The Role of Side Chains in the Fine-Tuning of the Metal-Binding Ability of Multihistidine Peptides.

The systematic studies of copper(II), nickel(II) and zinc(II) ion complexes of protected multihistidine peptides containing amino acids with different side chains (Ac-SarHAH-NH2, Ac-HADH-NH2, Ac-HDAH-NH2, Ac-HXHYH-NH2 X, Y = A, F, D or K, Ac-HXHAHXH-NH2, X = F or D) have provided information about the metal ion and protein interaction and have made it possible to draw conclusions regarding general trends in the coordination of metal complexes of multihistidine peptides. The stability of the metal complexes significantly depends on the position of the histidines and amino acids, which are present in the neighbourhood of the histidine amino acids as well. The most significant effect was observed on peptides containing aspartic acid or phenylalanine. The redox parameters of complexes, however, depend on the number and position of histidines, and the other side chain donor atoms have practically no effect on the electrochemical properties of imidazole-coordinated species. However, the presence of aspartic acid side chains results in a more distorted geometry of amide-coordinated species and increases the reducibility of these complexes.

Preterm Intraventricular Hemorrhage-Induced Inflammatory Response in Human Choroid Plexus Epithelial Cells.

Following an intraventricular hemorrhage (IVH), red blood cell lysis and hemoglobin (Hb) oxidation with the release of heme can cause sterile neuroinflammation. In this study, we measured Hb derivates and cellular adhesion molecules ICAM-1 and VCAM-1 with cell-free miRNAs in cerebrospinal fluid (CSF) samples obtained from Grade-III and Grade-IV preterm IVH infants (IVH-III and IVH-IV, respectively) at multiple time points between days 0-60 after the onset of IVH. Furthermore, human choroid plexus epithelial cells (HCPEpiCs) were incubated with IVH and non-IVH CSF (10 v/v %) for 24 h in vitro to investigate the IVH-induced inflammatory response that was investigated via: (i) HMOX1, IL8, VCAM1, and ICAM1 mRNAs as well as miR-155, miR-223, and miR-181b levels by RT-qPCR; (ii) nuclear translocation of the NF-κB p65 subunit by fluorescence microscopy; and (iii) reactive oxygen species (ROS) measurement. We found a time-dependent alteration of heme, IL-8, and adhesion molecules which revealed a prolonged elevation in IVH-IV vs. IVH-III with higher miR-155 and miR-181b expression at days 41-60. Exposure of HCPEpiCs to IVH CSF samples induced HMOX1, IL8, and ICAM1 mRNA levels along with increased ROS production via the NF-κB pathway activation but without cell death, as confirmed by the cell viability assay. Additionally, the enhanced intracellular miR-155 level was accompanied by lower miR-223 and miR-181b expression in HCPEpiCs after CSF treatment. Overall, choroid plexus epithelial cells exhibit an abnormal cell phenotype after interaction with pro-inflammatory CSF of IVH origin which may contribute to the development of later clinical complications in preterm IVH.

Oxidized hemoglobin forms contribute to NLRP3 inflammasome-driven IL-1ß production upon intravascular hemolysis.

Damage associated molecular patterns (DAMPs) are released form red blood cells (RBCs) during intravascular hemolysis (IVH). Extracellular heme, with its pro-oxidant, pro-inflammatory and cytotoxic effects, is sensed by innate immune cells through pattern recognition receptors such as toll-like receptor 4 and nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3), while free availability of heme is strictly controlled. Here we investigated the involvement of different hemoglobin (Hb) forms in hemolysis-associated inflammatory responses. We found that after IVH most of the extracellular heme molecules are localized in oxidized Hb forms. IVH was associated with caspase-1 activation and formation of mature IL-1ß in plasma and in the liver of C57BL/6 mice. We showed that ferrylHb (FHb) induces active IL-1ß production in LPS-primed macrophages in vitro and triggered intraperitoneal recruitment of neutrophils and monocytes, caspase-1 activation and active IL-1ß formation in the liver of C57BL/6 mice. NLRP3 deficiency provided a survival advantage upon IVH, without influencing the extent of RBC lysis or the accumulation of oxidized Hb forms. However, both hemolysis-induced and FHb-induced pro-inflammatory responses were largely attenuated in Nlrp3-/- mice. Taken together, FHb is a potent trigger of NLRP3 activation and production of IL-1ß in vitro and in vivo, suggesting that FHb may contribute to hemolysis-induced inflammation. Identification of RBC-derived DAMPs might allow us to develop new therapeutic approaches for hemolytic diseases.

Induction of NLRP3 Inflammasome Activation by Heme in Human Endothelial Cells.

Hemolytic or hemorrhagic episodes are often associated with inflammation even when infectious agents are absent suggesting that red blood cells (RBCs) release damage-associated molecular patterns (DAMPs). DAMPs activate immune and nonimmune cells through pattern recognition receptors. Heme, released from RBCs, is a DAMP and induces IL-1ß production through the activation of the nucleotide-binding domain and leucine-rich repeat-containing family and pyrin domain containing 3 (NLRP3) in macrophages; however, other cellular targets of heme-mediated inflammasome activation were not investigated. Because of their location, endothelial cells can be largely exposed to RBC-derived DAMPs; therefore, we investigated whether heme and other hemoglobin- (Hb-) derived species induce NLRP3 inflammasome activation in these cells. We found that heme upregulated NLRP3 expression and induced active IL-1ß production in human umbilical vein endothelial cells (HUVECs). LPS priming largely amplified the heme-mediated production of IL-1ß. Heme administration into C57BL/6 mice induced caspase-1 activation and cleavage of IL-1ß which was not observed in NLRP3-/- mice. Unfettered production of reactive oxygen species played a critical role in heme-mediated NLRP3 activation. Activation of NLRP3 by heme required structural integrity of the heme molecule, as neither protoporphyrin IX nor iron-induced IL-1ß production. Neither naive nor oxidized forms of Hb were able to induce IL-1ß production in HUVECs. Our results identified endothelial cells as a target of heme-mediated NLRP3 activation that can contribute to the inflammation triggered by sterile hemolysis. Thus, understanding the characteristics and cellular counterparts of RBC-derived DAMPs might allow us to identify new therapeutic targets for hemolytic diseases.