| 1. |
- Papareddy, Praveen, et al.
(author)
-
A human antithrombin isoform dampens inflammatory responses and protects from organ damage during bacterial infection
- 2019
-
In: Nature Microbiology. - : Springer Science and Business Media LLC. - 2058-5276. ; 4:12, s. 2442-2455
-
Journal article (peer-reviewed)abstract
- Severe infectious diseases are often characterized by an overwhelming and unbalanced systemic immune response to microbial infections. Human antithrombin (hAT) is a crucial coagulation inhibitor with anti-inflammatory activities. Here we identify three hAT-binding proteins (CD13, CD300f and LRP-1) on human monocytes that are involved in blocking the activity of nuclear factor-kappa B. We found that the modulating effect is primarily restricted to the less abundant beta-isoform (h beta AT) of hAT that lacks N-glycosylation at position 135. Individuals with a mutation at this position have increased production of h beta AT and analysis of their blood, which was stimulated ex vivo with lipopolysaccharide, showed a decreased inflammatory response. Similar findings were recorded when heterozygotic mice expressing hAT or h beta AT were challenged with lipopolysaccharide or infected with Escherichia coli bacteria. Our results finally demonstrate that in a lethal E. coli infection model, survival rates increased when mice were treated with h beta AT one hour and five hours after infection. The treatment also resulted in a reduction of the inflammatory response and less severe organ damage.
|
|
| 2. |
- Anders, Emma, et al.
(author)
-
Globular C1q receptor (p33) binds and stabilizes pro-inflammatory MCP-1 : a novel mechanism for regulation of MCP-1 production and function
- 2018
-
In: Biochemical Journal. - 0264-6021. ; 475:4, s. 775-786
-
Journal article (peer-reviewed)abstract
- The protein gC1qR (globular C1q receptor), also named p33, was originally identified as a binding partner of the globular heads of C1q in the complement system. gC1qR/p33 is abundantly expressed in many cell types, but the functional importance of this protein is not completely understood. Here, we investigate the impact of gC1qR/p33 on the production and function of the pathophysiologically important chemokine monocyte chemoattractant protein-1 (MCP-1) and the underlying molecular mechanisms. Knockdown of gC1qR/p33 negatively regulated the production of MCP-1, but had no effect on the expression of transcript for MCP-1 in human periodontal ligament cells, suggesting a translational/post-translational mechanism of action. Laser scanning confocal microscopy showed considerable cytosolic co-localization of gC1qR/p33 and MCP-1, and co-immunoprecipitation disclosed direct physical interaction between gC1qR/p33 and MCP-1. Surface plasmon resonance analysis revealed a high-affinity binding (KD = 10.9 nM) between gC1qR/p33 and MCP-1. Using a transwell migration assay, we found that recombinant gC1qR/p33 enhances MCP-1-induced migration of human THP-1 monocytes, pointing to a functional importance of the interaction between gC1qR/p33 and MCP-1. An in vitro assay revealed a rapid turnover of the MCP-1 protein and that gC1qR/ p33 stabilizes MCP-1, hence preventing its degradation. We propose that endogenous gC1qR/p33 physically interacts with MCP-1 causing stabilization of the MCP-1 protein and stimulation of its activity in human periodontal ligament cells, suggesting a novel gC1qR/p33-mediated pro-inflammatory mechanism of action.
|
|
| 3. |
- Neumann, Ariane, et al.
(author)
-
Immunoregulation of Neutrophil Extracellular Trap Formation by Endothelial-Derived p33 (gC1q Receptor)
- 2018
-
In: Journal of Innate Immunity. - : S. Karger AG. - 1662-811X .- 1662-8128. ; 10:1, s. 30-43
-
Journal article (peer-reviewed)abstract
- The formation of neutrophil extracellular traps (NETs) is a host defence mechanism, known to facilitate the entrapment and growth inhibition of many bacterial pathogens. It has been implicated that the translocation of myeloperoxidase (MPO) from neutrophilic granules to the nucleus is crucial to this process. Under disease conditions, however, excessive NET formation can trigger self-destructive complications by releasing pathologic levels of danger-associated molecular pattern molecules (DAMPs). To counteract such devastating immune reactions, the host has to rely on precautions that help circumvent these deleterious effects. Though the induction of DAMP responses has been intensively studied, the mechanisms that are used by the host to down-regulate them are still not understood. In this study, we show that p33 is an endothelial-derived protein that has the ability to annul NET formation. We found that the expression of human p33 is up-regulated in endothelial cells upon infections with Streptococcus pyogenes bacteria. Using tissue biopsies from a patient with streptococcal necrotising fasciitis, we monitored co-localisation of p33 with MPO. Further in vitro studies revealed that p33 is able to block the formation of DAMP-induced NET formation by inhibiting the enzymatic activity of MPO. Additionally, mice challenged with S. pyogenes bacteria demonstrated diminished MPO activity when treated with p33. Together, our results demonstrate that host-derived p33 has an important immunomodulating function that helps to counterbalance an overwhelming DAMP response.
|
|
| 4. |
- Oehmcke, Sonja, et al.
(author)
-
A Novel Role for Pro-Coagulant Microvesicles in the Early Host Defense against Streptococcus pyogenes
- 2013
-
In: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 9:8
-
Journal article (peer-reviewed)abstract
- Previous studies have shown that stimulation of whole blood or peripheral blood mononuclear cells with bacterial virulence factors results in the sequestration of pro-coagulant microvesicles (MVs). These particles explore their clotting activity via the extrinsic and intrinsic pathway of coagulation; however, their pathophysiological role in infectious diseases remains enigmatic. Here we describe that the interaction of pro-coagulant MVs with bacteria of the species Streptococcus pyogenes is part of the early immune response to the invading pathogen. As shown by negative staining electron microscopy and clotting assays, pro-coagulant MVs bind in the presence of plasma to the bacterial surface. Fibrinogen was identified as a linker that, through binding to the M1 protein of S. pyogenes, allows the opsonization of the bacteria by MVs. Surface plasmon resonance analysis revealed a strong interaction between pro-coagulant MVs and fibrinogen with a K-D value in the nanomolar range. When performing a mass-spectrometry-based strategy to determine the protein quantity, a significant up-regulation of the fibrinogen-binding integrins CD18 and CD11b on pro-coagulant MVs was recorded. Finally we show that plasma clots induced by pro-coagulant MVs are able to prevent bacterial dissemination and possess antimicrobial activity. These findings were confirmed by in vivo experiments, as local treatment with pro-coagulant MVs dampens bacterial spreading to other organs and improved survival in an invasive streptococcal mouse model of infection. Taken together, our data implicate that pro-coagulant MVs play an important role in the early response of the innate immune system in infectious diseases.
|
|
| 5. |
- Svensson, Daniel, et al.
(author)
-
Human endogenous peptide p33 inhibits detrimental effects of LL-37 on osteoblast viability
- 2015
-
In: Journal of Periodontal Research. - : John Wiley & Sons. - 0022-3484 .- 1600-0765. ; 50:1, s. 80-88
-
Journal article (peer-reviewed)abstract
- Background and Objective: High levels of the antimicrobial peptide, LL-37, are detected in gingival crevicular fluid from patients with chronic periodontitis. LL-37 not only shows antimicrobial activity but also affects host-cell viability. The objective of the present study was to identify endogenous mechanisms that antagonize the detrimental effects of LL-37 on osteoblast viability, focusing on the human peptide p33 expressed on the surface of various cell types. Material and Methods: Human osteoblast-like MG63 cells and human hFOB1.19 osteoblasts were treated with or without LL-37 in the presence or absence of p33. Recombinant human p33 was expressed in an Escherichia coli expression system. Lactate dehydrogenase (LDH) was assessed using an enzymatic spectrophotometric assay. DNA synthesis was determined by measuring [3H]-thymidine incorporation. Cell number was assessed by counting cells in a Bu€rker chamber. Intracellular Ca2+ was monitored by recording Fluo 4-AM fluorescence using a laser scanning confo- cal microscope. Cellular expression of p33 was determined by western blotting. Results: LL-37 caused a concentration-dependent release of LDH from human osteoblasts, showing a half-maximal response value (EC50) of 4 lM and a rapid and sustained rise in the intracellular Ca2+ concentration of osteoblasts, sug- gesting that LL-37 forms pores in the cell membrane. p33 (10 lM) inhibited the LL-37-induced LDH release and LL-37-evoked rise in intracellular Ca2+ con- centration, suggesting that p33 prevents LL-37-induced permeabilization of the cell membrane. Moreover, p33 blocked LL-37-induced attenuation of osteoblast numbers. Also, mucin antagonized, at concentrations representative for nonsti- mulated whole saliva, LL-37-evoked LDH release, whilst cationic endogenous polyamines had no impact on LL-37-induced LDH release from osteoblasts. Conclusions: The endogenous peptide p33 prevents LL-37-induced reduction of human osteoblast viability. Importantly, this mechanism may protect the osteo- blasts from LL-37-induced cell damage in patients suffering from chronic peri- odontitis associated with high levels of LL-37 locally.
|
|
| 6. |
- Wang, Yongzhi, et al.
(author)
-
Neutrophil extracellular trap-microparticle complexes enhance thrombin generation via the intrinsic pathway of coagulation in mice
- 2018
-
In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8:1
-
Journal article (peer-reviewed)abstract
- Abdominal sepsis is associated with dysfunctional hemostasis. Thrombin generation (TG) is a rate-limiting step in systemic coagulation. Neutrophils can expell neutrophil extracellular traps (NETs) and/or microparticles (MPs) although their role in pathological coagulation remains elusive. Cecal ligation and puncture (CLP)-induced TG in vivo was reflected by a reduced capacity of plasma from septic animals to generate thrombin. Depletion of neutrophils increased TG in plasma from CLP mice. Sepsis was associated with increased histone 3 citrullination in neutrophils and plasma levels of cell-free DNA and DNA-histone complexes and administration of DNAse not only eliminated NET formation but also elevated TG in sepsis. Isolated NETs increased TG and co-incubation with DNAse abolished NET-induced formation of thrombin. TG triggered by NETs was inhibited by blocking factor XII and abolished in factor XII-deficient plasma but intact in factor VII-deficient plasma. Activation of neutrophils simultaneously generated large amount of neutrophil-derived MPs, which were found to bind to NETs via histone-phosphatidylserine interactions. These findings show for the first time that NETs and MPs physically interact, and that NETs might constitute a functional assembly platform for MPs. We conclude that NET-MP complexes induce TG via the intrinsic pathway of coagulation and that neutrophil-derived MPs play a key role in NET-dependent coagulation.
|
|
| 7. |
- Westman, Johannes, et al.
(author)
-
Extracellular Histones Induce Chemokine Production in Whole Blood Ex Vivo and Leukocyte Recruitment In Vivo.
- 2015
-
In: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 11:12
-
Journal article (peer-reviewed)abstract
- The innate immune system relies to a great deal on the interaction of pattern recognition receptors with pathogen- or damage-associated molecular pattern molecules. Extracellular histones belong to the latter group and their release has been described to contribute to the induction of systemic inflammatory reactions. However, little is known about their functions in the early immune response to an invading pathogen. Here we show that extracellular histones specifically target monocytes in human blood and this evokes the mobilization of the chemotactic chemokines CXCL9 and CXCL10 from these cells. The chemokine induction involves the toll-like receptor 4/myeloid differentiation factor 2 complex on monocytes, and is under the control of interferon-γ. Consequently, subcutaneous challenge with extracellular histones results in elevated levels of CXCL10 in a murine air pouch model and an influx of leukocytes to the site of injection in a TLR4 dependent manner. When analyzing tissue biopsies from patients with necrotizing fasciitis caused by Streptococcus pyogenes, extracellular histone H4 and CXCL10 are immunostained in necrotic, but not healthy tissue. Collectively, these results show for the first time that extracellular histones have an important function as chemoattractants as their local release triggers the recruitment of immune cells to the site of infection.
|
|
| 8. |
- Westman, Johannes, et al.
(author)
-
p33 (gC1q Receptor) Prevents Cell Damage by Blocking the Cytolytic Activity of Antimicrobial Peptides.
- 2013
-
In: Journal of Immunology. - : The American Association of Immunologists. - 1550-6606 .- 0022-1767. ; 191:11, s. 5714-5721
-
Journal article (peer-reviewed)abstract
- The innate immune system is the first line of defense against invading microbes. Its specificity relies a great deal on host pattern recognition molecules that sense pathogen-associated molecular patterns of the invading pathogen. However, full protection is not always guaranteed, and some early defense mechanisms involved in bacterial killing, such as the complement system, can also exert cytolytic activity against host cells. Although these cascades are tightly regulated, the host has to take additional precautions to prevent its cell destruction. In this study, we describe that p33, a negatively charged surface protein found on endothelial cells also known as gC1q receptor, protects host cells from a cytolytic attack by antimicrobial peptides (AMPs), such as LL37 and β-defensin 3. To this end, we characterized the interaction of p33 with AMPs by biochemical and functional means. Our data show that p33 forms a doughnut-shaped trimer that can bind up to three AMPs, and we identified a segment in p33 forming a β-sheet that mediates the binding to all AMPs. Moreover, our results show that p33 abolishes the lytic activity of AMPs at an equimolar ratio, and it protects endothelial cells and erythrocytes from AMP-induced lysis. Taken together, our data suggest a novel protective mechanism of p33 in modulating innate immune response by neutralizing cytotoxic AMPs at the host cell surface.
|
|
| 9. |
- Westman, Johannes, et al.
(author)
-
Protein SIC secreted from Streptococcus pyogenes forms complexes with extracellular histones that boost cytokine production
- 2018
-
In: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 9:FEB, s. 1-14
-
Journal article (peer-reviewed)abstract
- Innate immunity relies on an effective recognition of the pathogenic microorganism as well as on endogenous danger signals. While bacteria in concert with their secreted virulence factors can cause a number of inflammatory reactions, danger signals released at the site of infection may in addition determine the amplitude of such responses and influence the outcome of the disease. Here, we report that protein SIC, Streptococcal Inhibitor of Complement, an abundant secreted protein from Streptococcus pyogenes, binds to extracellular histones, a group of danger signals released during necrotizing tissue damage. This interaction leads to the formation of large aggregates in vitro. Extracellular histones and SIC are abundantly expressed and seen colocalized in biopsies from patients with necrotizing soft-tissue infections caused by S. pyogenes. In addition, binding of SIC to histones neutralized their antimicrobial activity. Likewise, the ability of histones to induce hemolysis was inhibited in the presence of SIC. However, when added to whole blood, SIC was not able to block the pro-inflammatory effect of histones. Instead SIC boosted the histone-triggered release of a broad range of cytokines and chemokines, including IL-6, TNF-α, IL-8, IL-1β, IL-1ra, G-CSF, and IFN-γ. These results demonstrate that the interaction between SIC and histones has multiple effects on the host response to S. pyogenes infection.
|
|
| 10. |
- Westman, Johannes, et al.
(author)
-
Treatment with p33 Curtails Morbidity and Mortality in a Histone-Induced Murine Shock Model.
- 2014
-
In: Journal of Innate Immunity. - : S. Karger AG. - 1662-811X .- 1662-8128. ; 6:6, s. 819-830
-
Journal article (peer-reviewed)abstract
- Collateral damage caused by extracellular histones has an immediate impact on morbidity and mortality in many disease models. A significant increase in the levels of extracellular histones is seen in critically ill patients with trauma and sepsis. We showed that histones are released from necrotic cells in patients with invasive skin infections. Under in vitro conditions, endogenous p33, an endothelial surface protein also known as the gC1q receptor, interacts with histones released from damaged endothelial cells. Functional analyses have revealed that recombinantly expressed p33 completely neutralizes the harmful features of histones, i.e. hemolysis of erythrocytes, lysis of endothelial cells and platelet aggregation. We also noted that mice treated with a sublethal dose of histones developed severe signs of hemolysis, thrombocytopenia and lung tissue damage already 10 min after inoculation. These complications were fully counteracted when p33 was administered together with the histones. Moreover, application of p33 significantly improved survival in mice receiving an otherwise lethal dose of histones. Together, our data suggest that treatment with p33 is a promising therapeutic approach in severe infectious diseases. © 2014 S. Karger AG, Basel.
|
|
