Sensory neuronal STAT3 is critical for IL-31 receptor expression and inflammatory itch.

IL-31 receptor blockade suppresses pruritus of atopic dermatitis. However, cell-type-specific contributions of IL-31 receptor to itch, its expression mechanism, and the downstream signaling pathway to induce itch remain unknown. Here, using conditional knockout mice, we demonstrate that IL-31-induced itch requires sensory neuronal IL-31 receptor and STAT3. We find that IL-31 receptor expression is dependent on STAT3 in sensory neurons. In addition, pharmacological experiments suggest that STAT3 activation is important for the itch-inducing signaling downstream of the IL-31 receptor. A cutaneous IL-31 injection induces the nuclear accumulation of activated STAT3 first in sensory neurons that abundantly express IL-31 receptor and then in other itch-transmitting neurons. IL-31 enhances itch induced by various pruritogens including even chloroquine. Finally, pruritus associated with dermatitis is partially dependent on sensory neuronal IL-31 receptor and strongly on sensory neuronal STAT3. Thus, sensory neuronal STAT3 is essential for IL-31-induced itch and further contributes to IL-31-independent inflammatory itch.

Homeostatic pruning and activity of epidermal nerves are dysregulated in barrier-impaired skin during chronic itch development.

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 Ca2+ increases in epidermal nerves were induced in association with the disturbed pruning. Finally, TRPA1 inhibition suppressed aberrant Ca2+ 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.

Thymic stromal lymphopoietin drives the development of IL-13+ Th2 cells.

T helper 2 (Th2) cells are pivotal in the development of allergy. Allergen exposure primes IL-4+ Th2 cells in lymph node, but production of effector cytokines including IL-5 and IL-13 is thought to require additional signals from antigen and the environment. Here we report that a substantial proportion of naive CD4+ T cells in spleen and lymph node express receptors for the epithelium-derived inflammatory cytokine thymic stromal lymphopoietin (TSLP). Culture of naive CD4+ T cells in anti-(a)CD3, aCD28, and TSLP-supplemented Th2 conditions enabled the development of a unique population of IL-13-single positive (IL-13-SP) CD4+ T cells; TSLP and Th2 conditions were both required for their development. Sorting experiments revealed that IL-13-SP Th2 cells originated from IL-4-negative precursors and coexpressed transcripts for the Th2 cytokines IL-5 and IL-9. In vivo, high TSLP levels acted directly on CD4+ T cells to induce the development of IL-13-SP and IL-4+IL-13+ double-positive populations in lymph node. These cells were phenotypically similar to Th2 effector cells and were CXCR5lowPD1low and expressed low levels of Bcl6 and Il21 transcripts and high levels of Gata3, Il3, and Il5 Our findings suggest a role of TSLP in directly promoting Th2 cell effector function and support the notion of TSLP as a key driver of Th2 inflammation.

Th2 responses are primed by skin dendritic cells with distinct transcriptional profiles.

The dendritic cell signals required for the in vivo priming of IL-4-producing T cells are unknown. We used RNA sequencing to characterize DCs from skin LN of mice exposed to two different Th2 stimuli: the helminth parasite Nippostrongylus brasiliensis (Nb) and the contact sensitizer dibutyl phthalate (DBP)-FITC. Both Nb and DBP-FITC induced extensive transcriptional changes that involved multiple DC subsets. Surprisingly, these transcriptional changes were highly distinct in the two models, with only a small number of genes being similarly regulated in both conditions. Pathway analysis of expressed genes identified no shared pathways between Nb and DBP-FITC, but revealed a type-I IFN (IFN-I) signature unique to DCs from Nb-primed mice. Blocking the IFN-I receptor at the time of Nb treatment had little effect on DC migration and antigen transport to the LN, but inhibited the up-regulation of IFN-I-induced markers on DCs and effectively blunted Th2 development. In contrast, the response to DBP-FITC was not affected by IFN-I receptor blockade, a finding consistent with the known dependence of this response on the innate cytokine TSLP. Thus, the priming of Th2 responses is associated with distinct transcriptional signatures in DCs in vivo, reflecting the diverse environments in which Th2 immune responses are initiated.

CD326(lo)CD103(lo)CD11b(lo) dermal dendritic cells are activated by thymic stromal lymphopoietin during contact sensitization in mice.

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 CD326(lo)CD103(lo)CD11b(lo) 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.

Sustained in vivo depletion of splenic langerin(+) CD8α(+) dendritic cells is well-tolerated by lang-DTREGFP mice.

Splenic langerin(+) CD8α(+) dendritic cells (DCs) have exhibited a critical role in cross-priming CD8(+) T cell responses. To further study the roles of this DC subset, a protocol for the continuous depletion of langerin(+) CD8α(+) DCs was established using the pre-existing lang-DTREGFP mouse model. Due to the fast turnover rate of splenic CD8α(+) DCs, maintaining the depletion of langerin(+) CD8α(+) DCs required multiple diphtheria toxin (DT) treatments. We found that prolonged treatment with DT did not cause weight loss, or neutrophilia, as reported in some DT-based depletion models. Therefore, the in vivo depletion of murine langerin(+) CD8α(+) DCs can be maintained over time to analyse their function during the full course of an immune response.