Macrophage Activation Assays to Evaluate the Immunostimulatory Capacity of Avibacterium paragallinarum in A Multivalent Poultry Vaccine
"> Figure 1
<p>The macrophage-like HD11 cell line produces nitric oxide upon stimulation with a broad range of TLR agonists for 48 h. HD11 cells were stimulated with titrated concentrations of the TLR agonists Pam3CSK4 (<b>a</b>), zymosan (<b>b</b>), poly(I:C) (<b>c</b>), LPS (<b>d</b>), R848 (<b>e</b>), and CpG (<b>f</b>). The expected receptors for each agonist are shown above each panel. Nitric oxide production is expressed as the concentration of nitrite ions (NO<sub>2</sub><sup>−</sup>) in the cell culture supernatant, as measured by the Griess test. Four parameter logistic curves were plotted together with their confidence intervals (dotted lines). Unstimulated HD11 cells (UNST) were used as a negative control and HD11 cells stimulated with 300 ng/ml LPS (<b>d</b>) were used as a positive control in this and all subsequent experiments. The experiment was performed in triplicate. The error bars represent the SEM.</p> "> Figure 2
<p>Nitric oxide was produced upon exposure to octavalent vaccine (IBV + NDV + EDSV + 5x <span class="html-italic">Av</span><span class="html-italic">. paragallinarum)</span> for 48 h. (<b>a</b>) HD11 cells were exposed to titrated concentrations of the octavalent vaccine. Moreover, unstimulated HD11 cells (UNST) and HD11 cells stimulated with 300 ng/ml LPS are shown as negative and positive controls. The data comprises three independent technical replicates performed in triplicate. The error bars represent the SEM. A Kruskal–Wallis test combined with Dunn’s multiple comparisons test was used to test for significant induction of nitric oxide production upon stimulation. *** <span class="html-italic">p</span> < 0.001. (<b>b</b>) RT-qPCR was performed on HD11 cell samples stimulated with 1 µL/mL octavalent vaccine and harvested at the given time points between 0 and 48 h. <span class="html-italic">iNOS</span> expression is shown relative to t = 0 h and expressed as 2<sup>-ΔΔCt</sup> values as calculated using the Livak method and <span class="html-italic">GAPDH</span> and <span class="html-italic">28S</span> as a reference gene. The experiment was performed in triplicate. The error bars represent the SEM.</p> "> Figure 3
<p>The octavalent vaccine induced the expression of pro-inflammatory cytokines at an early time point, followed by expression of the anti-inflammatory cytokine IL-10 and finally IL-12p40. HD11 cells were stimulated with 1 µL/mL octavalent vaccine and harvested at the indicated time points between 0 and 48 h. RT-qPCR was performed for the cytokines<span class="html-italic">IL-1β</span> (<b>a</b>), <span class="html-italic">CXCLi1</span> (<b>b</b>), <span class="html-italic">CXCLi2</span> (<b>c</b>), <span class="html-italic">TNF</span> (<b>d</b>), <span class="html-italic">IL-6</span> (<b>e</b>), <span class="html-italic">IFN-α</span> (<b>f</b>), <span class="html-italic">IL-10</span> (<b>g</b>), <span class="html-italic">IL-12p40</span> (<b>h</b>) and <span class="html-italic">IFN-γ</span> (<b>i</b>). The relative gene expression levels were normalized against t = 0 h and expressed as 2<sup>-ΔΔCt</sup> values as calculated using the Livak method and both <span class="html-italic">GAPDH</span> and <span class="html-italic">28S</span> as reference genes. The experiment was performed in triplicate. The error bars represent the SEM.</p> "> Figure 4
<p>A trivalent vaccine without <span class="html-italic">Av</span><span class="html-italic">. paragallinarum</span> antigens did not induce nitric oxide production or expression of pro-inflammatory cytokines. (<b>a</b>) Nitric oxide production was determined for HD11 cells exposed to titrated doses of trivalent (IBV + NDV + EDSV) and octavalent vaccine (IBV + NDV + EDSV + 5x <span class="html-italic">Av</span><span class="html-italic">. paragallinarum</span>) for 48 h. (<b>b</b>) Antigenic fractions were extracted from the tri- and octavalent vaccines using isopropyl myristate and given to HD11 cells to determine nitric oxide production after 48 h. (<b>c</b>) The bacterial pellet was purified from the antigenic fraction of the octavalent vaccine and given to HD11 cells to determine nitric oxide production after 48 h. A four-parameter logistic curve could be calculated and was plotted. (<b>d</b>) The controls of the nitric oxide production assay included unstimulated HD11 cells (UNST) and HD11 cells stimulated with 300 ng/ml LPS. (<b>e</b>-<b>j</b>) Expression levels of <span class="html-italic">iNOS</span> (<b>e</b>), <span class="html-italic">IL-1β</span> (<b>f</b>), <span class="html-italic">TNF</span> (<b>g</b>), <span class="html-italic">CXCLi1</span> (<b>h</b>), <span class="html-italic">CXCLi2</span> (<b>i</b>), and <span class="html-italic">IFN-α</span> (<b>j</b>) by HD11 cells were determined 8 h after stimulation with 1.0 µL/mL tri- or octavalent vaccine. The values are expressed as 2<sup>-ΔΔCt</sup> values as calculated using the Livak method and both <span class="html-italic">GAPDH</span> and <span class="html-italic">28S</span> as reference genes. All figures show three independent technical replicates. The error bars represent the SEM. A Kruskal-Wallis test combined with Dunn’s multiple comparisons test was used to test for statistical significance of the data. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001. The relative gene expression data were log-transformed prior to the statistical analysis to generate normally distributed data.</p> "> Figure 5
<p>Nitric oxide production upon exposure to the antigenic fraction of the octavalent vaccine could be inhibited by the LPS-binding antibiotic polymyxin B. (<b>a</b>–<b>c</b>) HD11 cells were pre-incubated in cell culture media without or with 1, 10, or 100 μg/ml polymyxin B for 1 h at 37 °C and subsequently exposed to cell culture media containing 300 ng/ml LPS (<b>a</b>), 300 ng/ml CpG (<b>b</b>) or 0.5 µL/mL antigens extracted from the octavalent vaccine (<b>c</b>) for 48 h. Unstimulated controls are represented by the dotted lines. The first bar of panel <b>a</b> is the result of HD11 stimulated with 300 ng/ml without polymyxin B and thus represents the positive control. (<b>d</b>–<b>h</b>) Expression levels of <span class="html-italic">iNOS</span> (<b>d</b>), <span class="html-italic">IL-1β</span> (<b>e</b>), <span class="html-italic">CXCLi1</span> (<b>f</b>), <span class="html-italic">CXCLi2</span> (<b>g</b>), and <span class="html-italic">IL-10</span> (<b>h</b>) by HD11 cells were determined 8 h after stimulation with 1 µL/mL octavalent vaccine and, when indicated, 1 h pre-incubation with 100 µg/ml polymyxin B. The values are expressed as 2<sup>-ΔΔCt</sup> values as calculated using the Livak method and both <span class="html-italic">GAPDH</span> and <span class="html-italic">28S</span> as reference genes. The data comprises three independent technical replicates performed in triplicate. Unstimulated controls are represented by the dotted lines. The error bars represent the SEM. A Kruskal–Wallis test combined with Dunn’s multiple comparisons test was used to test for statistical significance of the data. * <span class="html-italic">p</span> < 0.05, ** <span class="html-italic">p</span> < 0.01, *** <span class="html-italic">p</span> < 0.001. The relative gene expression data were log-transformed prior to the statistical analysis to create normally distributed data.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. HD11 Cell Culture and Stimulation
2.2. Griess Test to Evaluate Nitric Oxide Production by Stimulated HD11 Cells
2.3. Relative Expression of iNOS and Cytokines Using Real-Time Quantitative PCR
2.4. Flow Cytometric Assessment of HD11 Cell Viability after Stimulation
2.5. Statistical Analysis
3. Results
3.1. The Inactivated Octavalent Poultry Vaccine Induces HD11 Cells to Produce Nitric Oxide
3.2. Stimulation with the Octavalent Vaccine Results in Enhanced Gene Expression of Cytokines and Chemokines
3.3. Av. Paragallinarum Antigens Contribute to the Stimulatory Capacity of the Octavalent Vaccine
3.4. Activation of HD11 Cells by the Octavalent Vaccine Largely Depends on Av. Paragallinarum Antigen-Associated LPS
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Gene | NCBI Reference | Type | Sequences (5′-3′) |
---|---|---|---|
iNOS | NM_204961.1 | Forward | TGGGTGGAAGCCGAAATA |
Reverse | GTACCAGCCGTTGAAAGGAC | ||
TNF | MF000729.1 | Forward | CGCTCAGAACGACGTCAA |
Reverse | GTCGTCCACACCAACGAG | ||
CXCLi1 | NM_205018.1 | Forward | CCAGTGCATAGAGACTCATTCCAAA |
Reverse | TGCCATCTTTCAGAGTAGCTATGACT | ||
GAPDH | NM_204305.1 | Forward | GTGGTGCTAAGCGTGTTATC |
Reverse | GCATGGACAGTGGTCATAAG | ||
IL-1β | NM_204524.1 | Forward | GCTCTACATGTCGTGTGTGATGAG |
Reverse | TGTCGATGTCCCGCATGA | ||
Probe | CCACACTGCAGCTGGAGGAAGCC | ||
IL-4 | NM_001007079.1 | Forward | AACATGCGTCAGCTCCTGAAT |
Reverse | TCTGCTAGGAACTTCTCCATTGAA | ||
Probe | AGCAGCACCTCCCTCAAGGCACC | ||
IL-6 | NM_204628.1 | Forward | GCTCGCCGGCTTCGA |
Reverse | GGTAGGTCTGAAAGGCGAACAG | ||
Probe | AGGAGAAATGCCTGACGAAGCTCTCCA | ||
CXCLi2 | NM_205498.1 | Forward | GCCCTCCTCCTGGTTTCA |
Reverse | TGGCACCGCAGCTCATT | ||
Probe | TCTTTACCAGCGTCCTACCTTGCGACA | ||
IL-10 | NM_001004414.2 | Forward | CATGCTGCTGGGCCTGAA |
Reverse | CGTCTCCTTGATCTGCTTGATG | ||
Probe | CGACGATGCGGCGCTGTCA | ||
IL-12p35 | NM_213588.1 | Forward | TGGCCGCTGCAAACG |
Reverse | ACCTCTTCAAGGGTGCACTCA | ||
Probe | CCAGCGTCCTCTGCTTCTGCACCTT | ||
IL-12p40 | NM_213571.1 | Forward | TGGGCAAATGATACGGTCAA |
Reverse | CTGAAAAGCTATAAAGAGCCAAGCAAGACGTTCT | ||
Probe | CAGAGTAGTTCTTTGCCTCACATTTT | ||
IFN-α | XM_015277440.2 | Forward | GACAGCCAACGCCAAAGC |
Reverse | GTCGCTGCTGTCCAAGCATT | ||
Probe | CTCAACCGGATCCACCGCTACACC | ||
IFN-γ | NM_205149.1 | Forward | GTGAAGAAGGTGAAAGATATCATGGA |
Reverse | GCTTTGCGCTGGATTCTCA | ||
Probe | TGGCCAAGCTCCCGATGAACGA | ||
28S | XR_003078040.1 | Forward | GGCGAAGCCAGAGGAAACT |
Reverse | GACGACCGATTTGCACGTC | ||
Probe | AGGACCGCTACGGACCTCCACCA |
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van den Biggelaar, R.H.G.A.; van Eden, W.; Rutten, V.P.M.G.; Jansen, C.A. Macrophage Activation Assays to Evaluate the Immunostimulatory Capacity of Avibacterium paragallinarum in A Multivalent Poultry Vaccine. Vaccines 2020, 8, 671. https://doi.org/10.3390/vaccines8040671
van den Biggelaar RHGA, van Eden W, Rutten VPMG, Jansen CA. Macrophage Activation Assays to Evaluate the Immunostimulatory Capacity of Avibacterium paragallinarum in A Multivalent Poultry Vaccine. Vaccines. 2020; 8(4):671. https://doi.org/10.3390/vaccines8040671
Chicago/Turabian Stylevan den Biggelaar, Robin H. G. A., Willem van Eden, Victor P. M. G. Rutten, and Christine A. Jansen. 2020. "Macrophage Activation Assays to Evaluate the Immunostimulatory Capacity of Avibacterium paragallinarum in A Multivalent Poultry Vaccine" Vaccines 8, no. 4: 671. https://doi.org/10.3390/vaccines8040671