-like receptor (TLR) 2 and TLR4 play important roles in the early, innate immune response to microbial challenge. TLR2 is preferentially involved in the inflammatory response to lipoteichoic acid, lipopeptides, and glycans from a variety of microbes, whereas TLR4 is essential for a complete response to LPSs. We report here that TLR2 transduces the response to phenol-soluble modulin, a factor secreted by Staphylococcus epidermidis. The TLR2-mediated response to this modulin was enhanced by TLR6 but inhibited by TLR1, indicating a functional interaction between these receptors. We also demonstrate that a response to phenol-soluble modulin mediated by TLR2 and TLR6 was more refractory to inhibition by TLR1 than one mediated by TLR2 alone.     

Crystal structure of mouse triggering receptor expressed on myeloid cells 1 (TREM-1) at 1.76 A

Triggering receptor expressed on myeloid cells (TREM) 1 is an activating receptor expressed on myeloid cells whose ligand(s) remain elusive. TREM-1 stimulation activates neutrophils and monocytes and induces the secretion of pro-inflammatory molecules, which amplifies the Toll-like receptor-initiated responses to invading pathogens. In addition, TREM-1 mediates the septic shock pathway, and thus represents a potential therapeutic target. We report the crystal structure of the mouse TREM-1 extracellular domain at 1.76A resolution. The mouse extracellular domain is monomeric, consistent with our previous human TREM-1 structure, and strongly supports the contention that the globular TREM-1 head is a monomer contrary to proposals of a symmetric dimer.     

Crystal structure of human triggering receptor expressed on myeloid cells 1 (TREM-1) at 1.47 A

The triggering receptor expressed on myeloid cells (TREM) family of single extracellular immunoglobulin receptors includes both activating and inhibitory isoforms whose ligands are unknown. TREM-1 activation amplifies the Toll-like receptor initiated responses to invading pathogens allowing the secretion of pro-inflammatory chemokines and cytokines. Hence, TREM-1 amplifies the inflammation induced by both bacteria and fungi, and thus represents a potential therapeutic target. We report the crystal structure of the human TREM-1 extracellular domain at 1.47 A resolution. The overall fold places it within the V-type immunoglobulin domain family and reveals close homology with Ig domains from antibodies, T-cell receptors and other activating receptors, such as NKp44. With the additional use of analytical ultracentrifugation and 1H NMR spectroscopy of both human and mouse TREM-1, we have conclusively demonstrated the monomeric state of this extracellular ectodomain in solution and, presumably, of the TREM family in general.     

Cutting edge: differential chemokine production by myeloid and plasmacytoid dendritic cells

To examine the different roles of myeloid dendritic cells (M-DCs) and plasmacytoid dendritic cells (P-DCs) in the induction and regulation of immune response, we have studied chemokine secretion by freshly isolated DC subsets in response to bacterial, viral, and T cell-derived stimuli. M-DCs selectively produced very high levels of the homeostatic chemokines CC chemokine ligand (CCL)17 and CCL22, while P-DCs produced very little if any. In contrast, the proinflammatory chemokine CCL3 was secreted mostly by P-DCs, whereas CCL4 and CXC chemokine ligand 8 were produced by both subsets. The selective production of CCL17 and CCL22 by M-DCs but not P-DCs was confirmed in vivo by immunohistology on human reactive lymph node sections. The high production of CCR4 ligands by M-DCs suggests their capacity to selectively recruit at sites of inflammation T cells with regulatory properties or with a Th2 phenotype, whereas P-DCs, by preferentially secreting CCR1/CCR5 ligands, would mostly recruit effector T cells and, in particular, Th1-type cells.     

Cutting edge: inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes

We have identified new activating receptors of the Ig superfamily expressed on human myeloid cells, called TREM (triggering receptor expressed on myeloid cells). TREM-1 is selectively expressed on blood neutrophils and a subset of monocytes and is up-regulated by bacterial LPS. Engagement of TREM-1 triggers secretion of IL-8, monocyte chemotactic protein-1, and TNF-alpha and induces neutrophil degranulation. Intracellularly, TREM-1 induces Ca2+ mobilization and tyrosine phosphorylation of extracellular signal-related kinase 1 (ERK1), ERK2 and phospholipase C-gamma. To mediate activation, TREM-1 associates with the transmembrane adapter molecule DAP12. Thus, TREM-1 mediates activation of neutrophil and monocytes, and may have a predominant role in inflammatory responses.     

Heterogeneous expression of the triggering receptor expressed on myeloid cells-2 on adult murine microglia

Microglial activation is an early and common feature of almost all neuropathologies, including multiple sclerosis, Alzheimer's disease and mechanical injury. To better understand the relative contributions microglia make toward neurodegeneration and neuroprotection, we used TOGA(R) to identify molecules expressed by microglia and regulated by inflammatory signals. Triggering receptor expressed on myeloid cells-2 (TREM-2) was among the mRNAs identified as being expressed by unactivated microglia, but down-regulated by lipopolysaccharide/interferon gamma. In the healthy CNS, not all microglia expressed TREM-2. Microglial expression of TREM-2 varied not only between brain regions but also within each brain region. Brain regions with an incomplete blood-brain barrier had the lowest percentages of TREM-2- expressing microglia, whereas the lateral entorhinal and cingulate cortex had the highest percentages. A novel form of TREM-2b that lacked a transmembrane domain was detected, perhaps indicating a soluble form of the protein. Taken together, these data suggest that (1) subsets of microglia are specialized to respond to defined extracellular signals; and (2) regional variations in TREM-2 expression may contribute to the varying sensitivities of different brain regions to similar pathological signals.     

IL-4 confers NK stimulatory capacity to murine dendritic cells: a signaling pathway involving KARAP/DAP12-triggering receptor expressed on myeloid cell 2 molecules

Dendritic cells (DC) regulate NK cell functions, but the signals required for the DC-mediated NK cell activation, i.e., DC-activated NK cell (DAK) activity, remain poorly understood. Upon acute inflammation mimicked by LPS or TNF-alpha, DC undergo a maturation process allowing T and NK cell activation in vitro. Chronic inflammation is controlled in part by Th2 cytokines. In this study, we show that IL-4 selectively confers to DC NK but not T cell stimulatory capacity. IL-4 is mandatory for mouse bone marrow-derived DC grown in GM-CSF (DC(GM/IL-4)) to promote NK cell activation in the draining lymph nodes. IL-4-mediated DAK activity depends on the KARAP/DAP12-triggering receptor expressed on myeloid cell 2 signaling pathway because: 1) gene targeting of the adaptor molecule KARAP/DAP12, a transmembrane polypeptide with an intracytoplasmic immunoreceptor tyrosine-based activation motif, suppresses the DC(GM/IL-4) capacity to activate NK cells, and 2) IL-4-mediated DAK activity is significantly blocked by soluble triggering receptor expressed on myeloid cell 2 Fc molecules. These data outline a novel role for Th2 cytokines in the regulation of innate immune responses through triggering receptors expressed on myeloid cells.     

Cutting edge: cell-extrinsic immune regulation by CTLA-4 expressed on conventional T cells

The CTLA-4 pathway is a key regulator of T cell activation and a critical failsafe against autoimmunity. Although early models postulated that CTLA-4 transduced a negative signal, in vivo evidence suggests that CTLA-4 functions in a cell-extrinsic manner. That multiple cell-intrinsic mechanisms have been attributed to CTLA-4, yet its function in vivo appears to be cell-extrinsic, has been an ongoing paradox in the field. Although CTLA-4 expressed on conventional T cells (Tconv) can mediate inhibitory function, it is unclear why this fails to manifest as an intrinsic effect. In this study, we show that Tconv-expressed CTLA-4 can function in a cell-extrinsic manner in vivo. CTLA-4(+/+) T cells, from DO11/rag(-/-) mice that lack regulatory T cells, were able to regulate the response of CTLA-4(-/-) T cells in cotransfer experiments. This observation provides a potential resolution to the above paradox and suggests CTLA-4 function on both Tconv and regulatory T cells can be achieved through cell-extrinsic mechanisms.     

Activation of triggering receptor expressed on myeloid cells-1 on human neutrophils by marburg and ebola viruses

Marburg virus (MARV) and Ebola virus (EBOV), members of the viral family Filoviridae, cause fatal hemorrhagic fevers in humans and nonhuman primates. High viral burden is coincident with inadequate adaptive immune responses and robust inflammatory responses, and virus-mediated dysregulation of early host defenses has been proposed. Recently, a novel class of innate receptors called the triggering receptors expressed in myeloid cells (TREM) has been discovered and shown to play an important role in innate inflammatory responses and sepsis. Here, we report that MARV and EBOV activate TREM-1 on human neutrophils, resulting in DAP12 phosphorylation, TREM-1 shedding, mobilization of intracellular calcium, secretion of proinflammatory cytokines, and phenotypic changes. A peptide specific to TREM-1 diminished the release of tumor necrosis factor alpha by filovirus-activated human neutrophils in vitro, and a soluble recombinant TREM-1 competitively inhibited the loss of cell surface TREM-1 that otherwise occurred on neutrophils exposed to filoviruses. These data imply direct activation of TREM-1 by filoviruses and also indicate that neutrophils may play a prominent role in the immune and inflammatory responses to filovirus infections.     

Developmental Regulation of TREM2 and DAP12 Expression in the Murine CNS: Implications for Nasu-Hakola Disease

Trem2 is an orphan, DAP12 associated receptor constitutively expressed in vivo by subsets of microglia in the healthy adult murine CNS and in vitro by subsets of oligodendrocytes in neonatal mixed glial cultures. Loss of a functional Trem2 signaling pathway is the genetic cause of Nasu-Hakola disease. Whether the early onset cognitive dementia and myelin-pallor associated with this disorder are due to deficits in functional Trem2 signaling in microglia and/or oligodendrocytes is still being debated. Here, we find that Trem2/DAP12 expression is detected in embryonic day 14 CNS mRNA. Using dual immunohistochemistry/in situ hybridization, we find that both Trem2 and DAP12 expression always co-localized with markers of microglia/macrophages. However, Trem2/DAP12 positive microglia are found in very close apposition with CNP+ oligodendrocytes prior to myelination (post-natal day 1). In addition, CNS expression of TREM2 and DAP12 are not detected in PU.1KO which lack microglia and macrophages. Our data provide continuing support for Nasu-Hakola disease being identified as a cognitive disorder caused by a primary dysfunction of CNS microglia. Special issue article in honor of Dr. George DeVries.     

Toll-like receptor (TLR) 2 and TLR4 are essential for Aspergillus-induced activation of murine macrophages

Aspergillus fumigatius is a ubiquitous saprophytic fungus that has become the most prevalent airborne fungal pathogen for immunocompromised patients during the last two decades. In this report we have analysed how macrophages recognize this microorganism. Using transfected human HEK 293 cells we demonstrate that NF-kappaB-dependent promoter activation triggered by A. fumigatus is mediated by Toll-like receptors TLR2 and TLR4, whereas no activation was observed in cells overexpressing other distinct TLR proteins (TLR1, TLR3, TLR5-10). Using macrophages derived from mice lacking TLR2 expression, expressing defective TLR4 or both we found that A. fumigatus conidia and hyphae induce NF-kappaB translocation, release of pro-inflammatory molecules, like TNFalpha, and the chemoattractant MIP-2 in a TLR2- and TLR4-dependent manner. Recognition of A. niger and A. fumigatus, was similar in terms of the parameters analysed, suggesting that pathogenic and non-pathogenic aspergilli are sensed by macrophages in a similar fashion. Finally, we found that recruitment of neutrophils is severely impaired in mice lacking both functional TLR2 and TLR4, but is less impaired in single TLR2- or TLR4-deficient mice, providing evidence that both receptors are required for an optimal immune response to Aspergillus in vivo.     

Mice lacking myeloid differentiation factor 88 display profound defects in host resistance and immune responses to Mycobacterium avium infection not exhibited by Toll-like receptor 2 (TLR2)- and TLR4-deficient animals

To assess the role of Toll-like receptor (TLR) signaling in host resistance to Mycobacterium avium infection, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88), as well as TLR2(-/-) and TLR4(-/-) animals, were infected with a virulent strain of M. avium, and bacterial burdens and immune responses were compared with those in wild-type (WT) animals. MyD88(-/-) mice failed to control acute and chronic M. avium growth and succumbed 9-14 wk postinfection. Infected TLR2(-/-) mice also showed increased susceptibility, but displayed longer survival and lower bacterial burdens than MyD88(-/-) animals, while TLR4(-/-) mice were indistinguishable from their WT counterparts. Histopathological examination of MyD88(-/-) mice revealed massive destruction of lung tissue not present in WT, TLR2(-/-), or TLR4(-/-) mice. In addition, MyD88(-/-) and TLR2(-/-), but not TLR4(-/-), mice displayed marked reductions in hepatic neutrophil infiltration during the first 2 h of infection. Although both MyD88(-/-) and TLR2(-/-) macrophages showed profound defects in IL-6, TNF, and IL-12p40 responses to M. avium stimulation in vitro, in vivo TNF and IL-12p40 mRNA induction was impaired only in infected MyD88(-/-) mice. Similarly, MyD88(-/-) mice displayed a profound defect in IFN-gamma response that was not evident in TLR2(-/-) or TLR4(-/-) mice or in animals deficient in IL-18. These findings indicate that resistance to mycobacterial infection is regulated by multiple MyD88-dependent signals in addition to those previously attributed to TLR2 or TLR4, and that these undefined elements play a major role in determining bacterial induced proinflammatory as well as IFN-gamma responses.     

Circulating soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) as diagnostic and prognostic marker in neonatal sepsis

ObjectiveTriggering receptor expressed on myeloid cells-1 (TREM-1) is an important receptor involved in the innate inflammatory response and sepsis. We assessed soluble TREM-1 (sTREM-1) in 112 septic neonates (63 culture-positive and 49 culture-negative) and 40 healthy controls as a potential early diagnostic and prognostic marker for neonatal sepsis (NS).MethodsStudied neonates were evaluated for early- or late-onset sepsis using clinical and laboratory indicators upon admission. sTREM-1 was measured on initial sepsis evaluation and at 48 h after antibiotic therapy. For ethical reasons, cord blood samples were collected from control neonates and only samples from neonates that proved to be healthy by clinical examination and laboratory analysis were further analyzed for sTREM-1.ResultsBaseline sTREM-1 levels were significantly elevated in culture-proven (1461.1 ± 523 pg/mL) and culture-negative sepsis (1194 ± 485 pg/mL) compared to controls (162.2 ± 61 pg/mL) with no significant difference between both septic groups. Culture-positive or negative septic preterm neonates had significantly higher sTREM-1 compared to full term neonates. sTREM-1 was significantly higher in neonates with early sepsis than late sepsis and was associated with high mortality. sTREM-1 was significantly decreased 48 h after antibiotic therapy compared to baseline or levels in neonates with persistently positive cultures. sTREM-1 was positively correlated to white blood cells (WBCs), absolute neutrophil count, immature/total neutrophil (I/T) ratio, C-reactive protein (hs-CRP) and sepsis score while negatively correlated to gestational age and weight. hs-CRP and sepsis score were independently related to sTREM-1 in multiregression analysis. sTREM-1 cutoff value of 310 pg/mL could be diagnostic for NS with 100% sensitivity and specificity (AUC, 1.0 and 95% confidence interval [CI], 0.696–1.015) while the cutoff value 1100 pg/mL was predictive of survival with 100% sensitivity and 97% specificity (AUC, 0.978 and 95% CI, 0.853–1.13). However, hs-CRP cutoff 13.5 mg/L could be diagnostic for NS with a sensitivity of 76% and specificity of 72% (AUC, 0.762 and 95% CI, 0.612–0.925) and levels were not related to survival as no significant difference was found between dead and alive septic neonates.ConclusionsElevated sTREM-1 could be considered an early marker for NS that reflects sepsis severity and poor prognosis.