A novel polarized cell culture model to study HCV infection

Transcription

1 A novel polarized cell culture model to study HCV infection Sandrine Belouzard Centre d Infection et d Immunité de Lille 15 ième réunion du réseau national hépatites Paris- 30 janvier 2015

2 HCV entry and hepatocyte polarity 2 26/07/2013

3 HepG2 and polarity Isolation of a HepG2 clone having the morphological characteristics of simple epithelium. 3 26/07/2013

4 HepG2-CD81 cells and polarity Kooistra M R H et al. J Cell Sci 2007;120:17-22 HepG2-CD81 cells ZO-1 staining 16 clones were isolated by limit dilution. 4 26/07/2013

5 Morphology of the isolated clones ZO-1 CD13 Clone 15 Clone /07/2013

6 Experimental conditions Cells are seeded on transwell. Transwell characteristics: Microporous membrane Pore size 3 µm Polyester (PET) Apical medium Basolateral medium Pore density pores/cm 2 When the cells are confluent, William s medium supplemented with 10% FBS and 1% DMSO is added to induce the polarization of the cells. (Day 0). To monitore the polarization properties of the clones : - immunofluorescence of tight junction and apical markers were performed - secretion of the human serum albumin (HSA) in each chamber (apical and basolateral) was measured 6 26/07/2013

7 Immunostaining of apical, basolateral and tight junctions markers 7 26/07/2013

8 Clone 12 Top Bottom CD13 Apical marker TJ ZO-1 Tight junction Nuclei 8 26/07/2013

9 Clone 12 Top Bottom CD13 Apical marker TJ ZO-1 Tight junction Nuclei 9 26/07/2013

10 Clone 12 Top Bottom CD13 Apical marker TJ ZO-1 Tight junction Nuclei 10 26/07/2013

11 Clone 1NV CD13 Apical marker ZO-1 Tight junction overlay 11

12 Clone 15 Top Bottom CD13 Apical marker TJ ZO-1 Tight junction TsfR Basolateral marker Nuclei 12

13 Clone 15 CD13 Apical marker CLDN1 Tight junction Nuclei 13

14 Clone 15 CD13 Apical marker ZO-1 Tight junction OCDN-GFP Nuclei 14

15 Secretion of human serum albumin 15

16 Vectorial secretion of human serum albumin Clone 15 and 1SC3 secreted up to 90% of HSA in the basolateral medium. 16

17 ApoE secretion At day 6 and 9, the clones 15 and 1SC3 secreted more than 80% of ApoE at the basolateral domain. 17

18 Based on the localization of apical and tight junction markers and the secretion of HSA, we selected two clones : clone 1SC3 and clone 15 to study HCV infection. We also determined that in these clones the maximum of polarization is achieved between day 6 and 9 after adding DMSO 18

19 Based on the localization of apical and tight junction markers and the secretion of HSA, we selected two clones : clone 1SC3 and clone 15 to study HCV infection. We also determined that in these clones the maximum of polarization is achieved between day 6 and 9 after adding DMSO Does infection occur at the apical and basolateral domain? 19

20 Based on the localization of apical and tight junction markers and the secretion of HSA, we selected two clones : clone 1SC3 and clone 15 to study HCV infection. We also determined that in these clones the maximum of polarization is achieved between day 6 and 9 after adding DMSO Does infection occur at the apical and basolateral domain? Is the virus secreted at the apical or basolateral domain? 20

21 HCV infection in polarized cells Cells were seeded on transwell. When the cells reach confluency, William s medium supplemented with 10% FBS, 1% DMSO is added to induce the polarization of the cells. Cells were infected 6 days later at the apical or basolateral domain Apical infection Basolateral infection VIRUS VIRUS 30 h later, cells were lysed and RNAs were extracted. HCV genome copies were quantified by RT-qPCR. 21

22 HCV infection in polarized cells Apical infection Basolateral infection HCV infects polarized cells predominantly at the basolateral domain 22

23 HCV secretion in polarized cells. Basolateral infection VIRUS Polarized cells were infected at the basolateral domain Apical medium Basolateral medium 48h later, apical and basolateral supernatants were collected and virus titers were determined. 23

24 HCV secretion in polarized cells. HCV secretion occurs at the basolateral domain of the cells. 24

25 Conclusions / Perspectives We selected two clones with good polarization properties : clones 15 and 1SC3 - correct localization of apical, basolateral and tight junction proteins - basolateral secretion of HSA Using this model, we were able to confirm that HCV enters the cells at the basolateral domain and to show that HCV secretion is polarized. This model offer the opportunity to study the different steps of HCV infection in a more relevant model: - HCV entry: to better understand the role of the tight junction - propagation of the virus in the cell monolayer - mechanisms of HCV secretion 25

26 Acknowledgements HCV team Adeline Danneels Lucie Fénéant Karin Séron Yves Rouillé Jean Dubuisson