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. Antibody-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 [Ca+2]i, 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.

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 well as 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

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. Using the inflammasome protocol, exposure of cardiomyocytes (CMs) to LPS followed by ATP or nigericin caused release of mature IL-1β. Immunostaining of left ventricular frozen sections before and 8 h after CLP revealed the presence of NLRP3 and IL-1β proteins in CMs. 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 mice showed reduced plasma levels of IL-1β and IL-6. In vitro exposure of wild-type CMs to recombinant C5a (rC5a) caused elevations 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 mice had reduced defects in echo/Doppler parameters in heart after CLP. 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.

There is accumulating evidence during sepsis that cardiomyocyte (CM) homeostasis is compromised, resulting in cardiac dysfunction. An important role for complement in these outcomes is now demonstrated. Addition of C5a to electrically-paced CMs caused prolonged elevations of [Ca2+]i during diastole, together with appearance of spontaneous Ca2+ transients. In polymicrobial sepsis in mice, we found that three key homeostasis-regulating proteins in CMs were reduced in amounts: Na+/K+ ATPase, which is vital for effective action potentials in CMs; and two [Ca2+]i regulatory proteins; SERCA2 and the Na+/Ca2+ exchanger (NCX). Sepsis caused reduced mRNA levels and reductions in protein concentrations in CMs for all three proteins. The absence of either C5a receptor mitigated sepsis-induced reductions in the three regulatory proteins. Absence of either C5a receptor (C5aR1, C5aR2) diminished development of defective systolic and diastolic ECHO/Doppler parameters developing in the heart (cardiac output, LV stroke volume, isovolumic relaxation, E’sa, E/E’sa, LV diastolic volume). We also found in CMs from septic mice the presence of defective current densities for Ik1, L-type calcium channel and NCX. These defects were accentuated in the co-presence of C5a. These data suggest complement-related mechanisms responsible for development of cardiac dysfunction during sepsis.