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

Complement C3b contributes to Escherichia coli-induced platelet aggregation in human whole blood

We found that compstatin, which inhibits the cleavage of complement component C3 to its components C3a and C3b, reduced the E. coli-induced platelet aggregation by 42%-76% (p = 0.0417). This C3-dependent aggregation was not C3a-mediated as neither inhibition of C3a using a blocking antibody or a C3a receptor antagonist, nor the addition of purified C3a had any effects. In contrast, a C3b-blocking antibody significantly reduced the E. coli-induced platelet aggregation by 67% (p = 0.0133). We could not detect opsonized C3b on platelets, indicating that the effect of C3 was not dependent on C3b-fragment deposition on platelets. Indeed, inhibition of glycoprotein IIb/IIIa (GPIIb/IIIa) and complement receptor 1 (CR1) showed that these receptors were involved in platelet aggregation. Furthermore, aggregation was more pronounced in hirudin whole blood than in hirudin platelet-rich plasma, indicating that E. coli-induced platelet aggregation involved other blood cells. In conclusion, the E. coli-induced platelet aggregation in human whole blood is partly C3b-dependent, and GPIIb/IIIa and CR1 are also involved in this process

Complement C3b contributes to Escherichia coli-induced platelet aggregation in human whole blood

IntroductionPlatelets have essential functions as first responders in the immune response to pathogens. Activation and aggregation of platelets in bacterial infections can lead to life-threatening conditions such as arterial thromboembolism or sepsis-associated coagulopathy.MethodsIn this study, we investigated the role of complement in Escherichia coli (E. coli)-induced platelet aggregation in human whole blood, using Multiplate® aggregometry, flow cytometry, and confocal microscopy.Results and DiscussionWe found that compstatin, which inhibits the cleavage of complement component C3 to its components C3a and C3b, reduced the E. coli-induced platelet aggregation by 42%-76% (p = 0.0417). This C3-dependent aggregation was not C3a-mediated as neither inhibition of C3a using a blocking antibody or a C3a receptor antagonist, nor the addition of purified C3a had any effects. In contrast, a C3b-blocking antibody significantly reduced the E. coli-induced platelet aggregation by 67% (p = 0.0133). We could not detect opsonized C3b on platelets, indicating that the effect of C3 was not dependent on C3b-fragment deposition on platelets. Indeed, inhibition of glycoprotein IIb/IIIa (GPIIb/IIIa) and complement receptor 1 (CR1) showed that these receptors were involved in platelet aggregation. Furthermore, aggregation was more pronounced in hirudin whole blood than in hirudin platelet-rich plasma, indicating that E. coli-induced platelet aggregation involved other blood cells. In conclusion, the E. coli-induced platelet aggregation in human whole blood is partly C3b-dependent, and GPIIb/IIIa and CR1 are also involved in this process

Functional characterisation of three human nicotinamide mononucleotide adenylyltransferases

Nicotinamide adenine dinucleotide, NAD, is an essential redox factor in many, primarily catabolic, hydrogen transfer reactions. While the redox reactions are not accompanied by a net loss of the pyridine nucleotides, NAD+ is actively degraded in a variety of ADP-ribosyl transfer reactions which are implicated, for examples, in cellular stress responses, calcium signalling and genomic stability. Thus, a continuous biosynthesis of NAD is essential for cell viability and metabolism. The final step in NAD biosynthesis, the reversible transfer of the AMP moiety from ATP onto a pyridine mononucleotide (NMN or its acidic form) is catalysed by nicotinamide mononucleotide adenylyltransferases (NMNATs) and is common to both the de novo and the known salvage pathways. In humans, three NMNAT isoforms have been described and partially characterised. The present study was aimed at a comprehensive characterisation of the human NMNATs, in particular, with regard to isoform-specific functions in order to gain further insight into the cellular NAD homeostasis and its regulation. Kinetic studies revealed that NMNAT1 possesses the highest specific activity both in the forward and reverse reactions, while NMNAT3 exhibited the lowest substrate selectivity. Importantly, NMNAT3 readily used the reduced nicotinamide substrates (NMNH or NADH in the forward and reverse reaction, respectively) thereby indicating that besides NAD+, NADH may be directly formed from a reduced precursor. No significant inhibition by physiological intermediates including nucleotides and nucleosides as well as poly(ADPribose), PAR, was found. However, we observed a considerable activation, particularly of NMNAT2, by the green tea polyphenol epigallocatechin 3-gallate (EGCG). Investigations of the subcellular distribution established distinct localisations of the three NMNAT isoforms in the nucleus, the Golgi complex and the mitochondria. Structural analyses revealed that all human NMNATs have insertions in their primary structures which are absent from the bacterial counterparts. While in NMNAT1 this region of the protein contains a known nuclear localisation signal, we revealed a targeting function of a corresponding domain in NMNAT2. The stretch of amino acids 109-192 was shown to direct the protein to the Golgi complex. Deletion of this domain resulted in a catalytically active, cytosolic protein. Moreover, we demonstrated that cysteine palmitoylation within this targeting domain is essential to anchor NMNAT2 at the cytosolic surface of the Golgi complex. The isoform NMNAT1 is known to be targeted to the nucleus by a nuclear localisation signal, which we highlight to reside within the corresponding domain. An important additional function of this targeting domain in NMNAT1 was also established. We found that this region (amino acids 102-149) mediates a phosphorylation-dependent association with poly(ADP-ribosyl)ated PARP1 which substantially enhances the poly(ADPribosyl) ating activity of PARP1. Interestingly, the catalytic activation of PARP1 was independent of the ability of NMNAT1 to synthesise NAD, as revealed using a catalytically dead mutant of NMNAT1. Collectively, our results suggest that the inserts into the sequences of the human NMNATs mediate subcellular targeting and interactions with other cellular factors. Therefore, we propose to denote this region as isoform-specific targeting and interaction domain (ISTID). It will be important to establish this function also for the corresponding domain in the mitochondrial isoform, NMNAT3. Interestingly, its amino acid sequence indeed lacks a classical N-terminal targeting sequence implying another mode of mitochondrial import

Isoform-specific Targeting and Interaction Domains in Human Nicotinamide Mononucleotide Adenylyltransferases

Lau C, Dölle C, Goßmann T, Agledal L, Niere M, Ziegler M. Isoform-specific Targeting and Interaction Domains in Human Nicotinamide Mononucleotide Adenylyltransferases. Journal of Biological Chemistry. 2010;285(24):18868-18876

Gene expression profiling of Gram-negative bacteria-induced inflammation in human whole blood: The role of complement and CD14-mediated innate immune response

Non-sterile pathogen-induced sepsis and sterile inflammation like in trauma or ischemia–reperfusion injury may both coincide with the life threatening systemic inflammatory response syndrome and multi-organ failure. Consequently, there is an urgent need for specific biomarkers in order to distinguish sepsis from sterile conditions. The overall aim of this study was to uncover putative sepsis biomarkers and biomarker pathways, as well as to test the efficacy of combined inhibition of innate immunity key players complement and Toll-like receptor co-receptor CD14 as a possible therapeutic regimen for sepsis. We performed whole blood gene expression analyses using microarray in order to profile Gram-negative bacteria-induced inflammatory responses in an ex vivo human whole blood model. The experiments were performed in the presence or absence of inhibitors of complement proteins (C3 and CD88 (C5a receptor 1)) and CD14, alone or in combination. In addition, we used blood from a C5-deficient donor. Anti-coagulated whole blood was challenged with heat-inactivated Escherichia coli for 2 h, total RNA was isolated and microarray analyses were performed on the Affymetrix GeneChip Gene 1.0 ST Array platform. The initial experiments were performed in duplicates using blood from two healthy donors. C5-deficiency is very rare, and only one donor could be recruited. In order to increase statistical power, a technical replicate of the C5-deficient samples was run. Subsequently, log2-transformed intensities were processed by robust multichip analysis and filtered using a threshold of four. In total, 73 microarray chips were run and analyzed. The normalized and filtered raw data have been deposited in NCBI's Gene Expression Omnibus (GEO) and are accessible with GEO Series accession number GSE55537. Linear models for microarray data were applied to estimate fold changes between data sets and the respective multiple testing adjusted p-values (FDR q-values). The interpretation of the data has been published by Lau et al. in an open access article entitled “CD14 and Complement Crosstalk and Largely Mediate the Transcriptional Response to Escherichia coli in Human Whole Blood as revealed by DNA Microarray” (Lau et al., 2015)

CD14 and complement crosstalk and largely mediate the transcriptional response to Escherichia coli in human whole blood as revealed by DNA microarray

Systemic inflammation like in sepsis is still lacking specific diagnostic markers and effective therapeutics. The first line of defense against intruding pathogens and endogenous damage signals is pattern recognition by e.g., complement and Toll-like receptors (TLR). Combined inhibition of a key complement component (C3 and C5) and TLR-co-receptor CD14 has been shown to attenuate certain systemic inflammatory responses. Using DNA microarray and gene annotation analyses, we aimed to decipher the effect of combined inhibition of C3 and CD14 on the transcriptional response to bacterial challenge in human whole blood. Importantly, combined inhibition reversed the transcriptional changes of 70% of the 2335 genes which significantly responded to heat-inactivated Escherichia coli by on average 80%. Single inhibition was less efficient (p<0.001) but revealed a suppressive effect of C3 on 21% of the responding genes which was partially counteracted by CD14. Furthermore, CD14 dependency of the Escherichia coli-induced response was increased in C5-deficient compared to C5-sufficient blood. The observed crucial distinct and synergistic roles for complement and CD14 on the transcriptional level correspond to their broad impact on the inflammatory response in human blood, and their combined inhibition may become inevitable in the early treatment of acute systemic inflammation

Human endothelial cell activation by Escherichia coli and staphylococcus aureus is mediated by TNF and IL-1β secondarily to activation of C5 and CD14 in whole blood

Endothelial cells (EC) play a central role in inflammation. E-selectin and ICAM-1 expression are essential for leukocyte recruitment and are good markers of EC activation. Most studies of EC activation are done in vitro using isolated mediators. The aim of the present study was to examine the relative importance of pattern recognition systems and downstream mediators in bacteria-induced EC activation in a physiological relevant human model, using EC incubated with whole blood. HUVEC were incubated with human whole blood. Escherichia coli– and Staphylococcus aureus–induced EC activation was measured by E-selectin and ICAM-1 expression using flow cytometry. The mAb 18D11 was used to neutralize CD14, and the lipid A analog eritoran was used to block TLR4/MD2. C5 cleavage was inhibited using eculizumab, and C5aR1 was blocked by an antagonist. Infliximab and canakinumab were used to neutralize TNF and IL-1β. The EC were minimally activated when bacteria were incubated in serum, whereas a substantial EC activation was seen when the bacteria were incubated in whole blood. E. coli–induced activation was largely CD14-dependent, whereas S. aureus mainly caused a C5aR1-mediated response. Combined CD14 and C5 inhibition reduced E-selectin and ICAM-1 expression by 96 and 98% for E. coli and by 70 and 75% for S. aureus. Finally, the EC activation by both bacteria was completely abolished by combined inhibition of TNF and IL-1β. E. coli and S. aureus activated EC in a CD14- and C5-dependent manner with subsequent leukocyte secretion of TNF and IL-1β mediating the effect

Combined inhibition of complement and CD14 improved outcome in porcine polymicrobial sepsis

Introduction Sepsis is an exaggerated and dysfunctional immune response to infection. Activation of innate immunity recognition systems including complement and the Toll-like receptor family initiate this disproportionate inflammatory response. The aim of this study was to explore the effect of combined inhibition of the complement component C5 and the Toll-like receptor co-factor CD14 on survival, hemodynamic parameters and systemic inflammation including complement activation in a clinically relevant porcine model of polymicrobial sepsis. Methods Norwegian landrace piglets (4 ± 0.5 kg) were blindly randomized to a treatment group (n = 12) receiving the C5 inhibitor coversin (OmCI) and anti-CD14 or to a positive control group (n = 12) receiving saline. Under anesthesia, sepsis was induced by a 2 cm cecal incision and the piglets were monitored in standard intensive care for 8 hours. Three sham piglets had a laparotomy without cecal incision or treatment. Complement activation was measured as sC5b-9 using enzyme immunoassay. Cytokines were measured with multiplex technology. Results Combined C5 and CD14 inhibition significantly improved survival (p = 0.03). Nine piglets survived in the treatment group and four in the control group. The treatment group had significantly lower pulmonary artery pressure (p = 0.04) and ratio of pulmonary artery pressure to systemic artery pressure (p < 0.001). Plasma sC5b-9 levels were significantly lower in the treatment group (p < 0.001) and correlated significantly with mortality (p = 0.006). IL-8 and IL-10 were significantly (p < 0.05) lower in the treatment group. Conclusions Combined inhibition of C5 and CD14 significantly improved survival, hemodynamic parameters and inflammation in a blinded, randomized trial of porcine polymicrobial sepsis