scholarly journals The Role of Bovine Cathelicidins in Host Defence

2021 ◽  
Author(s):  
◽  
Blake William Paget

<p>Cattle are constantly exposed to environmental pathogens and are susceptible to a number of diseases which cause significant economic loss or animal welfare concerns. The mucosal surface is a key barrier to infection and a greater understanding of bovine mucosal immunology may lead to improved disease management strategies. The cathelicidins are a family of host defence proteins which may play an important role in this defensive barrier.  The cathelicidins are composed of a globular cathelin-like domain (CLD) and a C-terminal antimicrobial peptide (AMP) domain. The CLD is highly conserved across all species in which cathelicidins are found, yet the role of the CLD during infection has not been firmly established. The first aim of this thesis was to produce functional recombinant bovine CLD as a precursor to further experiments. However, the recombinant protein bound less lipopolysaccharide, was unable to agglutinate microbes, and was unable to permeabilise neutrophil membranes when compared to the activity of a native CLD preparation. Although further studies were not carried out with recombinant CLD, these results demonstrated that cell death induced by the native CLD and the agglutination of microbes potentially contribute to a broad anti-inflammatory role for the CLD during infection.  In contrast to humans and mice where only one cathelicidin isoform is expressed, bovine express seven cathelicidins, with variable AMP domains. Therefore the second aim of this thesis was to profile the effect of bovine cathelicidin AMPs on neutrophil function. The bovine AMPs were able to modify a number of activities. Migration and reactive oxygen species (ROS) production were enhanced by several peptides while ROS production was inhibited by others. When investigated in further detail, linear Bac1 (bactenecin), Bac5 and BMAP-34 (bovine myeloid antimicrobial peptide) were able to dose-dependently induce or inhibit several key neutrophil functions including migration, degranulation, respiratory burst and phagocytosis, indicating significant roles in differential modulation of immune responses. In particular, Bac5 was able to differentially modify neutrophil respiratory burst without significant disruption to cellular homeostasis, which suggested Bac5 was acting via an intracellular mechanism.  The third aim of this thesis was to investigate the mechanism by which Bac5 modulated neutrophil function. The results demonstrated the ability of Bac5 to be internalised by neutrophils and that Bac5 inhibition of p47phox binding to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a possible explanation for the differential induction and inhibition of extracellular ROS production. β-actin was identified as a major intracellular target for Bac5 and is consistent with the ability of Bac5 to modulate multiple neutrophil functions.  In summary, this thesis demonstrates that the bovine cathelicidins have multiple roles in host defence. The conserved CLD appears to have an anti-inflammatory role through an as yet undefined mechanism. The variable AMPs together have multifaceted roles which may act in concert to promote elimination of pathogens and regulate excessive detrimental neutrophil activity. The combined effect of these roles will facilitate clearance of pathogens during infection and aid in the resolution of the innate inflammatory response at mucosal surfaces.</p>

2021 ◽  
Author(s):  
◽  
Blake William Paget

<p>Cattle are constantly exposed to environmental pathogens and are susceptible to a number of diseases which cause significant economic loss or animal welfare concerns. The mucosal surface is a key barrier to infection and a greater understanding of bovine mucosal immunology may lead to improved disease management strategies. The cathelicidins are a family of host defence proteins which may play an important role in this defensive barrier.  The cathelicidins are composed of a globular cathelin-like domain (CLD) and a C-terminal antimicrobial peptide (AMP) domain. The CLD is highly conserved across all species in which cathelicidins are found, yet the role of the CLD during infection has not been firmly established. The first aim of this thesis was to produce functional recombinant bovine CLD as a precursor to further experiments. However, the recombinant protein bound less lipopolysaccharide, was unable to agglutinate microbes, and was unable to permeabilise neutrophil membranes when compared to the activity of a native CLD preparation. Although further studies were not carried out with recombinant CLD, these results demonstrated that cell death induced by the native CLD and the agglutination of microbes potentially contribute to a broad anti-inflammatory role for the CLD during infection.  In contrast to humans and mice where only one cathelicidin isoform is expressed, bovine express seven cathelicidins, with variable AMP domains. Therefore the second aim of this thesis was to profile the effect of bovine cathelicidin AMPs on neutrophil function. The bovine AMPs were able to modify a number of activities. Migration and reactive oxygen species (ROS) production were enhanced by several peptides while ROS production was inhibited by others. When investigated in further detail, linear Bac1 (bactenecin), Bac5 and BMAP-34 (bovine myeloid antimicrobial peptide) were able to dose-dependently induce or inhibit several key neutrophil functions including migration, degranulation, respiratory burst and phagocytosis, indicating significant roles in differential modulation of immune responses. In particular, Bac5 was able to differentially modify neutrophil respiratory burst without significant disruption to cellular homeostasis, which suggested Bac5 was acting via an intracellular mechanism.  The third aim of this thesis was to investigate the mechanism by which Bac5 modulated neutrophil function. The results demonstrated the ability of Bac5 to be internalised by neutrophils and that Bac5 inhibition of p47phox binding to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a possible explanation for the differential induction and inhibition of extracellular ROS production. β-actin was identified as a major intracellular target for Bac5 and is consistent with the ability of Bac5 to modulate multiple neutrophil functions.  In summary, this thesis demonstrates that the bovine cathelicidins have multiple roles in host defence. The conserved CLD appears to have an anti-inflammatory role through an as yet undefined mechanism. The variable AMPs together have multifaceted roles which may act in concert to promote elimination of pathogens and regulate excessive detrimental neutrophil activity. The combined effect of these roles will facilitate clearance of pathogens during infection and aid in the resolution of the innate inflammatory response at mucosal surfaces.</p>


Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3324-3331 ◽  
Author(s):  
J Elsner ◽  
M Oppermann ◽  
W Czech ◽  
A Kapp

In contrast to C5a, which represents a well-established potent activator of the respiratory burst in polymorphonuclear neutrophilic granulocytes (PMN), the functional role of C3a in the activation of PMN is, so far, poorly understood. Herein, the potential role of human C3a in the activation of the respiratory burst in human PMN was investigated. The release of reactive oxygen species (ROS) of PMN from healthy donors was measured by lucigenin-dependent chemiluminescence. C3a dose-dependently induced the production of ROS in human PMN in the range between 10 ng/mL and 1,000 ng/mL, whereas C3a-desArg was inactive. Flow cytometric measurement of H2O2 by dihydrorhodamine-123 labeling of anti-CD16-stained PMN showed that predominantly neutrophilic PMN are responsible for the C3a-induced activation of the respiratory burst. To exclude that C3a stimulation was caused by contamination with C5a, the specificity of C3a-induced activation of PMN was shown using monoclonal antibodies (MoAbs). Accordingly, the effect of C3a was completely abolished in the presence of Fab fragments of a blocking anti-C3a MoAb. In addition, blockade of the C5a receptor by the anti-C5a receptor (anti-C5aR) MoAb, S5/1, totally inhibited the C5a-induced production of ROS, whereas the C3a response in the presence of the anti-C5aR MoAb was unaffected. The specificity of the response was further confirmed by homologous desensitization after restimulation with C3a. In contrast, no cross-desensitization was observed upon stimulation with C5a. The C3a-induced ROS production by PMN was inhibited by pertussis toxin, indicating the involvement of guanine nucleotide-binding proteins (Gi proteins) in the signal transduction process initiated by C3a. In addition, stimulation of PMN by C3a resulted in a transient increase in the cytosolic free calcium concentration ([Ca2+]i) in a dose-dependent manner. In contrast to C3a- induced ROS production, C3a did not induce a chemotactic response in PMN, indicating functional qualitative differences as compared with C5a. In summary, these results show that C3a is a potent activator of the respiratory burst in human PMN. Therefore, these findings point to a novel role of C3a in the pathogenesis of inflammatory diseases associated with increased C3a levels and PMN activation.


2008 ◽  
Vol 2008 ◽  
pp. 1-10 ◽  
Author(s):  
J. Stefanska ◽  
R. Pawliczak

Apocynin is a naturally occurring methoxy-substituted catechol, experimentally used as an inhibitor of NADPH-oxidase. It can decrease the production of superoxide (O2−) from activated neutrophils and macrophages while the ability of phagocytosis remains unaffected. The anti-inflammatory activity of apocynin has been demonstrated in a variety of cell and animal models of inflammation. Apocynin, after metabolic conversion, inhibits the assembly of NADPH-oxidase that is responsible for reactive oxygen species (ROS) production. It is, therefore, extensively used to reveal the role of this enzyme in cell and experimental models. Although some of the ROS serve as signaling molecules in the cells, excessive production is damaging and has been implicated to play an important role in the progression of many disease processes. This is why in many studies apocynin presents a promising potential treatment for some disorders; however, its utility with inflammatory diseases remains to be determined. Since its mode of action is not well defined, we tried to get a more precise insight into the mechanisms by which apocynin exerts its activity. Considering the anti-inflammatory activities of apocynin, we may conclude that this compound definitely deserves further study.


2019 ◽  
Vol 8 (1) ◽  
pp. 116 ◽  
Author(s):  
Youngmi Kim ◽  
So Park ◽  
Harry Jung ◽  
You Noh ◽  
Jae Lee ◽  
...  

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase [NOX] enzymes serve several hemostatic and host defense functions in various lung diseases, but the role of NOX4 signaling in tuberculous pleurisy is not well understood. The role of NOX4 signaling in tuberculous pleural fibrosis was studied using invitro pleural mesothelial cell (PMC) experiments and a murine model of Mycobacterium bovis bacillus Calmette–Guérin (BCG) pleural infection. The production of NOX4 reactive oxygen species (NOX4–ROS) and the epithelial mesenchymal transition (EMT) in PMCs were both induced by heat-killed mycobacterium tuberculosis (HKMT). In cultured PMCs, HKMT-induced collagen-1 synthesis and EMT were blocked by pretreatment with small interfering RNA (siRNA) NOX4. Moreover, NOX4–ROS production and subsequent fibrosis were reduced by treatment with losartan and the toll-like receptor 4 (TLR4) inhibitor TAK-242. The HKMT-induced EMT and intracellular ROS production were mediated by NOX4 via the activation of extracellular signal-regulated kinase (ERK) signaling. Finally, in a BCG-induced pleurisy model, recruitment of inflammatory pleural cells, release of inflammatory cytokines, and thickened mesothelial fibrosis were attenuated by SiNOX4 compared to SiCon. Our study identified that HKMT-induced pleural fibrosis is mediated by NOX4–ERK–ROS via TLR4 and Angiotensin II receptor type1 (AT1R). There results suggest that NOX4 may be a novel therapeutic target for intervention in tuberculous pleural fibrosis.


Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3324-3331 ◽  
Author(s):  
J Elsner ◽  
M Oppermann ◽  
W Czech ◽  
A Kapp

Abstract In contrast to C5a, which represents a well-established potent activator of the respiratory burst in polymorphonuclear neutrophilic granulocytes (PMN), the functional role of C3a in the activation of PMN is, so far, poorly understood. Herein, the potential role of human C3a in the activation of the respiratory burst in human PMN was investigated. The release of reactive oxygen species (ROS) of PMN from healthy donors was measured by lucigenin-dependent chemiluminescence. C3a dose-dependently induced the production of ROS in human PMN in the range between 10 ng/mL and 1,000 ng/mL, whereas C3a-desArg was inactive. Flow cytometric measurement of H2O2 by dihydrorhodamine-123 labeling of anti-CD16-stained PMN showed that predominantly neutrophilic PMN are responsible for the C3a-induced activation of the respiratory burst. To exclude that C3a stimulation was caused by contamination with C5a, the specificity of C3a-induced activation of PMN was shown using monoclonal antibodies (MoAbs). Accordingly, the effect of C3a was completely abolished in the presence of Fab fragments of a blocking anti-C3a MoAb. In addition, blockade of the C5a receptor by the anti-C5a receptor (anti-C5aR) MoAb, S5/1, totally inhibited the C5a-induced production of ROS, whereas the C3a response in the presence of the anti-C5aR MoAb was unaffected. The specificity of the response was further confirmed by homologous desensitization after restimulation with C3a. In contrast, no cross-desensitization was observed upon stimulation with C5a. The C3a-induced ROS production by PMN was inhibited by pertussis toxin, indicating the involvement of guanine nucleotide-binding proteins (Gi proteins) in the signal transduction process initiated by C3a. In addition, stimulation of PMN by C3a resulted in a transient increase in the cytosolic free calcium concentration ([Ca2+]i) in a dose-dependent manner. In contrast to C3a- induced ROS production, C3a did not induce a chemotactic response in PMN, indicating functional qualitative differences as compared with C5a. In summary, these results show that C3a is a potent activator of the respiratory burst in human PMN. Therefore, these findings point to a novel role of C3a in the pathogenesis of inflammatory diseases associated with increased C3a levels and PMN activation.


Author(s):  
Giuseppe Marruchella ◽  
Francesco Mosca ◽  
Jasmine Hattab ◽  
Abigail R. Trachtman ◽  
Pietro G. Tiscar

Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used in veterinary medicine. Additionally, interest regarding the anti-infammatory properties of phytochemicals has emerged in recent years. In the present study, we aim to investigate the in vitro effects of meloxicam, flunixin meglumine, and harpagoside on the respiratory burst of porcine polymorphonuclear neutrophils (PMNs). We observed that harpagoside was able to suppress the respiratory burst, similarly to flunixin meglumine. Conversely, meloxicam enhanced the PMNs response. However, these effects were only detected at concentrations higher than those achievable in plasma and tissues. The present study intends to offer insights into the role of these molecules on phagocytosis mechanisms in animals to complement what is already known regarding human PMNs.


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