Bactericidal activity of porcine neutrophil secretions

Scapinello, Sarah Elizabeth; Brooks, Andrew S; MacInnes, Janet I.; Hammermueller, Jutta; Clark, Mary E.; Caswell, Jeff

doi:10.1016/j.vetimm.2010.09.004

Cited by 20 publications

(18 citation statements)

References 23 publications

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“…For NET blockage, the following inhibitors were used: the NOX-inhibitor DPI [10 µM, Sigma-Aldrich, according to Farley et al (43)], the leukocyte elastase-inhibitor Suc-Ala-Ala-Pro-Val chloromethyl ketone [CMK; 1 mM, Sigma-Aldrich, according to Scapinello et al (44)], the MPO-inhibitor 4-aminobenzoic acid hydrazide [ABAH; 100 µM, Merck, according Parker et al (45)], the SOCE-inhibitor aminoethoxydiphenyl borate [2-APB; 100 µM, Sigma-Aldrich, according to Conejeros et al (46)], UO126 as inhibitor of ERK1/2 [50 µM; Sigma-Aldrich, according to Muñoz-Caro et al (12)], SB 202190 as specific inhibitor of p38 MAPK [10 µM; Sigma-Aldrich, according to Muñoz-Caro et al (12)], the G-protein-coupled receptor (GPCR) antagonist NF-449 for P2Y2 blockage (GPCR-NF-449, 10 µM; Santa Cruz Biotechnology), and N-α-benzoyl-N5-(2-chloro-1-iminoethyl)- l -Orn amide for PAD4 inhibition (Cl-amidine, 200 µM, Merck). For blocking experiments, PMN were pre-exposed with the corresponding inhibitor in serum-free medium RPMI 1640 without phenol red (RT, GPCR-NF-449, and Cl-amidine: 120 min, all other inhibitors: 30 min) prior to exposure to N. caninum tachyzoites.…”

Section: Methodsmentioning

confidence: 99%

Bovine Polymorphonuclear Neutrophils Cast Neutrophil Extracellular Traps against the Abortive Parasite Neospora caninum

et al. 2017

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Neospora caninum represents a relevant apicomplexan parasite causing severe reproductive disorders in cattle worldwide. Neutrophil extracellular trap (NET) generation was recently described as an efficient defense mechanism of polymorphonuclear neutrophils (PMN) acting against different parasites. In vitro interactions of bovine PMN with N. caninum were analyzed at different ratios and time spans. Extracellular DNA staining was used to illustrate the typical molecules of NETs [i.e., histones (H3), neutrophil elastase (NE), myeloperoxidase (MPO), pentraxin] via antibody-based immunofluorescence analyses. Functional inhibitor treatments were applied to reveal the role of several enzymes [NADPH oxidase (NOX), NE, MPO, PAD4], ATP-dependent P2Y2 receptor, store-operated Ca++entry (SOCE), CD11b receptor, ERK1/2- and p38 MAPK-mediated signaling pathway in tachyzoite-triggered NETosis. N. caninum tachyzoites triggered NETosis in a time- and dose-dependent manner. Scanning electron microscopy analyses revealed NET structures being released by bovine PMN and entrapping tachyzoites. N. caninum-induced NET formation was found not to be NOX-, NE-, MPO-, PAD4-, ERK1/2-, and p38 MAP kinase-dependent process since inhibition of these enzymes led to a slight decrease of NET formation. CD11b was also identified as a neutrophil receptor being involved in NETosis. Furthermore, N. caninum-triggered NETosis depends on Ca++ influx as well as neutrophil metabolism since both the inhibition of SOCE and of P2Y2-mediated ATP uptake diminished NET formation. Host cell invasion assays indicated that PMN-derived NETosis hampered tachyzoites from active host cell invasion, thereby inhibiting further intracellular replication. NET formation represents an early and effective mechanism of response of the innate immune system, which might reduce initial infection rates during the acute phase of cattle neosporosis.

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Section: Methodsmentioning

confidence: 99%

Bovine Polymorphonuclear Neutrophils Cast Neutrophil Extracellular Traps against the Abortive Parasite Neospora caninum

et al. 2017

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“…For NADPH oxidase-, NE- and MPO-inhibition assays, the following inhibitors were used: the NADPH oxidase inhibitor diphenylene iodonium (DPI, 10 μM, Sigma-Aldrich); the NE inhibitor Suc-Ala-Ala-Pro-Val chloromethyl ketone (CMK; 1 mM, Sigma-Aldrich), according to Scapinello et al [29] and the MPO inhibitor 4-aminobenzoic acid hydrazide (ABAH; 100 μM, Calbiochem), according to Parker et al [30]. In brief, PMN and sporozoites (1:1 ratio, n = 3) were incubated (30 min, 37 °C) in HBSS-buffer without phenol red (Gibco) for positive controls.…”

Section: Methodsmentioning

confidence: 99%

Eimeria bovis-triggered neutrophil extracellular trap formation is CD11b-, ERK 1/2-, p38 MAP kinase- and SOCE-dependent

Muñoz-Caro

Huertas

Conejeros

et al. 2015

Vet Res

121

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Eimeria bovis is an important coccidian parasite that causes high economic losses in the cattle industry. We recently showed that polymorphonuclear neutrophils (PMN) react upon E. bovis sporozoite exposure by neutrophil extracellular trap (NET) formation. We focused here on the molecular mechanisms that are involved in this process. The sporozoite encounter led to an enhanced surface expression of neutrophil CD11b suggesting a potential role of this receptor in E. bovis-mediated NETosis. Antibody-mediated blockage of CD11b confirmed this assumption and led to a significantly decreased sporozoite-triggered NET. In addition, E. bovis-induced NETosis was found to be Ca2+-dependent since the inhibition of store-operated calcium entry (SOCE) significantly diminished NET. Furthermore, NADPH oxidase, neutrophil elastase (NE) and myeloperoxidase (MPO) were confirmed as key molecules in sporozoite-triggered NETosis, as inhibition thereof blocked parasite-triggered NET. PMN degranulation analyses revealed a significant release of matrix metalloprotease-9 containing granules upon sporozoite exposure. We further show a significantly enhanced phosphorylation of ERK1/2 and p38 MAPK in sporozoite-exposed PMN indicating a key role of this signaling pathway in E. bovis-mediated NETosis. Accordingly, ERK 1/2 and p38 MAPK inhibition led to a significant decrease in NET formation. Finally, we demonstrate that sporozoite-induced NETosis is neither a stage-, species-, nor host-specific process.

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“…Protein spots of interest were excised from the gel automatically using an Ettan Spot Picker robot (GE Healthcare, Montreal, QC, Canada). Those spots were digested and analyzed by tandem mass spectrometry at the Mass Spectrometry Facility, Hospital for Sick Children, Toronto, ON, Canada [6], and proteins were identified (Scaffold Proteome Software, version 02, Portland, OR, USA). All MS/MS spectra were searched against the bovine protein database (downloaded 11 March 2011 from the National Center for Biotechnology Information), and peptides were identified based on the coverage, probability, peptide match, amino acid match, similarity of the observed and theoretical molecular weights as well as the pI and the unique total spectra.…”

Section: Methodsmentioning

confidence: 99%

Increased annexin A1 and A2 levels in bronchoalveolar lavage fluid are associated with resistance to respiratory disease in beef calves

Senthilkumaran

Clark

Abd-Elaziz

et al. 2013

Vet Res

Self Cite

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Strategies to control bovine respiratory disease depend on accurate classification of disease risk. An objective method to refine the risk classification of beef calves could be economically beneficial, improve welfare by preventing unexpected disease occurrences, refine and reduce the use of antibiotics in beef production, and facilitate alternative methods of disease control. The objective of this study was to identify proteins in bronchoalveolar lavage fluid (BALF) of stressed healthy calves that predict later disease outcome, serve as biomarkers of susceptibility to pneumonia, and play a role in pathogenesis. BALF was collected from 162 healthy beef calves 1–2 days after weaning and transportation. Difference in gel electrophoresis (DIGE) and mass spectrometry were used to compare proteins in samples from 7 calves that later developed respiratory disease compared to 7 calves that remained healthy. Calves that later developed pneumonia had significantly lower levels of annexin A1, annexin A2, peroxiredoxin I, calcyphosin, superoxide dismutase, macrophage capping protein and dihydrodiol dehydrogenase 3. Differences in annexin levels were partially confirmed by western blot analysis. Thus, lower levels of annexins A1 and A2 are potential biomarkers of increased susceptibility to pneumonia in recently weaned and transported feedlot cattle. Since annexins are regulated by glucocorticoids, this finding may reflect individual differences in the stress response that predispose to pneumonia. These findings also have implications in pathogenesis. Annexins A1 and A2 are known to prevent neutrophil influx and fibrin deposition respectively, and may thus act to minimize the harmful effects of the inflammatory response during development of pneumonia.

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Bactericidal activity of porcine neutrophil secretions

Cited by 20 publications

References 23 publications

Bovine Polymorphonuclear Neutrophils Cast Neutrophil Extracellular Traps against the Abortive Parasite Neospora caninum

Bovine Polymorphonuclear Neutrophils Cast Neutrophil Extracellular Traps against the Abortive Parasite Neospora caninum

Eimeria bovis-triggered neutrophil extracellular trap formation is CD11b-, ERK 1/2-, p38 MAP kinase- and SOCE-dependent

Increased annexin A1 and A2 levels in bronchoalveolar lavage fluid are associated with resistance to respiratory disease in beef calves

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