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Hepatitis C Virus Improves Human Tregs Suppressive Function and Promotes Their Recruitment to the Liver

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Hepatitis C Virus Improves Human Tregs Suppressive Function and Promotes Their Recruitment to the Liver

Laurissa Ouaguia et al. Cells.

Abstract

Background: The role of regulatory T cells (Tregs) is now well established in the progression of hepatocellular carcinoma (HCC) linked to Hepatitis C virus (HCV) infection. However, nothing is known about the potential interplay between Tregs and HCV. In this pilot study, we have investigated the ability of Tregs to hang HCV on and the subsequent effect on their suppressive function and phenotype. Moreover, we have evaluated how HCV could promote the recruitment of Tregs by infected primary human hepatocytes.

Methods: Tregs of healthy donors were incubated with JFH-1/HCVcc. Viral inoculation was assessed using adapted assays (RT-qPCR, Flow Citometry (FACS) and Western Blot (WB). Expression of Tregs phenotypic (CD4, CD25, CD127 and Foxp3) and functional (IL-10, GZMB, TGF-β1 and IL-2) markers was monitored by RT-qPCR, FACS and ELISA. Suppressive activity was validated by suppressive assays. Tregs recruitment by infected primary hepatic cells was evaluated using Boyden Chamber.

Results: Tregs express the classical HCV receptors (CD81, CLDN1 and LDLR) and some co-receptors (CD5). HCV inoculation significantly increases the suppressive phenotype and activity of Tregs, and raises their anergy by inducing an unexpected IL-2 production. Moreover, HCV infection induces the expression of chemokines (CCL17, CXCL16, and CCL20) by primary hepatic human hepatocytes and chemokine receptors (CCR4, CXCR6 and CCR6) by Tregs. Finally, infected hepatocytes have a significantly higher potential to recruit Tregs in a seemingly CCL20-dependent manner.

Conclusions: Direct interaction between HCV and Tregs represents a newly defined mechanism that could potentiate HCV immune evasion and favor intratumoral recruitment contributing to HCC progression.

Keywords: HCV; HCV/JFH-1; chemokines; immune escape; regulatory T cells.

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Tregs possess the classical hepatitis C virus (HCV) entry receptors: CD81, CLDN1 and LDLR. Gene expression analyses (qPCR) of receptors CD81 (A), CLDN1 (B), SCARB1 (C) and LDLR (D), associated with HCV entry in host cells (left panels). Results are expressed as relative expression and presented as means of four independent experiments ± standard error of the mean (SEM) bars. Protein expression Flow Cytometry (FACS) of receptors related to HCV entry (middle panels). Results are expressed in histograms displaying the percentage of cells positive for protein labeling compared to isotype control. Protein expression (Western Blot) of receptors linked to HCV entry (right panels). Analyses were performed on Huh7 (control), PBMC and Tregs. The images are representative of at least four independent experiments.
Figure 2
Figure 2
HCV inoculation increases the expression of its receptors on Tregs. HCV inoculation increases the mRNA expression of CLDN1 and SCARB1 at 3 h post-inoculation (3 h p.i) (A) and CD81, EGFR and CD5 at 24 h p.i (B). Gene expression is normalized using GADPH, beta-actin, 18s and Hypoxanthine-guanine phosphoribosyltransferase (HRPT) mRNA as a housekeeping-gene before being reported to non-inoculated Tregs (black bars). Results represent means of five independent experiments and are presented as fold change (2–ΔΔCt) ± SEM bars. p0.05 (*), p ≤ 0.001 (**), p ≤ 0.0001 (***) and p ≤ 0.00001 (****).
Figure 3
Figure 3
HCV inoculation increases the suppressive phenotype of Tregs. HCV inoculation affects CD4, CD25, CD127, FOXP3, CTLA4 and LAG3 expression in activated Tregs at 3 h post HCV inoculation (3 h p.i) (A) and 24 h p.i (B). Results are presented as means of four independent experiments of inoculated Tregs (light grey bars) versus non-inoculated Tregs (dark bars). Gene expressions are normalized by using GADPH, beta-actin, 18s and HRPT mRNA as housekeeping-genes and result are expressed in fold change (2–ΔΔCt) ± SEM bars. (C) Representative dot plot of double stained CD4+CD25high and CD25high CD127-/low Tregs 0 h post HCV inoculation (0 h p.i), 3 h p.i and 24 h p.i.
Figure 4
Figure 4
HCV inoculation increases the in vitro proliferative capacity of Tregs. Tregs proliferation was measured by using [3H]-thymidine incorporation assay and cell counting assays at 24 h (A), 48 h (B) and 72 h (C) after HCV inoculation. Results are expressed in index of proliferation of inoculated Tregs (hatched column) compared to non-inoculated (light column). Results are presented as mean values of triplicate ± SEM bars of four independent experiments. qPCR analyses showed that HCV inoculation increases IL-2, IL-4, BLIMP1, BCL6 and IL-15 gene expressions in Tregs at 3 h p.i, (D) and 24 h p.i (E). Results are presented as means of four independent experiments in inoculated Tregs (light grey bars) versus non-inoculated Tregs (dark bars). Gene expressions are normalized by using GADPH, beta-actin, 18s and HRPT mRNA as housekeeping-genes and the results are expressed in fold change (2–ΔΔCt) ± SEM bars. Secretion of the proliferative cytokine IL-2 by Tregs after HCV inoculation (F). Results are expressed as mean of three independent experiments and presented in pg/mL ± SEM bars comparing secretion by inoculated Tregs (light grey bars) versus non-inoculated Tregs (dark bars). p ≤ 0.05 (*), p ≤ 0.001 (**).
Figure 5
Figure 5
HCV inoculation increases the suppressive activity of Tregs. Tregs were pre-incubated for 24 h with HCV virions produced in cell culture (HCVcc) before co-cultured with autologous PBMC at 2:1 ratio in activated conditions (A). Results are expressed as mean values of triplicates of three independent experiments and presented in index of suppression ± SEM bars. Mechanistic analyses highlight an increase expression of IL-2RA, IL-10, IL-24, IL-12p35, EBI3, GZMB and TGF-β1 mRNA in inoculated Tregs at 3 h p.i (B) and 24 h p.i (C). Results are presented as means of four independent experiments in inoculated Tregs (light grey bars) versus non inoculated Tregs (dark bars). Gene expressions are normalized by using GADPH, beta-actin, 18s and HRPT mRNA as housekeeping-genes and the results are expressed in fold change (2–ΔΔCt) ± SEM bars. Secretion of immunosuppressive cytokine TGF-β1 (D) and IL-10 (E) was evaluated at different time point after HCV inoculation on three independent experiments. Results are expressed in pg/mL ± SEM bars comparing secretion by inoculated Tregs (light grey bars) versus non-inoculated Tregs (dark bars). p ≤ 0.05 (*).
Figure 6
Figure 6
Tregs are recruited by infected primary human hepatocytes supernatants. Tregs recruitment by primary human hepatocytes (PHH) was evaluated by a Boyden chamber assay. In vitro infected PHHs recruit more natural Tregs than non-infected PHHs (A). Results are expressed as mean values of quintuplicate of six independent experiments and presented in index of migration of Tregs ± SEM bars. qPCR analyses show an increase of the expression of several chemokines associated with Treg recruitment such as CCL20, CCL17 or CXCL16 by PHH (B). Results are presented as means of five independent experiments on infected PHH (light grey bars) versus non-infected PHH (dark bars). HCV inoculation also increases the expression of corresponding chemokines receptors by isolated Tregs at 3 h p.i and 24 h p.i (C). All gene expressions are normalized by using GADPH, beta-actin, 18s and HRPT mRNA as housekeeping-genes and the results are expressed in fold change (2–ΔΔCt) ± SEM bars of five independent experiments. Secretion of three chemokines CCL20, CCL17 and CXCL16 has been examined by ELISA (DF, left panels). Results are expressed as the mean of four independent experiments and presented in pg/mL ± SEM bars comparing secretion by infected PHH (light grey bars) versus non-infected PHH (dark bars). The chemotactic potential of PHHs on CD4+CD25highCD127−/low Tregs was investigated in a Boyden chamber assay, using DMEM medium as a negative control; h-rec-CCL20, h-rec-CCL17 and h-rec-CXCL16 as positive controls and PHH supernatant with specific blocking anti-chemokines (DF, right panels). Results are presented as means of quintuplicate of four independent experiments and presented in the index of migration related to DMEM medium ± SEM bars. When stated, statistical analysis were achieved by comparing the pointed condition to either infected PHH in the presence of non-inoculated (NI; $) or inoculated (I) Tregs (*), 50 ng of human recombinant (h) CLL20 with NI (+) or I (#) Tregs, h CCL17 in the presence of NI (•) or I (◦) Treg. p ≤ 0.05 (*), p ≤ 0.001 (**), p ≤ 0.0001 (***) and p ≤ 0.00001 (****).
Figure 6
Figure 6
Tregs are recruited by infected primary human hepatocytes supernatants. Tregs recruitment by primary human hepatocytes (PHH) was evaluated by a Boyden chamber assay. In vitro infected PHHs recruit more natural Tregs than non-infected PHHs (A). Results are expressed as mean values of quintuplicate of six independent experiments and presented in index of migration of Tregs ± SEM bars. qPCR analyses show an increase of the expression of several chemokines associated with Treg recruitment such as CCL20, CCL17 or CXCL16 by PHH (B). Results are presented as means of five independent experiments on infected PHH (light grey bars) versus non-infected PHH (dark bars). HCV inoculation also increases the expression of corresponding chemokines receptors by isolated Tregs at 3 h p.i and 24 h p.i (C). All gene expressions are normalized by using GADPH, beta-actin, 18s and HRPT mRNA as housekeeping-genes and the results are expressed in fold change (2–ΔΔCt) ± SEM bars of five independent experiments. Secretion of three chemokines CCL20, CCL17 and CXCL16 has been examined by ELISA (DF, left panels). Results are expressed as the mean of four independent experiments and presented in pg/mL ± SEM bars comparing secretion by infected PHH (light grey bars) versus non-infected PHH (dark bars). The chemotactic potential of PHHs on CD4+CD25highCD127−/low Tregs was investigated in a Boyden chamber assay, using DMEM medium as a negative control; h-rec-CCL20, h-rec-CCL17 and h-rec-CXCL16 as positive controls and PHH supernatant with specific blocking anti-chemokines (DF, right panels). Results are presented as means of quintuplicate of four independent experiments and presented in the index of migration related to DMEM medium ± SEM bars. When stated, statistical analysis were achieved by comparing the pointed condition to either infected PHH in the presence of non-inoculated (NI; $) or inoculated (I) Tregs (*), 50 ng of human recombinant (h) CLL20 with NI (+) or I (#) Tregs, h CCL17 in the presence of NI (•) or I (◦) Treg. p ≤ 0.05 (*), p ≤ 0.001 (**), p ≤ 0.0001 (***) and p ≤ 0.00001 (****).
Figure 7
Figure 7
Tregs cannot be recruited by primary human intra-hepatic fibroblasts even upon HCV inoculation. Tregs recruitment by in vitro inoculated primary human intra-hepatic fibroblast (IHF) was evaluated by a Boyden chamber assay (A). Results are expressed as mean values of quintuplicate of two independent experiments and presented in an index of migration of Tregs ± SEM bars. Secretion of chemokines CCL20, CCL17 and CXCL16 has been examined by ELISA (B). Results are expressed as the mean of two independent experiments and presented in pg/mL ± SEM bars comparing secretion by inoculated IHF (light grey bars) versus non-inoculated IHF (dark bars). p ≤ 0.05 (*).

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References

    1. Hadigan C., Kottilil S. Hepatitis C virus infection and coinfection with human immunodeficiency virus: Challenges and advancements in management. JAMA. 2011;306:294–301. doi: 10.1001/jama.2011.975. - DOI - PubMed
    1. Baumert T.F., Ito S., Wong D.T., Liang T.J. Hepatitis C virus structural proteins assemble into viruslike particles in insect cells. J. Virol. 1998;72:3827–3836. - PMC - PubMed
    1. Ding Q., von Schaewen M., Ploss A. The impact of hepatitis C virus entry on viral tropism. Cell Host Microbe. 2014;16:562–568. doi: 10.1016/j.chom.2014.10.009. - DOI - PMC - PubMed
    1. Lupberger J., Zeisel M.B., Xiao F., Thumann C., Fofana I., Zona L., Davis C., Mee C.J., Turek M., Gorke S., et al. EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy. Nat. Med. 2011;17:589–595. doi: 10.1038/nm.2341. - DOI - PMC - PubMed
    1. Sarhan M.A., Chen A.Y., Michalak T.I. Differential expression of candidate virus receptors in human T lymphocytes prone or resistant to infection with patient-derived hepatitis C virus. PLoS ONE. 2013;8:e62159 doi: 10.1371/journal.pone.0062159. - DOI - PMC - PubMed

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