Impact of varicella-zoster virus on dendritic cell subsets in human skin during natural infection

Abstract

Varicella-zoster virus (VZV) causes varicella and herpes zoster, diseases characterized by distinct cutaneous rashes. Dendritic cells (DC) are essential for inducing antiviral immune responses; however, the contribution of DC subsets to immune control during natural cutaneous VZV infection has not been investigated. Immunostaining showed that compared to normal skin, the proportion of cells expressing DC-SIGN (a dermal DC marker) or DC-LAMP and CD83 (mature DC markers) were not significantly altered in infected skin. In contrast, the frequency of Langerhans cells was significantly decreased in VZV-infected skin, whereas there was an influx of plasmacytoid DC, a potent secretor of type I interferon (IFN). Langerhans cells and plasmacytoid DC in infected skin were closely associated with VZV antigen-positive cells, and some Langerhans cells and plasmacytoid DC were VZV antigen positive. To extend these in vivo observations, both plasmacytoid DC (PDC) isolated from human blood and Langerhans cells derived from MUTZ-3 cells were shown to be permissive to VZV infection. In VZV-infected PDC cultures, significant induction of alpha IFN (IFN-alpha) did not occur, indicating the VZV inhibits the capacity of PDC to induce expression of this host defense cytokine. This study defines changes in the response of DC which occur during cutaneous VZV infection and implicates infection of DC subtypes in VZV pathogenesis.

FIG. 1.

Frequency and distribution of DC subsets in VZV-infected skin. Immunohistochemical staining (purple) of sections of uninfected skin (A, C, and E) and VZV-infected skin (B, D, and F) for the Langerhans cell markers CD1a (A and B) and langerin (C and D) and for the plasmacytoid DC marker CD123 (E and F). Sections were lightly counterstained by hematoxylin. Immunofluorescent staining (red) for the plasmacytoid DC (PDC) marker BDCA-2 on uninfected skin (G) and VZV-infected skin (H) is shown. Sections were stained with DAPI to reveal cell nuclei (blue). Examples of positively stained cells are indicated by arrows, with BDCA-2-positive PDC shown in panels F and H being deeper within the dermis. Images were captured under magnification ×40, with scale bars representing 50 μm. I and J plot the mean frequency (± SEM) of cells stained positive for a variety of DC markers in the epidermis and dermis, respectively, of uninfected skin (3 donors; white bars), varicella skin (5 donors; light purple bars), and herpes zoster lesions (5 donors; dark purple bars).

FIG. 2.

Distribution of VZV antigen during natural cutaneous VZV infection. Immunohistochemical staining (purple) for VZV glycoprotein E (gE) in skin biopsy specimens of a varicella lesion (A) or uninfected skin (C) is shown. Infiltrating cells staining positive for VZV gE in the dermis of skin from the varicella skin biopsy specimen are boxed. A consecutive section from the varicella skin biopsy specimen stained with an isotype control antibody is shown in panel B. Images were captured under magnification ×10, with scale bars representing 200 μm. (D) Mean frequency (± SEM) of VZV gE-positive cells in skin from uninfected (3 donors), varicella (5 donors), and herpes zoster (5 donors) samples within the epidermis (light purple bars) or dermis (dark purple bars) is plotted.

FIG. 3.

Detection of VZV antigens and DC subsets in skin during natural cutaneous VZV infection. Dually immunofluorescently stained sections of varicella skin lesion (A and B, cases V2 and V1, respectively) and herpes zoster skin lesion (C and D, cases HZ2 and HZ1, respectively) for combinations of DC markers and VZV antigen are shown. Panels A and C show staining for the Langerhans cell marker langerin (red) in combination with staining for VZV (green) in the epidermis. Panels B and D show staining for the plasmacytoid DC marker BDCA-2 (red) in combination with staining for the VZV (green) in the dermis, with panel B showing an area closer to the lesion and panel D showing an area deeper within the dermis. Panels E and F show isotype control stained sections from varicella and herpes zoster skin samples, respectively. Panels G and H show staining of uninfected skin for langerin and VZV in the epidermis (G) and for BDCA-2 and VZV in the dermis (H). All sections were stained with DAPI to mark cell nuclei (blue). Images were captured under magnification ×40, with scale bars representing 50 μm. Examples of langerin and BDCA-2-positive cells are indicated by white arrows, and examples of VZV-positive cells are indicated by orange arrows. The location of a VZV lesion (L) is indicated when it was present within the field of view.

FIG. 4.

VZV antigens detected in LC and PDC during natural cutaneous VZV infection. Sections of varicella and herpes zoster skin lesions were dually immunofluorescently stained for combinations of DC markers and VZV antigens, showing the presence of dually positive cells. (A to C) Sections stained for the plasmacytoid DC marker BDCA-2 (red) in combination with staining for VZV antigens IE62 (A) (case HZ1), ORF4 (B) (case HZ3), or gE (C) (case HZ3) (green). (D to F) Sections stained for the Langerhans cell marker langerin (red) in combination with staining for VZV antigens IE62 (D) (case V2), ORF4 (E) (case V2), or ORF29 (F) (case V2) (green) are shown. (G to I) Staining of uninfected skin for langerin and IE62 (G), langerin and ORF29 (H), or BDCA-2 and gE (I) is shown. All sections were stained with DAPI to mark cell nuclei (blue). The main images were captured under magnification ×40, with scale bars representing 50 μm. Examples of dually positive cells are boxed and shown at higher magnification as insets for each staining combination.

FIG. 5.

PDC in vitro are permissive to VZV infection. (A) Dual immunofluorescent staining of plasmacytoid DC exposed to VZV for the plasmacytoid DC marker BDCA-2 (red) and VZV antigen IE62, ORF4, ORF29, or gE (green). Staining with isotype control antibodies and mock-infected plasmacytoid DC stained for BDCA-2 and VZV IE62 are also shown. Scale bars, 20 μm. (B) The percentage (± SEM) of BDCA-2⁺ cells costaining for each VZV antigen from 4 independent replicate experiments.

FIG. 6.

MUTZ-3-derived LC are permissive to VZV infection. (A) Dual immunofluorescent staining of MUTZ-3-derived LC exposed to VZV for the LC marker langerin (red) and VZV antigen IE62, ORF4, ORF29, or gE (green). Staining with isotype control antibodies and mock-infected MUTZ-3 LC stained for langerin and VZV IE62 are also shown. Scale bars, 20 μm. (B) The percentage (± SEM) of langerin⁺ cells costaining for each VZV antigen from 3 independent replicate experiments.

FIG. 7.

VZV infection of PDC in vitro impacts IFN-α synthesis. Measurement by ELISA of IFN-α secreted from plasmacytoid DC cultures of either mock-infected PDC (white bars) or VZV-infected PDC (gray bars) infected for 24 h or infected for 12 h and then treated with a 2.5 μM concentration of the TLR agonist ODN2216 (ODN) to induce IFN-α production is shown. Panels A and B show results from 2 independent replicate experiments. Error bars show the range of values from duplicate samples for each replicate.

FIG. 8.

IFN-α synthesis by uninfected PDC exposed to supernatant from VZV-infected PDC cultures. Measurement by ELISA of IFN-α secreted from uninfected PDC or from uninfected PDC treated with supernatant from VZV-infected PDC after treatment with (gray bars) or without (white bars) 2.5 μM ODN2216 is shown. Treatment of uninfected PDC with supernatant from three independent VZV-infected PDC cultures is shown. The graph shows the level of IFN-α produced by each culture (in pg/ml) along the y axis against each culture condition along the x axis.