Morphologic characterization and cytokine response of chicken bone-marrow derived dendritic cells to infection with high and low pathogenic avian influenza virus

Abstract

Dendritic cells (DCs) are professional antigen-presenting cells, which are key components of the immune system and involved in early immune responses. DCs are specialized in capturing, processing, and presenting antigens to facilitate immune interactions. Chickens infected with avian influenza virus (AIV) demonstrate a wide range of clinical symptoms, based on pathogenicity of the virus. Low pathogenic avian influenza (LPAI) viruses typically induce mild clinical signs, whereas high pathogenic avian influenza (HPAI) induce more severe disease, which can lead to death. For this study, chicken bone marrow-derived DC (ckBM-DC)s were produced and infected with high and low pathogenic avian influenza viruses of H5N2 or H7N3 subtypes to characterize innate immune responses, study effect on cell morphologies, and evaluate virus replication. A strong proinflammatory response was observed at 8 hours post infection, via upregulation of chicken interleukin-1β and stimulation of the interferon response pathway. Microscopically, the DCs underwent morphological changes from classic elongated dendrites to a more general rounded shape that eventually led to cell death with the presence of scattered cellular debris. Differences in onset of morphologic changes were observed between H5 and H7 subtypes. Increases in viral titers demonstrated that both HPAI and LPAI are capable of infecting and replicating in DCs. The increase in activation of infected DCs may be indicative of a dysregulated immune response typically seen with HPAI infections.

Keywords: avian influenza; chicken; cytokines; dendritic cells; innate immunity; interferon.

Figure 1

Morphology of ckBM-DC. Bone marrow-derived cells were cultured in the presence of different levels of recombinant chicken granulocyte-macrophage stimulating factor (GM-CSF) and recombinant chicken interleukin-4 (IL-4) for 6 days and dendrite formation was observed by microscopy. (A) 0 ng/ml GM-CSF + 0 ng/ml IL-4. (B) 10 ng/ml GM-CSF + 10 ng/ml IL-4. (C) 25 ng/ml GM-CSF + 25 ng/ml IL-4. (D) 50 ng/ml GM-CSF + 50 ng/ml IL-4. A representative image is shown for each concentration at 200x magnification.

Figure 2

Morphology of immature ckBM-DC stimulated with LPS. Cells were cultured in the presence of 50 ng/ml GM-CSF + 50 ng/ml IL-4 for 6 days and then stimulated with LPS (500ng/ml). ckBM-DCs were observed by microscopy for 30 hours. Images show cells cultured at (A) 0 hours, (B) 10 hours, (C) 20 hours, and (D) 30 hours. A representative image is shown for each timepoint at 100x magnification. Differening levels of elongated dendrites are in black boxes.

Figure 3

Comparative analysis of surface markers on immature and mature ckBM-DCs. Cells were cultured in the presence of 50 ng/ml GM-CSF + 50 ng/ml IL-4 for 6 days, and then stimulated with 500 ng/ml LPS for 30 hours. (A) Immature cells (-LPS) are on the left (A1, B1, C1) and mature cells (+LPS) are on the right (A2, B2, C2). Immunofluorescence analysis was performed using a FITC labeled mouse-anti-chicken MHC-II antibody (green) (A1, A2). Cells were also stained with mouse anti-chicken CD11c (B1, B2) (red) and mouse anti-chicken CD40 (C1, C2) followed by a goat-anti-mouse secondary (green). A representative image is shown for each at 100x magnification. (B) Cellular RNA was extracted to measure expression levels of surface markers, via qPCR, in ckBM-DCs. RNA was normalized using the Ck 28S house-keeping gene. The data is expressed as the fold change in mRNA levels between immature and mature ckBM-DCs for MHC-II, CD11c, CD40, CD80, CD83, and CD86. The data shown is a representative of three independent experiments. Error bars represent the standard error.

Figure 4

Functionality of immature ckBM-DCs. Cells were cultured in the presence of 50 ng/ml GM-CSF + 50 ng/ml IL-4 for 6 days. (A) ckBM-DCs were incubated with 0.5 µm carboxylate modified fluorescent red latex beads or (B) FITC labeled-inactivated H5N9 avian influenza virus for 4 hours. Following incubation cells were counterstained with DAPI, washed 5x with 1X PBS, and visualized by immunofluorescence microscopy. A representative image is shown for each at 100x magnification.

Figure 5

Distribution of sialic acid receptors on ckBM-DCs and susceptibility to pandemic H1N1 and H5N9 viruses. Cells were cultured in the presence of 50 ng/ml GM-CSF + 50 ng/ml IL-4 for 6 days. Immature ckBM-DCs (A1, B1) were stained with FITC-labeled MAA (SA-α2,3-Gal) (A2) or TRITC-labeled SNA (SA-α2,6-Gal) (B2) and visualized by immunofluorescence microscopy. CkBM-DCs were infected at an MOI of 1 with A/turkey/Virginia/SEP-4/2009 H1N1 (SA-α2,6-Gal preference) and A/turkey/Wisconsin/68 H5N9 (SA-α2,3-Gal preference). At 20 hpi, viral-infected cells, H1N1 (C1) and H5N9 (C2), were washed 2x with 1X PBS, fixed with methanol, and observed by microscopy. Viral NP proteins, H1N1 (D1) and H5N9 (D2), were detected using a mouse-anti-NP antibody followed by a FITC-conjugated anti-mouse IgG secondary (D1,D2). A representative image is shown for each at 200x (A1,A2,B1,B2) and 100x (C1,C2,D1,D2) magnification.

Figure 6

Change in morphology and growth of ckBM-DCs infected with LPAIV and HPAIV. Cells were cultured in the presence of 50 ng/ml GM-CSF + 50 ng/ml IL-4 for 6 days. Immature ckBM-DCs were infected at a MOI of 1 with LPAIV (A/Chicken/Pennsylvania/21525/1983 H5N2 and A/Cinnamon Teal/Mexico/2817/2006 H7N3) and HPAIV (A/Chicken/Pennsylvania/1370/1983 H5N2 and A/Chicken/Jalisco/CPA1/2017 H7N3) viruses. (A) CPE (black arrows) and cellular morphological changes were observed via microscopy at 8 and 24 HPI. A representative image is shown for each at 100x and 200x magnification. (B) Supernatants were obtained at 2, 8, and 24 HPI and viral titers were evaluated by EID50. The data shown is a representative of three independent experiments. Error bars represent the standard error of triplicate samples.

Figure 7

Cytokine expression levels of ckBM-DCs infected with HPAIV or LPAIV. Cells were cultured in the presence of 50 ng/ml GM-CSF + 50 ng/ml IL-4 for 6 days. Immature ckBM-DCs were infected at a MOI of 1 with LPAIV (A/Chicken/Pennsylvania/21525/1983 H5N2 and A/Cinnamon Teal/Mexico/2817/2006 H7N3) and HPAIV (A/Chicken/Pennsylvania/1370/1983 H5N2 and A/Chicken/Jalisco/CPA1/2017 H7N3) viruses. Cellular RNA was extracted 8 hpi to measure relative gene expression levels of IFNα, Mx, TLR-7, TLR-3, MHC-1, IL-1B, IL-6, Casp-3, and Casp-8 (A–D). RNA was normalized using the Ck 28S house-keeping gene. The fold change (2^-ΔΔCT) was determined by the comparison of infected ckBM-DCs to sham-infected ckBM-DCs. Tukey one-way ANOVA analysis was performed to determine significant differences between LPAIV and HPAIV infected ckBM-DCs. * indicates a significant difference (p<0.05). The data shown is a representative of three independent experiments. Error bars represent the standard error of triplicate samples.