Eicosanoid signalling blockade protects middle-aged mice from severe COVID-19.

Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective, but vaccine efficacy is partly compromised by the emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially for aged populations. Here we describe the isolation of highly virulent mouse-adapted viruses and use them to test a new therapeutic drug in infected aged animals. Many of the alterations observed in SARS-CoV-2 during mouse adaptation (positions 417, 484, 493, 498 and 501 of the spike protein) also arise in humans in variants of concern2. Their appearance during mouse adaptation indicates that immune pressure is not required for selection. For murine SARS, for which severity is also age dependent, elevated levels of an eicosanoid (prostaglandin D2 (PGD2)) and a phospholipase (phospholipase A2 group 2D (PLA2G2D)) contributed to poor outcomes in aged mice3,4. mRNA expression of PLA2G2D and prostaglandin D2 receptor (PTGDR), and production of PGD2 also increase with ageing and after SARS-CoV-2 infection in dendritic cells derived from human peripheral blood mononuclear cells. Using our mouse-adapted SARS-CoV-2, we show that middle-aged mice lacking expression of PTGDR or PLA2G2D are protected from severe disease. Furthermore, treatment with a PTGDR antagonist, asapiprant, protected aged mice from lethal infection. PTGDR antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, suggesting that the PLA2G2D-PGD2/PTGDR pathway is a useful target for therapeutic interventions.

The prostaglandin D2 antagonist asapiprant ameliorates clinical severity in young hosts infected with invasive Streptococcus pneumoniae.

Streptococcus pneumoniae (pneumococcus) remains a serious cause of pulmonary and systemic infections globally, and host-directed therapies are lacking. The aim of this study was to test the therapeutic efficacy of asapiprant, an inhibitor of prostaglandin D2 signaling, against pneumococcal infection. Treatment of young mice with asapiprant after pulmonary infection with invasive pneumococci significantly reduced systemic spread, disease severity, and host death. Protection was specific against bacterial dissemination from the lung to the blood but had no effect on pulmonary bacterial burden. Asapiprant-treated mice had enhanced antimicrobial activity in circulating neutrophils, elevated levels of reactive oxygen species (ROS) in lung macrophages/monocytes, and improved pulmonary barrier integrity indicated by significantly reduced diffusion of fluorescein isothiocyanate (FITC)-dextran from lungs into the circulation. These findings suggest that asapiprant protects the host against pneumococcal dissemination by enhancing the antimicrobial activity of immune cells and maintaining epithelial/endothelial barrier integrity in the lungs.

The discovery of novel and potent indazole NLRP3 inhibitors enabled by DNA-encoded library screening.

NLRP3 is an intracellular sensor protein that detects a broad range of danger signals and environmental insults. Its activation results in a protective pro-inflammatory response designed to impair pathogens and repair tissue damage via the formation of the NLRP3 inflammasome. Assembly of the NLRP3 inflammasome leads to caspase 1-dependent secretory release of the pro-inflammatory cytokines IL-1ß and IL-18 as well as to gasdermin d-mediated pyroptotic cell death. Herein, we describe the discovery of a novel indazole series of high affinity, reversible inhibitors of NLRP3 activation through screening of DNA-encoded libraries and the potent lead compound 3 (BAL-0028, IC50 = 25 nM) that was identified directly from the screen. SPR studies showed that compound 3 binds tightly (KD range 104-123 nM) to the NACHT domain of NLRP3. A CADD analysis of the interaction of compound 3 with the NLRP3 NACHT domain proposes a binding site that is distinct from those of ADP and MCC950 and includes specific site interactions. We anticipate that compound 3 (BAL-0028) and other members of this novel indazole class of neutral inhibitors will demonstrate significantly different physical, biochemical, and biological properties compared to NLRP3 inhibitors previously identified.

N-Oleoyl dopamine induces IL-10 via central nervous system TRPV1 and improves endotoxemia and sepsis outcomes.

BACKGROUND: The transient receptor potential vanilloid 1 (TRPV1) participates in thermosensation and inflammatory pain, but its immunomodulatory mechanisms remain enigmatic. N-Oleoyl dopamine (OLDA), an endovanilloid and endocannabinoid, is a TRPV1 agonist that is produced in the central nervous system and the peripheral nervous system. We studied the anti-inflammatory effects and TRPV1-dependent mechanisms of OLDA in models of inflammation and sepsis. METHODS: Mice were challenged intratracheally or intravenously with LPS, or intratracheally with S. aureus to induce pneumonia and sepsis, and then were treated intravenously with OLDA. Endpoints included plasma cytokines, leukocyte activation marker expression, mouse sepsis scores, lung histopathology, and bacterial counts. The role of TRPV1 in the effects of OLDA was determined using Trpv1-/- mice, and mice with TRPV1 knockdown pan-neuronally, in peripheral nervous system neurons, or in myeloid cells. Circulating monocytes/macrophages were depleted using clodronate to determine their role in the anti-inflammatory effects of OLDA in endotoxemic mice. Levels of exogenous OLDA, and of endovanilloids and endocannabinoids, at baseline and in endotoxemic mice, were determined by LC-MS/MS. RESULTS: OLDA administration caused an early anti-inflammatory response in endotoxemic and septic mice with high serum levels of IL-10 and decreased levels of pro-inflammatory cytokines. OLDA also reduced lung injury and improved mouse sepsis scores. Blood and lung bacterial counts were comparable between OLDA- and carrier-treated mice with S. aureus pneumonia. OLDA's effects were reversed in mice with pan-neuronal TRPV1 knockdown, but not with TRPV1 knockdown in peripheral nervous system neurons or myeloid cells. Depletion of monocytes/macrophages reversed the IL-10 upregulation by OLDA in endotoxemic mice. Brain and blood levels of endovanilloids and endocannabinoids were increased in endotoxemic mice. CONCLUSIONS: OLDA has strong anti-inflammatory actions in mice with endotoxemia or S. aureus pneumonia. Prior studies focused on the role of peripheral nervous system TRPV1 in modulating inflammation and pneumonia. Our results suggest that TRPV1-expressing central nervous system neurons also regulate inflammatory responses to endotoxemia and infection. Our study reveals a neuro-immune reflex that during acute inflammation is engaged proximally by OLDA acting on neuronal TRPV1, and through a multicellular network that requires circulating monocytes/macrophages, leads to the systemic production of IL-10.

N-Arachidonoyl Dopamine Modulates Acute Systemic Inflammation via Nonhematopoietic TRPV1.

N-Arachidonoyl dopamine (NADA) is an endogenous lipid that potently activates the transient receptor potential vanilloid 1 (TRPV1), which mediates pain and thermosensation. NADA is also an agonist of cannabinoid receptors 1 and 2. We have reported that NADA reduces the activation of cultured human endothelial cells by LPS and TNF-α. Thus far, in vivo studies using NADA have focused on its neurologic and behavioral roles. In this article, we show that NADA potently decreases in vivo systemic inflammatory responses and levels of the coagulation intermediary plasminogen activator inhibitor 1 in three mouse models of inflammation: LPS, bacterial lipopeptide, and polymicrobial intra-abdominal sepsis. We also found that the administration of NADA increases survival in endotoxemic mice. Additionally, NADA reduces blood levels of the neuropeptide calcitonin gene-related peptide but increases the neuropeptide substance P in LPS-treated mice. We demonstrate that the anti-inflammatory effects of NADA are mediated by TRPV1 expressed by nonhematopoietic cells and provide data suggesting that neuronal TRPV1 may mediate NADA's anti-inflammatory effects. These results indicate that NADA has novel TRPV1-dependent anti-inflammatory properties and suggest that the endovanilloid system might be targeted therapeutically in acute inflammation.

The endocannabinoid/endovanilloid N-arachidonoyl dopamine (NADA) and synthetic cannabinoid WIN55,212-2 abate the inflammatory activation of human endothelial cells.

Although cannabinoids, such as Δ(9)-tetrahydrocannabinol, have been studied extensively for their psychoactive effects, it has become apparent that certain cannabinoids possess immunomodulatory activity. Endothelial cells (ECs) are centrally involved in the pathogenesis of organ injury in acute inflammatory disorders, such as sepsis, because they express cytokines and chemokines, which facilitate the trafficking of leukocytes to organs, and they modulate vascular barrier function. In this study, we find that primary human ECs from multiple organs express the cannabinoid receptors CB1R, GPR18, and GPR55, as well as the ion channel transient receptor potential cation channel vanilloid type 1. In contrast to leukocytes, CB2R is only minimally expressed in some EC populations. Furthermore, we show that ECs express all of the known endocannabinoid (eCB) metabolic enzymes. Examining a panel of cannabinoids, we demonstrate that the synthetic cannabinoid WIN55,212-2 and the eCB N-arachidonoyl dopamine (NADA), but neither anandamide nor 2-arachidonoylglycerol, reduce EC inflammatory responses induced by bacterial lipopeptide, LPS, and TNFα. We find that endothelial CB1R/CB2R are necessary for the effects of NADA, but not those of WIN55,212-2. Furthermore, transient receptor potential cation channel vanilloid type 1 appears to counter the anti-inflammatory properties of WIN55,212-2 and NADA, but conversely, in the absence of these cannabinoids, its inhibition exacerbates the inflammatory response in ECs activated with LPS. These data indicate that the eCB system can modulate inflammatory activation of the endothelium and may have important implications for a variety of acute inflammatory disorders that are characterized by EC activation.

Regulation of hemidesmosome disassembly by growth factor receptors.

Hemidesmosomes (HDs) promote the stable adhesion of basal epithelial cells to the underlying basement membrane (BM). Critical for the mechanical stability of the HD is the interaction between integrin alpha6beta4 and plectin, which is destabilized when HD disassembly is required, for instance, to allow keratinocyte migration during wound healing. Growth factors such as epidermal growth factor (EGF) can trigger HD disassembly and induce phosphorylation of the beta4 intracellular domain. Whereas tyrosine phosphorylation appears to mediate cooperation with growth factor signaling pathways and invasion in carcinoma cells, serine phosphorylation seems the predominant mechanism for regulating HD destabilization. Here, we discuss recent advances that shed light on the residues involved, the identity of the kinases that phosphorylate them, and the interactions that become disrupted by these phosphorylations.

ERK5 protein promotes, whereas MEK1 protein differentially regulates, the Toll-like receptor 2 protein-dependent activation of human endothelial cells and monocytes.

Endothelial cell (EC) Toll-like receptor 2 (TLR2) activation up-regulates the expression of inflammatory mediators and of TLR2 itself and modulates important endothelial functions, including coagulation and permeability. We defined TLR2 signaling pathways in EC and tested the hypothesis that TLR2 signaling differs in EC and monocytes. We found that ERK5, heretofore unrecognized as mediating TLR2 activation in any cell type, is a central mediator of TLR2-dependent inflammatory signaling in human umbilical vein endothelial cells, primary human lung microvascular EC, and human monocytes. Additionally, we observed that, although MEK1 negatively regulates TLR2 signaling in EC, MEK1 promotes TLR2 signaling in monocytes. We also noted that activation of TLR2 led to the up-regulation of intracellularly expressed TLR2 and inflammatory mediators via NF-κB, JNK, and p38-MAPK. Finally, we found that p38-MAPK, JNK, ERK5, and NF-κB promote the attachment of human neutrophils to lung microvascular EC that were pretreated with TLR2 agonists. This study newly identifies ERK5 as a key regulator of TLR2 signaling in EC and monocytes and indicates that there are fundamental differences in TLR signaling pathways between EC and monocytes.

Bacterial lipoprotein TLR2 agonists broadly modulate endothelial function and coagulation pathways in vitro and in vivo.

TLR2 activation induces cellular and organ inflammation and affects lung function. Because deranged endothelial function and coagulation pathways contribute to sepsis-induced organ failure, we studied the effects of bacterial lipoprotein TLR2 agonists, including peptidoglycan-associated lipoprotein, Pam3Cys, and murein lipoprotein, on endothelial function and coagulation pathways in vitro and in vivo. TLR2 agonist treatment induced diverse human endothelial cells to produce IL-6 and IL-8 and to express E-selectin on their surface, including HUVEC, human lung microvascular endothelial cells, and human coronary artery endothelial cells. Treatment of HUVEC with TLR2 agonists caused increased monolayer permeability and had multiple coagulation effects, including increased production of plasminogen activator inhibitor-1 (PAI-1) and tissue factor, as well as decreased production of tissue plasminogen activator and tissue factor pathway inhibitor. TLR2 agonist treatment also increased HUVEC expression of TLR2 itself. Peptidoglycan-associated lipoprotein induced IL-6 production by endothelial cells from wild-type mice but not from TLR2 knockout mice, indicating TLR2 specificity. Mice were challenged with TLR2 agonists, and lungs and plasmas were assessed for markers of leukocyte trafficking and coagulopathy. Wild-type mice, but not TLR2 mice, that were challenged i.v. with TLR2 agonists had increased lung levels of myeloperoxidase and mRNAs for E-selectin, P-selectin, and MCP-1, and they had increased plasma PAI-1 and E-selectin levels. Intratracheally administered TLR2 agonist caused increased lung fibrin levels. These studies show that TLR2 activation by bacterial lipoproteins broadly affects endothelial function and coagulation pathways, suggesting that TLR2 activation contributes in multiple ways to endothelial activation, coagulopathy, and vascular leakage in sepsis.

Alveolar macrophages and Toll-like receptor 4 mediate ventilated lung ischemia reperfusion injury in mice.

BACKGROUND: Ischemia-reperfusion (I-R) injury is a sterile inflammatory process that is commonly associated with diverse clinical situations such as hemorrhage followed by resuscitation, transient embolic events, and organ transplantation. I-R injury can induce lung dysfunction whether the I-R occurs in the lung or in a remote organ. Recently, evidence has emerged that receptors and pathways of the innate immune system are involved in recognizing sterile inflammation and overlap considerably with those involved in the recognition of and response to pathogens. METHODS: The authors used a mouse surgical model of transient unilateral left pulmonary artery occlusion without bronchial involvement to create ventilated lung I-R injury. In addition, they mimicked nutritional I-R injury in vitro by transiently depriving cells of all nutrients. RESULTS: Compared with sham-operated mice, mice subjected to ventilated lung I-R injury had up-regulated lung expression of inflammatory mediator messenger RNA for interleukin-1ß, interleukin-6, and chemokine (C-X-C motif) ligand-1 and -2, paralleled by histologic evidence of lung neutrophil recruitment and increased plasma concentrations of interleukin-1ß, interleukin-6, and high-mobility group protein B1 proteins. This inflammatory response to I-R required toll-like receptor-4 (TLR4). In addition, the authors demonstrated in vitro cooperativity and cross-talk between human macrophages and endothelial cells, resulting in augmented inflammatory responses to I-R. Remarkably, the authors found that selective depletion of alveolar macrophages rendered mice resistant to ventilated lung I-R injury. CONCLUSIONS: The data reveal that alveolar macrophages and the pattern recognition receptor toll-like receptor-4 are involved in the generation of the early inflammatory response to lung I-R injury.

EGF-induced MAPK signaling inhibits hemidesmosome formation through phosphorylation of the integrin {beta}4.

Migration of keratinocytes requires a regulated and dynamic turnover of hemidesmosomes (HDs). We and others have previously identified three serine residues on the integrin ß4 cytoplasmic domain that play a critical role in the regulation of HD disassembly. In this study we show that only two of these residues (Ser-1356 and Ser-1364) are phosphorylated in keratinocytes after stimulation with either PMA or EGF. Furthermore, in direct contrast to previous studies performed in vitro, we found that the PMA- and EGF-stimulated phosphorylation of ß4 is not mediated by PKC, but by ERK1/2 and its downstream effector kinase p90RSK1/2. EGF-stimulated phosphorylation of ß4 increased keratinocyte migration, and reduced the number of stable HDs. Furthermore, mutation of the two serines in ß4 to phospho-mimicking aspartic acid decreased its interaction with the cytoskeletal linker protein plectin, as well as the strength of α6ß4-mediated adhesion to laminin-332. During mitotic cell rounding, when the overall cell-substrate area is decreased and the number of HDs is reduced, ß4 was only phosphorylated on Ser-1356 by a distinct, yet unidentified, kinase. Collectively, these data demonstrate an important role of ß4 phosphorylation on residues Ser-1356 and Ser-1364 in the formation and/or stability of HDs.

ERK1/2 Has Divergent Roles in LPS-Induced Microvascular Endothelial Cell Cytokine Production and Permeability.

ABSTRACT: Endothelial cells play a major role in inflammatory responses to infection and sterile injury. Endothelial cells express Toll-like receptor 4 (TLR4) and are activated by LPS to express inflammatory cytokines/chemokines, and to undergo functional changes, including increased permeability. The extracellular signal-regulated kinase 1/2 (ERK1/2) mediates pro-inflammatory signaling in monocytes and macrophages, but the role of ERK1/2 in LPS-induced activation of microvascular endothelial cells has not been defined. We therefore studied the role of ERK1/2 in LPS-induced inflammatory activation and permeability of primary human lung microvascular endothelial cells (HMVEC). Inhibition of ERK1/2 augmented LPS-induced IL-6 and vascular cell adhesion protein (VCAM-1) production by HMVEC. ERK1/2 siRNA knockdown also augmented IL-6 production by LPS-treated HMVEC. Conversely, ERK1/2 inhibition abrogated permeability and restored cell-cell junctions of LPS-treated HMVEC. Consistent with the previously described pro-inflammatory role for ERK1/2 in leukocytes, inhibition of ERK1/2 reduced LPS-induced cytokine/chemokine production by primary human monocytes. Our study identifies a complex role for ERK1/2 in TLR4-activation of HMVEC, independent of myeloid differentiation primary response gene (MyD88) and TIR domain-containing adaptor inducing IFN-ß (TRIF) signaling pathways. The activation of ERK1/2 limits LPS-induced IL-6 production by HMVEC, while at the same time promoting HMVEC permeability. Conversely, ERK1/2 activation promotes IL-6 production by human monocytes. Our results suggest that ERK1/2 may play an important role in the nuanced regulation of endothelial cell inflammation and vascular permeability in sepsis and injury.

EGFR-dependent tyrosine phosphorylation of integrin ß4 is not required for downstream signaling events in cancer cell lines.

In epithelial cancers, the epidermal growth factor receptor (EGFR) and integrin α6ß4 are frequently overexpressed and found to synergistically activate intracellular signaling pathways that promote cell proliferation and migration. In cancer cells, the ß4 subunit is phosphorylated at tyrosine residues not normally recognized as kinase substrates; however, the function of these phosphotyrosine residues in cancer cells is a subject of much debate. In EGFR-overexpressing carcinoma cells, we found that the Src family kinase (SFK) inhibitor PP2 reduces ß4 tyrosine phosphorylation following the activation of EGFR. However, siRNA mediated knockdown of the SFKs Src, Fyn, Yes and Lyn, individually or in combination, did not affect the EGF-induced phosphorylation of ß4. Using phospho-peptide affinity chromatography and mass spectrometry, we found that PLCγ1 binds ß4 at the phosphorylated residues Y1422/Y1440, but were unable to verify this interaction in A431 carcinoma cells that overexpress the EGFR. Furthermore, using A431 cells devoid of ß4 or reconstituted with phenylalanine specific mutants of ß4, the activation of several downstream signaling pathways, including PLCγ/PKC, MAPK and PI3K/Akt, were not substantially affected. We conclude that tyrosine-phosphorylated ß4 does not enhance EGFR-mediated signaling in EGFR-overexpressing cells, despite the fact that this integrin subunit is highly tyrosine phosphorylated in these cells.

Eicosanoid signaling as a therapeutic target in middle-aged mice with severe COVID-19.

Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Resolution of the COVID-19 pandemic has been advanced by the recent development of SARS-CoV-2 vaccines, but vaccine efficacy is partly compromised by the recent emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially in aged populations. Here, we describe the isolation of a new set of highly virulent mouse-adapted viruses and use them to test a novel therapeutic drug useful in infections of aged animals. Initially, we show that many of the mutations observed in SARS-CoV-2 during mouse adaptation (at positions 417, 484, 501 of the spike protein) also arise in humans in variants of concern (VOC)2. Their appearance during mouse adaptation indicates that immune pressure is not required for their selection. Similar to the human infection, aged mice infected with mouse-adapted SARS-CoV-2 develop more severe disease than young mice. In murine SARS, in which severity is also age-dependent, we showed that elevated levels of an eicosanoid, prostaglandin D2 (PGD2) and of a phospholipase, PLA2G2D, contributed to poor outcomes in aged mice3,4. Using our virulent mouse-adapted SARS-CoV-2, we show that infection of middle-aged mice lacking expression of DP1, a PGD2 receptor, or PLA2G2D are protected from severe disease. Further, treatment with a DP1 antagonist, asapiprant, protected aged mice from a lethal infection. DP1 antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, and demonstrates that the PLA2G2D-PGD2/DP1 pathway is a useful target for therapeutic interventions.

Nesprin-3, a novel outer nuclear membrane protein, associates with the cytoskeletal linker protein plectin.

Despite their importance in cell biology, the mechanisms that maintain the nucleus in its proper position in the cell are not well understood. This is primarily the result of an incomplete knowledge of the proteins in the outer nuclear membrane (ONM) that are able to associate with the different cytoskeletal systems. Two related ONM proteins, nuclear envelope spectrin repeat (nesprin)-1 and -2, are known to make direct connections with the actin cytoskeleton through their NH2-terminal actin-binding domain (ABD). We have now isolated a third member of the nesprin family that lacks an ABD and instead binds to the plakin family member plectin, which can associate with the intermediate filament (IF) system. Overexpression of nesprin-3 results in a dramatic recruitment of plectin to the nuclear perimeter, which is where these two molecules are colocalized with both keratin-6 and -14. Importantly, plectin binds to the integrin alpha6beta4 at the cell surface and to nesprin-3 at the ONM in keratinocytes, suggesting that there is a continuous connection between the nucleus and the extracellular matrix through the IF cytoskeleton.

A RNA interference screen identifies the protein phosphatase 2A subunit PR55gamma as a stress-sensitive inhibitor of c-SRC.

Protein Phosphatase type 2A (PP2A) represents a family of holoenzyme complexes with diverse biological activities. Specific holoenzyme complexes are thought to be deregulated during oncogenic transformation and oncogene-induced signaling. Since most studies on the role of this phosphatase family have relied on the use of generic PP2A inhibitors, the contribution of individual PP2A holoenzyme complexes in PP2A-controlled signaling pathways is largely unclear. To gain insight into this, we have constructed a set of shRNA vectors targeting the individual PP2A regulatory subunits for suppression by RNA interference. Here, we identify PR55gamma and PR55delta as inhibitors of c-Jun NH(2)-terminal kinase (JNK) activation by UV irradiation. We show that PR55gamma binds c-SRC and modulates the phosphorylation of serine 12 of c-SRC, a residue we demonstrate to be required for JNK activation by c-SRC. We also find that the physical interaction between PR55gamma and c-SRC is sensitive to UV irradiation. Our data reveal a novel mechanism of c-SRC regulation whereby in response to stress c-SRC activity is regulated, at least in part, through loss of the interaction with its inhibitor, PR55gamma.

Serine phosphorylation of the integrin beta4 subunit is necessary for epidermal growth factor receptor induced hemidesmosome disruption.

Hemidesmosomes (HDs) are multiprotein adhesion complexes that promote attachment of epithelial cells to the basement membrane. The binding of alpha6beta4 to plectin plays a central role in their assembly. We have defined three regions on beta4 that together harbor all the serine and threonine phosphorylation sites and show that three serines (S1356, S1360, and S1364), previously implicated in HD regulation, prevent the interaction of beta4 with the plectin actin-binding domain when phosphorylated. We have also established that epidermal growth factor receptor activation, which is known to function upstream of HD disassembly, results in the phosphorylation of only one or more of these three residues and the partial disassembly of HDs in keratinocytes. Additionally, we show that S1360 and S1364 of beta4 are the only residues phosphorylated by PKC and PKA in cells, respectively. Taken together, our studies indicate that multiple kinases act in concert to breakdown the structural integrity of HDs in keratinocytes, which is primarily achieved through the phosphorylation of S1356, S1360, and S1364 on the beta4 subunit.

Phorbol 12-myristate 13-acetate-induced release of the colony-stimulating factor 1 receptor cytoplasmic domain into the cytosol involves two separate cleavage events.

The colony-stimulating factor 1 (CSF-1) receptor is a protein-tyrosine kinase that regulates cell division, differentiation, and development. In response to phorbol 12-myristate 13-acetate (PMA), the CSF-1 receptor is subject to proteolytic processing. Use of chimeric receptors indicates that the CSF-1 receptor is cleaved at least two times, once in the extracellular domain and once in the transmembrane domain. Cleavage in the extracellular domain results in ectodomain shedding while the cytoplasmic domain remains associated with the membrane. Intramembrane cleavage depends on the sequence of the transmembrane domain and results in the release of the cytoplasmic domain. This process can be blocked by gamma-secretase inhibitors. The cytoplasmic domain localizes partially to the nucleus, displays limited stability, and is degraded by the proteosome. CSF-1 receptors are continuously subject to down-modulation and regulated intramembrane proteolysis (RIP). RIP is stimulated by granulocyte-macrophage-CSF, CSF-1, interleukin-2 (IL-2), IL-4, lipopolysaccharide, and PMA and may provide the CSF-1 receptor with an additional mechanism for signal transduction.

C-Cbl binds the CSF-1 receptor at tyrosine 973, a novel phosphorylation site in the receptor's carboxy-terminus.

The colony-stimulating factor-1 (CSF-1) receptor is a protein-tyrosine kinase that regulates the proliferation and differentiation of monocyte and macrophage precursors. Binding of CSF-1 to its receptor results in activation of the kinase domain and autophosphorylation on a number of tyrosine residues. Phosphorylated tyrosine residues function as binding sites for SH2 domain-containing signaling proteins. It is known that activated receptors are internalized and degraded, but the mechanics of this process remain largely unknown. Recently, evidence has started to emerge that the ubiquitin-protein ligase c-Cbl is involved in CSF-1 receptor degradation. In addition, there is evidence that the CSF-1 receptor carboxy-terminus is involved in down regulation of the receptor. Here we show that the c-Cbl tyrosine kinase-binding (TKB) domain binds in vitro and in vivo to the CSF-1 receptor. Binding is dependent on the receptor's protein-kinase activity. Deletion of the carboxy-terminus or mutation of Tyr 973 blocks binding. We further provide evidence that the CSF-1 receptor's carboxy-terminus is a substrate for autophosphorylation. Our observations are consistent with a model in which receptor autophosphorylation at Tyr 973 creates a binding site for c-Cbl. Association of c-Cbl with the receptor leads to ubiquitination, followed by receptor degradation.

Vascular endothelial cell Toll-like receptor pathways in sepsis.

The endothelium forms a vast network that dynamically regulates vascular barrier function, coagulation pathways and vasomotor tone. Microvascular endothelial cells are uniquely situated to play key roles during infection and injury, owing to their widespread distribution throughout the body and their constant interaction with circulating blood. While not viewed as classical immune cells, endothelial cells express innate immune receptors, including the Toll-like receptors (TLRs), which activate intracellular inflammatory pathways mediated through NF-κB and the MAP kinases. TLR agonists, including LPS and bacterial lipopeptides, directly upregulate microvascular endothelial cell expression of inflammatory mediators. Intriguingly, TLR activation also modulates microvascular endothelial cell permeability and the expression of coagulation pathway intermediaries. Microvascular thrombi have been hypothesized to trap microorganisms thereby limiting the spread of infection. However, dysregulated activation of endothelial inflammatory pathways is also believed to lead to coagulopathy and increased vascular permeability, which together promote sepsis-induced organ failure. This article reviews vascular endothelial cell innate immune pathways mediated through the TLRs as they pertain to sepsis, highlighting links between TLRs and coagulation and permeability pathways, and their role in healthy and pathologic responses to infection and sepsis.