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Histones appear in plasma during infectious or non-infectious sepsis and are associated with multiorgan injury. In the current studies, intravenous infusion of histones resulted in their localization in major organs. In vitro exposure of mouse macrophages to histones caused a buildup of histones on cell membranes followed by localization into cytosol and into the nucleus. After polymicrobial sepsis (cecal ligation and puncture), histones appeared in plasma as well as in a multiorgan pattern, peaking at 8 h followed by decline. In lungs, histones and neutrophils appeared together, with evidence for formation of neutrophil extracellular traps (NETs), which represent an innate immune response to trap and kill bacteria and other infectious agents. In liver, there was intense NET formation, featuring linear patterns containing histones and strands of DNA. When neutrophils were activated in vitro with C5a or phorbol myristate acetate, NET formation ensued. While formation of NETs represents entrapment and killing of infectious agents, the simultaneous release from neutrophils of histones often results in tissue/organ damage.

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., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Sepsis, both in humans and in rodents, is associated with persistent immunosuppression accompanied by defects in innate immunity during the acute phase of sepsis. Mice were rendered septic by cecal ligation and puncture (CLP) followed by the induction of acute lung injury, employing distal airway deposition of IgG immune complexes, in order to quantitatively evaluate innate immune responses following the induction of sepsis. Suppression of innate immune responses in the lung occurred as early as 12 h after CLP and up to 21 days thereafter. The mechanism of innate immune defects included a reduced leak of albumin into the lungs together with reduced levels of tumor necrosis factor in bronchoalveolar lavage fluids and increased levels of interleukin-10 that were persistent. Bone marrow-derived neutrophils (polymorphonuclear neutrophils; PMNs) from CLP mice also had reduced levels of the activation marker CD11b and a depressed respiratory burst following stimulation in vitro. These results were not observed in mice with endotoxemia, where the innate inflammatory response was preserved. However, sustained lymphopenia was present in both models, suggesting differential regulation of innate and adaptive immunity in the two sepsis models. These data indicate that CLP induced a prolonged suppression of inflammatory responses both in the lung and systemically, as defined by bone marrow-derived PMN dysfunction.

Delayed neutrophil apoptosis is characteristic of sepsis and may accentuate organ injury. It has been shown that PI-3K and MAPK pathways provide survival signaling in neutrophils. In this study, we demonstrate that neutrophils isolated from septic rats are resistant to apoptosis in comparison with the cells from normal animals. In contrast to normal serum, septic sera induced strong phosphorylation of AKT and p44/42 in neutrophils obtained from normal rats, resulting in marked resistance of these cells to apoptosis. Protection from apoptosis by septic sera was abrogated completely by inhibition of PI-3K and partially diminished by MEK inhibition. Increased neutrophil survival in septic rats was associated with increased levels of Bcl-xL in neutrophils and decreased levels of Bim expression. In vivo blockade of C5a in cecal ligation and puncture rats by anti-C5a antibody markedly restored the susceptibility of neutrophils to undergo apoptosis. C5a activated AKT and p44/42 and also enhanced X-linked inhibitor of apoptosis expression in neutrophils. LPS and C5a were able to induce Bcl-xL expression. Thus, neutrophil survival signals derived from effects of septic sera could be linked to activation of ERK1/2 and PI-3K, increased antiapoptotic protein expression, and ultimately, delayed neutrophil apoptosis.

Neutrophil accumulation in the lung plays a pivotal role in the pathogenesis of acute lung injury during sepsis. Directed movement of neutrophils is mediated by a group of chemoattractants, especially CXC chemokines. Local lung production of CXC chemokines is intensified during experimental sepsis induced by cecal ligation and puncture (CLP), as reflected by rising levels of MIP-2 and cytokine-induced neutrophil chemoattractant-1 in bronchoalveolar lavage fluids. Alveolar macrophages are primed and blood neutrophils are down-regulated for production of MIP-2 and cytokine-induced neutrophil chemoattractant production in response to LPS and C5a. Under these conditions of stimulation, activation of MAPKs (p38, p42/p44) occurs in sham neutrophils but not in CLP neutrophils, while under the same conditions phosphorylation of p38 and p42/p44 occurs in both sham and CLP alveolar macrophages. These data indicate that, under septic conditions, there is impaired signaling in neutrophils and enhanced signaling in alveolar macrophages, resulting in CXC chemokine production, and C5a appears to play a pivotal role in this process. As a result, CXC chemokines increase in lung, setting the stage for neutrophil accumulation in lung during sepsis.

There is evidence that C5a and macrophage migration inhibitory factor (MIF) both play important roles in experimental sepsis. Humans with sepsis also show elevated levels of both mediators in the blood. Regulation of MIF during sepsis is poorly understood. We now demonstrate that neutrophil depletion greatly reduced serum MIF levels in rats and mice during the onset of sepsis after cecal ligation and puncture. In vitro, C5a induced MIF release from rat and mouse neutrophils. In vivo blockade of C5aR or absence of C5aR led to significantly reduced MIF generation during the onset of sepsis. C5a-induced release in vitro of MIF from neutrophils appeared to be due to up-regulation of MIF in cytoplasmic granules of neutrophils via activation of the protein kinase B signaling pathway together with involvement of PI3K. Our data suggest that C5a plays a role in enhancing MIF release from neutrophils in vitro and during sepsis. These findings represent a previously unrecognized function of C5a and neutrophils in the appearance of MIF in sepsis.

Complement fragment 5a (C5a)-C5a receptor (C5aR) signaling plays an essential role in neutrophil innate immunity. Blockade of either the ligand or the receptor improves survival rates in experimental sepsis. In the current study, sepsis was induced in rats by cecal ligation/puncture. Early in sepsis C5aR content on neutrophils significantly dropped, reached the nadir at 24 h after onset of sepsis, and progressively elevated thereafter. Western-blot, RT-PCR, and confocal microscopy analyses revealed that the loss and re-expression of C5aR during sepsis might be due, at least in part, to the receptor internalization and reconstitution. The reduction and reconstitution of C5aR correlate with the loss and restoration of innate immune functions of blood neutrophils (chemotaxis and reactive oxygen species production), respectively. Quantitative measurements of C5aR on blood neutrophils are highly predictive of survival or death during sepsis. These data suggest that neutrophil C5aR content represents an essential component of an efficient defense system in sepsis and may serve as a prognostic marker for the outcome.

In sepsis, there is evidence that excessive C5a generation leads to compromised innate immune functions, being associated with poor outcome. We now report that in vitro exposure of neutrophils to C5a causes increased levels of IkappaBalpha, decreased NF-kappaB-dependent gene transcription of TNFalpha, and decreased lipopolysaccharide (LPS)-induced TNFalpha production. Similar findings were obtained with neutrophils from cecal ligation/puncture (CLP)-induced septic rats. Such changes were reversed by antibody-induced in vivo blockade of C5a. In contrast, in vitro exposure of alveolar macrophages to C5a and LPS resulted in enhanced production of TNFalpha and no increase in IkappaBalpha. These data suggest that CLP-induced sepsis causes a C5a-dependent dysfunction of neutrophils, which is characterized by altered signaling associated with NF-kappaB activation.

In polymicrobial sepsis, the extracellular histones, mainly released from activated neutrophils, significantly contribute to cardiac dysfunction (septic cardiomyopathy), as demonstrated in our previous studies using Echo-Doppler measurements. This study aims to elucidate the roles of extracellular histones and their interactions with Toll-like receptors (TLRs) in cardiac dysfunction. Through ex vivo assessments of ECG, left ventricle (LV) function parameters, and in vivo Echo-Doppler studies in mice perfused with extracellular histones, we aim to provide comprehensive insights into the mechanisms underlying sepsis-induced cardiac dysfunction. Langendorff-perfused hearts from both wild-type and TLR2, TLR3, or TLR4 knockout (KO) mice were examined. Paced mouse hearts were perfused with histones to assess contractility and relaxation. Echo-Doppler studies evaluated cardiac dysfunction after intravenous histone injection. Histone perfusion caused defects in contractility and relaxation, with TLR2 and TLR3 KO mice being partially protected. Specifically, TLR2 KO mice exhibited the greatest reduction in Echo-Doppler abnormalities, while TLR4 KO exacerbated cardiac dysfunction. Among individual histones, H1 induced the most pronounced abnormalities in cardiac function, apoptosis of cardiomyocytes, and LDH release. Our data highlight significant interactions between histones and TLRs, providing insights into histones especially H1 as potential therapeutic targets for septic cardiomyopathy. Further studies are needed to explore specific histone–TLR interactions and their mechanisms. [ABSTRACT FROM AUTHOR]

These studies were undertaken to extend emerging evidence that β2 adrenergic receptor (β2AR) agonists, in addition to their bronchorelaxing effects, may have broad anti-inflammatory effects in the lung following onset of experimental acute lung injury (ALI). Young male C57BL/6 mice (25 g) developed ALI following airway deposition of bacterial LPS or IgG immune complexes in the absence or presence of appropriate stereo-isomers (enantiomers) of β2AR agonists, albuterol or formoterol. Endpoints included albumin leak into lung and buildup of polymorphonuclear neutrophils and cytokines/chemokines in bronchoalveolar fluids. Both β2AR agonists suppressed lung inflammatory parameters (IC50 = 10-7 M). Similar effects of β2AR agonists on mediator release were found when mouse macrophages were stimulated in vitro with LPS. The protective effects were associated with reduced activation (phosphorylation) of JNK but not of other signaling proteins. Collectively, these data suggest that2AR agonists have broad anti-inflammatory effects in the setting of ALI. While β2AR agonists suppress JNK activation, the extent to which this can explain the blunted lung inflammatory responses in the ALI models remains to be determined. [ABSTRACT FROM AUTHOR]

Acute lung injury (ALI) in mouse lung occurs after distal airway deposition of IgG immune complexes (IgGICs), resulting in a breakdown of the vascular-airway barrier, causing intrapulmonary edema, hemorrhage, and accumulation of neutrophils [polymorphonuclear leukocytes (PMNs)] in the alveolar compartment, these changes being complement (C5a) and C5a receptor (C5aR) dependent. In this ALI model, C5aR expression (protein) was found to occur on upper (bronchial) and lower (alveolar) airway epithelial cells. An adenovirus construct (siRNA) was used to silence mRNA for C5aR in the lung. Under such conditions, C5aR protein was markedly reduced on lung epithelial cells, resulting in much reduced leakage of albumin into the lung, diminished buildup of PMNs, and lower levels of proinflammatory mediators in bronchoalveolar lavage fluids. These studies indicate that bronchial and alveolar epithelial cell CSaR is up-regulated and greatly contributes to inflammation and injury in the lung. The use of siRNA administered into the airways avoids systemic suppression of C5aR, which might compromise innate immunity. It is possible that such an intervention might be employed in humans with ALI or acute respiratory distress syndrome as well as in upper-airway inflammatory diseases, such as chronic obstructive pulmonary disease and asthma, where there is evidence for complement activation and buildup of PMNs. [ABSTRACT FROM AUTHOR]

Pathogen-pattern-recognition by Toll-like receptors (TLRs) and pathogen clearance after immune complex formation via engagement with Fc receptors (FcRs) represent central mechanisms that trigger the immune and inflammatory responses. In the present study, a linkage between TLR4 and FcγR was evaluated in vitro and in vivo. Most strikingly, in vitro activation of phagocytes by IgG immune complexes (IgGIC) resulted in an association of TLR4 with FcγRIII (CD16) based on coimmunoprecipitation analyses. Neutrophils and macrophages from TLR4 mutant (mut) mice were unresponsive to either lipopolysaccharide (LPS) or IgGIC in vitro, as determined by cytokine production. This phenomenon was accompanied by the inability to phosphorylate tyrosine residues within immunoreceptor tyrosine-based activation motifs (ITAMs) of the FcRγ-subunit. To transfer these findings in vivo, two different models of acute lung injury (ALI) induced by intratracheal administration of either LPS or IgGIC were employed. As expected, LPS-induced ALI was abolished in TLR4 mut and TLR4-/- mice. Unexpectedly, TLR4 mut and TLR4-/- mice were also resistant to development of ALI following IgGIC deposition in the lungs. In conclusion, our findings suggest that TLR4 and FcγRIII pathways are structurally and functionally connected at the receptor level and that TLR4 is indispensable for FccRIII signaling via FcRγ-subunit activation. [ABSTRACT FROM AUTHOR]

Presents findings of a study which investigated the presence and regulation of the second C5a receptor on neutrophils and various organs during sepsis. Evaluation the role for C5L2 nucleotide sequence in balancing the biological responses to C5a; Comparison of 5′-RACE polymerase chain reaction, cyclic DNA cloning and sequence in the rat C5L2 receptor; Contrast between C5L2 and C5aR expression in neutrophils during sepsis; Expression of nRNA for C5L2 during sepsis; Effects of αC5aR and αC5L2 on in vitro and in vivo production of interleukin-6.

Presents a study on the effects of C5a on lipopolysaccharide(LPS)-induced interleukin(IL)-6 generation in neutrophils in vitro and on in vivo IL-6 production in septic rodents. Effects of C5a on LPS-induced IL-6 production and gene transcription; Effects of blockade of C5a/C5a receptor activation on serum IL-6 levels during sepsis; Effects of signaling pathway inhibitors on IL-6 production in neutrophils; Conclusions of the study.

Signaling in neutrophils involving the powerful complement anaphylatoxin (C5a) and its receptor (C5aR) plays an essential role in innate immunity. We have previously shown in rodents that blockade of either C5a or C5aR dramatically improves survival rates in experimental sepsis. We have also shown that defensive functions of neutrophils are compromised during sepsis in a C5a-dependent manner. The main aim of the current study was to elucidate the mechanisms for C5aR alteration on neutrophils during sepsis and to evaluate the relationship between C5aR levels on neutrophils and survival after the onset of sepsis. [ABSTRACT FROM AUTHOR]