Connor et al. show that transcriptomic profiling of DCs exposed to two different Th2 stimuli in vivo reveals large numbers of differentially expressed genes but few similarities between conditions.

The cytokine thymic stromal lymphopoietin (TSLP) is produced by epithelia exposed to the contact sensitizer dibutyl phthalate (DBP), and it is critical for the induction of Th2 immune responses by DBP-FITC. TSLP is thought to act on dendritic cells (DC), but the precise DC subsets involved in the response to TSLP remain to be fully characterized. In this study we show that a subset of CD326loCD103loCD11blo dermal DC, which we termed “triple-negative (TN) DC,” is highly responsive to TSLP. In DBP-FITC–treated mice, TN DC upregulated expression of CD86 and rapidly migrated to the draining lymph node to become the most abundant skin-derived DC subset at 24 and 48 h after sensitization. None of these responses was observed in TSLPR-deficient mice. In contrast, TN DC numbers were not increased after treatment with the allergen house dust mite or the bacteria Escherichia coli and bacillus Calmette–Guérin, which increased other DC subsets. In vivo, treatment with rTSLP preferentially increased the numbers of TN DC in lymph nodes. In vitro, TN DC responded to rTSLP treatment with a higher level of STAT5 phosphorylation compared with other skin-derived DC subsets. The TN DC subset shared the morphology, phenotype, and developmental requirements of conventional DC, depending on FLT3 expression for their optimal development from bone marrow precursors, and CCR7 for migration to the draining lymph node. Thus, TN DC represent a dermal DC subset that should be considered in future studies of TSLP-dependent contact sensitization and skin immune responses.

SignificanceT helper 2 (Th2) cells are defined by their ability to produce the hallmark cytokine IL-4. However, to mediate allergic inflammation in tissues, Th2 cells must secrete additional cytokines including IL-13 and IL-5. We used IL-4 and IL-13 dual-reporter mice to show that naive CD4+ T cells cultured in the presence of IL-4 and thymic stromal lymphopoietin (TSLP) generate a population of IL-4negIL-13pos Th2 cells that develop from IL-4neg precursors and express the Th2 effector cytokines IL-5 and IL-9. In vivo, high TSLP levels promote the development of a similar population of IL-4negIL-13pos T cells that also express Gata3, Il5, and Il3 transcripts. Thus, TSLP drives the early differentiation of a distinct population of effector Th2 cells with pro-inflammatory properties.

The epidermal barrier is thought to protect sensory nerves from overexposure to environmental stimuli, and barrier impairment leads to pathological conditions associated with itch, such as atopic dermatitis (AD). However, it is not known how the epidermal barrier continuously protects nerves for the sensory homeostasis during turnover of the epidermis. Here we show that epidermal nerves are contained underneath keratinocyte tight junctions (TJs) in normal human and mouse skin, but not in human AD samples or mouse models of chronic itch caused by epidermal barrier impairment. By intravital imaging of the mouse skin, we found that epidermal nerve endings were frequently extended and retracted, and occasionally underwent local pruning. Importantly, the epidermal nerve pruning took place rapidly at intersections with newly forming TJs in the normal skin, whereas this process was disturbed during chronic itch development. Furthermore, aberrant Ca 2+ increases in epidermal nerves were induced in association with the disturbed pruning. Finally, TRPA1 inhibition suppressed aberrant Ca 2+ increases in epidermal nerves and itch. These results suggest that epidermal nerve endings are pruned through interactions with keratinocytes to stay below the TJ barrier, and that disruption of this mechanism may lead to aberrant activation of epidermal nerves and pathological itch.

No abstract

Type II immune responses are associated with protection against helminth infections, as well as the pathology of allergic diseases that are initiated against innocuous antigens. Two integral cytokines associated with type II immunity are interleukin (IL-)4 and IL-13. The 4C13R transgenic dual reporter mouse has been created to allow identification of IL-4 and IL-13 producing cells by the production of two intracellular fluorescent molecules, AmCyan and DS-Red respectively. The expression of the distinct reporter proteins are under the normal transcriptional control of the Il4 or Il13 genes. This technology allows for the analysis of in situ IL-4 and/or IL-13 production by the relevant differentiated immune cell types without any effect on the endogenous cytokine genes or their effector activities in the mouse. Using this reporter system we have identified that in vitro generated CD4+ Th2 cells have divergent expression of IL-4 and IL-13, suggesting cytokine specific Th2 subsets. Additionally, IL-13 expression is delayed compared with IL-4 in this system. Reporter expression in vivo has also highlighted that IL-4/IL-13 double producers are only a subpopulation of CD4+ T cells, and cytokine expression profiles differ significantly between lymph node and effector tissues during type II responses. Studying the expression and regulation of these cytokines will allow us to understand their contribution in both disease and protection.

We have examined the role of dendritic cells using different models of Th2 allergic immune responses in the skin. In the first model we used the nematode parasite Nippostrongylus brasiliensis, which infects rodent hosts by penetrating through the skin. Material from labelled N. brasiliensis is taken up by migratory dendritic cells and transported to the draining lymph node where it initiates a strong Th2 immune response. We have characterized the surface phenotype of these dendritic cells in terms of lineage markers, and for expression of markers associated with Th2 immune responses. We find that these dendritic cells express markers that are compatible with a steady-state population of dermal dendritic cells. In addition, we have carried out dendritic cell transfer experiments to show that these migratory dendritic cells maintain the full capacity to prime Th2 immune responses in vivo. Additional models of Th2 immune responses, such as those initiated by the powerful allergen House Dust Mite and by application of the vitamin D analogue MC903, are currently being investigated. Our data indicate that dendritic cells can carry a sufficient set of signals to instruct the initiation of Th2 immune responses in secondary lymphoid organs.