Technical advance: live-imaging analysis of human dendritic cell migrating behavior under the influence of immune-stimulating reagents in an organotypic model of lung.

This manuscript describes technical advances allowing manipulation and quantitative analyses of human DC migratory behavior in lung epithelial tissue. DCs are hematopoietic cells essential for the maintenance of tissue homeostasis and the induction of tissue-specific immune responses. Important functions include cytokine production and migration in response to infection for the induction of proper immune responses. To design appropriate strategies to exploit human DC functional properties in lung tissue for the purpose of clinical evaluation, e.g., candidate vaccination and immunotherapy strategies, we have developed a live-imaging assay based on our previously described organotypic model of the human lung. This assay allows provocations and subsequent quantitative investigations of DC functional properties under conditions mimicking morphological and functional features of the in vivo parental tissue. We present protocols to set up and prepare tissue models for 4D (x, y, z, time) fluorescence-imaging analysis that allow spatial and temporal studies of human DCs in live epithelial tissue, followed by flow cytometry analysis of DCs retrieved from digested tissue models. This model system can be useful for elucidating incompletely defined pathways controlling DC functional responses to infection and inflammation in lung epithelial tissue, as well as the efficacy of locally administered candidate interventions.

Detection of IL-17A-producing peripheral blood monocytes in Langerhans cell histiocytosis patients.

Langerhans cell histiocytosis (LCH) is a rare disease of unknown cause with manifestations ranging from isolated granulomatous lesions to life-threatening multi-system organ involvement. This disorder is further characterized by infiltration of immune cells in affected tissues and an association with interleukin (IL)-17A has been reported. Here, we investigated the presence of IL-17A-producing cells among peripheral blood mononuclear cells isolated from LCH patients and observed a high percentage of IL-17A(+) monocytes in peripheral blood of LCH patients compared to controls. The IL-17A(+) monocytes were also positive for the transcription factor retinoic acid orphan receptor (ROR) γt and showed increased mRNA levels for both IL-17A and RORγt. Notably, IL-17A was produced by all monocyte subsets and the expression level was positively associated with LCH disease activity. These data support a role for monocytes in the pathogenesis of LCH. Future therapeutic approaches may consider identification of patients who may benefit from IL-17A-targeted interventions.