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Journal of General Virology (2006), 87, 3177–3181 Short Communication DOI 10.1099/vir.0.82238-0 Maturation and function of human dendritic cells are inhibited by orf virus-encoded interleukin-10 Anna Chan, Margaret Baird, Andrew A. Mercer and Stephen B. Fleming Correspondence Stephen Fleming Department of Microbiology and Immunology, University of Otago, PO Box 56, Dunedin, New Zealand stephen.fleming@stonebow. otago.ac.nz Received 25 May 2006 Accepted 11 July 2006 Orf virus (ORFV) is a parapoxvirus that infects sheep, goats and man. In humans, the virus induces acute, pustular skin lesions that can develop into a progressive disease. Humans are susceptible to reinfection with ORFV and rare cases of persistent infection have been reported. ORFV encodes several immunomodulators, including a homologue of interleukin-10 (ORFV IL-10), that may explain these phenomena. The immunosuppressive effects of ORFV IL-10 on immature human dendritic cells (DCs) cultured from blood-derived monocytes (MoDCs) were investigated. MoDCs exposed simultaneously to lipopolysaccharide and ORFV IL-10 showed enhanced ovalbumin–FITC uptake and reduced IL-12 expression, indicating inhibition of maturation. Moreover, ORFV IL-10 inhibited the upregulation of DC cell-surface activation and maturation markers MHC II, CD80, CD83 and CD86 and inhibited the capacity of MoDCs to activate CD4+ T cells in an oxidative mitogenesis assay. These findings suggest that ORFV IL-10 may influence the development of acquired immunity in humans by impairing DC function. Orf virus (ORFV) is a parapoxvirus that infects sheep and goats, but is transmissible to man. In humans, the virus induces acute, pustular skin lesions that can develop into a progressive disease in the form of multiple lesions (Sanchez et al., 1985). In immune-impaired individuals, large, highly vascularized, tumour-like lesions of the skin have been reported (Savage & Black, 1972; Tan et al., 1991). ORFV infections can cause complications such as erythema multiforme reactions (Agger & Webster, 1983; Blakemore et al., 1948; Fastier, 1957; Mourtada et al., 2000) and severe forms of erythema multiforme have been reported (Erickson et al., 1975). In addition, ORFV can readily reinfect its hosts, in spite of apparently normal host antivirus immune and inflammatory responses (Haig & McInnes, 2002; Robinson & Lyttle, 1992). This phenomenon has raised questions about the mechanisms underlying this apparent escape from immunity. ORFV encodes a number of immunomodulators, which include a homologue of interleukin-10 (ORFV IL-10) (Fleming et al., 1997; Mercer et al., 2006). Mammalian IL10 is a multifunctional cytokine that is a potent suppressor of inflammation and cross-regulates a type 1 cell-mediated response (Moore et al., 2001). ORFV IL-10 is 80 % identical at the amino acid level to ovine IL-10, suggesting that the gene has been captured from sheep and has adapted to this A figure showing characterization of MoDCs and a table showing the effect of ORFV IL-10 on IL-12 production from MoDCs are available as supplementary material in JGV Online. 0008-2238 G 2006 SGM species. In comparison, ORFV IL-10 is 67 % identical at the amino acid level to human IL-10 (hIL-10), with differences in amino acids that are likely to be critical for interaction of ORFV IL-10 with the human IL-10 receptor (Fickenscher et al., 2002; Josephson et al., 2001). We have previously characterized ORFV IL-10 activities in ovine cells and murine cells and shown that it is functionally similar to mammalian IL-10 (Fleming et al., 1997, 2000; Haig et al., 2002; Imlach et al., 2002; Lateef et al., 2003). The acquired immune response is initiated by dendritic cells (DCs) that also regulate the quality and magnitude of the immune response. DCs are disrupted at multiple stages by IL-10 (De Smedt et al., 1997; Faulkner et al., 2000; Moore et al., 2001). IL-10 inhibits the activation and maturation of DCs, manifested as the inability to upregulate major histocompatibility complex (MHC) class II and costimulatory molecules such as CD80, CD83 and CD86, reduced IL12 production and reduced ability to activate and present antigen to T cells. Here, we report the immunosuppressive effects of ORFV IL-10 on human monocyte-derived DCs (MoDCs) in vitro. Human immature MoDCs were generated from peripheral blood mononuclear cells (PBMCs) from healthy donors (approved by the Otago University Ethics Committee). PBMCs were isolated as described previously (Copland et al., 2003). The adherent monocytes were cultured in DC medium containing 25 ng ml21 each of recombinant human granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-4 (R&D Systems). On day 2, a further Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 13:39:55 Printed in Great Britain 3177 A. Chan and others 2 ml DC medium was added to cells. After 5 days culture, non-adherent immature MoDCs were seeded at 66105 cells ml21 before use. MoDCs that were generated in vitro were analysed by using flow cytometry as described previously (Copland et al., 2003). MoDCs exhibited forward (FSC-H) and side (SSCH) scatter characteristics of DCs and the majority of the cells lay in the R1 gate (see Supplementary Fig. S1, available in JGV Online). MoDCs in the R1 gate were further analysed for expression of CD11c, a specific marker for MoDCs (Freudenthal & Steinman, 1990; Shortman & Liu, 2002), with allophycocyanin-conjugated anti-CD11c (S-HCL-3; IgG2b; BD Biosciences). MoDCs were gated on this marker for all subsequent analyses. Over a number of donors (more than eight), the proportion of cells in culture that were CD11c+ (MoDCs) was between 46 and 89 % and >95 % of cells in the R1 gate were CD11c+. The MoDCs generated had a typical immature phenotype, as shown by the low percentage of cells expressing the cell-surface activation markers CD80, CD83 and CD86, and almost all MoDCs expressed MHC II. The mouse anti-human mAbs used were: phycoerythrin-conjugated anti-HLA-DR (MHC II) (clone L243; isotype IgG2a; BD Biosciences), anti-CD80 (L307.4; IgG1k Pharmingen), anti-CD83 (HB15e; IgG1; Caltag) and anti-CD86 (BU63; IgG1; Serotech). As DCs mature, they lose their ability to acquire antigen. We examined whether ORFV IL-10 was able to inhibit MoDC maturation induced by lipopolysaccharide (LPS) by quantifying the uptake of the antigen fluorescein isothiocyanatelabelled ovalbumin (FITC–OVA) (Copland et al., 2003). Immature MoDCs were treated with hIL-10 (R&D Systems) or affinity-purified FLAG-tagged ORFV IL-10 (Imlach et al., 2002) and with or without LPS (Escherichia coli O26 : B6; Sigma) for 24 h. The optimal amount of ORFV IL-10 used in these assays (50 ng ml21) was determined in preliminary experiments (data not shown) and in addition by examining its ability to inhibit cytokine synthesis in LPS-activated THP1 cells (human monocytes) (L. M. Wise, C. A. McCaughan & S. B. Fleming, unpublished data). MoDCs were then incubated with 40 mg FITC–OVA ml21 (Lateef et al., 2003), labelled for CD11c and analysed by flow cytometry. CD11c+ MoDCs treated with ORFV IL-10 in the presence of LPS retained their ability to take up antigen and the levels determined were similar to those for immature MoDCs not exposed to LPS (Fig. 1). Statistical analysis (single-factor analysis of variance and Tukey’s test) using the combined data for the three donors showed a statistically significant difference for antigen uptake between cells treated with ORFV IL-10/LPS or hIL-10/LPS and cells exposed to LPS only (P<0?05). There was no significant difference between the effects of ORFV IL-10/LPS and hIL-10/LPS (P>0?05). Supernatants collected from the above cells, prior to flowcytometry analysis, were analysed for IL-12p70 by ELISA (Pharmingen). IL-12 was detected (305–631 pg ml21) where cells were activated with LPS and then exposed to FITC–OVA, but not where ORFV IL-10 or hIL-10 was added 3178 Fig. 1. Effect of ORFV IL-10 on antigen uptake by MoDCs. MoDCs were treated simultaneously with hIL-10 or ORFV IL10 (50 ng ml”1) in the presence or absence of LPS (10 ng ml”1) for 24 h. MoDCs were then incubated with FITC– OVA for 2 h and analysed by flow cytometry. MoDCs that were not exposed to hIL-10, ORFV IL-10, LPS or FITC–OVA are shown as MoDC. MoDCs that were only exposed to FITC– OVA are shown as FITC control. Antigen uptake is shown as median fluorescence intensity (MFI). Filled bars, donor R; shaded bars, donor V; empty bars, donor Y. to cells in addition to LPS/FITC–OVA (see Supplementary Table S1, available in JGV Online). As we were detecting IL12 from a heterogeneous population of cells, we could not be certain whether IL-12 was produced by CD11c+ cells or from other cell types. Nevertheless, it was apparent that both IL-10s were potent inhibitors of IL-12 production. We then examined whether ORFV IL-10 inhibited the maturation of human DCs in the presence of an activation signal by examining changes in cell-surface phenotypic markers that characterize mature DCs. MoDCs were cultured for 24 h with or without ORFV IL-10 in the presence of LPS, double-stained with antibodies against CD11c and MHC II, CD80, CD83 and CD86 and analysed by flow cytometry. Ten thousand cells were collected from each sample and analysed by using CellQuest Pro software (BD Biosciences). Addition of ORFV IL-10 during LPS activation appeared to inhibit the upregulation of MHC II and was most marked in cells from donor W, which showed a 4?8-fold reduction in MHC II expression in cells treated with LPS and ORFV IL10 compared with cells treated with LPS only (Fig. 2a). MoDCs derived from donor R showed a 2?2-fold reduction in MHC II expression, whilst MoDCs derived from donor T showed readily distinguishable, but lower, inhibition. There was little difference in the percentage of cells that stained CD11c+ MHC II+ for all treatments (data not shown). There was no significant difference (P>0?05) for the combined data from the three donors for CD11c+ MHC II expression for cells treated with LPS compared with cells treated with LPS/ORFV IL-10, which is due to the smaller differences seen in donor T. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 13:39:55 Journal of General Virology 87 ORFV IL-10 inhibits DC maturation and function over 90 % of cells treated with LPS expressed this marker. Where cells were treated simultaneously with LPS and ORFV IL-10, the proportion of cells that stained positive for CD80 varied from 37 % (donor W) to 88 % (donor R). The proportion of untreated immature MoDCs expressing the CD83 activation marker was <5 %, but increased to between 32 and 48 % on stimulation with LPS alone. With cells treated simultaneously with IL-10 and LPS, the proportion of cells expressing the activation marker CD83 was <17 %. The proportion of untreated immature cells expressing CD86 was also low (<8 %), but increased to 58–81 % when cells were activated with LPS alone. In contrast, with cells treated simultaneously with LPS and ORFV IL-10, the proportion of cells expressing CD86 was between 5 and 22 %. Statistical analysis for the combined data of the three donors showed that there was no significant difference in CD80 expression for cells treated with LPS compared with cells treated with ORFV IL-10/LPS or hIL10/LPS (P>0?05), whereas there was a significant difference for CD83 and CD86 expression (P<0?05). Overall, ORFV IL-10 and hIL-10 had similar inhibitory effects on MoDC phenotypic-marker expression. We then investigated the effects of ORFV IL-10 on the ability of MoDCs to activate T cells in a functional oxidative mitogenesis assay. This assay measures the capacity of MoDCs to induce CD4+ T-cell proliferation (Gunzer et al., 2000; Thoeni et al., 2005). CD4+ T cells were purified from PBMCs by using BD IMag anti-human CD4 particles-DM (clone L200) according to the manufacturer’s instructions (BD Biosciences) (final suspension, >84 % CD4+). CD4+ T cells (16107 ml21) were treated with 0?25 mg sodium periodate ml21 (Sigma), washed twice and seeded at 2?56105 per well. Pre-treated MoDCs were harvested, washed twice and 2?56104 pre-treated MoDCs were then added to CD4+ T cells and incubated for 24 h. The amount of [3H]thymidine incorporated was determined 20 h after addition to the culture by using a 1450 Microbeta liquid scintillation counter. Fig. 2. Effect of ORFV IL-10 on the phenotype of LPS-activated MoDCs. MoDCs were treated simultaneously with hIL-10 or ORFV IL-10 (50 ng ml”1) in the presence or absence of LPS (10 ng ml”1) for 24 h and analysed by flow cytometry. (a) Degree of MHC II expression (MFI). Filled bars, donor R; shaded bars, donor T; empty bars, donor W. (b) Percentage of CD11c+ MoDCs expressing CD80, CD83 and CD86. MoDC indicates cells not exposed to hIL-10, ORFV IL-10 or LPS (control). Empty bars, CD80; shaded bars, CD83; filled bars, CD86. The inhibitory effects of ORFV IL-10 were also seen in the numbers of cells expressing CD80, CD83 and CD86 (Fig. 2b). The proportion of untreated MoDCs expressing CD80 varied from 20 to 46 % over the three donors, whereas http://vir.sgmjournals.org In preliminary experiments, LPS-treated MoDCs were titrated against purified CD4+ T cells to obtain the optimal ratio for the oxidative mitogenesis assay. From the titrations of MoDCs, a ratio of 1 : 10 (MoDCs : CD4+ T cells) was used for subsequent assays (data not shown). The ability of ORFV IL-10 to modulate CD4+ T-cell activation by MoDCs indirectly was then examined. Our results showed that whereas LPS-matured MoDCs stimulated significantly higher proliferation than did untreated MoDCs (P<0?05, Student’s t-test), those MoDCs pretreated with ORFV IL-10 were unable to do this (P>0?05; Fig. 3). The CD4+ T-cell proliferation induced by these MoDCs was comparable to that induced by MoDCs treated with hIL-10 and LPS or to that induced by untreated MoDCs. Similar results were obtained by using autologous T cells or allogeneic T cells. These results suggest that ORFV IL-10-mediated inhibition of DC maturation impairs their ability to stimulate T cells. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 13:39:55 3179 A. Chan and others persistent infection in some normal individuals (Pether et al., 1986; Rogers et al., 1989) and East Fresian sheep (S. B. Fleming & A. A. Mercer, unpublished data) suggests that this is the case. The effects of ORFV IL-10 on DCs could affect both the innate responses and the acquired immune response in several ways. It could impair the production of IL-12 by DCs, which in turn will result in poor production of IFN-c by NK and NKT cells. It could lead to a delay in the development of acquired immunity, skew the immune response from an antiviral Th1 response towards a Th2 response (Chang et al., 2004) or lead to immunological tolerance (Lutz & Schuler, 2002) or anergy (Raftery et al., 2004). Fig. 3. Effect of ORFV IL-10 on the ability of MoDCs to activate CD4+ T cells. MoDCs (2?56104) treated with hIL-10 or ORFV IL-10 (50 ng ml”1), with and without LPS (10 ng ml”1) for 24 h, were co-cultured with 2?56105 CD4+ T cells for 24 h. Experiments were performed with autologous cells except where indicated. Assays were carried out in triplicate. Proliferation was measured by incorporation of [3H]thymidine during the last 20 h of culture. Results are expressed as mean c.p.m.±SD. Significant differences were relative to LPS control. *P<0?05. MoDC indicates cells not exposed to hIL-10, ORFV IL-10 or LPS (control). The results of our study showed unequivocally that ORFV IL-10 has inhibitory effects on the maturation of human MoDCs, despite some variation in responses that may reflect genetic differences, assay-to-assay variation or maturation status of cells at the time of isolation (Morel et al., 1997). In all assays performed, with the exception of MHC II expression and CD80 expression, statistically significant differences were demonstrated between donors. Surprisingly, differences in the polypeptide sequence between ORFV IL-10 and hIL-10 did not translate into functional differences for these cytokines and will be discussed in detail elsewhere. DCs represent a strategically important target for immune evasion by viruses that cause persistent infections, such as herpesviruses, which produce secreted versions of IL-10. It is unusual for viruses that cause acute infections to produce factors that target DCs specifically; however, the fact that ORFV reinfects sheep and humans and is able to establish 3180 We predict that ORFV IL-10 is suppressing inflammation early in infection, in conjunction with other anti-inflammatory viral factors, and thus delaying the recruitment of DCs to the site of infection. Whilst there is no evidence that Langerhans cells are involved in ORFV infection, other DC subsets, such as dermal or blood-derived DCs, could play important roles (Lear et al., 1996; Villadangos & Heath, 2005). Immature DCs that reach the site of infection and take up antigen could be prevented from maturing by the presence of ORFV IL-10. The additional consequence of this effect could be that it also serves to clear inflammatory stimuli from the site of inflammation (Grütz, 2005). In addition, there could be other mechanisms acting that destroy DCs. Cytomegalovirus IL-10 has been shown to increase apoptosis associated with DC maturation (Chang et al., 2004; Raftery et al., 2004). We have observed similar effects when human MoDCs were treated with ORFV IL-10 over an extended period (data not shown). In cases of reinfection, it is possible that the same mechanisms operate, as there is no evidence that Th1 memory is impaired in ovine species, as shown by a strong delayed hypersensitivity response to ORFV antigen (Buddle & Pulford, 1984). In conclusion, we have shown, by demonstrating the inhibitory effects of ORFV IL-10 on human DC maturation and its indirect effects on T-cell activation, that it has the potential to impair DCs over many stages of functionality. We propose that ORFV IL-10, in conjunction with other ORFV immunomodulators that have activity on human cells and signalling molecules, causes a delay in the development of cell-mediated immunity during primary exposure and reinfection. 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Immunity 15, 35–46. http://vir.sgmjournals.org aminobiopterin attenuates dendritic cell-induced T cell priming independently from inducible nitric oxide synthase. J Immunol 174, 7584–7591. Villadangos, J. A. & Heath, W. R. (2005). Life cycle, migration and antigen presenting functions of spleen and lymph node dendritic cells: limitations of the Langerhans cells paradigm. Semin Immunol 17, 262–272. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 16 Jun 2017 13:39:55 3181