Relay of Herpes Simplex Virus between Langerhans Cells and Dermal Dendritic Cells in Human Skin

<div><p>The mechanism by which immunity to Herpes Simplex Virus (HSV) is initiated is not completely defined. HSV initially infects mucosal epidermis prior to entering nerve endings. In mice, epidermal Langerhans cells (LCs) are the first dendritic cells (DCs) to encounter HSV, but it is CD103⁺ dermal DCs that carry viral antigen to lymph nodes for antigen presentation, suggesting DC cross-talk in skin. In this study, we compared topically HSV-1 infected human foreskin explants with biopsies of initial human genital herpes lesions to show LCs are initially infected then emigrate into the dermis. Here, LCs bearing markers of maturation and apoptosis formed large cell clusters with BDCA3⁺ dermal DCs (thought to be equivalent to murine CD103⁺ dermal DCs) and DC-SIGN⁺ DCs/macrophages. HSV-expressing LC fragments were observed inside the dermal DCs/macrophages and the BDCA3⁺ dermal DCs had up-regulated a damaged cell uptake receptor CLEC9A. No other infected epidermal cells interacted with dermal DCs. Correspondingly, LCs isolated from human skin and infected with HSV-1 <i>in vitro</i> also underwent apoptosis and were taken up by similarly isolated BDCA3⁺ dermal DCs and DC-SIGN⁺ cells. Thus, we conclude a viral antigen relay takes place where HSV infected LCs undergo apoptosis and are taken up by dermal DCs for subsequent antigen presentation. This provides a rationale for targeting these cells with mucosal or perhaps intradermal HSV immunization.</p></div

HSV-1 infected LCs in human skin.

<p>(A) LCs in the epidermis of mock or HSV-1 infected inner foreskin explants. The right panel quantifies density of total LCs in the epidermis of mock and HSV-1 infected explants at 24 hr p.i. in 20 representative fields per sample at 60x magnification. n = 3, mean ± SEM, *p<0.05. (B) ICP27 expression of the HSV-1 infected LC emigrated into the dermis of the inner foreskin explant. ICP27: immediate early protein of HSV, D: dermis. Representative result of 3 different donors is shown. (C) LCs in the primary penile herpetic lesion. E: epidermis, gD1: HSV-1 glycoprotein D. Representative result of 2 different donors. Scale bar indicates 20 μm. Maximum projections of Z-series are presented.</p

Fate of human LCs infected with HSV-1.

<p>Inner foreskin explants (A, B, C, D & E) or isolated skin LCs (F) were infected with v-UL37GFP for 24 hr (A, D, & E), 48 hr (B & C), or 18 hr (F). (A) LCs in the dermis of inner foreskin explants. Dotted line represents basement membrane. (B) Comparison of LCs migrating into the dermis in mock and infected inner foreskin explants expressed as % LCs per total no. of dermal cells. n = 6, mean ± SEM, *p<0.05. (C) Proportion of LCs in the dermis of inner foreskin explants expressing GFP, n = 3, mean ± SEM, ***p<0.001. (B) (C) LCs with or without GFP expression were quantified in 20 representative fields per sample at 60x magnification. (D) (E) Infected LCs in the dermis of inner foreskin explants were tested for the expression of a maturation marker CD80 (D) or an apoptosis marker caspase 3 (E). (F) Infected LCs isolated from abdominal skin were examined for the expression of caspase 3. Right-hand panels show quantification of each marker for (D), (E), (F) as in (B) and (C), n = 3, mean ± SEM, ***p<0.001. Maximum projections of Z-series are presented (D, E & F). E: epidermis, D: dermis. Scale bar indicates 15 μm.</p

Migration and/or interaction of HSV infected human LCs with dermal DCs.

<p>(A) Migration of LCs and dermal DCs out of skin after topical HSV application to inner foreskin explants. (B) Summary diagram of interaction between HSV infected LCs and dermal DCs in human skin.</p

CLEC9A expression by BDCA3⁺ dermal DCs in HSV-1 infected foreskin explants.

<p>Inner foreskin explants were infected with or without v-UL37GFP for 48 hr. (A) Representative image from 3 different donors is shown. D: dermis. Scale bar indicates 15 μm. (B) CLEC9A⁺BDCA3⁺ cells were quantified in 20 representative fields per sample at 60x magnification from 3 donors, mean ± SEM, p***<0.001.</p

Interaction of HSV-1 infected LCs with DC-SIGN⁺ dermal cells (dermal DCs/macrophages).

<p>(A) (B) (C) Foreskin explants, 48 hr p.i. (A) LCs and DC-SIGN⁺ dermal cells interacted in clusters. (B) Proportion of clusters GFP⁺LC/DC-SIGN⁺ dermal cells containing >10 cells, n = 3, mean ± SEM, p***>0.001. (C) DC-SIGN⁺ dermal cells with or without GFP expression were quantified in 20 representative fields per sample at 60x magnification from 3 separate samples. Mean ± SEM, p***>0.001 (D) Primary penile herpetic lesion, blue: DAPI, orange: langerin, red: DC-SIGN, green: gD1. E: epidermis, D: dermis. The dotted line represents the basement membrane. Maximum projections of Z-series are presented (A & D). Scale bar in (A) indicates 15 μm and scale bars in (D) indicate 50 μm (white) and 15 μm (yellow), respectively.</p

The Microvesicle Component of HIV-1 Inocula Modulates Dendritic Cell Infection and Maturation and Enhances Adhesion to and Activation of T Lymphocytes

<div><p>HIV-1 is taken up by immature monocyte derived dendritic cells (iMDDCs) into tetraspanin rich caves from which the virus can either be transferred to T lymphocytes or enter into endosomes resulting in degradation. HIV-1 binding and fusion with the DC membrane results in low level <i>de novo</i> infection that can also be transferred to T lymphocytes at a later stage. We have previously reported that HIV-1 can induce partial maturation of iMDDCs at both stages of trafficking. Here we show that CD45⁺ microvesicles (MV) which contaminate purified HIV-1 inocula due to similar size and density, affect DC maturation, <i>de novo</i> HIV-1 infection and transfer to T lymphocytes. Comparing iMDDCs infected with CD45-depleted HIV-1_BaL or matched non-depleted preparations, the presence of CD45⁺ MVs was shown to enhance DC maturation and ICAM-1 (CD54) expression, which is involved in DC∶T lymphocyte interactions, while restricting HIV-1 infection of MDDCs. Furthermore, in the DC culture HIV-1 infected (p24⁺) MDDCs were more mature than bystander cells. Depletion of MVs from the HIV-1 inoculum markedly inhibited DC∶T lymphocyte clustering and the induction of alloproliferation as well as limiting HIV-1 transfer from DCs to T lymphocytes. The effects of MV depletion on these functions were reversed by the re-addition of purified MVs from activated but not non-activated SUPT1.CCR5-CL.30 or primary T cells. Analysis of the protein complement of these MVs and of these HIV-1 inocula before and after MV depletion showed that Heat Shock Proteins (HSPs) and nef were the likely DC maturation candidates. Recombinant HSP90α and β and nef all induced DC maturation and ICAM-1 expression, greater when combined. These results suggest that MVs contaminating HIV-1 released from infected T lymphocytes may be biologically important, especially in enhancing T cell activation, during uptake by DCs <i>in vitro</i> and <i>in vivo</i>, particularly as MVs have been detected in the circulation of HIV-1 infected subjects.</p></div

Recombinant HSP90β and recombinant Nef induce MDDC maturation and ICAM-1 expression.

<p>MDDCs (0.5×10⁶ cells/mL) were treated with recombinant proteins, A) HSP90β and B) nef at variable concentrations for 48 hours. The effects on the maturation markers, CD80, CD83 and CD86, were measured by flow cytometry and expressed as log (2) fold change in MFI compared to mock treated MDDCs (mean +/− SEM, n = 3, *p<0.05, **p<0.01). C) MDDCs (0.5×10⁶ cells/mL) were treated with recombinant nef at 10 nM (lowest significant effective concentration) and recombinant HSP90β at 0.5 nM (completely ineffective concentration on MDDC maturation alone) or maturation mix for 48 hours and expression of CD80, CD83 and CD86 measured by flow cytometry and expressed as log (2) fold change in MFI compared to mock treatment (mean +/− SEM, n = 3, *p<0.05, **p<0.001).</p

HIV-1_BaL(CD45⁻) induces less maturation than HIV-1_BaL(pellet).

<p>MDDCs were exposed to high titre HIV-1_BaL(pellet), HIV-1_BaL(CD45⁻) at a MOI of 3 or a potent maturation cocktail for 48 hours and expression of maturation determined by flow cytometry. A) Representative histograms for CD83 and CD86 shown (isotype grey tint, immature solid grey line, HIV-1_BaL(CD45⁻) dashed line, HIV-1_BaL(pellet) dotted line and mature solid black line). B) The levels of surface maturation marker expression with flow cytometry MFI results expressed as a ratio of normalised to untreated (mock/immature) controls as log (2) fold change (mean +/− SEM, n = 12, *p<0.05 **p<0.01). C) Kinetics over time of ICAM-1 expression determined by flow cytometry MFI for HIV-1_BaL(pellet) treated MDDCs (mean +/− SEM, n = 3). D) Productively infected MDDCs are more mature than uninfected MDDCs for both virus stocks. Infected cells were identified by p24 antigen staining and the expression of CD83, CD86 and ICAM-1 was compared to that for the HIV-1 exposed uninfected cells. Representative histograms for the markers of p24⁺ (dashed line) and p24⁻ (dotted line) compared to isotype control (grey tint) for HIV-1_BaL(pellet) and HIV-1_BaL (CD45⁻). Bar graphs delineating the expression of normalised maturation and co-stimulatory marker expressed as log (2) fold change to mock treated cells (mean +/− SEM, n = 5, *p<0.05).</p

Addition of microvesicles to HIV-1_BaL(CD45⁻) restores MDDC maturation.

<p>MDDCs were either treated with HIV-1_BaL(pellet), HIV-1_BaL(CD45⁻) alone, CD3/CD28 activated MVs (20 µl/3.5×10⁵ cells) alone or HIV-1_BaL(CD45⁻) with the addition of CD3/CD28 activated MVs for 48 hours. To calculate the MV ‘inoculum’ the CD45 concentration of the MVs preparation was matched to the CD45 concentration of the HIV-1_BaL(pellet) stock (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003700#ppat-1003700-t001" target="_blank">Table 1</a>). A) The expression of maturation markers was determined by flow cytometry and compared to the maturation seen with the HIV-1_BaL(pellet) stock (mean +/− SEM, n = 3, *p<0.05 **p<0.01). B) The expression of CD83 and CD86 was assessed and there was a positive correlation between amount of MVs added and the level of maturation seen in HIV-1 exposed MDDCs (mean +/− SEM, n = 3, CD83: p = 0.78, r = 0.9 and CD86: p = 0.27, r = 0.87). The level of p24⁺ cells was also measured by flow cytometry. C) The proportion of HIV-1 infected MDDCs at 120 hours was reduced by addition of MVs to the HIV-1_BaL(CD45⁻) inoculum (mean +/− SEM, n = 3, *p<0.05). D) MVs from anti CD3/CD28 activated primary T lymphocytes also induce MDDC maturation. MDDCs (0.5×10⁶ cells/mL) were treated with MVs from CD3/CD28 activated or non-activated primary T lymphocytes at the same concentration as in panel A for 48 hours and expression of CD80, 83 and 86 measured by flow cytometry (mean +/−SEM, n = 3, *p<0.05, **p<0.01 compared to mock or, where bracketed, activated MVs are compared to non-activated MVs). E) HIV-1_BAL(pellet) and MVs+HIV-1_BAL(CD45⁻) also induce maturation of primary blood myeloid DCs. Blood myeloid BDCA1 DCs (0.5×10⁶ cells/mL) were isolated from blood and exposed to maturation mix, HIV-1_BAL(pellet), HIV-1_BAL(CD45⁻) and HIV-1_BAL(CD45⁻)+MVs from activated SUPT1.CCR5-CL.30 cells at similar concentrations to panel A. CD83 expression was measured by flow cytometry. Representative histogram of three separate experiments.</p