Miriam Kalbitz

The inflammasome is a key factor in innate immunity and senses soluble pathogen and danger-associated molecular patterns as well as biological crystals (urate, cholesterol, etc.), resulting in expression of IL-1β and IL-18. Using a standard model of acute lung injury (ALI) in mice featuring airway instillation of LPS, ALI was dependent on availability of NLRP3 as well as caspase-1, which are known features of the NLRP3 inflammasome. The appearance of IL-1β, a product of NLRP3 inflammasome activation, was detected in bronchoalveolar lavage fluids (BALF) in a macrophage- and neutrophil-dependent manner. Neutrophil-derived extracellular histones appeared in the BALF during ALI and directly activated the NLRP3 inflammasome. Ab-mediated neutralization of histones significantly reduced IL-1β levels in BALF during ALI. Inflammasome activation by extracellular histones in LPS-primed macrophages required NLRP3 and caspase-1 as well as extrusion of K(+), increased intracellular Ca(2+) concentration, and generation of reactive oxygen species. NLRP3 and caspase-1 were also required for full extracellular histone presence during ALI, suggesting a positive feedback mechanism. Extracellular histone and IL-1β levels in BALF were also elevated in C5a-induced and IgG immune complex ALI models, suggesting a common inflammatory mechanism. These data indicate an interaction between extracellular histones and the NLRP3 inflammasome, resulting in ALI. Such findings suggest novel targets for treatment of ALI, for which there is currently no known efficacious drug.

ABSTRACT Cardiac dysfunction develops during sepsis in humans and rodents. In the model of polymicrobial sepsis induced by cecal ligation and puncture (CLP), we investigated the role of the NLRP3 inflammasome in the heart. Mouse heart homogenates from sham‐procedure mice contained high mRNA levels of NLRP3 and IL‐1β. Usingthe inflamm a some protocol, exposure of cardiomyocytes (CMs) to LPS followed by ATP or nigericin caused release of mature IL‐1β. Immuno staining of left ventricular frozen sections before and 8 h after CLP revealed the presence of NLRP3 and IL‐1β proteins inCMs. CLP caused substantial increases in mRNAs for IL‐1β and NLRP3 in CMs which are reduced in the absence of either C5aR1 or C5aR2. After CLP, NLRP3 2/2 mice showed reduced plasma levels of IL‐1βand IL‐6. In vitro exposure of wild‐type CMs to recombinant C5a (rC5a) cause delevations in both cytosolic and nuclear/mitochondrial reactive oxygen species (ROS), which were C5a‐receptor dependent. Use of a selective NOX2 inhibitor prevented increased cytosolic and nuclear/mitochondrial ROS levels and release of IL‐1β. Finally, NLRP3 2/2 mice had reduced defects in echo/Doppler parameters in heart afterCLP. These studies establish that the NLRP3 inflammasome contributes to the cardiomyopathy of polymicrobial sepsis.—Kalbitz, M., Fattahi, F., Grailer, J. J., Jajou, L., Malan, E. A., Zetoune, F. S., Huber‐Lang, M., Russell, M. W., Ward, P. A. Complement‐induced activation of the cardiac NLRP3 inflammasome in sepsis. FASEB J. 30, 3997–4006 (2016). www.fasebj.org

ABSTRACT The purpose of this study was to define the relationship in polymicrobial sepsis (in adult male C57BL/6 mice) between heart dysfunction and the appearance in plasma of extracellular histones. Procedures included induction of sepsis by cecal ligation and puncture and measurement of heart function using echocardiogram/Doppler parameters. We assessed the ability of histones to cause disequilibrium in the redox status and intracellular [Ca 2+ ] i levels in cardiomyocytes (CMs) (from mice and rats). We also studied the ability of histones to disturb both functional and electrical responses of hearts perfused with histones. Main findings revealed that extracellular histones appearing in septic plasma required C5a receptors, polymorphonuclear leukocytes (PMNs), and the Nacht‐, LRR‐, and PYD‐domains‐containing protein 3 (NLRP3) inflammasome. In vitro exposure of CMs to histones caused loss of homeostasis of the redox system and in [Ca 2+ ] i , as wellas defects in mitochondrial function. Perfusion of hearts with histones caused electrical and functional dysfunction. Finally, in vivo neutralization of histones in septic mice markedly reduced the parameters of heart dysfunction. Histones caused dysfunction in hearts during polymicrobial sepsis. These events could be attenuated by histone neutralization, suggesting that histones may be targets in the setting of sepsis to reduce cardiac dysfunction.—Kalbitz, M., Grailer, J. J., Fattahi, F., Jajou, L., Herron, T. J., Campbell, K. F., Zetoune, F. S., Bosmann, M., Sarma, J. V., Huber‐Lang, M., Gebhard, F., Loaiza, R., Valdivia, H. H., Jalife, J., Russell, M. W., Ward, P. A. Role of extracellular histones in the cardiomyopathy of sepsis. FASEB J. 29, 2185‐2193 (2015). www.fasebj.org

Trauma is the leading cause of mortality in humans below the age of 40. Patients injured by accidents frequently suffer severe multiple trauma, which is life-threatening and leads to death in many cases. In multiply injured patients, thoracic trauma constitutes the third most common cause of mortality after abdominal injury and head trauma. Furthermore, 40-50% of all trauma-related deaths within the first 48 h after hospital admission result from uncontrolled hemorrhage. Physical trauma and hemorrhage are frequently associated with complex pathophysiological and immunological responses. To develop a greater understanding of the mechanisms of single and/or multiple trauma, reliable and reproducible animal models, fulfilling the ethical 3 R's criteria (Replacement, Reduction and Refinement), established by Russell and Burch in 'The Principles of Human Experimental Technique' (published 1959), are required. These should reflect both the complex pathophysiological and the immunological alterations induced by trauma, with the objective to translate the findings to the human situation, providing new clinical treatment approaches for patients affected by severe trauma. Small animal models are the most frequently used in trauma research. Rattus norvegicus was the first mammalian species domesticated for scientific research, dating back to 1830. To date, there exist numerous well-established procedures to mimic different forms of injury patterns in rats, animals that are uncomplicated in handling and housing. Nevertheless, there are some physiological and genetic differences between humans and rats, which should be carefully considered when rats are chosen as a model organism. The aim of this review is to illustrate the advantages as well as the disadvantages of rat models, which should be considered in trauma research when selecting an appropriate in vivo model. Being the most common and important models in trauma research, this review focuses on hemorrhagic shock, blunt chest trauma, bone fracture, skin and soft-tissue trauma, burns, traumatic brain injury and polytrauma.

Abstract The complement and neutrophil defence systems, as major components of innate immunity, are activated during inflammation and infection. For neutrophil migration to the inflamed region, we hypothesized that the complement activation product C5a induces significant changes in cellular morphology before chemotaxis. Exposure of human neutrophils to C5a dose‐ and time‐dependently resulted in a rapid C5a receptor‐1 (C5aR1)‐dependent shape change, indicated by enhanced flow cytometric forward‐scatter area values. Similar changes were observed after incubation with zymosan‐activated serum and in blood neutrophils during murine sepsis, but not in mice lacking the C5aR1. In human neutrophils, Amnis high‐resolution digital imaging revealed a C5a‐induced decrease in circularity and increase in the cellular length/width ratio. Biomechanically, microfluidic optical stretching experiments indicated significantly increased neutrophil deformability early after C5a stimulation. The C5a‐induced shape changes were inhibited by pharmacological blockade of either the ‐exchanger or the Cl − ‐channel. Furthermore, actin polymerization assays revealed that C5a exposure resulted in a significant polarization of the neutrophils. The functional polarization process triggered by ATP –P2X/Y‐purinoceptor interaction was also involved in the C5a‐induced shape changes, because pretreatment with suramin blocked not only the shape changes but also the subsequent C5a‐dependent chemotactic activity. In conclusion, the data suggest that the anaphylatoxin C5a regulates basic neutrophil cell processes by increasing the membrane elasticity and cell size as a consequence of actin‐cytoskeleton polymerization and reorganization, transforming the neutrophil into a migratory cell able to invade the inflammatory site and subsequently clear pathogens and molecular debris.