Vaccinia virus induces rapid necrosis in keratinocytes by a STAT3-dependent mechanism.

RATIONALE: Humans with a dominant negative mutation in STAT3 are susceptible to severe skin infections, suggesting an essential role for STAT3 signaling in defense against cutaneous pathogens. METHODS: To focus on innate antiviral defenses in keratinocytes, we used a standard model of cutaneous infection of severe combined immunodeficient mice with the current smallpox vaccine, ACAM-2000. In parallel, early events post-infection with the smallpox vaccine ACAM-2000 were investigated in cultured keratinocytes of human and mouse origin. RESULTS: Mice treated topically with a STAT3 inhibitor (Stattic) developed larger vaccinia lesions with higher virus titers and died more rapidly than untreated controls. Cultured human and murine keratinocytes infected with ACAM-2000 underwent rapid necrosis, but when treated with Stattic or with inhibitors of RIP1 kinase or caspase-1, they survived longer, produced higher titers of virus, and showed reduced activation of type I interferon responses and inflammatory cytokines release. Treatment with inhibitors of RIP1 kinase and STAT3, but not caspase-1, also reduced the inflammatory response of keratinocytes to TLR ligands. Vaccinia growth properties in Vero cells, which are known to be defective in some antiviral responses, were unaffected by inhibition of RIP1K, caspase-1, or STAT3. CONCLUSIONS: Our findings indicate that keratinocytes suppress the replication and spread of vaccinia virus by undergoing rapid programmed cell death, in a process requiring STAT3. These data offer a new framework for understanding susceptibility to skin infection in patients with STAT3 mutations. Interventions which promote prompt necroptosis/pyroptosis of infected keratinocytes may reduce risks associated with vaccination with live vaccinia virus.

Hemoglobin-driven pathophysiology is an in vivo consequence of the red blood cell storage lesion that can be attenuated in guinea pigs by haptoglobin therapy.

Massive transfusion of blood can lead to clinical complications, including multiorgan dysfunction and even death. Such severe clinical outcomes have been associated with longer red blood cell (rbc) storage times. Collectively referred to as the rbc storage lesion, rbc storage results in multiple biochemical changes that impact intracellular processes as well as membrane and cytoskeletal properties, resulting in cellular injury in vitro. However, how the rbc storage lesion triggers pathophysiology in vivo remains poorly defined. In this study, we developed a guinea pig transfusion model with blood stored under standard blood banking conditions for 2 (new), 21 (intermediate), or 28 days (old blood). Transfusion with old but not new blood led to intravascular hemolysis, acute hypertension, vascular injury, and kidney dysfunction associated with pathophysiology driven by hemoglobin (Hb). These adverse effects were dramatically attenuated when the high-affinity Hb scavenger haptoglobin (Hp) was administered at the time of transfusion with old blood. Pathologies observed after transfusion with old blood, together with the favorable response to Hp supplementation, allowed us to define the in vivo consequences of the rbc storage lesion as storage-related posttransfusion hemolysis producing Hb-driven pathophysiology. Hb sequestration by Hp might therefore be a therapeutic modality for enhancing transfusion safety in severely ill or massively transfused patients.

Transcriptome analysis revealed unique genes as targets for the anti-inflammatory action of activated protein C in human macrophages.

BACKGROUND: Activated protein C (APC) has been introduced as a therapeutic agent for treatment of patients with severe sepsis due to its unique anticoagulant and anti-inflammatory properties in the vascular system. In this study we investigated novel targets for the anti-inflammatory action of APC in human macrophages. METHODS: Using a genome-wide approach, effects of APC on the expression profile in inflammatory activated human macrophages were analyzed. RESULTS: We identified, for the first time, genes that are specifically regulated by APC under inflammatory conditions, such as chromatin binding protein 4B (CHMP4B) and p300/CBP-associated factor (PCAF), thus indicating a role of APC in the epigenetic control of gene transcription. A functional assay showed the influence of APC in the acetyltransferase/deacetylase activity of nuclear extracts from inflamed macrophages. CONCLUSION: Our data sheds new light on APC targets in inflammation and opens new lines of investigation that may be explored in order to further elucidate its unique molecule properties.

Quantitative mass spectrometry defines an oxidative hotspot in hemoglobin that is specifically protected by haptoglobin.

The reaction of hemoglobin (Hb) with hydrogen peroxide (H(2)O(2)) results in free radicals generated at the heme iron, followed by radical transfer to the porphyrin/globin. In the present work, we employed isobaric tagging for relative and absolute quantification (iTRAQ) and a LC-MALDI-MS/MS-based proteomic approach to identify the extent of oxidative changes within tetrameric Hb and dimeric Hb-haptoglobin (Hb-Hp) complexes. Extensive oxidative modifications were found to be restricted to peptides containing alphaTyr42, betaTyr145, and betaCys93. The protein region composed of these peptides appears to define an area of oxidative activity within the Hb tetramer that extends across the critical alpha1beta2/alpha2beta1 interface. Extensive oxidative modifications occurring at betaCys93 indicate that this surface amino acid is an important end point for free radical induced protein oxidation within Hb. Conversely when Hp 1-1 or 2-2 was complexed with dissociable Hb, oxidative changes in Hp complexed dimeric Hb were prevented. This protection was not observed in a stabilized tetrameric Hb, which displays a weak binding affinity for Hp. Therefore, dimerization of Hb and Hp binding may interfere with free radical translocation and play an important role in the overall antioxidant mechanism of Hp. Interestingly, the prevention of peroxide induced Hb amino acid oxidation in purified Hb-Hp1-1 and Hb-Hp2-2 was found to be equal, indicating a phenotype independent specificity in the process of oxidative protection. Taken together, these data suggest differences in oxidative modifications resulting from peroxide induced heme emanated free radical distribution in tetrameric compared to Hp1-1/Hp2-2 stabilized dimeric Hb.

Hemoglobin can attenuate hydrogen peroxide-induced oxidative stress by acting as an antioxidative peroxidase.

Hemoglobin is considered a potentially toxic molecule when released from erythrocytes during hemolysis, inflammation, or tissue injury. The mechanisms of toxicity involve reduced nitric oxide bioavailability and oxidative processes both occurring at the heme prosthetic groups. When the endogenous oxidant H(2)O(2) reacts with Hb, transient radicals are generated during the peroxidative consumption of H(2)O(2). If not neutralized, these radicals can lead to tissue toxicity. The net biologic effect of extracellular Hb in an H(2)O(2)-rich environment will therefore be determined by the balance of H(2)O(2) decomposition (potential protective effect) and radical generation (potential damaging effect). Here we show that Hb can protect different cell types from H(2)O(2)-mediated cell death and the associated depletion of intracellular glutathione and ATP. Importantly, Hb blunts the transcriptional oxidative-stress response induced by H(2)O(2) in human vascular smooth muscle cells (VSMCs). Based on spectrophotometric and quantitative mass spectrometry analysis, we suggested a novel mechanism in which Hb redox-cycles H(2)O(2) and simultaneously internalizes the radical burden, with irreversible structural globin changes starting with specific amino acid oxidation involving the heme proximate betaCys93 and ultimately ending with protein precipitation. Our results suggest that complex interactions determine whether extracellular Hb, under certain circumstances, acts a protective or a damaging factor during peroxidative stress conditions.

Sequestration of extracellular hemoglobin within a haptoglobin complex decreases its hypertensive and oxidative effects in dogs and guinea pigs.

Release of hemoglobin (Hb) into the circulation is a central pathophysiologic event that contributes to morbidity and mortality in chronic hemolytic anemias and severe malaria. These toxicities arise from Hb-mediated vasoactivity, possibly due to NO scavenging and localized tissue oxidative processes. Currently, there is no established treatment that targets circulating extracellular Hb. Here, we assessed the role of haptoglobin (Hp), the primary scavenger of Hb in the circulation, in limiting the toxicity of cell-free Hb infusion. Using a canine model, we found that glucocorticoid stimulation of endogenous Hp synthesis prevented Hb-induced hemodynamic responses. Furthermore, guinea pigs administered exogenous Hp displayed decreased Hb-induced hypertension and oxidative toxicity to extravascular environments, such as the proximal tubules of the kidney. The ability of Hp to both attenuate hypertensive responses during Hb exposure and prevent peroxidative toxicity in extravascular compartments was dependent on Hb-Hp complex formation, which likely acts through sequestration of Hb rather than modulation of its NO- and O2-binding characteristics. Our data therefore suggest that therapies involving supplementation of endogenous Hb scavengers may be able to treat complications of acute and chronic hemolysis, as well as counter the adverse effects associated with Hb-based oxygen therapeutics.

The wnt pathway: a macrophage effector molecule that triggers inflammation.

Wnt proteins are members of the highly conserved wingless family of proteins responsible for cell differentiation and development and for neoplastic and degenerative processes. Recently, Toll-like receptor-mediated Wnt signaling was found to be associated with innate immunity in Drosophila. Upregulation of Wnt5A in human macrophages upon microbial challenge indicated a similar mechanism. Toll-like receptor-mediated Wnt5A expression is a key process for sustained inflammatory macrophage activation through autocrine and paracrine signaling. Downregulation of Wnt5A expression and subsequent attenuation of inflammatory macrophage responses by activated protein C supports the link between inflammation and coagulation, another highly conserved biologic system. Direct evidence for the relevance of Wnt5A in severe systemic inflammation is provided by the finding of higher Wnt5A levels in patients with sepsis than in healthy individuals. The fact that Wnt5A signaling can be modulated by anti-inflammatory mediators makes this effector molecule an attractive target for therapeutic intervention in inflammatory diseases.

Characterization of high molecular weight multimeric states of human haptoglobin and hemoglobin-based oxygen carriers by high-mass MALDI MS.

High-mass MALDI-TOF mass spectrometry (MS) is a novel analytical approach to study large biomolecules and their interactions. It is a powerful alternative method to gel electrophoresis (GE) and size exclusion chromatography (SEC) for obtaining information on the molecular weights of macromolecules and for determining protein complexes. The precision of mass measurements (mass accuracy), high sensitivity, speed of the analysis, and tolerance toward sample heterogeneity are the major features of this MS-based approach. Remarkably, MS provides direct stoichiometric information of macromolecular protein complexes, when noncovalent interactions are stabilized during desorption/ionization by use of chemical cross-linking reagents. In this study, high-mass MALDI-TOF MS was applied to characterize the multimeric state of the human plasma protein haptoglobin (Hp), which is in the mass range of 150-300 kDa. Also, higher order structures of hemoglobin-based oxygen carriers (HBOCs) and their interactions with human haptoglobin were analyzed. These investigations are of clinical importance and contribute to the overall understanding of specific toxicity and clearance of HBOCs.

Haptoglobin preserves the CD163 hemoglobin scavenger pathway by shielding hemoglobin from peroxidative modification.

Detoxification and clearance of extracellular hemoglobin (Hb) have been attributed to its removal by the CD163 scavenger receptor pathway. However, even low-level hydrogen peroxide (H(2)O(2)) exposure irreversibly modifies Hb and severely impairs Hb endocytosis by CD163. We show here that when Hb is bound to the high-affinity Hb scavenger protein haptoglobin (Hp), the complex protects Hb from structural modification by preventing alpha-globin cross-links and oxidations of amino acids in critical regions of the beta-globin chain (eg, Trp15, Cys93, and Cys112). As a result of this structural stabilization, H(2)O(2)-exposed Hb-Hp binds to CD163 with the same affinity as nonoxidized complex. Endocytosis and lysosomal translocation of oxidized Hb-Hp by CD163-expressing cells were found to be as efficient as with nonoxidized complex. Hp complex formation did not alter Hb's ability to consume added H(2)O(2) by redox cycling, suggesting that within the complex the oxidative radical burden is shifted to Hp. We provide structural and functional evidence that Hp protects Hb when oxidatively challenged with H(2)O(2) preserving CD163-mediated Hb clearance under oxidative stress conditions. In addition, our data provide in vivo evidence that unbound Hb is oxidatively modified within extravascular compartments consistent with our in vitro findings.

The reaction of hydrogen peroxide with hemoglobin induces extensive alpha-globin crosslinking and impairs the interaction of hemoglobin with endogenous scavenger pathways.

Cell-free hemoglobin (Hb) enhances the oxidation-related toxicity associated with inflammation, ischemia, and hemolytic disorders. Hb is highly vulnerable to oxidative damage, and irreversible structural changes involving iron/heme oxidation, heme-adduct products, and amino acid oxidation have been reported. Specific structural features of Hb, such as unconstrained alpha-chains and molecular size, determine the efficiency of interactions between the endogenous Hb scavengers haptoglobin (Hp) and CD163. Using HPLC, mass spectrometry, and Western blotting, we show that H(2)O(2)-mediated Hb oxidation results in the formation of covalently stabilized globin multimers, with prominent intramolecular crosslinking between alpha-globin chains. These structural alterations are associated with reduced Hp binding, reduced CD163 interaction, and severely impaired endocytosis of oxidized Hb by the Hp-CD163 pathway. As a result, when exposed to oxidized Hb, CD163-positive HEK293 cells and human macrophages do not increase hemeoxygenase-1 (HO-1) expression, the physiological anti-oxidative macrophage response to Hb exposure. Failed Hb clearance, inadequate HO-1 expression, and the subsequent accumulation of oxidatively damaged Hb species might thus contribute to pathologies related to oxidative stress.