Endometrial cells sense and react to tissue damage during infection of the bovine endometrium via interleukin 1

Cells generate inflammatory responses to bacteria when pattern recognition receptors bind pathogen-associated molecules such as lipopolysaccharide. Cells may also respond to tissue damage by sensing damage-associated molecules. Postpartum bacterial infections of the bovine uterus cause endometritis but the risk of disease is increased by tissue trauma triggered by dystocia. Animals that suffered dystocia had increased concentrations of inflammatory mediators IL-8, IL-1β and IL-1α in vaginal mucus 3 weeks postpartum, but they also had more bacteria than normal animals. Ex vivo organ cultures of endometrium, endometrial cells and peripheral blood monocytes did not generate inflammatory responses to prototypical damage molecules, HMGB1 or hyaluronan, or to necrotic cells; although they secreted IL-6 and IL-8 in a concentration-dependent manner when treated with IL-1α. However, necrotic endometrial cells did not accumulate intracellular IL-1α or release IL-1α, except when pre-treated with lipopolysaccharide or bacteria. Endometrial cell inflammatory responses to IL-1α were dependent on the cognate receptor IL-1R1, and the receptor adaptor protein MyD88, and the inflammatory response to IL-1α was independent of the response to lipopolysaccharide. Rather than a typical damage-associated molecule, IL-1α acts to scale the inflammatory response in recognition that there is a combination of pathogen challenge followed by endometrial cell damage

Targeting colorectal cancer stem cells with inducible caspase-9

Colorectal cancer stem cells (CSCs) drive tumor growth and are suggested to initiate distant metastases. Moreover, colon CSCs are reportedly more resistant to conventional chemotherapy, which is in part due to upregulation of anti-apoptotic Bcl-2 family members. To determine whether we could circumvent this apoptotic blockade, we made use of an inducible active caspase-9 (iCasp9) construct to target CSCs. Dimerization of iCasp9 with AP20187 in HCT116 colorectal cancer cells resulted in massive and rapid induction of apoptosis. In contrast to fluorouracil (5-FU)-induced apoptosis, iCasp9-induced apoptosis was independent of the mitochondrial pathway as evidenced by Bax/Bak double deficient HCT116 cells. Dimerizer treatment of colon CSCs transduced with iCasp9 (CSC-iCasp9) also rapidly induced high levels of apoptosis, while these cells were unresponsive to 5-FU in vitro. More importantly, injection of the dimerizer into mice that developed a colon CSC-iCasp9-induced tumor resulted in a strong decrease in tumor size, an increase in tumor cell apoptosis and a clear loss of CD133+ CSCs. Taken together, our data indicate that dimerization of iCasp9 circumvents the apoptosis block in CSCs, which results in effective tumor regression in vivo

Identification of Mammalian Protein Quality Control Factors by High-Throughput Cellular Imaging

Protein Quality Control (PQC) pathways are essential to maintain the equilibrium between protein folding and the clearance of misfolded proteins. In order to discover novel human PQC factors, we developed a high-content, high-throughput cell-based assay to assess PQC activity. The assay is based on a fluorescently tagged, temperature sensitive PQC substrate and measures its degradation relative to a temperature insensitive internal control. In a targeted screen of 1591 siRNA genes involved in the Ubiquitin-Proteasome System (UPS) we identified 25 of the 33 genes encoding for 26S proteasome subunits and discovered several novel PQC factors. An unbiased genome-wide siRNA screen revealed the protein translation machinery, and in particular the EIF3 translation initiation complex, as a novel key modulator of misfolded protein stability. These results represent a comprehensive unbiased survey of human PQC components and establish an experimental tool for the discovery of genes that are required for the degradation of misfolded proteins under conditions of proteotoxic stress

Beyond reduction of atherosclerosis: PON2 provides apoptosis resistance and stabilizes tumor cells

Major contributors to atherosclerosis are oxidative damage and endoplasmic reticulum (ER) stress-induced apoptosis; both of which can be diminished by the anti-oxidative protein paraoxonase-2 (PON2). ER stress is also relevant to cancer and associated with anti-cancer treatment resistance. Hence, we addressed, for the first time, whether PON2 contributes to tumorigenesis and apoptotic escape. Intriguingly, we found that several human tumors upregulated PON2 and such overexpression provided resistance to different chemotherapeutics (imatinib, doxorubicine, staurosporine, or actinomycin) in cell culture models. This was reversed after PON2 knock-down. Remarkably, just deficiency of PON2 caused apoptosis of selective tumor cells per se, demonstrating a previously unanticipated oncogenic function. We found a dual mechanistic role. During ER stress, high PON2 levels lowered redox-triggered induction of pro-apoptotic CHOP particularly via the JNK pathway, which prevented mitochondrial cell death signaling. Apart from CHOP, PON2 also diminished intrinsic apoptosis as it prevented mitochondrial superoxide formation, cardiolipin peroxidation, cytochrome c release, and caspase activation. Ligand-stimulated apoptosis by TRAIL or TNFα remained unchanged. Finally, PON2 knock-down caused vast reactive oxygen species formation and stimulated JNK-triggered CHOP expression, but inhibition of JNK signaling did not prevent cell death, demonstrating the pleiotropic, dominating anti-oxidative effect of PON2. Therefore, targeting redox balance is powerful to induce selective tumor cell death and proposes PON2 as new putative anti-tumor candidate

The IL-1-Like Cytokine IL-33 Is Constitutively Expressed in the Nucleus of Endothelial Cells and Epithelial Cells In Vivo: A Novel ‘Alarmin’?

BACKGROUND: Interleukin-33 (IL-33) is an IL-1-like cytokine ligand for the IL-1 receptor-related protein ST2, that activates mast cells and Th2 lymphocytes, and induces production of Th2-associated cytokines in vivo. We initially discovered IL-33 as a nuclear factor (NF-HEV) abundantly expressed in high endothelial venules from lymphoid organs, that associates with chromatin and exhibits transcriptional regulatory properties. This suggested that, similarly to IL-1alpha and chromatin-associated cytokine HMGB1, IL-33 may act as both a cytokine and a nuclear factor. Although the activity of recombinant IL-33 has been well characterized, little is known yet about the expression pattern of endogenous IL-33 in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that IL-33 is constitutively and abundantly expressed in normal human tissues. Using a combination of human tissue microarrays and IL-33 monoclonal and polyclonal antibodies, we found that IL-33 is a novel nuclear marker of the endothelium widely expressed along the vascular tree. We observed abundant nuclear expression of IL-33 in endothelial cells from both large and small blood vessels in most normal human tissues, as well as in human tumors. In addition to endothelium, we also found constitutive nuclear expression of IL-33 in fibroblastic reticular cells of lymphoid tissues, and epithelial cells of tissues exposed to the environment, including skin keratinocytes and epithelial cells of the stomach, tonsillar crypts and salivary glands. CONCLUSIONS/SIGNIFICANCE: Together, our results indicate that, unlike inducible cytokines, IL-33 is constitutively expressed in normal human tissues. In addition, they reveal that endothelial cells and epithelial cells constitute major sources of IL-33 in vivo. Based on these findings, we speculate that IL-33 may function, similarly to the prototype 'alarmin' HMGB1, as an endogenous 'danger' signal to alert the immune system after endothelial or epithelial cell damage during trauma or infection

YopJ-Induced Caspase-1 Activation in Yersinia-Infected Macrophages: Independent of Apoptosis, Linked to Necrosis, Dispensable for Innate Host Defense

Yersinia outer protein J (YopJ) is a type III secretion system (T3SS) effector of pathogenic Yersinia (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis) that is secreted into host cells. YopJ inhibits survival response pathways in macrophages, causing cell death. Allelic variation of YopJ is responsible for differential cytotoxicity in Yersinia strains. YopJ isoforms in Y. enterocolitica O:8 (YopP) and Y. pestis KIM (YopJKIM) strains have high cytotoxic activity. In addition, YopJKIM-induced macrophage death is associated with caspase-1 activation and interleukin-1β (IL-1β secretion. Here, the mechanism of YopJKIM-induced cell death, caspase-1 activation, and IL-1β secretion in primary murine macrophages was examined. Caspase-3/7 activity was low and the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Y. pestis KIM5-infected macrophages. In addition, cytotoxicity and IL-1β secretion were not reduced in the presence of a caspase-8 inhibitor, or in B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 homologous antagonist/killer (Bak) knockout macrophages, showing that YopJKIM-mediated cell death and caspase-1 activation occur independent of mitochondrial-directed apoptosis. KIM5-infected macrophages released high mobility group protein B1 (HMGB1), a marker of necrosis, and microscopic analysis revealed that necrotic cells contained active caspase-1, indicating that caspase-1 activation is associated with necrosis. Inhibitor studies showed that receptor interacting protein 1 (RIP1) kinase and reactive oxygen species (ROS) were not required for cytotoxicity or IL-β release in KIM5-infected macrophages. IL-1β secretion was reduced in the presence of cathepsin B inhibitors, suggesting that activation of caspase-1 requires cathepsin B activity. Ectopically-expressed YopP caused higher cytotoxicity and secretion of IL-1β in Y. pseudotuberculosis-infected macrophages than YopJKIM. Wild-type and congenic caspase 1 knockout C57BL/6 mice were equally susceptible to lethal infection with Y. pseudotuberculosis ectopically expressing YopP. These data suggest that YopJ-induced caspase-1 activation in Yersinia-infected macrophages is a downstream consequence of necrotic cell death and is dispensable for innate host resistance to a strain with enhanced cytotoxicity

Diffusion is capable of translating anisotropic apoptosis initiation into a homogeneous execution of cell death

<p>Abstract</p> <p>Background</p> <p>Apoptosis is an essential cell death process throughout the entire life span of all metazoans and its deregulation in humans has been implicated in many proliferative and degenerative diseases. Mitochondrial outer membrane permeabilisation (MOMP) and activation of effector caspases are key processes during apoptosis signalling. MOMP can be subject to spatial coordination in human cancer cells, resulting in intracellular waves of cytochrome-c release. To investigate the consequences of these spatial anisotropies in mitochondrial permeabilisation on subsequent effector caspase activation, we devised a mathematical reaction-diffusion model building on a set of partial differential equations.</p> <p>Results</p> <p>Reaction-diffusion modelling suggested that even if strong spatial anisotropies existed during mitochondrial cytochrome c release, these would be eliminated by free diffusion of the cytosolic proteins that instantiate the apoptosis execution network. Experimentally, rapid sampling of mitochondrial permeabilisation and effector caspase activity in individual HeLa cervical cancer cells confirmed predictions of the reaction-diffusion model and demonstrated that the signalling network of apoptosis execution could efficiently translate spatial anisotropies in mitochondrial permeabilisation into a homogeneous effector caspase response throughout the cytosol. Further systems modelling suggested that a more than 10,000-fold impaired diffusivity would be required to maintain spatial anisotropies as observed during mitochondrial permeabilisation until the time effector caspases become activated.</p> <p>Conclusions</p> <p>Multi-protein diffusion efficiently contributes to eliminating spatial asynchronies which are present during the initiation of apoptosis execution and thereby ensures homogeneous apoptosis execution throughout the entire cell body. For previously reported biological scenarios in which effector caspase activity was shown to be targeted selectively to specific subcellular regions additional mechanisms must exist that limit or spatially coordinate caspase activation and/or protect diffusing soluble caspase substrates from unwanted proteolysis.</p

Induction of autophagy by spermidine promotes longevity.

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Kupffer Cells Hasten Resolution of Liver Immunopathology in Mouse Models of Viral Hepatitis

Kupffer cells (KCs) are widely considered important contributors to liver injury during viral hepatitis due to their pro-inflammatory activity. Herein we utilized hepatitis B virus (HBV)-replication competent transgenic mice and wild-type mice infected with a hepatotropic adenovirus to demonstrate that KCs do not directly induce hepatocellular injury nor do they affect the pathogenic potential of virus-specific CD8 T cells. Instead, KCs limit the severity of liver immunopathology. Mechanistically, our results are most compatible with the hypothesis that KCs contain liver immunopathology by removing apoptotic hepatocytes in a manner largely dependent on scavenger receptors. Apoptotic hepatocytes not readily removed by KCs become secondarily necrotic and release high-mobility group box 1 (HMGB-1) protein, promoting organ infiltration by inflammatory cells, particularly neutrophils. Overall, these results indicate that KCs resolve rather than worsen liver immunopathology

Senescent cells as a source of inflammatory factors for tumor progression

Cellular senescence, which is associated with aging, is a process by which cells enter a state of permanent cell cycle arrest, therefore constituting a potent tumor suppressive mechanism. Recent studies show that, despite the beneficial effects of cellular senescence, senescent cells can also exert harmful effects on the tissue microenvironment. The most significant of these effects is the acquisition of a senescent-associated secretory phenotype (SASP), which entails a striking increase in the secretion of pro-inflammatory cytokines. Here, we summarize our knowledge of the SASP and the impact it has on tissue microenvironments and ability to stimulate tumor progression