Hepatitis E Virus-induced antiviral response by plasmacytoid dendritic cells is modulated by the ORF2 protein

  1. Garima Joshi
  2. Elodie Décembre
  3. Jacques Brocard
  4. Claire Montpellier
  5. Martin Ferrié
  6. Omran Allatif
  7. Ann-Kathrin Mehnert
  8. Johann Pons
  9. Delphine Galiana
  10. Viet Loan Dao Thi
  11. Nolwenn Jouvenet
  12. Laurence Cocquerel
  13. Marlène Dreux

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Abstract

Type I and III interferons (IFN-I/III) are critical to protect the host during viral infection. Previous studies have shown that IFN-mediated antiviral responses against hepatitis E virus (HEV) are suppressed and defeated by viral escape mechanisms at play in infected hepatocytes. Here, we studied the anti-HEV function of IFN secreted by plasmacytoid dendritic cells (pDCs), which are specialized producers of IFNs. We showed that pDCs co-cultured with HEV-replicating cells secreted IFN in a cell-to-cell contact-dependent manner. Pharmacological inhibitor and antibodies targeting contact proteins revealed that pDC response against HEV required the endosomal nucleic-acid sensor TLR7 and adhesion molecules, such as ICAM-I and α L β 2 -integrin. IFNs secreted by pDCs reduced viral spread. Intriguingly, ORF2, the capsid protein of HEV, can be produced in various forms by the infected cells. During infection, a fraction of the intracellular ORF2 protein localizes into the nucleus while another ORF2 fraction packages viral genomes to produce infectious virions. In parallel, glycosylated forms of ORF2 are also massively secreted by infected cells. Using viral genome expressing ORF2 mutants, we showed that glycosylated ORF2 forms contribute to better recognition of infected cells by pDCs via regulation of contacts between infected cells and pDCs. ORF2 forms may thus modulate pDC-mediated anti-HEV response. Together, our results suggest that liver-resident pDCs, which exhibit comparable IFN-producing ability as blood-derived pDCs, may be essential to control HEV replication.

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    Reply to the reviewers

    point-by-point response is included in the submitted figure; named: Joshi et al response to Reviewer

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    Referee #2

    Evidence, reproducibility and clarity

    Joshi et al. discovered that human pDC isolated from blood of healthy donors incubated in coculture with hepatitis E virus (HEV) infected hepatoma cell lines were triggered in a cell-cell contact-dependent manner to mount interferon-alpha responses. This conclusion was supported by experiments in a Transwell setting in which pDC were separated from infected cells by a permeable membrane and under which conditions pDC were not triggered to mount interferon-alpha responses. Treatment of the coculture system with an TLR7 inhibitor or with antibodies against the cell adhesion receptors ICAM-I and alphaLbeta2-integrin either entirely inhibited or reduced the induction of interferon-alpha responses, respectively. HepG2/C3A cells infected with different ORF2 mutants induced overall similar interferon-alpha responses in pDC. In contrast, PLC3 cells infected with an ORF2 variant that is not properly translocated into the nucleus did not induce interferon-alpha responses in pDC, whereas the other variants induced normal responses. This observation indicated that in the context of PLC3 cells the subcellular localization of ORF2 in the nucleus is critical to induce interferon-alpha responses in pDC. Finally, quantification of contact points between pDC and infected cells supported the conclusion that enhanced cell-cell contact was necessary to efficiently induce interferon-alpha responses in pDC.

    Overall, the study was carefully carried out and shows interesting results. It is appreciated that responses of human pDC isolated from blood of healthy donors were analyzed. Furthermore, the stimulation of pDC with infected hepatoma cell lines is interesting. Earlier studies showed that infected cells might be better pDC stimulators than free virus. Furthermore, in line 140 of the manuscript the authors correctly state that pDCs are resistant to virtually all viruses. Thus, they should have included experiments in which they stimulate pDC with free HEV. Comparative analysis of the data presumably would further highlight the relevance of pDC triggering by infected cells.

    Despite interesting observations are presented regarding differences in the induction of pDC responses with PLC3 and HepG2/C3A cells infected with an ORF2 variant that is not properly translocated into the nucleus, no experiments were offered to explain this phenomenon. It is highly recommended to include additional experiments that support concepts either in one or the other direction as presented in the discussion.

    The paper is written in an unnecessarily complicated way. The authors should try to arrange the manuscript in a manner that the readability is enhanced. In the end, the observations that have to be communicated are not very complicated. Showing only controls in Fig. 1 is a cumbersome start in the manuscript. The authors should consider moving such results in a supplementary Figure. The authors do not thoroughly describe the experiment in Fig. 2I in the results section, although it seems to be rather interesting. The authors should give more explanations in the results section how they quantified cell-to-cell HEV infection in presence or absence of pDC.

    Minor comments

    In the legend of Fig. 1D and E it should be clarified what "Cont cells" and "HEV cells" means. I assume it is the mock control and HEV infected cells. It should be mentioned in the figure caption with which multiplicity of infections the cells were treated.

    For reasons of consistency, in Fig. 2 controls should be identified also by "Cont cells" and not by "cont cell" (in A and B) and "cont cells" (in C and D).

    In Fig. 2H, above "Fold-change in ORF2+ cells" there is indicated PLC3 in small letters. Should this be better moved above the panel, as done in F and G?

    The schematic depiction of different cell types in Fig. 2H and J is not very helpful, if not explained in the figure caption.

    In Fig. 3B the labels shown on the left side of A should be repeated.

    Why above the second row in A and B it is indicated "ORF2-AF488", but everywhere else in the manuscript it is referred to ORF2. If the AF488 addition is necessary (presumably it refers to an Alexa Fluor 488 conjugate of the antibody used to detect ORF2), this should be introduced either in the Materials and Methods section or in the figure caption.

    Significance

    The study is of high relevance. The presented phenomena are clearly described. Mechanistic aspects are not fully covered, yet. This study will be interesting for a relatively broad audience of virologists and immunologists. Currently, the manuscript is written in a manner that it is relatively difficult to read. This should be improved to reach the relatively divergent audience.

  3. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Plasmacytoid dendritic cells (pDCs) are the major producers of type I interferon after viral infections and play key role in antiviral immune response. This article by Joshi et al. investigates the role of pDCs in regulating the Hepatitis E virus (HEV) infection. In Fig. 1, the authors investigated the immunocompetence of different cell lines and HepG2/C3A and PLC3 were chosen for further studies. By utilizing a combination of flow cytometry, RT-qPCR and other techniques, the authors showed in Fig. 2 that the cell-cell contacts between pDCs and HEV infected cells induce the pDCs to secrete interferon (IFN). This interaction is mediated by cell adhesion molecules and is dependent on TLR7 signaling. The authors then went on to show that the IFN produced by pDCs controlled the viral spread. Further, using several mutant forms of ORF2 protein and utilizing imaging, RT-qPCR and other techniques, in Fig. 3 and 4 the authors elucidated the importance of the glycosylation pattern, localization of different forms of HEV ORF2 protein, cell-cell contact in triggering the immune response. Overall, this study provided insights in the pDC mediated IFN response against HEV.

    Major comments:

    1. The authors report that in the PLC3 cells, STOP mutation significantly reduced IFN⍺ production (Fig. 3f), significantly reduced pDC contact with infected cells (Fig. 4c) and thus concluded that the ORF2g/c is involved in pDC-infected cell interaction and IFN⍺ production. However, in the HepG2/C3A cells, the STOP mutation does not decrease the IFN⍺ production (Fig. 3e). In the manuscript, one of the key conclusions is that the glycosylated form of ORF2 leads to better recognition of the infected cells by pDC. So, it is critical that the difference in the IFN⍺ production between these two cell lines with STOP mutation is addressed with further details.
    2. The authors show that the IFN⍺ response was reduced in 5R/5A mutant HepG2/C3A cells (Fig. 3e), whereas the IFN⍺ response was completely absent in 5R/5A mutant PLC3 cells (Fig. 3f). The authors suggested that the difference in IFN⍺ response may be due to lack of ORF2i in PLC3 and other cell specific regulation in HepG2/C3A. Further evidence for this differential regulation would strengthen the claim.
    3. In the PLC3-pDC co-culture experiment (Fig. 2b), there is already an induction of IFN-1 (Interferon Lambda 1) in the uninfected PLC3-pDC co-culture (right panel, Fig. 2b). An explanation for the IFN-1 (Interferon Lambda 1) expression in the uninfected state would be helpful.

    Additional comments:

    1. Authors checked the expression of two ISGs- MXA, ISG15 in Fig. 1a-c, 2a-b. Were the expressions of other ISGs, such as members of OAS family (OAS1, OAS2 etc.), IFITM family or any other ISGs checked? This may be helpful, since in the Fig. 2c there is IFN⍺ production in pDC-infected PLC3 co-culture, but the ISGs (MXA, ISG15) are not upregulated significantly in Fig. 2b.
    2. In the HepG2/C3A-pDC co-culture experiment (Fig. 2a), there is not much difference in IFN-1 (Interferon Lambda 1) level in the infected HepG2/C3A-pDC co-culture (right panel, Fig. 2a) in comparison to infected HepG2/C3A alone (left panel, Fig. 2b), and also this outcome is different from that in the PLC3 experiment (Fig. 2b). Further clarification would help to support the conclusion regarding the IFN-1 (Interferon Lambda 1) upregulation in HEV infected cells-pDC co-culture.
    3. The authors show that in the pDC-PLC3 co-culture system, IFN⍺ was induced at 18h (Fig. 2c-2e), but the viral replication was not decreased in PLC3 cells (Fig. 2g). But, the HepG2/C3A-pDC co-culture has reduced viral replication at 18h (Fig. 2f). An explanation for the difference in the observation in two different cell lines at the same timepoints would strengthen the antiviral role of pDCs on HEV infected cells.
    4. The authors quantified the fold change in HEV infected PLC3+ cells in Fig. 2h. Was it performed by flow cytometry? It would be helpful to mention it in the figure legend. Also, if the said quantitation was done by flow cytometry, performing similar assay with HEPG2/C3A cells at 48h would provide the readers a better idea about the antiviral response across the cell lines at
      comparable timepoints.

    Minor comments:

    1. Was it expected to observe the increased induction of IL6 (Fig. 1b) in HepG2/C3A cells (but not in other cell lines) after IFN- (Interferon Lambda) treatment?
    2. In Fig. 3e, for the WT cells, 4 datapoints are visible while in the legend it is mentioned n=5.
    3. Typo: IRS661 in line 263, 699, Figure 2e.
    4. Typo: 200l in line 579.
    5. Catalogue number for ELISA kit is missing (Line 584).
    6. It would be helpful if the color code for the imaging in Supplementary figure 2f is provided on the top of the images, as it is provided in other images.

    Significance

    This article by Joshi et al. provides insight about the role of pDCs in controlling the HEV infection. However, the importance of pDC-infected cell contact mediated IFN-I secretion in antiviral response has been previously shown by the authors' group (Assil et al., 2019, Cell Host & Microbe) and others as well (E.g., Yun et al., 2021, Sci. Immunol.). The involvement of integrin mediated cell adhesion and TLR signaling in mediating this response was also shown. Though this manuscript does not advance the field of pDC biology or virology significantly, it does provide better understanding of the pDC antiviral response in the landscape of HEV infection. Although, it is out of the scope of this manuscript, elucidation of the mechanistic regulation how ORF2g/c controls the pDC-infected cell contact would be of great interest and significance. Overall, this study could be of interest to a general audience, especially to the virologists and researchers working in pDC biology.

  4. Version published to 10.1101/2024.04.12.589258v2 on bioRxiv
  5. Version published to 10.1101/2024.04.12.589258v1 on bioRxiv