Requirement of Complement C6 for Intact Innate Immune Responses in Mice.

Over the first days of polymicrobial sepsis, there is robust activation of the innate immune system, causing the appearance of proinflammatory cytokines and chemokines, along with the appearance of extracellular histones, which are highly proinflammatory and prothrombotic. In the current study, we studied different innate immune responses in mice with knockout (KO) of complement protein 6 (C6). Polymorphonuclear neutrophils (PMNs) from these KO mice had defective innate immune responses, including defective expression of surface adhesion molecules, generation of superoxide anion, and appearance of reactive oxygen species and histone release after activation of PMNs, along with defective phagocytosis. In addition, in C6-/- mice, the NLRP3 inflammasome was defective both in PMNs and in macrophages. When these KO mice were subjected to polymicrobial sepsis, their survival was improved, associated with reduced levels in the plasma of proinflammatory cytokines and chemokines and lower levels of histones in plasma. In addition, sepsis-induced cardiac dysfunction was attenuated in these KO mice. In a model of acute lung injury induced by LPS, C6-/- mice showed reduced PMN buildup and less lung epithelial/endothelial cell dysfunction (edema and hemorrhage). These data indicate that C6-/- mice have reduced innate immune responses that result in less organ injury and improved survival after polymicrobial sepsis.

Complement Destabilizes Cardiomyocyte Function In Vivo after Polymicrobial Sepsis and In Vitro.

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 intracellular Ca(2+) concentrations during diastole, together with the appearance of spontaneous Ca(2+) transients. In polymicrobial sepsis in mice, we found that three key homeostasis-regulating proteins in CMs were reduced: Na(+)/K(+)-ATPase, which is vital for effective action potentials in CMs, and two intracellular Ca(2+) concentration regulatory proteins, that is, sarcoplasmic/endoplasmic reticulum calcium ATPase 2 and the Na(+)/Ca(2+) exchanger. 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 or C5aR2) diminished development of defective systolic and diastolic echocardiographic/Doppler parameters developing in the heart (cardiac output, left ventricular stroke volume, isovolumic relaxation, E' septal annulus, E/E' septal annulus, left ventricular diastolic volume). We also found in CMs from septic mice the presence of defective current densities for Ik1, l-type calcium channel, and Na(+)/Ca(2+) exchanger. These defects were accentuated in the copresence of C5a. These data suggest complement-related mechanisms responsible for development of cardiac dysfunction during sepsis.

Selective Biological Responses of Phagocytes and Lungs to Purified Histones.

Histones invoke strong proinflammatory responses in many different organs and cells. We assessed biological responses to purified or recombinant histones, using human and murine phagocytes and mouse lungs. H1 had the strongest ability in vitro to induce cell swelling independent of requirements for toll-like receptors (TLRs) 2 or 4. These responses were also associated with lactate dehydrogenase release. H3 and H2B were the strongest inducers of [Ca2+]i elevations in phagocytes. Cytokine and chemokine release from mouse and human phagocytes was predominately a function of H2A and H2B. Double TLR2 and TLR4 knockout (KO) mice had dramatically reduced cytokine release induced in macrophages exposed to individual histones. In contrast, macrophages from single TLR-KO mice showed few inhibitory effects on cytokine production. Using the NLRP3 inflammasome protocol, release of mature IL-1ß was predominantly a feature of H1. Acute lung injury following the airway delivery of histones suggested that H1, H2A, and H2B were linked to alveolar leak of albumin and the buildup of polymorphonuclear neutrophils as well as the release of chemokines and cytokines into bronchoalveolar fluids. These results demonstrate distinct biological roles for individual histones in the context of inflammation biology and the requirement of both TLR2 and TLR4.

Abl tyrosine kinase promotes dendrogenesis by inducing actin cytoskeletal rearrangements in cooperation with Rho family small GTPases in hippocampal neurons.

Nonreceptor tyrosine kinase Abl is an actin-binding protein and a key regulator of neuronal axonal development. Although Abl family kinases also are localized in dendrites and are implicated in postsynaptic functions, it is not clear how Abl kinases regulate dendritic morphogenesis. Using a developing hippocampal culture as a model, we found that the inhibition of Abl kinases by STI571 leads to a remarkable simplification of dendritic branching similar to the phenotype caused by an increased activity of small GTPase RhoA. Time-lapse microscopic imaging reveals a prominent reduction of dendritic branching. In contrast, neurons expressing a constitutively active v-abl construct (CA-Abl) show an exuberant microtubule-associated protein 2-positive (MAP2-positive) dendrite outgrowth, suggesting that Abl modulates dendritic growth. Biochemical assays using a glutathione S-transferase pull-down method to determine GTP-bound active Rho GTPases demonstrate that Abl inhibition increases RhoA activity but has no effect on the activity of Rac1 or Cdc42. At the cellular level the alteration of Abl also changes actin organization consistent with RhoA inhibition. Suppression of the RhoA downstream effector Rho kinase reverses STI571-induced dendritic simplification, demonstrating that activity of the Rho pathway is responsible for the Abl-induced changes in dendrogenesis. Furthermore, CA-Abl-induced neurite outgrowth is blocked by the expression of a constitutively active RhoA construct. The CA-Abl phenotype is not affected by destabilization of microtubules but is reversed partially when actin filaments are stabilized with jasplakinolide. Together, these studies support a critical role for Abl kinases in regulating dendrogenesis by inducing actin cytoskeletal rearrangements in cooperation with Rho GTPases.

Increased DJ-1 expression under oxidative stress and in Alzheimer's disease brains.

Mutations in the DJ-1 gene have been linked to autosomal recessive familial Parkinson's disease. To understand the function of DJ-1, we determined the DJ-1 expression in both zebrafish and post mortem human brains. We found that DJ-1 was expressed early during zebrafish development and throughout adulthood. Knock down (KD) of DJ-1 by injection of morpholino did not cause dramatic morphologic alterations during development, and no loss of dopaminergic neurons was observed in embryos lacking DJ-1. However, DJ-1 KD embryos were more susceptible to programmed cell death. While a slight reduction in staining for islet-1 positive neurons was observed in both DJ-1 KD and H2O2 treated embryos, the number of apoptotic cells was significantly increased in both KD and H2O2 treated embryos. Interestingly, DJ-1 expression was increased in brains of zebrafish under conditions of oxidative stress, indicating that DJ-1 is a part of stress-responsive machinery. Since oxidative stress is one of the major contributors to the development of Alzheimer's disease (AD), we also examined DJ-1 expression in AD brains. Using DJ-1 specific antibodies, we failed to detect a robust staining of DJ-1 in brain tissues from control subjects. However, DJ-1 immunoreactivity was detected in hippocampal pyramidal neurons and astrocytes of AD brains. Therefore, our results strongly suggest that DJ-1 expression is not necessary during zebrafish development but can be induced in zebrafish exposed to oxidative stress and is present in human AD brains.