H5N1 Influenza a Virus Replicates Productively in Pancreatic Cells and Induces Apoptosis and Pro-Inflammatory Cytokine Response

Abstract

The inflammatory response and apoptosis have been proved to have a crucial role in the pathogenesis of the influenza A virus (IAV). Previous studies indicated that while IAV commonly causes pancreatitis and pancreatic damage in naturally and experimentally infected animals, the molecular mechanisms of the pathogenesis of IAV infection are less reported. In the present study, we showed for the first time that both avian-like (α-2,3-linked) and human-like (α-2,6-linked) sialic acid (SA) receptors were expressed by the mouse pancreatic cancer cell line PAN02 and the human pancreatic cancer cell line PANC-1. Using growth kinetics experiments, we also showed that PAN02 and PANC-1 cells supported the productive replication of the H5N1 highly pathogenic avian influenza while exhibited the limited replication of IAV subtypes H1N1 and H7N2 in vitro. The in vivo infection of H5N1 in pancreatic cells was confirmed by the histopathological and immunohistochemical staining of pancreas tissue from mice. Other than H1N1 and H7N2, severe damage and extensive positive signals were observed in pancreas of H5N1 infected mice. All three virus subtypes induced apoptosis but also triggered the infected PAN02 and PANC-1 cells to release pro-inflammatory cytokines and chemokines including interferon (IFN)-α, IFN-β, IFN-γ, chemokine (C-C motif) ligand 2 (CCL2), tumor necrosis factor (TNF)-α, and interleukin (IL)-6. Notably, the subtypes of H5N1 could significantly upregulate these cytokines and chemokines in both two cells when compared with H1N1 and H7N2. The present data provide further understanding of the pathogenesis of H5N1 IAV in pancreatic cells derived from humans and mammals and may also benefit the development of new treatment against H5N1 influenza virus infection.

Keywords: H5N1 influenza A virus; apoptosis; inflammatory response; pancreatic cells; pathogenesis.

Figure 1

Pancreatic cells express α-2,3- and α-2,6-linked sialic acid (SA) receptors. (A,B) The pancreatic cell lines PAN02 and PANC-1 were placed on polylysine-coated slides and stained with fluorescein isothiocyanate (FITC)-conjugated Sambucus nigra bark lectin (SNA) or Maackia amurensis lectin I (MAA-I) (green), and 4′,6′-diamidine-2-phenylindole (DAPI; blue) for nuclei. (C,D) Trypsinized PAN02 and PANC-1 cells were incubated with FITC-conjugated SNA or MAA-I (concentrations from left to right are 5, 10, and 20 μg/mL) and analyzed using flow cytometry to determine the relative percentages of cells expressing α-2,3-SA (MAA, yellow) or α-2,6-SA (SNA, blue) compared to unstained cells (red). (E) Representative pancreas sections from mock-treated mice were analyzed by immunohistochemical staining using SNA and MAA-I antibody, respectively. Black arrows indicate positive signals.

Figure 2

H5N1 IAV could productively infect pancreatic cells while H1N1 and H7N2 showed limited replication. PAN02 and PANC-1 cells were infected with the three IAV subtypes at an equal multiplicity of infection (MOI) of 1 for the periods specified. (A) Cells were homogenized in Trizol reagent and the viral non-structural protein 1 (NS1) gene was quantified using real time quantitative PCR (RT-qPCR). (B) The culture media supernatant was used to determine viral titers by plaque assay. The results shown here were pooled from three independent replicates (ns, not significantly and ^***P < 0.001).

Figure 3

Pancreatic cells release infectious virus following H5N1 IAV infection. (A) PAN02 and (B) PANC-1 cells were mock-treated or infected with three IAV subtypes at an MOI of 1 and transmission electron microscopy was used to observe the viruses released from the cell surface. Arrows denote the virus particles.

Figure 4

H5N1 IAV could infect pancreatic cells in vivo. Representative pancreas sections from control or IAV-infected mice. (A) Sections were analyzed by H & E staining. Black arrows indicate hemorrhage. Hollow arrows indicate necrosis of pancreatic cells. Triangles indicate dilatation and exudate in pancreatic duct. (B) Sections were analyzed by IHC staining. Black arrows indicate positive signals.

Figure 5

Cytopathic effects were induced in H5N1 IAV-infected pancreatic cells. (A) PAN02 and (B) PANC-1 cells were mock-treated or infected with H1N1, H5N1, or H7N2 at an MOI of 1 for 12 h. Morphologic analysis of the effect of IAV infection on cell death were taken. Cell morphology was visualized by light microscopy. Black arrows indicate numerous rounded cells. Hollow arrows indicate dead cells. Bar = 25 μm.

Figure 6

Apoptosis was induced in H5N1 IAV-infected pancreatic cells. (A) PAN02 and (B) PANC-1 cells were mock-treated or infected with H1N1, H5N1, or H7N2 at an MOI of 1 for 12 h. A terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay was used to measure apoptosis in PAN02 and PANC-1 cells with DNase I as a positive control. Blue showed nucleus and green showed positive TUNEL signals.

Figure 7

Pancreatic cell apoptosis was measured at specified times following IAV infection. PAN02 and PANC-1 cells were mock-treated or infected with IAV (H1N1, H5N1, or H7N2) at an MOI of 1, taxol served as a positive control. Then the cells were harvested at 6, 12, and 24 h post-infection. The total number of apoptotic cells in early stage was analyzed by flow cytometry. Asterisks indicate statistically significantly values compared with mock-treated cells (^**P < 0.01, and ^***P < 0.001).

Figure 8

H5N1 IAV infection increased the release of pro-inflammatory cytokines and chemokines outside the cells to function. (A) PAN02 and (B) PANC-1 cells were treated or infected as described in Figure 5 and the culture media supernatant were harvested at 6, 12, and 24 h post-infection. The expression of interferon (IFN)-α, IFN-β, IFN-γ, chemokine (C-C motif) ligand 2 (CCL2), and tumor necrosis factor (TNF)-α was analyzed by RT-qPCR. Graphs shown are the mean ± SD of three independent replicates. Asterisks indicate statistically significant increases compared to mock-treated cells (ns, not significantly and ^***P < 0.001).

Figure 9

H5N1 IAV upregulates the mRNA expression of Toll-like receptor 3 (TLR3), cytolytic RNA helicases retinoic acid-inducible gene I (RIG-I), and melanoma differentiation-associated gene 5 (MDA-5). (A) PAN02 and (B) PANC-1 cells were treated or infected as described in Figure 5 and harvested at 6, 12, and 24 h post-infection. Total RNA was isolated at the designated times and examined by RT-qPCR. The mRNA expression of TLR3, RIG-I, and MDA-5 is shown. The data are presented as the relative fold change over mock treatment. Graphs shown are the mean ± standard deviation (SD) of three independent replicates. Asterisks indicate statistically significant increases compared with mock-treated cells (^*P < 0.05, ^**P < 0.01, and ^***P < 0.001).