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P01.07

TLR-3 and TLR-7/8 Ligands Indirectly Activate Langerhans Cells When Intradermally Injected by Triggering the Recruitment of Inflammatory Cells


  TLR-3 and TLR-7/8 ligands indirectly activate Langerhans cells when intradermally injected by triggering the recruitment of inflammatory cells

Olivier Epaulard, Lucille Adam, Roger Le Grand, Frédéric Martinon

CEA, Division of Immuno-virology, Institute for Emerging Diseases and Innovative Therapies, DSV, Fontenay aux Roses, France; UMR E1, Université Paris-Sud, Orsay, France; and Vaccine Research Institute, Créteil, France

INTRODUCTION

Skin is an important site of vaccine delivery, however, the role of the skin APC in the vaccination process is poorly understood, and little is known about the early events following vaccine injection in the skin. It is likely that vaccine antigen is loaded onto APC activated and/or recruited by the pathogen itself (for attenuated vaccines) or by the associated adjuvant (for subunit vaccines). The toll-like receptors (TLR) ligands have been regarded as particularly promising in the field of vaccine adjuvants. These receptors bind pathogen-associated molecular patterns (PAMP) such as double-stranded RNA (TLR-3), or single-stranded RNA (TLR-7&8), inducing the activation of TLR-expressing cells, such as polymorphonuclear neutrophils (PMN), macrophages, and dendritic cells (DC). In the present work, we describe the modifications in Langerhans cells (LC) and other leucocytes populations induced by intradermal injection of a TLR-7/8 agonist (R848) and a TLR-3 agonist (Poly(I:C)) in non-human primates, and we explore how PMN and macrophage recruitment may be crucial for LC activation and migration.

  Macaque skin at steady state

Skin Is organized in 3 main parts: epidermis, dermis and hypodermis(Fig.1A). We will focus on epidermis and dermis sheets which contain several immune cell populations. Immunohistofluorescence analysis of macaque skin biopsies revealed the presence of DC (expressing CD209 (DC-SIGN)) in the dermis and LC (expressing CD207 (langerin)) in the epidermis. LC also express high level of CD1a and network the epidermis with 300 to 1000 cells per cm2 (Fig.1B). In order to further characterize skin APC, cells were extracted from biopsies by using a dispase-based separation of dermis from epidermis, followed by a trypsin-based extraction for epidermis, and a collagenase-based extraction for dermis. This strategy allows collecting most of the cells from both sheets including macrophages and granulocytes, without addition of any cytokine. Flow cytometry analysis indicated that epidermis cell suspension contained more than 75% of keratinocytes, expressing cytokeratin (Fig.1C). LC were characterised by the expression of CD45 and high levels of HLA-DR and CD1a. They also highly expressed CD207 and CD11b. Expression of CD11c by LC was heterogeneous between animals. LC were negative for CD14 and CD163, and the expression of CD209 was very low. LC represent 0.71±0.23% (n=12) of total epidermis cells at steady state (Fig.1C). In the dermis, CD45+ HLA-DR+ cells were analysed for the expression of CD11c, CD163, CD14 and CD1a (Fig.1D). Dermis macrophages were characterized by the CD11clow CD163+ phenotype. Further analysis of the CD11c+ CD163- dermal classical DC shows the differential CD1a and CD14 expression (Fig.1D). Note that CD1a positive dermal DC have a lower CD1a expression than LC. At steady state, the proportion of macrophages was 1.48±0.70% (mean±SD, n=18) of the total living cells in the dermis cell suspension. Plotting CD66abce versus HLA-DR in the CD45+ cells allowed identifying CD66+, HLA-DR- PMN (Fig.1D). At steady state, the PMN represented 0.88±0.45% (mean±SD, n=13) of the total living cells in the dermis cell suspension.

 Skin modification induced by i.d. injection of TLR agonists

Immunohistofluorescence analysis showed a dramatic decrease of LC density at 72 hours post-injection (Fig.2A). LC were identified with both anti-CD207 and anti-CD1a staining, to exclude that the non-detection of LC was not due to a downregulation of the expression of these markers. By contrast, dermal DC, visualized with the anti-CD209, remained unchanged after TLR ligand injections. Flow cytometry analysis indicated a significant decrease of LC by a factor 1.47±0.21, 72h after the injection of R848 when compared to PBS (Fig.2B). Mean LC proportion among epidermal cells was 0.51±0.16% 72 hours after PBS injection, and 0.28±0.13% 72h after R848 injection (p=0.0156). Intradermal injection of 200 μg of poly(I:C) induced a decrease by a mean factor 1.36±0.09 of epidermal LC density within 72h. Mean LC proportion among epidermal cells was 0.59±0.11%, 72 hours after PBS injection, and 0.40±0.06% 72h after poly(I:C) injection (p=0.0313) (Fig.2B). The decrease in LC number was associated with a decrease in CD207 membrane expression and a significant increase in the expression of the activation/maturation markers CD80, CD83 and CD86 (Fig.2B). Intradermal injection of 200 μg of R848 or poly(I:C) induced a recruitment of PMN and macrophages in the dermis. Dermal cell suspension contained a mean of 1.4% and 0.9% of PMN 24 hours and 72 hours after PBS injection, respectively; a mean of 8.9% and 9.0% of PMN 24 hours and 72 hours after R848 injection, respectively; and 5.1% and 5.3% of PMN 24 hours and 72 hours after poly(I:C) injection, respectively (Fig.2C). Dermal cell suspension contained a mean of 3.0% and 3.7% of macrophages 24 hours and 72 hours after PBS injection, respectively; a mean of 8.4% and 9.3% of macrophages 24 hours and 72 hours after R848 injection, respectively; and 13.9% and 10.7% of macrophages 24 hours and 72 hours after poly(I:C) injection, respectively (Fig.2C). The intensity of neutrophil recruitment was correlated with LC number and status after injection of PBS, R848 or poly(I:C). Indeed, PMN dermal density and macrophage dermal density was significantly positively correlated with the expression of the activation/maturation markers CD80, CD83, and CD86 by LC at 24 hours (Fig.2D). Moreover, there was a significant negative correlation between PMN dermal density at 24 hours and LC epidermal density at 72 hours and a negative correlation between macrophages dermal density at 24 hours and LC epidermal density at 72 hours (Fig.2D). 

 CONCLUSION

Intradermal injection of R848 or poly(I:C) induces a local inflammation characterised by the recruitment of PMN and macrophages in the dermis. This inflammation induces the maturation and activation of LC which result in their migration from the epidermis. The indirect targeting of LC with such adjuvants has implication for the design of future vaccines.

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