| 1. |
- Johnson, Christina, et al.
(författare)
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Thrombin Differentially Modulates the Acute Inflammatory Response to Escherichia coli and Staphylococcus aureus in Human Whole Blood
- 2022
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Ingår i: Journal of Immunology. - American Association of Immunologists : AMER ASSOC IMMUNOLOGISTS. - 0022-1767 .- 1550-6606. ; 208:12, s. 2771-2778
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Tidskriftsartikel (refereegranskat)abstract
- 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 degrees 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-1 alpha, and mip-1 beta were potentiated in the thrombin-active GPRP model, whereas the levels of IP-10, TNF, IL-6, and IL-1 beta were elevated in the thrombin-inactive lepirudin model. Complement CS-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.
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| 2. |
- Landsem, Anne, et al.
(författare)
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Complement C3b contributes to Escherichia coli-induced platelet aggregation in human whole blood
- 2022
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Ingår i: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Platelets 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. Methods: In this study, we investigated the role of complement in Escherichia coli (E. coli)-induced platelet aggregation in human whole blood, using Multiplate(R) aggregometry, flow cytometry, and confocal microscopy. Results and Discussion: 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.
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| 3. |
- Nymo, Stig, et al.
(författare)
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Human Endothelial Cell Activation by Escherichia coli and Staphylococcus aureus Is Mediated by TNF and IL-1 beta Secondarily to Activation of C5 and CD14 in Whole Blood
- 2016
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Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 196:5, s. 2293-2299
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Tidskriftsartikel (refereegranskat)abstract
- 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-1 beta. 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-1 beta. E. coli and S. aureus activated EC in a CD14- and C5-dependent manner with subsequent leukocyte secretion of TNF and IL-1 beta mediating the effect.
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| 4. |
- Thomas, Anub Mathew, et al.
(författare)
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Combined Inhibition of C5 and CD14 Attenuates Systemic Inflammation in a Piglet Model of Meconium Aspiration Syndrome.
- 2018
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Ingår i: Neonatology. - : S. Karger. - 1661-7800 .- 1661-7819. ; 113:4, s. 322-330
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Meconium aspiration syndrome (MAS) is a severe lung condition affecting newborns and it can lead to a systemic inflammatory response. We previously documented complement activation and cytokine release in a piglet MAS model. Additionally, we showed ex vivo that meconium-induced inflammation was dependent on complement and Toll-like receptors.OBJECTIVES: To assess the efficacy of the combined inhibition of complement (C5) and CD14 on systemic inflammation induced in a forceful piglet MAS model.METHODS: Thirty piglets were randomly allocated to a treatment group receiving the C5-inhibitor SOBI002 and anti-CD14 (n = 15) and a nontreated control group (n = 15). MAS was induced by intratracheal meconium instillation, and the piglets were observed for 5 h. Complement, cytokines, and myeloperoxidase (MPO) were measured by ELISA.RESULTS: SOBI002 ablated C5 activity and the formation of the terminal complement complex in vivo. The combined inhibition attenuated the inflammasome cytokines IL-1β and IL-6 by 60 (p = 0.029) and 44% (p = 0.01), respectively, and also MPO activity in the bronchoalveolar fluid by 42% (p = 0.017). Ex vivo experiments in human blood revealed that the combined regimen attenuated meconium-induced MPO release by 64% (p = 0.008), but there was only a negligible effect with single inhibition, indicating a synergic cross-talk between the key molecules C5 and CD14.CONCLUSION: Combined inhibition of C5 and CD14 attenuates meconium-induced inflammation in vivo and this could become a future therapeutic regimen for MAS.
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| 5. |
- Thomas, Anub M., et al.
(författare)
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Complement Component C5 and TLR Molecule CD14 Mediate Heme-Induced Thromboinflammation in Human Blood
- 2019
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Ingår i: Journal of Immunology. - : American Association for Immunologists. - 0022-1767 .- 1550-6606. ; 203:6, s. 1571-1578
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Tidskriftsartikel (refereegranskat)abstract
- Heme is a critical danger molecule liberated from hemeproteins in various conditions, including from hemoglobin in hemolytic diseases. Heme may cause thromboinflammatory damage by activating inflammatory and hemostatic pathways, such as complement, the TLRs, coagulation, and platelets. In this study, we explored the effect of single and dual inhibition of complement component C5 and TLR coreceptor CD14 on heme-induced thromboinflammation in an ex vivo human whole blood model. Heme induced a dose-dependent activation of complement via the alternative pathway. Single inhibition of C5 by eculizumab attenuated the release of IL-6, IL-8, TNF, MCP-1, MIP-1 alpha, IFN-gamma, LTB-4, MMP-8 and -9, and IL-1Ra with more than 60% (p < 0.05 for all) reduced the upregulation of CD11b on granulocytes and monocytes by 59 and 40%, respectively (p < 0.05), and attenuated monocytic tissue factor expression by 33% (p < 0.001). Blocking CD14 attenuated IL-6 and TNF by more than 50% (p < 0.05). In contrast to single inhibition, combined C5 and CD14 was required for a significantly attenuated prothrombin cleavage (72%, p < 0.05). Markers of thromboinflammation were also quantified in two patients admitted to the hospital with sickle cell disease (SCD) crisis. Both SCD patients had pronounced hemolysis and depleted plasma hemopexin and haptoglobin. Plasma heme and complement activation was markedly increased in one patient, a coinciding observation as demonstrated ex vivo. In conclusion, heme-induced thromboinflammation was largely attenuated by C5 inhibition alone, with a beneficial effect of adding a CD14 inhibitor to attenuate prothrombin activation. Targeting C5 has the potential to reduce thromboinflammation in SCD crisis patients.
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