Thrombin Differentially Modulates the Acute Inflammatory Response to Escherichia coli and Staphylococcus aureus in Human Whole Blood

Thrombin plays a central role in thromboinflammatory responses, but its activity is blocked in the common ex vivo human whole blood models, making an ex vivo study of thrombin effects on thromboinflammatory responses unfeasible. In this study, we exploited the anticoagulant peptide Gly-Pro-Arg-Pro (GPRP) that blocks fibrin polymerization to study the effects of thrombin on acute inflammation in response to Escherichia coli and Staphylococcus aureus. Human blood was anticoagulated with either GPRP or the thrombin inhibitor lepirudin and incubated with either E. coli or S. aureus for up to 4 h at 37°C. In GPRP-anticoagulated blood, there were spontaneous elevations in thrombin levels and platelet activation, which further increased in the presence of bacteria. Complement activation and the expression of activation markers on monocytes and granulocytes increased to the same extent in both blood models in response to bacteria. Most cytokines were not elevated in response to thrombin alone, but thrombin presence substantially and heterogeneously modulated several cytokines that increased in response to bacterial incubations. Bacterial-induced releases of IL-8, MIP-1α, and MIP-1β were potentiated in the thrombin-active GPRP model, whereas the levels of IP-10, TNF, IL-6, and IL-1β were elevated in the thrombin-inactive lepirudin model. Complement C5-blockade, combined with CD14 inhibition, reduced the overall cytokine release significantly, both in thrombin-active and thrombin-inactive models. Our data support that thrombin itself marginally induces leukocyte-dependent cytokine release in this isolated human whole blood but is a significant modulator of bacteria-induced inflammation by a differential effect on cytokine patterns

Virus-specific and shared gene expression signatures in immune cells after vaccination in response to influenza and vaccinia stimulation

BackgroundIn the vaccine era, individuals receive multiple vaccines in their lifetime. Host gene expression in response to antigenic stimulation is usually virus-specific; however, identifying shared pathways of host response across a wide spectrum of vaccine pathogens can shed light on the molecular mechanisms/components which can be targeted for the development of broad/universal therapeutics and vaccines.MethodWe isolated PBMCs, monocytes, B cells, and CD8+ T cells from the peripheral blood of healthy donors, who received both seasonal influenza vaccine (within <1 year) and smallpox vaccine (within 1 - 4 years). Each of the purified cell populations was stimulated with either influenza virus or vaccinia virus. Differentially expressed genes (DEGs) relative to unstimulated controls were identified for each in vitro viral infection, as well as for both viral infections (shared DEGs). Pathway enrichment analysis was performed to associate identified DEGs with KEGG/biological pathways.ResultsWe identified 2,906, 3,888, 681, and 446 DEGs in PBMCs, monocytes, B cells, and CD8+ T cells, respectively, in response to influenza stimulation. Meanwhile, 97, 120, 20, and 10 DEGs were identified as gene signatures in PBMCs, monocytes, B cells, and CD8+ T cells, respectively, upon vaccinia stimulation. The majority of DEGs identified in PBMCs were also found in monocytes after either viral stimulation. Of the virus-specific DEGs, 55, 63, and 9 DEGs occurred in common in PBMCs, monocytes, and B cells, respectively, while no DEGs were shared in infected CD8+ T cells after influenza and vaccinia. Gene set enrichment analysis demonstrated that these shared DEGs were over-represented in innate signaling pathways, including cytokine-cytokine receptor interaction, viral protein interaction with cytokine and cytokine receptor, Toll-like receptor signaling, RIG-I-like receptor signaling pathways, cytosolic DNA-sensing pathways, and natural killer cell mediated cytotoxicity.ConclusionOur results provide insights into virus-host interactions in different immune cells, as well as host defense mechanisms against viral stimulation. Our data also highlights the role of monocytes as a major cell population driving gene expression in ex vivo PBMCs in response to viral stimulation. The immune response signaling pathways identified in this study may provide specific targets for the development of novel virus-specific therapeutics and improved vaccines for vaccinia and influenza. Although influenza and vaccinia viruses have been selected in this study as pathogen models, this approach could be applicable to other pathogens

Synthetic Oligodeoxynucleotide CpG Motifs Activate Human Complement through Their Backbone Structure and Induce Complement-Dependent Cytokine Release

Bacterial and mitochondrial DNA, sharing an evolutionary origin, act as danger-associated molecular patterns in infectious and sterile inflammation. They both contain immunomodulatory CpG motifs. Interactions between CpG motifs and the complement system are sparsely described, and mechanisms of complement activation by CpG remain unclear. Lepirudin-anticoagulated human whole blood and plasma were incubated with increasing concentrations of three classes of synthetic CpGs: CpG-A, -B, and -C oligodeoxynucleotides and their GpC sequence controls. Complement activation products were analyzed by immunoassays. Cytokine levels were determined via 27-plex beads-based immunoassay, and CpG interactions with individual complement proteins were evaluated using magnetic beads coated with CpG-B. In whole blood and plasma, CpG-B and CpG-C (p 0.8 for all), led to time- and dose-dependent increase of soluble C5b-9, the alternative complement convertase C3bBbP, and the C3 cleavage product C3bc. GpC-A, -B, and -C changed soluble fluid-phase C5b-9, C3bBbP, and C3bc to the same extent as CpG-A, -B, and -C, indicating a DNA backbone–dependent effect. Dose-dependent CpG-B binding was found to C1q (r = 0.83; p = 0.006) and factor H (r = 0.93; p < 0.001). The stimulatory complement effect was partly preserved in C2-deficient plasma and completely preserved in MASP-2–deficient serum. CpG-B increased levels of IL-1β, IL-2, IL-6, IL-8, MCP-1, and TNF in whole blood, which were completely abolished by inhibition of C5 and C5aR1 (p < 0.05 for all). In conclusion, synthetic analogs of bacterial and mitochondrial DNA activate the complement system via the DNA backbone. We suggest that CpG-B interacts directly with classical and alternative pathway components, resulting in complement-C5aR1–dependent cytokine release

COMPLEMENT ACTIVATION BY GOLD NANOPARTICLES: MECHANISM AND APPLICATION

Ph.DDOCTOR OF PHILOSOPH

Narrowband Interference Detection and Mitigation for Indoor Ultra-Wideband Communication Systems

libraries of this University may make it freely available for inspection. The author further agrees that permission for copying of this thesis in any manner, in whole or in part for scholarly purposes may be granted by the professor who supervised this thesis work or, in his absence, by the Head of the Department or the Dean of the College of Graduate Studies and Research at the University of Saskatchewan. Any copying, publication, or use of this thesis, or parts thereof, for financial gain without the author’s written permission is strictly prohibited. Proper recognition shall be given to the author and the University of Saskatchewan in any scholarly use which may be made of any material in this thesis. Request for permission to copy or to make any other use of material in this thesis in whole or part should be addressed to

Non-specific adsorption of complement proteins affects complement activation pathways of gold nanomaterials

<p>The complement system is a key humoral component of innate immunity, serving as the first line of defense against intruders, including foreign synthetic nanomaterials. Although gold nanomaterials (AuNMs) are widely used in nanomedicine, their immunological response is not well understood. Using AuNMs of three shapes commonly used in biomedical applications: spherical gold nanoparticles, gold nanostars and gold nanorods, we demonstrated that AuNMs activated whole complement system, leading to the formation of SC5b-9 complex. All three complement pathways were simultaneously activated by all the AuNMs. Recognition molecules of the complement system interacted with all AuNMs <i>in vitro</i>, except for l-ficolin, but the correlation between these interactions and corresponding complement pathway activation was only observed in the classical and alternative pathways. We also observed the mediating role of complement activation in cellular uptake of all AuNMs by human U937 promonocytic cells, which expresses complement receptors. Taken together, our results highlighted the potential immunological challenges for clinical applications of AuNMs that were often overlooked.</p

Platelet-depletion of whole blood reveals that platelets potentiate the release of IL-8 from leukocytes Into plasma in a thrombin-dependent manner

Objective In a recent study, we found an elevated level of interleukin 8 (IL-8) in response to bacterial incubation in thrombin-sufficient human whole blood anticoagulated by the fibrin polymerization blocking peptide GPRP. Whether thrombin directly activated leukocytes or mediated the release via thrombin-dependent activation of platelets remains unresolved. Herein, we addressed the role of thrombin and platelets in IL-8 release. Methods We separated platelets from whole blood using a combination of 0.7% (w/v) citrate and GPRP for attenuating the hemostatic response during the separation of platelets. Cytokine responses were compared in whole blood and platelet-depleted blood upon Escherichia coli incubation. Cytokine responses were also profiled with and without reconstitution of either platelets or the supernatant from activated platelets. Results Platelets were not activated during the separation process but responded to stimuli upon re-calcification. Plasma levels of IL-1β, IL-1Ra, IL-6, IL-8, IP-10, MIP-1α, and MIP-1β were significantly reduced in platelet-depleted blood compared to whole blood, but recovered in the presence of platelets, or with the supernatant of activated platelets. The leukocyte fraction and platelets were each found to contribute to the elevation of IL-8 at around 5 ng/ml; however, if combined, the release of IL-8 increased to 26 ng/ml. This process was dependent on thrombin since the levels of IL-8 remained at 5 ng/ml in whole blood if thrombin was blocked. Intracellular staining revealed that monocytes were the main source for IL-8 expression. Conclusion Our findings suggest that the release of IL-8 is mediated by the leukocytes, mainly monocytes, but potentiated via thrombin-dependent activation of platelets

Platelet-Depletion of Whole Blood Reveals That Platelets Potentiate the Release of IL-8 From Leukocytes Into Plasma in a Thrombin-Dependent Manner

ObjectiveIn a recent study, we found an elevated level of interleukin 8 (IL-8) in response to bacterial incubation in thrombin-sufficient human whole blood anticoagulated by the fibrin polymerization blocking peptide GPRP. Whether thrombin directly activated leukocytes or mediated the release via thrombin-dependent activation of platelets remains unresolved. Herein, we addressed the role of thrombin and platelets in IL-8 release. MethodsWe separated platelets from whole blood using a combination of 0.7% (w/v) citrate and GPRP for attenuating the hemostatic response during the separation of platelets. Cytokine responses were compared in whole blood and platelet-depleted blood upon Escherichia coli incubation. Cytokine responses were also profiled with and without reconstitution of either platelets or the supernatant from activated platelets. ResultsPlatelets were not activated during the separation process but responded to stimuli upon re-calcification. Plasma levels of IL-1 beta, IL-1Ra, IL-6, IL-8, IP-10, MIP-1 alpha, and MIP-1 beta were significantly reduced in platelet-depleted blood compared to whole blood, but recovered in the presence of platelets, or with the supernatant of activated platelets. The leukocyte fraction and platelets were each found to contribute to the elevation of IL-8 at around 5 ng/ml; however, if combined, the release of IL-8 increased to 26 ng/ml. This process was dependent on thrombin since the levels of IL-8 remained at 5 ng/ml in whole blood if thrombin was blocked. Intracellular staining revealed that monocytes were the main source for IL-8 expression. ConclusionOur findings suggest that the release of IL-8 is mediated by the leukocytes, mainly monocytes, but potentiated via thrombin-dependent activation of platelets

Complement Activation by PEGylated Gold Nanoparticles

Gold nanoparticles (AuNPs) are widely used in biomedical applications, but much less is known about their immunological properties, particularly their interaction with the complement system, a key component of innate immunity serving as an indicator of their biocompatibility. Using a library of different-sized AuNPs (10, 20, 40, and 80 nm) passivated with polyethylene glycol (PEG) of different molecular weight (<i>M</i>_w = 1, 2, 5, and 10 kDa), we demonstrated that citrate-capped AuNPs activated the whole complement system in a size-dependent manner, characterized by the formation of the end-point activation product, SC5b-9, in human serum. Although PEGylation of AuNPs mitigated, but did not abolish, the activation level, complement activation by PEGylated AuNPs was independent of both the core size of AuNPs and the molecular weight of PEG. The cellular uptake of both citrate-capped and PEGylated AuNPs by human U937 promonocytic cells which expresses complement receptors were highly correlated to the level of complement activation. Taken together, our results provided new insights on the innate complement activation by PEGylated AuNPs that are widely considered to be inert biocompatible nanomaterials