Mechanism of Tumor-Platelet Communications in Cancer.

BACKGROUND: Thrombosis is one of the main complications in cancer patients often leading to mortality. However, the mechanisms underlying platelet hyperactivation are poorly understood. METHODS: Murine and human platelets were isolated and treated with small extracellular vesicles (sEVs) from various cancer cell lines. The effects of these cancer-sEVs on platelets were evaluated both in vitro and in vivo using various approaches, including the detection of cancer-sEV-specific markers in murine platelets and patient samples, measurement of platelet activation and thrombosis assays. Signaling events induced by cancer-sEVs and leading to platelet activation were identified, and the use of blocking antibodies to prevent thrombosis was demonstrated. RESULTS: We demonstrate that platelets very effectively take up sEVs from aggressive cancer cells. The process of uptake is fast, proceeds effectively in circulation in mice, and is mediated by the abundant sEV membrane protein-CD63. The uptake of cancer-sEVs leads to the accumulation of cancer cell-specific RNA in platelets in vitro and in vivo. The human prostate cancer-sEV-specific RNA marker PCA3 is detected in platelets of ~70% of prostate cancer patients. This was markedly reduced after prostatectomy. In vitro studies showed that platelet uptake of cancer-sEVs induces strong platelet activation in a CD63-RPTPα (receptor-like protein tyrosine phosphatase alpha)-dependent manner. In contrast to physiological agonists ADP and thrombin, cancer-sEVs activate platelets via a noncanonical mechanism. Intravital studies demonstrated accelerated thrombosis both in murine tumor models and in mice that received intravenous injections of cancer-sEVs. The prothrombotic effects of cancer-sEVs were rescued by blocking CD63. CONCLUSIONS: Tumors communicate with platelets by means of sEVs, which deliver cancer markers and activate platelets in a CD63-dependent manner leading to thrombosis. This emphasizes the diagnostic and prognostic value of platelet-associated cancer markers and identifies new pathways for intervention.

EGFR-mediated tyrosine phosphorylation of STING determines its trafficking route and cellular innate immunity functions.

STING (STimulator of INterferon Genes) mediates protective cellular response to microbial infection and tissue damage, but its aberrant activation can lead to autoinflammatory diseases. Upon ligand stimulation, the endoplasmic reticulum (ER) protein STING translocates to endosomes for induction of interferon production, while an alternate trafficking route delivers it directly to the autophagosomes. Here, we report that phosphorylation of a specific tyrosine residue in STING by the epidermal growth factor receptor (EGFR) is required for directing STING to endosomes, where it interacts with its downstream effector IRF3. In the absence of EGFR-mediated phosphorylation, STING rapidly transits into autophagosomes, and IRF3 activation, interferon production, and antiviral activity are compromised in cell cultures and mice, while autophagic activity is enhanced. Our observations illuminate a new connection between the tyrosine kinase activity of EGFR and innate immune functions of STING and suggest new experimental and therapeutic approaches for selective regulation of STING functions.

A new mechanism of interferon's antiviral action: Induction of autophagy, essential for paramyxovirus replication, is inhibited by the interferon stimulated gene, TDRD7.

The interferon (IFN) system represents the first line of defense against a wide range of viruses. Virus infection rapidly triggers the transcriptional induction of IFN-ß and IFN Stimulated Genes (ISGs), whose protein products act as viral restriction factors by interfering with specific stages of virus life cycle, such as entry, transcription, translation, genome replication, assembly and egress. Here, we report a new mode of action of an ISG, IFN-induced TDRD7 (tudor domain containing 7) inhibited paramyxovirus replication by inhibiting autophagy. TDRD7 was identified as an antiviral gene by a high throughput screen of an ISG shRNA library for blocking IFN's protective effect against Sendai virus (SeV) replication. The antiviral activity of TDRD7 against SeV, human parainfluenza virus 3 and respiratory syncytial virus was confirmed by its genetic ablation or ectopic expression in several types of mouse and human cells. TDRD7's antiviral action was mediated by its ability to inhibit autophagy, a cellular catabolic process which was robustly induced by SeV infection and required for its replication. Mechanistic investigation revealed that TDRD7 interfered with the activation of AMP-dependent kinase (AMPK), an enzyme required for initiating autophagy. AMPK activity was required for efficient replication of several paramyxoviruses, as demonstrated by its genetic ablation or inhibition of its activity by TDRD7 or chemical inhibitors. Therefore, our study has identified a new antiviral ISG with a new mode of action.

Constitutively Bound EGFR-Mediated Tyrosine Phosphorylation of TLR9 Is Required for Its Ability To Signal.

Mammalian TLRs recognize microbial infection or cell death-associated danger signals and trigger the appropriate cellular response. These responses determine the strength and the outcome of the host-microbe interaction. TLRs are transmembrane proteins located on the plasma or the endosomal membrane. Their ectodomains recognize specific microbial or endogenous ligands, and the cytoplasmic domains interact with specific proteins to activate intracellular signaling pathways. TLR9, an endosomal TLR, is activated by endocytosed DNA. Activated TLR9 recruits the cytoplasmic adapter MyD88 and other signaling proteins to induce the synthesis of inflammatory cytokines and IFN. Uncontrolled activation of TLR9 leads to the undesired overproduction of inflammatory cytokines and consequent pathogenesis. Therefore, appropriate activation and the regulation of TLR9 signaling are critical. Tyrosine (Tyr) phosphorylation of TLR9 is essential for its activation; however, the role of specific Tyr kinases is not clear. In this article, we report that epidermal growth factor receptor (EGFR), a membrane-bound protein Tyr kinase, is essential for TLR9 signaling. Genetic ablation of EGFR or pharmacological inhibition of its kinase activity attenuates TLR9-mediated induction of genes in myeloid and nonmyeloid cell types. EGFR is constitutively bound to TLR9; upon ligand stimulation, it mediates TLR9 Tyr phosphorylation, which leads to the recruitment of MyD88, activation of the signaling kinases and transcription factors, and gene induction. In mice, TLR9-mediated liver injury and death are blocked by an EGFR inhibitor or deletion of the EGFR gene from myeloid cells, which are the major producers of inflammatory cytokines.

EGFR kinase activity is required for TLR4 signaling and the septic shock response.

Mammalian Toll-like receptors (TLR) recognize microbial products and elicit transient immune responses that protect the infected host from disease. TLR4--which signals from both plasma and endosomal membranes--is activated by bacterial lipopolysaccharides (LPS) and induces many cytokine genes, the prolonged expression of which causes septic shock in mice. We report here that the expression of some TLR4-induced genes in myeloid cells requires the protein kinase activity of the epidermal growth factor receptor (EGFR). EGFR inhibition affects TLR4-induced responses differently depending on the target gene. The induction of interferon-ß (IFN-ß) and IFN-inducible genes is strongly inhibited, whereas TNF-α induction is enhanced. Inhibition is specific to the IFN-regulatory factor (IRF)-driven genes because EGFR is required for IRF activation downstream of TLR--as is IRF co-activator ß-catenin--through the PI3 kinase/AKT pathway. Administration of an EGFR inhibitor to mice protects them from LPS-induced septic shock and death by selectively blocking the IFN branch of TLR4 signaling. These results demonstrate a selective regulation of TLR4 signaling by EGFR and highlight the potential use of EGFR inhibitors to treat septic shock syndrome.

Role of interferon regulatory factor 3-mediated apoptosis in the establishment and maintenance of persistent infection by Sendai virus.

Infection of cultured cells by paramyxoviruses causes cell death, mediated by a newly discovered apoptotic pathway activated by virus infection. The key proapoptotic protein in this pathway is interferon regulatory factor 3 (IRF-3), which upon activation by virus infection binds BAX, translocates it to mitochondria, and triggers apoptosis. When IRF-3-knockdown cells were infected with Sendai virus (SeV), persistent infection (PI) was established. The PI cells produced infectious SeV continuously and constitutively expressed many innate immune genes. Interferon signaling was blocked in these cells. The elevated levels of IRF-3-driven genes in the PI cells indicated that the amount of residual IRF-3 activated by endogenous SeV was high enough to drive the transcriptional effects of IRF-3 but too low to trigger its apoptotic activity. We confirmed this IRF-3 threshold idea by generating a tetracycline (Tet)-inducible cell line for IRF-3 expression, which enabled us to express various levels of IRF-3. PI could be established in the Tet-off cell line, and as expected, when doxycycline was withdrawn, the cells underwent apoptosis. Finally, we tested for PI establishment in 12 mouse embryo fibroblasts by natural selection. Eleven lines became persistently infected; although seven out of them had low IRF-3 levels, four did not. When one of the latter four was further analyzed, we observed that it expressed a very low level of caspase 3, the final executor protease of the apoptotic pathway. These results demonstrated that SeV PI can arise from infection of normal wild-type cells, but only if they can find a way to impair the IRF-3-dependent apoptotic pathway.

A controlled trial of acute effects of human exposure to traffic particles on pulmonary oxidative stress and heart rate variability.

BACKGROUND: For many individuals, daily commuting activities on roadways account for a substantial proportion of total exposure, as well as peak-level exposures, to traffic-related air pollutants (TRAPS) including ultrafine particles, but the health impacts of these exposures are not well-understood. We sought to determine if exposure to TRAPs particles during commuting causes acute oxidative stress in the respiratory tract or changes in heart rate variability (HRV), a measure of autonomic activity. METHODS: We conducted a randomized, cross-over trial in which twenty-one young adults took two 1.5-hr rides in a passenger vehicle in morning rush-hour traffic. The subjects wore a powered-air-purifying respirator, and were blinded to high-efficiency particulate air (HEPA) filtration during one of the rides. At time points before and after the rides, we measured HRV and markers of oxidative stress in exhaled breath condensate (EBC) including nitrite, the sum of nitrite and nitrate, malondialdehyde, and 8-isoprostane. We used mixed linear models to evaluate the effect of exposure on EBC and HRV outcomes, adjusting for pre-exposure response levels. We used linear models to examine the effects of particle concentrations on EBC outcomes at post-exposure time points. RESULTS: Mean EBC nitrite and the sum of nitrite and nitrate were increased from baseline at immediately post-exposure comparing unfiltered to filtered rides (2.11 µM vs 1.70 µM, p = 0.02 and 19.1 µM vs 10.0 µM, p = 0.02, respectively). Mean EBC malondialdehyde (MDA) concentrations were about 10% greater following the unfiltered vs. filtered exposures, although this result was not statistically significant. We found no significant associations between exposure to traffic particles and HRV outcomes at any of the time points. At immediately post-exposure, an interquartile range increase in particle number concentration was associated with statistically significant increases in nitrite (99.4%, 95% CI 32.1% to 166.7%) and nitrite + nitrate (75.7%, 95% CI 21.5% to 130.0%). CONCLUSIONS: Increases in markers of oxidative stress in EBC may represent early biological responses to widespread exposures to TRAPs particles that affect passengers in vehicles on heavily trafficked roadways.

Interferon-stimulated gene TDRD7 interacts with AMPK and inhibits its activation to suppress viral replication and pathogenesis.

IMPORTANCE: Virus infection triggers induction of interferon (IFN)-stimulated genes (ISGs), which ironically inhibit viruses themselves. We identified Tudor domain-containing 7 (TDRD7) as a novel antiviral ISG, which inhibits viral replication by interfering with autophagy pathway. Here, we present a molecular basis for autophagy inhibitory function of TDRD7. TDRD7 interacted with adenosine monophosphate (AMP)-activated protein kinase (AMPK), the kinase that initiates autophagy, to inhibit its activation. We identified domains required for the interaction; deleting AMPK-interacting domain blocked antiAMPK and antiviral activities of TDRD7. We used primary cells and mice to evaluate the TDRD7-AMPK antiviral pathway. TDRD7-deficient primary mouse cells exhibited enhanced AMPK activation and viral replication. Finally, TDRD7 knockout mice showed increased susceptibility to respiratory virus infection. Therefore, our study revealed a new antiviral pathway of IFN and its contribution to host response. Our results have therapeutic potential; a TDRD7-derived peptide may be an effective AMPK inhibitor with application as antiviral agent.

STING-Mediated Interferon Induction by Herpes Simplex Virus 1 Requires the Protein Tyrosine Kinase Syk.

The nature and the intensity of innate immune response to virus infection determine the course of pathogenesis in the host. Among the many pathogen-associated molecular pattern recognition receptors, STING, an endoplasmic reticulum (ER)-associated protein, plays a pivotal role in triggering responses to microbial or cellular cytoplasmic DNA. Herpes simplex virus 1 (HSV-1), a common human pathogen, activates STING signaling, and the resultant induction of type I interferon causes inhibition of virus replication. In this context, we have observed that phosphorylation of Tyr245 of STING by epidermal growth factor receptor kinase is necessary for interferon induction. Here, we report that phosphorylation of Tyr240 by the tyrosine kinase Syk is essential for all signaling activities of STING. Our analysis showed that upon ligand-binding, STING dimerizes and interacts with membrane-bound EGFR, which autophosphorylates and provides the platform for the recruitment of cytoplasmic Syk to the signaling complex and its activation. Activated Syk phosphorylates Tyr240 of STING, followed by phosphorylation of Tyr245 by epidermal growth factor receptor (EGFR). Pharmacological or genetic ablation of Syk activity resulted in an arrest of STING in the ER compartment and a complete block of gene induction. Consequently, in the absence of Syk, HSV-1 could not induce interferon, and it replicated more robustly. IMPORTANCE The innate immune response to virus infection leads to interferon production and inhibition of viral replication. STING, an ER-bound protein, mediates such a response to cytoplasmic cellular or microbial DNA. HSV-1, a DNA virus, activates STING, and it replicates more efficiently in the absence of STING signaling. We demonstrate that phosphorylation of Tyr240 of STING by the protein tyrosine kinase Syk is essential for STING-mediated gene induction. To signal, ligand-activated STING recruits two kinases, Syk and EGFR, which phosphorylate Tyr240 and Tyr245, respectively. The dependence of STING signaling on Syk has broad significance, because STING plays a major role in many microbial, mitochondrial, and autoimmune diseases as well as in cancer development and therapy.

Endothelial TLR2 promotes proangiogenic immune cell recruitment and tumor angiogenesis.

Toll-like receptor 2 (TLR2) is implicated in various pathologies, mainly in terms of its function within innate immune cells. However, TLR2 is also present in endothelial cells. Here, we explored the physiological and pathophysiological roles of endothelial TLR2 signaling. We found that TLR2 was highly abundant in the endothelium within various tissues using TLR2-IRES-EGFP reporter mice and was required for proinflammatory endothelial cell function. Endothelial cells lacking TLR2 exhibited reduced proinflammatory potential at the protein, cell, and tissue levels. Loss of endothelial TLR2 blunted the inflammatory response to both exogenous and endogenous danger signals in endothelial cells in culture and in vivo. Endothelial TLR2 promoted tumor growth, angiogenesis, and protumorigenic immune cell recruitment in a mouse model of prostate cancer. Furthermore, the cell surface localization of P-selectin and the subsequent production of other critical cell adhesion molecules (such as E-selectin, ICAM-1 and VCAM-1) that recruit immune cells required endothelial TLR2. Our findings demonstrate that endothelial cells actively contribute to innate immune pathways and propose that endothelial TLR2 has a pathological role in proinflammatory conditions.

Pulmonary effects of inhaled diesel exhaust in aged mice.

Pulmonary morbidity and mortality resulting from exposure to fine particulate matter (PM) increases with age. The present studies analyzed potential mechanisms underlying increased susceptibility of the elderly to PM using diesel exhaust (DE) as a model. Mice (2 m and 18 m) were exposed to DE (0, 300, and 1000 microg/m(3)) for 3 h once (single) or 3 h/day for 3 days (repeated). Bronchoalveolar lavage fluid (BAL), serum and lung tissue were collected 0 and 24 h later. Exposure to DE resulted in structural alterations in the lungs of older but not younger mice, including patchy thickening of the alveolar septa and inflammatory cell localization in alveolar spaces. These effects were most pronounced 24 h after a single exposure to the higher dose of DE. Significant increases in BAL nitrogen oxides were also noted in older mice, as well as expression of lipocalin 24p3, an oxidative stress marker in the lung with no effects in younger mice. Following DE inhalation, expression of Tumor Necrosis Factor alpha (TNFalpha) was upregulated in lungs of both younger and older mice; however, this was attenuated in older animals. Whereas exposure to DE resulted in increases in lung Interleukin-6 (IL-6) expression in both older and younger mice, IL-8 increased only in older animals. In younger mice, constitutive expression of manganese superoxide dismutase (MnSOD) decreased after DE exposure, while in older mice, constitutive MnSOD was not detectable and DE had no effect on expression of this antioxidant. Taken together, these results suggest that altered generation of inflammatory mediators and MnSOD may contribute to increased susceptibility of older mice to inhaled DE.

Inhibition of viral pathogenesis and promotion of the septic shock response to bacterial infection by IRF-3 are regulated by the acetylation and phosphorylation of its coactivators.

Interferon (IFN) is required for protecting mice from viral pathogenesis; reciprocally, it mediates the deleterious septic shock response to bacterial infection. The critical transcription factor for IFN induction, in both cases, is IRF-3, which is activated by TLR3 or RIG-I signaling in response to virus infection and TLR4 signaling in response to bacterial infection. Here, we report that IRF-3's transcriptional activity required its coactivators, ß-catenin and CBP, to be modified by HDAC6-mediated deacetylation and protein kinase C isozyme ß (PKC-ß)-mediated phosphorylation, respectively, so that activated nuclear IRF-3 could form a stable transcription initiation complex at the target gene promoters. ß-Catenin bridges IRF-3 and CBP, and the modifications were required specifically for the interaction between ß-catenin and CBP but not ß-catenin and IRF-3. Consequently, like IRF-3(-/-) mice, HDAC6(-/-) mice were resistant to bacterial lipopolysaccharide-induced septic shock. Conversely, they were highly susceptible to pathogenesis caused by Sendai virus infection. Thus, HDAC6 is an essential component of the innate immune response to microbial infection.