Human Type 2 Myeloid Dendritic Cells Produce Interferon-λ and Amplify Interferon-α in Response to Hepatitis C Virus Infection  Shuye Zhang, Karen Kodys, Kui Li, Gyongyi Szabo  Gastroenterology  Volume 144, Issue 2, Pages 414-425.e7 (February 2013) DOI: 10.1053/j.gastro.2012.10.034 Copyright © 2013 AGA Institute Terms and Conditions

Figure 1 All 3 types of IFNs (IFN-α, IFN-γ, and IFN-λs [IL-28 and IL-29]) are produced from co-cultures of human PBMCs and HCV-infected cells. (A) Huh7.5 cells or HCVcc/Huh7.5 cells were co-cultured with PBMCs for 24 hours. IFNs in the supernatants were measured by enzyme-linked immunosorbent assay (mean ± SD; n = 6). (B) HCVcc/Huh7.5 cells with different infection percentages (NS3 expression level) were co-cultured with human PBMCs for 24 hours. IFN levels were measured by enzyme-linked immunosorbent assay. Mean ± SD is shown, and every dot represents one data point. (C) Human PBMCs and hepatoma cells were collected before co-culture as 0 hour control, or 4, 8 and 16 hours later. Total RNA was extracted from co-cultured cells, and IFN or MxA (MX1) expression were examined by real-time polymerase chain reaction, using glyceraldehyde-3-phosphate dehydrogenase as internal control (mean ± SD; n = 3). (D) Huh7.5 cells or HCVcc/Huh7.5 cells were co-cultured with PBMCs. Supernatants were collected 4, 8, 16, and 24 hours later and IFNs or IP-10 levels determined by enzyme-linked immunosorbent assay (mean ± SD; n = 3–6). Gastroenterology 2013 144, 414-425.e7DOI: (10.1053/j.gastro.2012.10.034) Copyright © 2013 AGA Institute Terms and Conditions

Figure 2 IFN-α is produced by pDC, IFN-γ is produced by NK/NKT, and IFN-λ is produced by mDC2. (A) HCVcc/Huh7.5 cells were co-cultured with human PBMCs, purified pDCs, PBMCs depleted of pDCs (PBMC-pDC), PBMCs depleted of NK/NKT (PBMC-CD56), or purified NK/NKT (CD56+) cells. IFNs in the supernatants were measured by enzyme-linked immunosorbent assay after 24 hours (mean ± SD; n = 3–6; *P < .05). (B) HCVcc/Huh7.5 cells were co-cultured with PBMCs, purified CD1c+ mDC1, monocyte derived DCs (MoDC), purified monocytes (CD14+ monocytes), CD16+ monocytes, platelet-associated cells (CD61+), PBMCs depleted of granulocytes (PBMC-CD15), PBMCs depleted of monocytes (PBMC-CD14), PBMCs depleted of pDC (PBMC-pDC), PBMCs depleted of platelet-associated cells (PBMC-CD61), or PBMCs depleted of lymphocytes (PBMC-[T, B, NK, pDC]). Co-cultured cells were preserved before co-culture as 0 hour control or collected after 4 hours. Total RNA was extracted from the cells and IL-28 or IL-29 expression was examined by real-time polymerase chain reaction (mean ± SD; n = 2). (C) HCVcc/Huh7.5 cells were co-cultured with PBMCs, PBMCs depleted of mDC2 cells (PBMC-mDC2), or purified mDC2s. IL-28 and IL-29 expression from co-cultures was examined by real-time polymerase chain reaction (mean ± SD; n = 2). Gastroenterology 2013 144, 414-425.e7DOI: (10.1053/j.gastro.2012.10.034) Copyright © 2013 AGA Institute Terms and Conditions

Figure 3 mDC2s specifically express TLR3 and produce IFN-λs in response to HCV-dsRNA or poly I:C stimulation. (A and B) Human PBMCs, purified pDCs, or mDC2s were co-cultured with HCVcc/Huh7.5 cells, or stimulated with different TLR ligands (Invivogen, San Diego, CA): TLR3-Poly I:C (10 μg/mL), TLR7-Gardiquimod (1 μg/mL), TLR7/8-CL075 (2.5 μg/mL), and TLR9-CpG-A (2 μmol/L) for 24 hours. IFNs in the supernatants were measured by enzyme-linked immunosorbent assay (mean ± SD; n = 3). (C) mRNA expression of TLRs (TLR3, 7, 8, and 9) and RLRs (DEAD box polypeptide 58 [DDX58] and interferon induced with helicase C domain 1 [IFIH1]) was examined by real-time polymerase chain reaction from different immune subsets in PBMCs using glyceraldehyde-3-phosphate dehydrogenase as internal control. Relative mRNA expressions are shown (mean ± SD; n = 3). (D) Gel electrophoresis of in vitro synthesized HCV-ssRNA and -dsRNA derived from the indicated HCV genome region. M, 1-kb DNA ladder (New England Biolabs Inc, Ipswich, MA). (E and F) Isolated pDCs or mDC2s were stimulated by 10 μg/mL HCV-ssRNA or -dsRNA mixed with Lipofectamine 2000 (Invitrogen, Grand Island, NY) for 24 hours. IFN levels were measured by enzyme-linked immunosorbent assay (n = 3). (E) IFN-α production in pDCs, and (F) IL-28 and IL-29 production in mDC2s, are shown. Gastroenterology 2013 144, 414-425.e7DOI: (10.1053/j.gastro.2012.10.034) Copyright © 2013 AGA Institute Terms and Conditions

Figure 4 Functional dichotomy of pDCs and mDC2s in IFN production. (A) Purified pDC or mDC2 populations were co-cultured with HCVcc/Huh7.5 cells, and culture supernatants were collected at different time points for IFN-α and IL-29 measurement by enzyme-linked immunosorbent assay (mean ± SD; n = 2). (B) PBMC-IFN production per cell was artificially set as 1 unit, then the average IFN release from pDCs or mDC2s was calculated and compared (mean ± SD; n = 3). (C) Purified pDCs or mDC2s were co-cultured with HCVcc/Huh7.5 cells, or stimulated with CpG-A or Poly I:C, respectively. Twenty-four hours later, IL-28 or IL-29 in culture supernatants was measured by enzyme-linked immunosorbent assay (mean ± SD; n = 3). (D) JFH-1–infected PHHs were collected 48 hours after infection for co-culture with human pDCs or mDC2s. Co-culture supernatants were collected at different time points and IL-28 or IL-29 levels were measured by enzyme-linked immunosorbent assay. Representative data from one experiment is shown. Gastroenterology 2013 144, 414-425.e7DOI: (10.1053/j.gastro.2012.10.034) Copyright © 2013 AGA Institute Terms and Conditions

Figure 5 HCV-induced IFN-λ induction requires cell-to-cell contact and endosomal acidification. (A) HCVcc/Huh7.5 cells were co-cultured with human PBMCs or purified mDC2s in a normal plate or a plate with a Transwell insert. IL-29 in the supernatants was measured by enzyme-linked immunosorbent assay after 24 hours (mean ± SD; n = 3). (B) Huh7.5 cells and HCV replicon cells, subgenomic replicon (BB7) and full-length genomic replicon (FL) cells, were co-cultured with human PBMCs or purified mDC2s. IL-29 in culture supernatants was measured by enzyme-linked immunosorbent assay (mean ± SD; n = 2). (C) HCVcc/Huh7.5 cells were co-cultured with purified mDC2 in the absence or presence of dimethyl amiloride (DMA), cytochalasin D (CCD), or chlorpromazine (CLP). IL-29 production was measured by enzyme-linked immunosorbent assay (mean ± SD; n = 2). (D) Purified human mDC2s were co-cultured with HCVcc/Huh7.5 cells or stimulated with Poly I:C in the presence of chloroquine or bafilomycin as indicated. IL-29 production was measured by enzyme-linked immunosorbent assay after 24 hours (mean ± SD; n = 2). Gastroenterology 2013 144, 414-425.e7DOI: (10.1053/j.gastro.2012.10.034) Copyright © 2013 AGA Institute Terms and Conditions

Figure 6 Cross-regulations exist between IFN induction in co-cultures. (A) HCVcc/Huh7.5 cells were co-cultured with human PBMCs in the absence or presence of recombinant IL-29 (25 ng/mL). IFN-α and IFN-γ levels were measured by enzyme-linked immunosorbent assay (mean ± SD; n = 3). (B) Human PBMCs or PBMCs depleted of mDC2s (PBMC-mDC2) were co-cultured with HCVcc/Huh7.5 cells or stimulated with CpG-A. IFN-α production in the supernatants was measured by enzyme-linked immunosorbent assay (mean ± SD; n = 3–4). (C) Purified human pDCs were co-cultured with HCVcc/Huh7.5 cells or stimulated with CpG-A in the absence or presence of IL-29. IFN-α production was measured by enzyme-linked immunosorbent assay (mean ± SD; n = 3). (D) Total RNA was extracted from different immune subsets in human PBMCs. IL-28RA expression was examined by real-time polymerase chain reaction and normalized to glyceraldehyde-3-phosphate dehydrogenase (mean ± SD; n = 3). (E) Human PBMCs, Huh7.5 cells, HCVcc/Huh7.5 cells, co-culture of PBMCs and Huh7.5 cells, or co-culture of PBMCs and HCVcc/Huh7.5 cells were preserved before culture as 0-hour control or collected after 4 hours culturing in the absence or presence of IFN-α-2a (100 U/mL). IL-28, IL-29, and MX1 induction were measured by real-time polymerase chain reaction; 1 representative experiment of 3 is shown. (F) Human PBMCs or purified mDC2s were co-cultured with HCVcc/Huh7.5 cells or stimulated with Poly I:C in the absence or presence of IFN-α-2a. IL-29 production in the supernatants was measured by enzyme-linked immunosorbent assay (mean ± SD; n = 3) (*P < .05). PBS, phosphate-buffered saline. Gastroenterology 2013 144, 414-425.e7DOI: (10.1053/j.gastro.2012.10.034) Copyright © 2013 AGA Institute Terms and Conditions

Figure 7 Genotype of rs12979860 does not correlate with IL-28 and IL-29 induction in human PBMCs by HCV-infected cells. (A) Human PBMCs were co-cultured with JFH-1–infected Huh7.5 cells or stimulated with PolyI:C (20 μg/mL) or CpG-A (2 μmol/L) for 24 hours. Co-culture supernatants were collected and IL-28 or IL-29 levels were determined by enzyme-linked immunosorbent assay. Each dot represents one individual. (B) Circulating mDC1, mDC2, and pDC frequencies from normal control (NC) or naive HCV patients (HCV) were determined by flow cytometry. (C) BDCA1 (CD1C), BDCA2 (CLEC4C), BDCD3 (THBD), and BDCA4 (NRP1) mRNA expression in human livers were determined by real-time polymerase chain reaction using glyceraldehyde-3-phosphate dehydrogenase as internal control. Gastroenterology 2013 144, 414-425.e7DOI: (10.1053/j.gastro.2012.10.034) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 1 Interferon-inducible cytokines were secreted from co-cultures of human PBMCs and HCV-infected cells. (A–E) HCVcc/Huh7.5 cells with different infection percentage (NS3-positive percentage was measured by flow cytometry) were co-cultured with human PBMCs. Cytokine production was measured by Bioplex. Each dot represents one data point. The induction of IL-1β, IL-10, IL-12, and tumor necrosis factor-α in the co-culture supernatants was minimal and less than 20 pg/mL (data not shown). Gastroenterology 2013 144, 414-425.e7DOI: (10.1053/j.gastro.2012.10.034) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 2 CXCL10, IL-29, and not inflammatory cytokine genes were induced in co-cultures between human PBMCs and HCV-infected cells. Human PBMCs were co-cultured with Huh7.5 cells or HCVcc/Huh7.5 cells. Co-cultured cells were preserved before culture as 0-hour control or collected at 8, 16, or 24 hours later. Gene induction was measured by real-time PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control (n = 3). Gastroenterology 2013 144, 414-425.e7DOI: (10.1053/j.gastro.2012.10.034) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 3 mDC2 and pDC respond to HCV-dsRNA and HCV-ssRNA, respectively, and increased expression of costimulatory molecules. mDC2s and pDCs were collected after HCV-ssRNA or HCV-dsRNA stimulation. Expression of CD80 and CD86 on DCs were determined by flow cytometry. Poly I:C and CpG-A were used as positive control for mDC2 and pDC stimulation, respectively. Gastroenterology 2013 144, 414-425.e7DOI: (10.1053/j.gastro.2012.10.034) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 4 PHHs produce IL-28 and IL-29 in response to JFH-1 HCVcc infection in vitro. PHHs were infected with concentrated JFH-1 virions or stimulated with PolyI:C (20 μg/mL), PolyI:C/Lyovec (1 μg/mL), or R848 (2 μg/mL). PHHs were maintained in hepatocyte maintenance medium (PMM; Lonza), and half of the medium was changed every 24 hours during the experiment. Culture supernatants were collected at different time points, and IL-28 or IL-29 levels were determined by ELISA. Representative data from one experiment is shown. Gastroenterology 2013 144, 414-425.e7DOI: (10.1053/j.gastro.2012.10.034) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 5 Concentrated JFH-1 HCV virions alone do not induce IL-29 production. (A) Human PBMCs were co-cultured with concentrated JFH-1 virions (HCVcc), and IL-29 induction was compared with control PBMCs after 4 hours by real-time PCR (mean ± SD; n = 3). (B) PBMCs or mDC2 were co-cultured with HCVcc/Huh7.5 cells or stimulated with concentrated HCVcc. IL-29 levels in culture supernatants were measured after 24 hours by ELISA (mean ± SD; n = 3). Gastroenterology 2013 144, 414-425.e7DOI: (10.1053/j.gastro.2012.10.034) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 6 The IL-29 gene is induced in co-cultures of human mDC2 and HCV replicon cells. Human PBMCs were co-cultured with HCV subgenomic or full-length replicon cells (BB7 and FL) and co-cultured cells were collected at the beginning and 4 hours later. IL-29 mRNA levels were quantified using real-time-PCR (mean ± SD; n = 2). Gastroenterology 2013 144, 414-425.e7DOI: (10.1053/j.gastro.2012.10.034) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 7 pDCs express the functional isoform of IL-28RA mRNA. Total cellular RNA was extracted from PBMC, pDC, Huh7, and Huh7.5 cells, and cDNA was synthesized. DNA segments spanning the spliced gap were amplified by PCR using cDNA as template to determine the alternatively spliced form of IL-28RA mRNA in different cell types. Gastroenterology 2013 144, 414-425.e7DOI: (10.1053/j.gastro.2012.10.034) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 8 PBMCs from chronic HCV-infected individuals produced a comparable amount of IL-29 in response to HCV-infected hepatoma cells with normal controls. IL-29 production by human PBMCs in response to JFH-1/Huh7.5 or Poly I:C (20 μg/mL). PBMC cells (4 × 106) from normal control (NC) and HCV-infected patients (HCV) were co-cultured with 2 × 105 JFH-1–infected Huh7.5 cells. Co-culture supernatants were collected after 24 hours, and IL-29 was measured by ELISA. No significant changes were observed between the NC and HCV groups (P > .05). Gastroenterology 2013 144, 414-425.e7DOI: (10.1053/j.gastro.2012.10.034) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 9 ISG mRNA expression was increased significantly in HCV-infected livers. ISG15, ISG56, MX1, and CXCL10 gene expression in livers were determined by a 2-step real-time PCR using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control. Gastroenterology 2013 144, 414-425.e7DOI: (10.1053/j.gastro.2012.10.034) Copyright © 2013 AGA Institute Terms and Conditions

Supplementary Figure 10 (A) Examination of HCV infection in JFH-1 infected Huh7.5 cells. Uninfected or infected Huh7.5 cells were collected at different time points after JFH-1 inoculation and intracellularly stained with anti-HCV-NS3 antibody. The HCV-NS3 expression representing the HCV-infection percentage was determined by flowcytometry. (B) Multicolor FACS-analysis strategy of human DC subsets in PBMCs. Total DC population was first gated out using Lin1 and HLA-DR, then mDC1, mDC2, and pDC were examined using CD1c, BDCA3, and CD123 as their specific markers as indicated. Gastroenterology 2013 144, 414-425.e7DOI: (10.1053/j.gastro.2012.10.034) Copyright © 2013 AGA Institute Terms and Conditions