Using gold nanoparticles for enhanced intradermal delivery of poorly soluble auto-antigenic peptides.

Ultra-small 1-2 nm gold nanoparticles (NP) were conjugated with a poorly-soluble peptide auto-antigen, associated with type 1 diabetes, to modify the peptide pharmacokinetics, following its intradermal delivery. Peptide distribution was characterized, in vivo, after delivery using either conventional intradermal injection or a hollow microneedle device. The poorly-soluble peptide was effectively presented in distant lymph nodes (LN), spleen and draining LN when conjugated to the nanoparticles, whereas peptide alone was only presented in the draining LN. By contrast, nanoparticle conjugation to a highly-soluble peptide did not enhance in vivo distribution. Transfer of both free peptide and peptide-NPs from the skin to LN was reduced in mice lacking lymphoid homing receptor CCR7, suggesting that both are actively transported by migrating dendritic cells to LN. Collectively, these data demonstrate that intradermally administered ultra-small gold nanoparticles can widen the distribution of poorly-soluble auto-antigenic peptides to multiple lymphoid organs, thus enhancing their use as potential therapeutics.

Unveiling skin macrophage dynamics explains both tattoo persistence and strenuous removal.

Here we describe a new mouse model that exploits the pattern of expression of the high-affinity IgG receptor (CD64) and allows diphtheria toxin (DT)-mediated ablation of tissue-resident macrophages and monocyte-derived cells. We found that the myeloid cells of the ear skin dermis are dominated by DT-sensitive, melanin-laden cells that have been missed in previous studies and correspond to macrophages that have ingested melanosomes from neighboring melanocytes. Those cells have been referred to as melanophages in humans. We also identified melanophages in melanocytic melanoma. Benefiting of our knowledge on melanophage dynamics, we determined the identity, origin, and dynamics of the skin myeloid cells that capture and retain tattoo pigment particles. We showed that they are exclusively made of dermal macrophages. Using the possibility to delete them, we further demonstrated that tattoo pigment particles can undergo successive cycles of capture-release-recapture without any tattoo vanishing. Therefore, congruent with dermal macrophage dynamics, long-term tattoo persistence likely relies on macrophage renewal rather than on macrophage longevity.

Hapten-Specific T Cell-Mediated Skin Inflammation: Flow Cytometry Analysis of Mouse Skin Inflammatory Infiltrate.

Hapten-specific T cell-mediated skin inflammation also known as contact hypersensitivity (CHS) is characterized by a strong influx of CD8+ cytotoxic T cells within the skin upon reexposure of sensitized individuals to the same hapten. As many other leukocytes are also recruited during this elicitation phase, we attempted to revisit the skin infiltrate and characterize the inflammatory pattern. Recent improvement in the isolation in conventional as well as inflammatory dendritic cell and macrophage subsets from tissues and in the use of appropriate surface markers unraveling their heterogeneity should allow to determinate their specific functions in the CHS model. Here, we describe procedures to extract those cells from the skin and to analyze them by flow cytometry using a combination of appropriate surface markers allowing further transcriptomic analysis and functional assays.

Isolation of Mouse Dendritic Cell Subsets and Macrophages from the Skin.

The improvement of dendritic cell subset isolation from tissues and the use of appropriate surface markers allowed to decipher their heterogeneity but also allowed to unravel some specific functions that are valuable for vaccine design as well as for a better understanding of the in situ pathophysiology upon infection. Here, we describe the procedures to extract those cells from the skin and to analyze them by flow cytometry using a combination of appropriate surface markers allowing further transcriptomic analysis and functional assays.

Dynamics and Transcriptomics of Skin Dendritic Cells and Macrophages in an Imiquimod-Induced, Biphasic Mouse Model of Psoriasis.

Psoriasis is a chronic inflammatory skin disease of unknown etiology. Previous studies showed that short-term, 5-7 d-long application of imiquimod (IMQ), a TLR7 agonist, to the skin of mice triggers a psoriasis-like inflammation. In the current study, by applying IMQ for 14 consecutive d, we established an improved mouse psoriasis-like model in that it recapitulated many of the clinical and cellular hallmarks observed in human patients during both the early-onset and the late-stable phase of psoriasis. Although macrophages and dendritic cells (DCs) have been proposed to drive the psoriatic cascade, their largely overlapping phenotype hampered studying their respective role. Based on our ability to discriminate Langerhans cells (LCs), conventional DCs, monocytes, monocyte-derived DCs, macrophages, and plasmacytoid DCs in the skin, we addressed their dynamics during both phases of our biphasic psoriasis-like model. Plasmacytoid DCs were not detectable during the whole course of IMQ treatment. During the early phase, neutrophils infiltrated the epidermis, whereas monocytes and monocyte-derived DCs were predominant in the dermis. During the late phase, LCs and macrophage numbers transiently increased in the epidermis and dermis, respectively. LC expansion resulted from local proliferation, a conclusion supported by global transcriptional analysis. Genetic depletion of LCs permitted to evaluate their function during both phases of the biphasic psoriasis-like model and demonstrated that their absence resulted in a late phase that is associated with enhanced neutrophil infiltration. Therefore, our data support an anti-inflammatory role of LCs during the course of psoriasis-like inflammation.

Laser-assisted intradermal delivery of adjuvant-free vaccines targeting XCR1+ dendritic cells induces potent antitumoral responses.

The development of vaccines inducing efficient CD8(+) T cell responses is the focus of intense research. Dendritic cells (DCs) expressing the XCR1 chemokine receptor, also known as CD103(+) or CD8α(+) DCs, excel in the presentation of extracellular Ags to CD8(+) T cells. Because of its high numbers of DCs, including XCR1(+) DCs, the skin dermis is an attractive site for vaccine administration. By creating laser-generated micropores through the epidermis, we targeted a model protein Ag fused to XCL1, the ligand of XCR1, to dermal XCR1(+) DCs and induced Ag-specific CD8(+) and CD4(+) T cell responses. Efficient immunization required the emigration of XCR1(+) dermal DCs to draining lymph nodes and occurred irrespective of TLR signaling. Moreover, a single intradermal immunization protected mice against melanoma tumor growth in prophylactic and therapeutic settings, in the absence of exogenous adjuvant. The mild inflammatory milieu created in the dermis by skin laser microporation itself most likely favored the development of potent T cell responses in the absence of exogenous adjuvants. The existence of functionally equivalent XCR1(+) dermal DCs in humans should permit the translation of laser-assisted intradermal delivery of a tumor-specific vaccine targeting XCR1(+) DCs to human cancer immunotherapy. Moreover, considering that the use of adjuvants in vaccines is often associated with safety issues, the possibility of inducing protective responses against melanoma tumor growth independently of the administration of exogenous adjuvants should facilitate the development of safer vaccines.

Origins and functional specialization of macrophages and of conventional and monocyte-derived dendritic cells in mouse skin.

In the skin, the lack of markers permitting the unambiguous identification of macrophages and of conventional and monocyte-derived dendritic cells (DCs) complicates understanding of their contribution to skin integrity and to immune responses. By combining CD64 and CCR2 staining, we successfully identified each of these cell types and studied their origin, transcriptomic signatures, and migratory and T cell stimulatory properties. We also analyzed the impact of microbiota on their development and their contribution to skin inflammation during contact hypersensitivity. Dermal macrophages had a unique scavenging role and were unable to migrate and activate T cells. Conventional dermal DCs excelled both at migrating and activating T cells. In the steady-state dermis, monocyte-derived DCs are continuously generated by extravasated Ly-6C(hi) monocytes. Their T cell stimulatory capacity combined with their poor migratory ability made them particularly suited to activate skin-tropic T cells. Therefore, a high degree of functional specialization occurs among the mononuclear phagocytes of the skin.