Response of Primary Hepatocytes to Hepatitis AV iral Infection In

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Response of Primary Hepatocytes to Hepatitis AV iral Infection In Powered By Docstoc
					                                                                                           Response of Primary Hepatocytes to Hepatitis A Viral Infection In-Vitro
                                                                                           Lilly A. Bayouth, Erika A. Eksioglu, Jennifer Bess, Haizhen Zhu, Xiaoxi Zhang, and Chen Liu
                                                                                                      Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL



                                                                                                                                                                                                                                                                                                                                                                     HAV
                                   ABSTRACT                                                                                                                                      RESULTS                                                                                                                                                                                                                                               589bp
Background and Aims: Currently, it is not known how primary hepatocytes respond to the
                                                                                                                                                                                                                                                                                                                                                               GAPDH
hepatitis A virus (HAV). The purpose of this experiment is to observe the innate immune                                                                                                                                                                                                                                                                                       C    D2 D4 D6 D8 D10 D12 -
response of primary hepatocytes when infected with HAV LSH/S (ATCC # VR – 2266), as                                                                                                                                                                                                                                           Figure 5. RT-PCR results suggest intracellular replication of HAV in primary hepatocyte host cells. Following HAV PCR,
well as demonstrate viral replication in the cells.                                                                                                                                                                                                                                                                           samples were loaded in a 1.5% agarose gel and run at 80V. Results at 589bp, corresponding to hepatitis A virus, show high
Methods: Primary hepatocytes, represented by the Hu793 cell line, were treated with                                                                                                                                                                                                                                           concentrations of HAV present on day 2 (directly after infection), as well as days 10 and 12, suggesting an intracellular viral
                                                                                                                                                                                                                                                                                                                              replication mechanism. GAPDH used as housekeeping gene.
infectious HAV in a cell culture environment; their immune response was monitored for 11
days post infection. RNA was extracted from the infected cells as well as the HAV negative
                                                                                                                                                                                                                                                                                                                                           A                                                                               B




                                                                                                                                                                                                                                                                                                                                                                                                        Relative Expression of
                                                                                                                                                                                                                                                                                                                           Relative Expression of
control Hu793 cells bi-daily for 11 days. Viral replication was tested by HAV PCR and gel
electrophoresis. Real-Time PCR was used to test for DR4 and DR5 receptors, INFβ                                                                                                                                                                                                                                                                                                                                                  1.2
                                                                                                                                                                                                                                                                                                                                                    40
production in the host cells, and GAPDH, a housekeeping gene. Apoptosis was also                                                                                                                                                                                                                                                                    35                                                                             1
                                                                                                                                                40X           Control                                                                   40X HAV Day 8
confirmed via light microscopy and flow cytometry.
                                                                                                                                                                                               A                                                                                                     B                                              30
                                                                                                                                                                                                                                                                                                                                                    25
                                                                                                                                                                                                                                                                                                                                                                                                                                 0.8




                                                                                                                                                                                                                                                                                                                                                                                                                 DR4
                                                                                                                                                                                                                                                                                                                                    IFNb
Results: The presence of HAV was most evident on days 1, 9, and 11, post-infection. This                                                                                                                                                                                                                                                            20                                                                           0.6
increase in concentration of the infectious agent is consistent with evidence of intracellular                                                                                                                                                                                                                                                      15                                                                           0.4
viral replication and induction of apoptosis of the host cells. The relative expression of INFβ,                                                                                                                                                                                                                                                    10
                                                                                                                                                                                                                                                                                                                                                     5                                                                           0.2
DR4, and DR5 attest to the proposed immune response.
                                                                                                                                                                                                                                                                                                                                                     0                                                                             0
Conclusions: Our data show that HAV can replicate in primary hepatocytes and induces
Type I INF production and apoptosis in primary hepatocytes.

                                                                                                                                                                                                                                                                                                                                                                Days Post HAV Infection                                                        Days Post HAV Infection
                               INTRODUCTION                                                                                                     100X Control                                                                            100X HAV Day 8
                                                                                                                                                                                               C                                                                                                D                                          C8




                                                                                                                                                                                                                                                                                                                           Relative Expression of
                                                                                                                                                                                                                                                                                                                                                                                                             Figure 6. HAV has cytopathic effects in human primary
Hepatitis A Virus is a small (27nm diameter) spherical nonenveloped single stranded positive                            Figure 1. Light microscopy pictures show a decrease over time in cell confluence after HAV                                                                                                                          7                                                                hepatocytes: induction of IFNβ production and DR4 and
RNA virus whose protein capsid retains icosahedral symmetry. It belongs to the Picornaviridae                           infection, suggesting apoptosis. At 40x resolution, Figures 1A and 1B show plates of HAV- control                                                                                                                            6                                                       DR5 response in TRAIL pathway. Cells were cultured with
family, along with well known viruses such as poliovirus, human rhinovirus, and human                                   cells, approximately 90-100% confluent, and cells 8 days after viral infection, 30-40 % confluent,                                                                                                                           5                                                       HAV LSH/S for 12 days and collected bi-daily for total RNA
                                                                                                                        respectively. This difference in confluence suggests the possibility of cell death. The same cell plates                                                                                                                     4                                                       isolation. Real time RT-PCR was performed with SYBR green
enteroviruses; however, it is the only species classified in the genus Hepatovirus, because of




                                                                                                                                                                                                                                                                                                                                    DR5
                                                                                                                        are shown at 100x resolution in Figures 1C and 1D.                                                                                                                                                                                                                                   on TRAIL and its two receptors, DR4 and DR5, compared
its unique characteristics, specifically relating to structural aspects of its methods of                                                                                                                                                                                                                                                            3
                                                                                                                                                                                                                                                                                                                                                                                                             against uninfected controls and normalized against the
                                                                                                                                                                                                                                                                                                                                                     2
polyprotein processing and virion morphogenesis.                                                                                                                                                                                                                                                                                                                                                             housekeeping gene GAPDH. Bars represent the standard
                                                                                                                                                                                                                                                                                                                                                     1
HAV, first identified approximately 35 years ago, causes acute infectious hepatitis; unlike other
hepatotropic viruses such as Hepatitis B and C Viruses (HBV and HCV), it is not associated             A    10
                                                                                                                 4



                                                                                                                 3
                                                                                                                       Hu793 Control.006

                                                                                                                      0.33%                                  0.07%            10
                                                                                                                                                                                     4
                                                                                                                                                                                         Hu793 D1 HAV.001

                                                                                                                                                                                         0.30%                                     0.05%
                                                                                                                                                                                                                                                      10
                                                                                                                                                                                                                                                              4
                                                                                                                                                                                                                                                                   Hu793 D3 HAV.002

                                                                                                                                                                                                                                                                   2.57%                                     0.20%
                                                                                                                                                                                                                                                                                                                                                     0
                                                                                                                                                                                                                                                                                                                                                                                                             error (SEM) of three separate experiments. A) IFNβ response
                                                                                                                                                                                                                                                                                                                                                                                                             to infected cells B) DR4 response to infected cells C) DR5
                                                                                                                                                                                                                                                                                                                                                                                                             response to infected cells.
with chronic liver disease. HAV is transmitted through the fecal-oral route and absorbed by the             10
                                                                                                                                                                              10
                                                                                                                                                                                     3                                                                10
                                                                                                                                                                                                                                                              3



stomach or small intestine where it then travels to the liver to replicate and is later secreted in
                                                                                                       PI




                                                                                                                 2
                                                                                                                                                                                                                                                                                                                                                                Days post HAV infection




                                                                                                                                                                                                                                                PI
                                                                                                            10                                                                                                                                                2




                                                                                                                                                                       PI
                                                                                                                                                                                     2                                                                10
                                                                                                                                                                              10
the bile and excreted in stool. It is present in the feces for several weeks, corresponding to the               1                                                                                                                                            1
                                                                                                            10                                                                       1                                                                10
incubation period (15-50 days) and the beginning of illness. Fecal shedding is then followed by                                                                               10

hepatocellular injury.                                                                                      10
                                                                                                              10
                                                                                                                 0
                                                                                                                 0
                                                                                                                      99.48%

                                                                                                                               10
                                                                                                                                     1
                                                                                                                                           10
                                                                                                                               Annexin V-FITC
                                                                                                                                                2
                                                                                                                                                    10
                                                                                                                                                         3
                                                                                                                                                             0.12%

                                                                                                                                                              10
                                                                                                                                                                   4          10
                                                                                                                                                                                10
                                                                                                                                                                                   0
                                                                                                                                                                                     0   99.63%
                                                                                                                                                                                              10
                                                                                                                                                                                                   1
                                                                                                                                                                                                             10
                                                                                                                                                                                                                  2
                                                                                                                                                                                                                       10
                                                                                                                                                                                                                               3
                                                                                                                                                                                                                                   0.01%
                                                                                                                                                                                                                                     10
                                                                                                                                                                                                                                            4
                                                                                                                                                                                                                                                      10
                                                                                                                                                                                                                                                        10
                                                                                                                                                                                                                                                           0
                                                                                                                                                                                                                                                              0    94.11%
                                                                                                                                                                                                                                                                       10
                                                                                                                                                                                                                                                                               1
                                                                                                                                                                                                                                                                                    10
                                                                                                                                                                                                                                                                                            2
                                                                                                                                                                                                                                                                                                 10
                                                                                                                                                                                                                                                                                                         3
                                                                                                                                                                                                                                                                                                              3.13%
                                                                                                                                                                                                                                                                                                               10
                                                                                                                                                                                                                                                                                                                       4                                                          CONCLUSIONS
Due to the mechanism of transmission, HAV is most prevalent in underdeveloped countries                                                                                                       Annexin V-FITC                                                           Annexin V-FITC
                                                                                                                                                                                                                                                                                                                                                    Currently, little is known about the mechanisms of innate immune response to Hepatitis A viral
with poor sanitation, but is also present in industrialized countries among persons living in
                                                                                                                                                                                                                                                                                                                                                    infection. In the in vitro cell culture environment, HAV does not normally induce cytopathic
similar conditions as a result of poor socioeconomic status. Within the body, the virus can                           Hu793 D5 HAV.003                                               Hu793 HAV D7.015                                                         Hu793 HCV Day 9.005
                                                                                                            10
                                                                                                                 4
                                                                                                                                                                            10
                                                                                                                                                                                 4
                                                                                                                                                                                                                                                     10
                                                                                                                                                                                                                                                          4
                                                                                                                                                                                                                                                                                                                                                    effects. Presence of NK cells and cytotoxic T lymphocytes (CD8+), an immunopathologic
withstand a variety of seemingly stressful conditions such as extremely low pH. Outside the                           0.62%                                  0.23%                   5.52%                                     0.84%                              2.53%                                  0.24%
                                                                                                                 3                                                               3                                                                        3                                                                                         response to infection, has lead to the current accepted assumption that liver injury is a result of
body, HAV survives on hands and inanimate objects long enough to cause infection. This                      10                                                              10                                                                       10
                                                                                                                                                                                                                                                                                                                                                    the cells’ killing action rather than a direct effect of the virus. In our experiment, we have
explains why in the United States, HAV transmission is primarily associated with day care
                                                                                                       PI




                                                                                                                                                                PI




                                                                                                                                                                                                                                            PI
                                                                                                                 2                                                               2                                                                        2
                                                                                                            10                                                              10                                                                       10
                                                                                                                                                                                                                                                                                                                                                    demonstrated that HAV can have cytopathic effects in primary hepatocytes through the
centers. The highest rates of diagnosed HAV are in children; with age, personal hygiene
                                                                                                            10
                                                                                                                 1
                                                                                                                                                                            10
                                                                                                                                                                                 1
                                                                                                                                                                                                                                                     10
                                                                                                                                                                                                                                                          1
                                                                                                                                                                                                                                                                                                                                                    induction of IFNβ and the TRAIL pathway. Primary hepatocytes were treated with infectious
improves and acquired immunity (anti-HAV) increases, so the virus is less common among
                                                                                                                 0    98.43%                                 0.72%               0   93.10%                                    0.54%                      0       97.22%                                     0.01%                                  HAV in a cell culture environment; their immune response was monitored for 11 days post
healthy adults. According to data produced by the Viral Hepatitis Surveillance Program, adults              10
                                                                                                                 0                   1          2        3         4
                                                                                                                                                                            10
                                                                                                                                                                                 0             1             2             3            4
                                                                                                                                                                                                                                                     10
                                                                                                                                                                                                                                                          0                1            2            3             4
                                                                                                              10               10          10
                                                                                                                               Annexin V-FITC
                                                                                                                                                    10        10              10          10
                                                                                                                                                                                          Annexin V-FITC
                                                                                                                                                                                                        10            10           10                  10             10           10
                                                                                                                                                                                                                                                                      Annexin V-FITC
                                                                                                                                                                                                                                                                                                10            10
                                                                                                                                                                                                                                                                                                                                                    infection. RNA was extracted from the infected cells as well as the HAV negative control bi-
diagnosed with HAV in the US likely acquired the virus through direct contact with an infected
                                                                                                                                                                                                                                                                                                                                                    daily for 11 days. The presence of HAV was most evident on days 1, 9, and 11, post-infection.
person; other possible sources of infection include international travel, contaminated needles
                                                                                                                 Percentage of Annexin
                                                                                                                 V positive cells out of




                                                                                                                                           3.5                                                                                     Figure 2: Flow cytometry confirms apoptosis                                                                      This increase in concentration of the infectious agent is consistent with evidence of
used with injected drugs, homosexual activity, and a food/waterborne outbreak. HAV infection
is easily prevented by immunization with formalin inactivated hepatitis A vaccines such as             B                                     3                                                                                     by an increase of ANNEXIN V after HAV
                                                                                                                                                                                                                                   LSH/S infection. One million hepatocytes
                                                                                                                                                                                                                                                                                                                                                    intracellular viral replication and induction of apoptosis of the host cells. Viral replication was
                                                                                                                     30000 events




                                                                                                                                           2.5                                                                                     infected with HAV LSH/S were collected every                                                                     tested by HAV PCR and gel electrophoresis. Our RT-PCR results suggest intracellular
VAQTA and HAVRIX.
                                                                                                                                             2                                                                                     two days and stained for ANNEXIN V and                                                                           replication of HAV in primary hepatocyte host cells. Results at 589bp, corresponding to
Little is known about the mechanisms of innate immune response to HAV. In the in vitro cell                                                                                                                                        Propidium Iodide (PI). Cells were then analyzed
                                                                                                                                           1.5                                                                                                                                                                                                      hepatitis A virus, show high concentrations of HAV present on day 2 (directly after infection),
culture environment, HAV does not normally induce cytopathic effects. Presence of NK cells                                                                                                                                         by flow cytometry on a Facscalibur flow
                                                                                                                                             1                                                                                                                                                                                                      as well as days 10 and 12, suggesting an intracellular viral replication mechanism. Light
and cytotoxic T lymphocytes (CD8+), an immunopathologic response to infection, leads to the                                                                                                                                        cytometer. A) Plots show the analysis of each
                                                                                                                                           0.5                                                                                                                                                                                                      microscopy pictures show a decrease over time in cell confluence after HAV infection,
current accepted assumption that liver injury is a result of the cells’ killing action rather than                                                                                                                                 time point collected. Percentage values of the
                                                                                                                                             0                                                                                     amount of cells per quadrant is shown. B) Plot                                                                   suggesting the possibility of programmed cell death, or apoptosis. This was confirmed by our
direct effect of the virus. In our experiment, we demonstrate that HAV can have cytopathic                                                                                                                                         representing the percentage of ANNEXIN V
                                                                                                                                                         0         2         4            6            8          10                                                                                                                                flow cytometry data: there is an evident increase of ANNEXIN V after HAV LSH/S infection.
effects in primary hepatocytes through the induction of IFNβ and the TRAIL pathway.                                                                                                                                                positive cells in Huh 7.5 cells showing an
                                                                                                                                                                                                                                                                                                                                                    RNA recovery data correlates with HAV infection, also suggesting increased cell death. Real-
                                                                                                                                                                                                                                   increase when RIG-I is expressed in these cells.
                                                                                                                                                             Days post infection                                                                                                                                                                    Time PCR was used to test for expression of DR4 and DR5 receptors, INFβ production in the
           EXPERIMENTAL APPROACH                                                                                                                                                                                                                                                                                                                    host cells, and GAPDH, a housekeeping gene. The relative expression of INFβ, DR4, and
                                                                                                                                                                                                                                                                                                                                                    DR5 attest to the proposed immune response. Our data show that HAV undergoes
                                                                                                                                                                                               Recovery of RNA




                                                                                                                                                                                                                                                                                                                                                    intracellular viral replication and has cytopathic effects in human primary hepatocytes; HAV
                                                            Test for viral replication by HAV                                                                                                                         70
                                                           PCR and gel electrophoresis. Use                                                                                                                                                                                                                                                         induces Type I IFN and TRAIL expression as well as an upregulation of the DR4 and DR5
                                                                                                                                           Figure 3. RNA recovery                                                     60                                                                                                                            receptors.
                                                           GAPDH as house keeping gene.                                                    correlates       with     HAV                                              50
                                                                                                                                                                                                    (ug)




                                  Extract RNA                                                                                              infection also suggesting
 Investigate response of                                                                                                                                                                                              40
  primary hepatocytes to
 hepatitis A viral infection
                                 from infected
                                    cells and               Test for expression of DR4 and
                                                                                                                                           increased cell death. RNA
                                                                                                                                           was extracted bi-daily with
                                                                                                                                                                                                                      30                                                                                                                                                       Acknowledgements
                                     HAV -                 DR5 receptors by Real-Time PCR.                                                 trizol and purified for further                                            20
   using human primary            control cells                                                                                                                                                                       10
  hepatocytes and HAV
                                                                                                                                           experimental use via RT-                                                                                                                                                                             We would like to express our gratitude to members of the Liu, Nelson, and Brantley
                                   bi-daily 11                                                                                             PCR.                                                                        0                                                                                                                        laboratories for their continued support. We would also like to acknowledge the Center for
   LSH/S (ATCC # VR –                 days.
2266). Culture cells for 12                                 Test for INF β production in host                                                                                                                                       0           2    4            6    8 10 12                                                                  Mammalian Genetics and Dr. Terada’s laboratory for allowing us the use of their equipment
 days (experiment run in                                      cells using Real-Time PCR.                                                                                                                                                                                                                                                        and Mr. Yingda Xi for his support with the Real Time PCR. This work was funded in part by
         triplicate)                                                                                                                                                                                                                                                                                                                        IH to CL.
                                                                                                                                                                                                                       Days Post HAV Infection                                                                                                  grants NIH-DK002958 and NIH-A1061158 from NIH to Dr. Chen Liu. Ms. Bayouth is a HHMI
                                 Check for cell death in                                                                                                                                                                                                                                                                                        Science for Life Undergraduate Research awardee. Ms. Eksioglu is an NIH Ruth Kirshchtein
                                   host cells via light         Check for apoptosis via
                                                                   flow cytometry.                                                                                                                                                                                                                                                              predoctoral fellowship recipient: 1F31AI071353-01.
                                     microscopy.

				
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