Neutrophil apoptosis during experimentally induced Staphylococcus aureus mastitis

Abstract -The objective of this study was to determine whether neutrophil apoptosis and their consequent elimination by macrophages from the mammary gland is modulated by an infection caused by Staphylococcus aureus (S. aureus). The study was performed on twenty mammary glands of 5 virgin heifers. A buffered physiological solution (PBS) was administered as a means of control into the mammary glands of the heifers and after 168 h, the glands were inoculated with S. aureus. The samples of cell populations were obtained by lavages of the mammary glands in 4 intervals (24, 48, 72 and 168 h) after the experimental infection. Flow cytometry was used for determination of Annexin-V positivity and propidium iodide (PI) negativity of neutrophils. Light microscopy was used for determination of neutrophil karyopyknosis. Cytochemistry was used for the detection of myeloperoxidase-positive (MPO+) macrophages. Instillation of S. aureus resulted in an intramammary infection which persisted during the following experimental period. The total number of both Annexin-V-positive and PI negative neutrophils and karyopyknotic neutrophils peaked at 24 h after both of PBS and S. aureus administration. The highest percentages of Annexin-V-positive and PI negative neutrophils and karyopyknotic neutrophils were detected 48 and 168 h after PBS and S. aureus administration, respectively. The total number of MPO+ macrophages was the highest 24 h and 48 h after PBS and S. aureus administration, respectively; the percentage of MPO+ macrophages was the highest at 72 h in both cases. The dynamics of resolution of mastitis caused by S. aureus was very similar to the resolution of inflammatory response of the mammary gland after PBS administration. Mechanisms of cell pathogen elimination as well as inflammation resolution were very intensively involved; nevertheless, the mammary gland infection persisted. An early inclusion of the mechanisms of an acute inflammatory resolution thus paradoxically led to chronic infection

Cytokine expression by CD163+ monocytes in healthy and Actinobacillus pleuropneumoniae-infected pigs

Distinct monocyte subpopulations have been previously described in healthy pigs and pigs experimentally infected with Actinobacillus pleuropneumoniae (APP). The CD163+ subpopulation of bone marrow (BM), peripheral blood (PB) and lung monocytes was found to play an important role in the inflammatory process. The inflammation is accompanied by elevation of inflammatory cytokines. The aim of the study was to evaluate the contribution of CD163+ monocytes and macrophages to cytokine production during APP-induced lung inflammation. Cytokine production was assessed by flow cytometry (FC) and quantitative PCR (qPCR) in CD163+ monocytes and by qPCR, immunohistochemistry/fluorescence in lungs and tracheobronchial lymph nodes (TBLN). Despite the systemic inflammatory response after APP infection, BM and PB CD163+ monocytes did not express elevated levels of a wide range of cytokines compared to control pigs. In contrast, significant amounts of IL-1β, IL-6, IL-8 and TNF-α were produced in lung lesions and IL-1β in the TBLN. At the protein level, TNF-α was expressed by both CD163+ monocytes and macrophages in lung lesions, whereas IL-1β, IL-6 and IL-8 expression was found only in CD163+ monocytes; no CD163+ macrophages were found to produce these cytokines. Furthermore, the quantification of CD163+ monocytes expressing the two cytokines IL-1β and IL-8 that were most elevated was performed. In lung lesions, 36.5% IL-1β positive CD163+ monocytes but only 18.3% IL-8 positive CD163+ monocytes were found. In conclusion, PB and BM CD163+ monocytes do not appear to contribute to the elevated cytokine levels in plasma. On the other hand, CD163+ monocytes contribute to inflammatory cytokine expression, especially IL-1β at the site of inflammation during the inflammatory process.Peer reviewe

Apoptoza neutrofilnich granulocytu mlecne zlazy skotu.

Available from STL, Prague, CZ / NTK - National Technical LibrarySIGLECZCzech Republi

Neutrophil apoptosis during experimentally induced Staphylococcus aureus mastitis

The objective of this study was to determine whether neutrophil apoptosis and their consequent elimination by macrophages from the mammary gland is modulated by an infection caused by Staphylococcus aureus (S. aureus). The study was performed on twenty mammary glands of 5 virgin heifers. A buffered physiological solution (PBS) was administered as a means of control into the mammary glands of the heifers and after 168 h, the glands were inoculated with S. aureus. The samples of cell populations were obtained by lavages of the mammary glands in 4 intervals (24, 48, 72 and 168 h) after the experimental infection. Flow cytometry was used for determination of Annexin-V positivity and propidium iodide (PI) negativity of neutrophils. Light microscopy was used for determination of neutrophil karyopyknosis. Cytochemistry was used for the detection of myeloperoxidase‑positive (MPO+) macrophages. Instillation of S. aureus resulted in an intramammary infection which persisted during the following experimental period. The total number of both Annexin-V-positive and PI negative neutrophils and karyopyknotic neutrophils peaked at 24 h after both of PBS and S. aureus administration. The highest percentages of Annexin-V‑positive and PI negative neutrophils and karyopyknotic neutrophils were detected 48 and 168 h after PBS and S. aureus administration, respectively. The total number of MPO+ macrophages was the highest 24 h and 48 h after PBS and S. aureus administration, respectively; the percentage of MPO+ macrophages was the highest at 72 h in both cases. The dynamics of resolution of mastitis caused by S. aureus was very similar to the resolution of inflammatory response of the mammary gland after PBS administration. Mechanisms of cell pathogen elimination as well as inflammation resolution were very intensively involved; nevertheless, the mammary gland infection persisted. An early inclusion of the mechanisms of an acute inflammatory resolution thus paradoxically led to chronic infection

Morphology of Apoptotic, necrotic and oncotic cells.

<p><b>A.</b> Characteristic apoptotic, necrotic and oncotic cells in multimodal holographic microscope, simulated DIC (differential interference contrast). 20 × magnification was used in MHM. Annexin V staining for the verification of cell membrane alteration. Red arrow indicates annexin V-positive “advanced” oncotic cell. Apoptotic cells displayed in initial step (left) with the typically round-shaped cells and in advanced step with the formation of apoptotic bodies. <b>B.</b> Scheme of typical apoptotic, necrotic and oncotic cells. Typical characteristics visible by MHM phase image. For a detailed description of the characteristic features of apoptotic, necrotic, and oncotic cells, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121674#pone.0121674.t001" target="_blank">Table 1</a>.</p

Characteristic features of apoptosis, oncosis, and necrosis.

<p>Characteristic features of apoptosis, oncosis, and necrosis.</p

Estimation of oncosis progression by multimodal holographic microscopy.

<p>Annexin V (green) and propidium iodide (PI, red) staining. Initial step of oncosis (first row, red arrow) is annexin V−/PI− and thus distinguishable only by native morphology, see typical cytoplasmic bleb in the phase image. This causes false-negativity by flow-cytometry. Second, early oncotic cells feature larger blebs and are annexin V+/PI−. Late oncosis is double positive for staining, with no apparent karyolysis. Advanced oncosis/necrosis transition is typical by double-positive staining and karyolysis.</p

Differences in cell death estimation between flow cytometry and holographic microscopy.

<p><b>A.</b> Plumbagin treatment, no annexin V/PI staining, used for gating set-up. Upper dot plot indicates annexin V/PI fluorescence, lower dot plot indicates FSC/SSC of these cells colour-coded according to gating regions. <b>B.</b> Annexin V/PI staining, untreated cells. 92% are double negative for staining. <b>C.</b> Annexin V/PI staining after 3 h of the experiment. See increased populations of annexin V-positive (green gating) and double positive (red staining). In FSC/SSC scatter plot, arrows indicate two populations (gating regions) of annexin V+/PI− cells: (R1) smaller cells (lower FSC) and (R2) larger cells (higher FSC). See the Results section for details. <b>D.</b> Multimodal holographic microscope, phase image. 10 × magnification 3 h after 2 μM plumbagin treatment. Red-outlined cells show size increase and oncotic phenotype, green-outlined cells show surface area decrease and apoptotic phenotype, blue-outlined cells show no changes during 2 h monitoring. For typical morphological criteria of oncotic/apoptic cells see (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121674#pone.0121674.t001" target="_blank">Table 1</a>) <b>E</b>. Relative change of cell surface in individual cells (relative to initial time point). Colour coding of individual cells is based on (D). <b>F</b>. Mass of individual cells in pg. Note a significantly higher mass of the “decrease-size” cell population. <b>G</b>. Time-lapse of typical oncotic “increase size” cell indicated by red arrow in (D), simulated digital interference microscopy <b>H.</b> Time-lapse of typical “decrease size” apoptotic cell indicated by green arrow in (D). Simulated digital interference microscopy. FSC—forward scatter, SSC—side scatter, PI—propidium iodide.</p

Holographic mode setup in Multimodal holographic microscope is based on the Mach-Zehnder-type interferometer.

<p>The light is divided into two separate optical paths—object arm and interferometer reference arm. Both arms consist of condenser (C), objective (O) and tube lens (TL). In the reference arm, a diffraction grating (DG) is placed. The object beam and the reference beam recombine in the output plane and create interference fringes pattern, which is captured by the camera (D). S—source; CL—collector lens; BS—beam splitter; M—mirror; C—condenser; O-objective; TL—tube lens; DG—diffraction grating; OL—output lens; D—detector.</p