Brant M. Wagener

Protein C and acute inflammation: a clinical and biological perspective

The protein C system plays an active role in modulating severe systemic inflammatory processes such as sepsis, trauma, and acute respiratory distress syndrome (ARDS) via its anticoagulant and anti-inflammatory properties. Plasma levels of activated protein C (aPC) are lower than normal in acute inflammation in humans, except early after severe trauma when high plasma levels of aPC may play a mechanistic role in the development of posttraumatic coagulopathy. Thus, following positive results of preclinical studies, a clinical trial (PROWESS) with high continuous doses of recombinant human aPC given for 4 days demonstrated a survival benefit in patients with severe sepsis. This result was not confirmed by subsequent clinical trials, including the recently published PROWESS-SHOCK trial in patients with septic shock and a phase II trial with patients with nonseptic ARDS. A possible explanation for the major difference in outcome between PROWESS and PROWESS-SHOCK trials is that lung-protective ventilation was used for the patients included in the recent PROWESS-SHOCK, but not in the original PROWESS trial. Since up to 75% of sepsis originates from the lung, aPC treatment may not have added enough to the beneficial effect of lung-protective ventilation to show lower mortality. Thus whether aPC will continue to be used to modulate the acute inflammatory response in humans remains uncertain. Because recombinant human aPC has been withdrawn from the market, a better understanding of the complex interactions between coagulation and inflammation is needed before considering the development of new drugs that modulate both coagulation and acute inflammation in humans.

The glycocalyx and Trauma: A Review

ABSTRACT In the United States trauma is the leading cause of mortality among those under the age of 45, claiming approximately 192,000 lives each year. Significant personal disability, lost productivity, and long-term healthcare needs are common and contribute 580 billion dollars in economic impact each year. Improving resuscitation strategies and the early acute care of trauma patients has the potential to reduce the pathological sequelae of combined exuberant inflammation and immune suppression that can co-exist, or occur temporally, and adversely affect outcomes. The endothelial and epithelial glycocalyx has emerged as an important participant in both inflammation and immunomodulation. Constituents of the glycocalyx have been used as biomarkers of injury severity and have the potential to be target(s) for therapeutic interventions aimed at immune modulation. In this review, we provide a contemporary understanding of the physiologic structure and function of the glycocalyx and its role in traumatic injury with a particular emphasis on lung injury.

HMGB1 Accelerates Alveolar Epithelial Repair via an IL-1β- and αvβ6 Integrin-dependent Activation of TGF-β1

High mobility group box 1 (HMGB1) protein is a danger-signaling molecule, known to activate an inflammatory response via TLR4 and RAGE. HMGB1 can be either actively secreted or passively released from damaged alveolar epithelial cells. Previous studies have shown that IL-1β, a critical mediator acute lung injury in humans that is activated by HMGB1, enhances alveolar epithelial repair, although the mechanisms are not fully understood. Herein, we tested the hypothesis that HMGB1 released by wounded alveolar epithelial cells would increase primary rat and human alveolar type II cell monolayer wound repair via an IL-1β-dependent activation of TGF-β1. HMGB1 induced in primary cultures of rat alveolar epithelial cells results in the release of IL-1β that caused the activation of TGF-β1 via a p38 MAPK-, RhoA- and αvβ6 integrin-dependent mechanism. Furthermore, active TGF-β1 accelerated the wound closure of primary rat epithelial cell monolayers via a PI3 kinase α-dependent mechanism. In conclusion, this study demonstrates that HMGB1 released by wounded epithelial cell monolayers, accelerates wound closure in the distal lung epithelium via the IL-1β-mediated αvβ6-dependent activation of TGF-β1, and thus could play an important role in the resolution of acute lung injury by promoting repair of the injured alveolar epithelium.

Red blood cell washing, nitrite therapy, and antiheme therapies prevent stored red blood cell toxicity after trauma–hemorrhage

Transfusion of stored red blood cells (RBCs) is associated with increased morbidity and mortality in trauma patients. Pro-oxidant, pro-inflammatory, and nitric oxide (NO) scavenging properties of stored RBCs are thought to underlie this association. In this study we determined the effects of RBC washing and nitrite and antiheme therapy on stored RBC-dependent toxicity in the setting of trauma-induced hemorrhage. A murine (C57BL/6) model of trauma-hemorrhage and resuscitation with 1 or 3 units of RBCs stored for 0-10 days was used. Tested variables included washing RBCs to remove lower MW components that scavenge NO, NO-repletion therapy using nitrite, or mitigation of free heme toxicity by heme scavenging or preventing TLR4 activation. Stored RBC toxicity was determined by assessment of acute lung injury indices (airway edema and inflammation) and survival. Transfusion with 5 day RBCs increased acute lung injury indexed by BAL protein and neutrophil accumulation. Washing 5 day RBCs prior to transfusion did not decrease this injury, whereas nitrite therapy did. Transfusion with 10 day RBCs elicited a more severe injury resulting in ~90% lethality, compared to <15% with 5 day RBCs. Both washing and nitrite therapy significantly protected against 10 day RBC-induced lethality, suggesting that washing may be protective when the injury stimulus is more severe. Finally, a spectral deconvolution assay was developed to simultaneously measure free heme and hemoglobin in stored RBC supernatants, which demonstrated significant increases of both in stored human and mouse RBCs. Transfusion with free heme partially recapitulated the toxicity mediated by stored RBCs. Furthermore, inhibition of TLR4 signaling, which is stimulated by heme, using TAK-242, or hemopexin-dependent sequestration of free heme significantly protected against both 5 day and 10 day mouse RBC-dependent toxicity. These data suggest that RBC washing, nitrite therapy, and/or antiheme and TLR4 strategies may prevent stored RBC toxicities.

Postexposure aerosolized heparin reduces lung injury in chlorine-exposed mice

Chlorine (Cl2) is a highly reactive oxidant gas that, when inhaled, may cause acute lung injury culminating in death from respiratory failure. In this study, we tested the hypothesis that exposure of mice to Cl2 causes intra-alveolar and systemic activation of the coagulation cascade that plays an important role in development of lung injury. C57Bl/6 mice were exposed to Cl2 (400 for 30 min or 600 ppm for 45 min) in environmental chambers and then returned to room air for 1 or 6 h. Native coagulation (NATEM) parameters such as blood clotting time and clot formation time were measured in whole blood by the viscoelastic technique. D-dimers and thrombin-anti-thrombin complexes were measured in both plasma and bronchoalveolar lavage fluid (BALF) by ELISA. Our results indicate that mice exposed to Cl2 gas had significantly increased clotting time, clot formation time, and D-dimers compared with controls. The thrombin-anti-thrombin complexes were also increased in the BALF of Cl2 exposed animals. To test whether increased coagulation contributed to the development of acute lung injury, mice exposed to Cl2 and returned to room air were treated with aerosolized heparin or vehicle for 20 min. Aerosolized heparin significantly reduced protein levels and the number of inflammatory cells in the BALF at 6 h postexposure. These findings highlight the importance of coagulation abnormities in the development of Cl2-induced lung injury.

Synergistic Inhibition of β2-adrenergic Receptor–mediated Alveolar Epithelial Fluid Transport by Interleukin-8 and Transforming Growth Factor-β

Background: Patients with acute respiratory distress syndrome who retain maximal alveolar fluid clearance (AFC) have better clinical outcomes. The release of endogenous catecholamines associated with shock or the administration of &bgr;2-adrenergic receptor (&bgr;2AR) agonists enhances AFC via a 3′-5′-cyclic adenosine monophosphate–dependent mechanism. The authors have previously reported that transforming growth factor-&bgr;1 (TGF-&bgr;1) and interleukin-8 (IL-8), two major mediators of alveolar inflammation associated with the early phase of acute respiratory distress syndrome, inhibit AFC upregulation by &bgr;2AR agonists via a phosphoinositol-3-kinase (PI3K)–dependent mechanism. However, whether TGF-&bgr;1 and IL-8 cause an additive or synergistic inhibition of AFC is unclear. Thus, the central hypothesis of the study was to determine whether they synergistically inhibit the &bgr;2AR-stimulated AFC by activating two different isoforms of PI3K. Methods: The effects of TGF-&bgr;1 or IL-8 on &bgr;2AR agonist–stimulated net alveolar fluid transport were studied using short-circuit current studies. Molecular pathways of inhibition were confirmed by pharmacologic inhibitors and Western blotting of p-Akt, G-protein–coupled receptor kinase 2, protein kinase C-&zgr;, and phospho-&bgr;2AR. Finally, our observations were confirmed by an in vivo model of AFC. Results: Combined exposure to TGF-&bgr;1 and IL-8/cytokine-induced neutrophil chemoattractant-1 caused synergistic inhibition of &bgr;2AR agonist–stimulated vectorial Cl− across alveolar epithelial type II cells (n = 12 in each group). This effect was explained by activation of different isoforms of PI3K by TGF-&bgr;1 and IL-8/cytokine-induced neutrophil chemoattractant-1 (n = 12 in each group). Furthermore, the inhibitory effect of TGF-&bgr;1 on 3′-5′-cyclic adenosine monophosphate–stimulated alveolar epithelial fluid transport required the presence of IL-8/cytokine-induced neutrophil chemoattractant-1 (n = 12 in each group). Inhibition of cytokine-induced neutrophil chemoattractant-1 prevented TGF-&bgr;1–mediated heterologous &bgr;2AR downregulation and restored physiologic &bgr;2AR agonist–stimulated AFC in rats (n = 6 in each group). Conclusions: TGF-&bgr;1 and IL-8 have a synergistic inhibitory effect on &bgr;2AR-mediated stimulation of pulmonary edema removal by the alveolar epithelium. This result may, in part, explain why a large proportion of the patients with acute respiratory distress syndrome have impaired AFC.

Acute brain trauma, lung injury, and pneumonia: more than just altered mental status and decreased airway protection

Traumatic brain injury (TBI) is a major cause of mortality and morbidity worldwide. Even when patients survive the initial insult, there is significant morbidity and mortality secondary to subsequent pulmonary edema, acute lung injury (ALI), and nosocomial pneumonia. Whereas the relationship between TBI and secondary pulmonary complications is recognized, little is known about the mechanistic interplay of the two phenomena. Changes in mental status secondary to acute brain injury certainly impair airway- and lung-protective mechanisms. However, clinical and translational evidence suggests that more specific neuronal and cellular mechanisms contribute to impaired systemic and lung immunity that increases the risk of TBI-mediated lung injury and infection. To better understand the cellular mechanisms of that immune impairment, we review here the current clinical data that support TBI-induced impairment of systemic and lung immunity. Furthermore, we also review the animal models that attempt to reproduce human TBI. Additionally, we examine the possible role of damage-associated molecular patterns, the chlolinergic anti-inflammatory pathway, and sex dimorphism in post-TBI ALI. In the last part of the review, we discuss current treatments and future pharmacological therapies, including fever control, tracheostomy, and corticosteroids, aimed to prevent and treat pulmonary edema, ALI, and nosocomial pneumonia after TBI.

Histone-Complexed DNA Fragments Levels are Associated with Coagulopathy, Endothelial Cell Damage, and Increased Mortality after Severe Pediatric Trauma

Background: The release of damage-associated molecular pattern molecules in the extracellular space secondary to injury has been shown to cause systemic activation of the coagulation system and endothelial cell damage. We hypothesized that pediatric trauma patients with increased levels of histone-complexed DNA fragments (hcDNA) would have evidence of coagulopathy and endothelial damage that would be associated with poor outcomes. Methods: We conducted a prospective observational study of 149 pediatric trauma patients and 62 control patients at two level 1 pediatric trauma centers from 2013 to 2016. Blood samples were collected upon arrival and at 24 h, analyzed for hcDNA, coagulation abnormalities, endothelial damage, and clinical outcome. Platelet aggregation was assessed with impedance aggregometry (Multiplate) and coagulation parameters were assessed by measuring prothrombin time ratio in plasma and the use of viscoelastic techniques (Rotational Thromboelastometry) in whole blood. Results: The median age was 8.3 years, the median injury severity score (ISS) was 20, and overall mortality was 10%. Significantly higher levels of hcDNA were found on admission in patients with severe injury (ISS > 25), coagulopathy, and/or abnormal platelet aggregation. Patients with high hcDNA levels also had significant elevations in plasma levels of syndecan-1, suggesting damage to the endothelial glycocalyx. Finally, significantly higher hcDNA levels were found in non-survivors. Conclusion: hcDNA is released following injury and correlates with coagulopathy, endothelial glycocalyx damage, and poor clinical outcome early after severe pediatric trauma. These results indicate that hcDNA may play an important role in development of coagulation abnormalities and endothelial glycocalyx damage in children following trauma.

Heat-shock Response Increases Lung Injury Caused by Pseudomonas aeruginosa via an Interleukin-10-dependent Mechanism in Mice

Background:The heat-shock response (HSR) protects from insults, such as ischemia–reperfusion injury, by inhibiting signaling pathways activated by sterile inflammation. However, the mechanisms by which the HSR activation would modulate lung damage and host response to a bacterial lung infection remain unknown. Methods:HSR was activated with whole-body hyperthermia or by intraperitoneal geldanamycin in mice that had their lungs instilled with Pseudomonas aeruginosa 24 h later (at least six mice per experimental group). Four hours after instillation, lung endothelial and epithelial permeability, bacterial counts, protein levels in bronchoalveolar lavage fluid, and lung myeloperoxidase activity were measured. Mortality rate 24 h after P. aeruginosa instillation was recorded. The HSR effect on the release of interleukin-10 and killing of P. aeruginosa bacteria by a mouse alveolar macrophage cell line and on neutrophil phagocytosis was also examined. Results:HSR activation worsened lung endothelial (42%) and epithelial permeability (50%) to protein, decreased lung bacterial clearance (71%), and increased mortality (50%) associated with P. aeruginosa pneumonia, an effect that was not observed in heat-shock protein–72-null mice. HSR-mediated decrease in neutrophil phagocytosis (69%) and bacterial killing (38%) by macrophages was interleukin-10 dependent, a mechanism confirmed by increased lung bacterial clearance and decreased mortality (70%) caused by P. aeruginosa pneumonia in heat-shocked interleukin-10-null mice. Conclusions:Prior HSR activation worsens lung injury associated with P. aeruginosa pneumonia in mice via heat-shock protein–72- and interleukin-10-dependent mechanisms. These results provide a novel mechanism for the immunosuppression observed after severe trauma that is known to activate HSR in humans.

Neuronal Wiskott-Aldrich syndrome protein regulates TGF-β1-mediated lung vascular permeability.

TGF‐β1 induces an increase in paracellular permeability and actin stress fiber formation in lung microvascular endothelial and alveolar epithelial cells via small Rho GTPase. The molecular mechanism involved is not fully understood. Neuronal Wiskott‐Aldrich syndrome protein (N‐WASP) has an essential role in actin structure dynamics. We hypothesized that N‐WASP plays a critical role in these TGF‐β1‐induced responses. In these cell monolayers, we demonstrated that N‐WASP down‐regulation by short hairpin RNA prevented TGF‐β1‐mediated disruption of the cortical actin structure, actin stress filament formation, and increased permeability. Furthermore, N‐WASP down‐regulation blocked TGF‐β1 activation mediated by IL‐1β in alveolar epithelial cells, which requires actin stress fiber formation. Control short hairpin RNA had no effect on these TGF‐β1‐induced responses. TGF‐β1‐induced phosphorylation of Y256 of N‐WASP via activation of small Rho GTPase and focal adhesion kinase mediates TGF‐β1‐induced paracellular permeability and actin cytoskeleton dynamics. In vivo, compared with controls, N‐WASP down‐regulation increases survival and prevents lung edema in mice induced by bleomycin exposure—a lung injury model in which TGF‐β1 plays a critical role. Our data indicate that N‐WASP plays a crucial role in the development of TGF‐β1‐mediated acute lung injury by promoting pulmonary edema via regulation of actin cytoskeleton dynamics.—Wagener, B. M., Hu, M., Zheng, A., Zhao, X., Che, P., Brandon, A., Anjum, N., Snapper, S., Creighton, J., Guan, J.‐L., Han, Q., Cai, G.‐Q., Han, X., Pittet, J.‐F., Ding, Q. Neuronal Wiskott‐Aldrich syndrome protein regulates TGF‐β1‐mediated lung vascular permeability. FASEB J. 30, 2557‐2569 (2016). www.fasebj.org