BackgroundGroup 2 innate lymphoid cells (ILC2s) are a potential innate source of type 2 cytokines in the pathogenesis of allergic conditions. Epithelial cytokines (IL-33, IL-25, and thymic stromal lymphopoietin [TSLP]) and mast cell mediators (prostaglandin D2 [PGD2]) are critical activators of ILC2s. Cysteinyl leukotrienes (cysLTs), including leukotriene (LT) C4, LTD4, and LTE4, are metabolites of arachidonic acid and mediate inflammatory responses. Their role in human ILC2s is still poorly understood.ObjectivesWe sought to determine the role of cysLTs and their relationship with other ILC2 stimulators in the activation of human ILC2s.MethodsFor ex vivo studies, fresh blood from patients with atopic dermatitis and healthy control subjects was analyzed with flow cytometry. For in vitro studies, ILC2s were isolated and cultured. The effects of cysLTs, PGD2, IL-33, IL-25, TSLP, and IL-2 alone or in combination on ILC2s were defined by using chemotaxis, apoptosis, ELISA, Luminex, quantitative RT-PCR, and flow cytometric assays. The effect of endogenous cysLTs was assessed by using human mast cell supernatants.ResultsHuman ILC2s expressed the LT receptor CysLT1, levels of which were increased in atopic subjects. CysLTs, particularly LTE4, induced migration, reduced apoptosis, and promoted cytokine production in human ILC2s in vitro. LTE4 enhanced the effect of PGD2, IL-25, IL-33, and TSLP, resulting in increased production of type 2 and other proinflammatory cytokines. The effect of LTE4 was inhibited by montelukast, a CysLT1 antagonist. Interestingly, addition of IL-2 to LTE4 and epithelial cytokines significantly amplified ILC2 activation and upregulated expression of the receptors for IL-33 and IL-25.ConclusionCysLTs, particularly LTE4, are important contributors to the triggering of human ILC2s in inflammatory responses, particularly when combined with other ILC2 activators.

Human type-2 CD8 T cells are a cell population with potentially important roles in allergic disease. We investigated this in the context of severe asthma with persistent airway eosinophilia-a phenotype associated with high exacerbation risk and responsiveness to type-2 cytokine-targeted therapies. In two independent cohorts we show that, in contrast to Th2 cells, type-2 cytokine-secreting CD8CRTH2 (Tc2) cells are enriched in blood and airways in severe eosinophilic asthma. Concentrations of prostaglandin D (PGD) and cysteinyl leukotriene E (LTE) are also increased in the airways of the same group of patients. In vitro PGD and LTE function synergistically to trigger Tc2 cell recruitment and activation in a TCR-independent manner. These lipids regulate diverse genes in Tc2 cells inducing type-2 cytokines and many other pro-inflammatory cytokines and chemokines, which could contribute to eosinophilia. These findings are consistent with an important innate-like role for human Tc2 cells in severe eosinophilic asthma and suggest a potential target for therapeutic intervention in this and other diseases.

Freshwater planarians are unique in their ability to regenerate a complete Central Nervous System (CNS) from almost any small piece of their body in just a few days. The planarian CNS contains a pair of anterior cephalic ganglia lying on top of two ventral nerve cords that extend along the length of the animal. Studies of planarian CNS regeneration have generally used pan-neural markers, which provide only a general overview of the process. Nevertheless, some reports have started to characterize the genes that are required for this process. In this study, to obtain a more detailed description of planarian neural regeneration, we monitored the regeneration of neuronal populations specifically labelled with antibodies against serotonin, allatostatin, neuropeptide F, GYRFamide and FMRFamide. We also characterized the regeneration of dopaminergic and octopaminergic cell populations by in situ hybridization. Finally, we characterized the expression pattern of a set of receptors for neurotransmitters, neuropeptides and hormones that are suggested to play a role in the regeneration process itself. Together, these data provide a more detailed description of the cellular events occurring during anterior and posterior CNS regeneration in planarians and provide the foundations for future mechanistic studies into the regeneration process in this important model system.

TSLP.To test the hypothesis that absence of keratins in KtyI 2/2 cells upregulates TSLP through stimulation of EGFR, growth factor-starved WT and KtyI 2/2 cells were treated for 3 and 7 hours with 200 ng/mL recombinant AREG, followed by TSLP mRNA and protein analysis. This revealed a strong induction of Tslp mRNA in WT and KtyI 2/2 cells caused by the AREG stimulus (Fig 2 , A), with greater Tslp mRNA levels in KtyI 2/2 cells. Interestingly, AREG-EGFR stimulation of TSLP protein by 4-fold was restricted to KtyI 2/2 cells, indicating increased sensitization of keratin-deficient cells toward TSLP induction through EGFR signaling (Fig 2 , B). Furthermore, we show that stable re-expression of keratin 14 using lentiviral transduction in keratin-free keratinocytes prevents a strong induction of TSLP by AREG compared with green fluorescent protein (GFP)-expressing keratinocytes, suggesting that keratins repress an AREG-EGFR-TSLP signaling pathway (Fig 2 , C).To substantiate the finding that AREG-mediated TSLP induction depended on EGFR and ERK1/2 (Fig 2, A and B), Western blot analysis of Raf and ERK1/2 kinases was performed. This confirmed their involvement in AREG-mediated TSLP induction (Fig 2 , D). Nuclear factor kB signaling, as measured based on p65 phosphorylation, remained unaltered between WT and KtyI 2/2 cells and was not induced upon AREG-EGFR stimulation (Fig 2 , D9). This suggests that nuclear factor kB is not significantly involved in the keratin-mediated TSLP induction in keratinocytes. Intriguingly, mRNA levels of the EGFR-regulated cytokine CCL20, which are known to be increased in blister fluid from patients with EBS, 7 were also upregulated strongly after AREG-EGFR stimulation in KtyI 2/2 keratinocytes (Fig 2, E). Other inflammatory mediators, such as CCL2, CXCL1, CXCL10, IL-1A, Defensin b4, and S100A7 remained uninduced or were regulated equally on AREG-EGFR stimulation in WT and KtyI 2/2 cells (see Fig E1 , B, in this article's Online Repository at www.jacionline.org). This indicates that lack of keratins triggers expression of a distinct set of cytokines, chemokines, and antimicrobial peptides at the level of AREG-EGFR to induce a distinct inflammatory response.To examine whether AREG is also involved in TSLP upregulation in patients with EBS, AREG and TSLP serum protein levels in sera from patients with EBS were analyzed (Fig 2 , F). Six of 7 tested patients with EBS had increased TSLP serum protein levels (> _50 pg/mL), of whom 3 (50%) exhibited detectable AREG serum protein levels. Moreover, increased epidermal AREG levels were recently identified in a microarray analysis of 6 patients with EBS, 8 which is in line with the known release of AREG from wound-activated keratinocytes. 9 We hypothesize that increased AREG levels in patients with EBS can trigger TSLP upregulation in keratinocytes with keratin defects to drive itch and T H 2 inflammation in patients with EBS.Collectively, we present a new mechanism for TSLP induction in keratin-deficient keratinocytes mediated by an autocrine AREG-EGFR...

This article was originally published under standard licence, but has now been made available under a [CC BY 4.0] license. The PDF and HTML versions of the paper have been modified accordingly.

The functions and in vivo roles of type-2 CD8 + T cells in humans have not been well defined and this cell type has been largely overlooked in models of disease. We investigated this in the context of severe asthma with persistent airway eosinophilia -a phenotype associated with high exacerbation risk and responsiveness to type-2 cytokine-targeted therapies. In two independent cohorts we show that, in contrast to Th2 cells, type-2 cytokine-secreting CD8 + CRTH2 + (Tc2) cells are enriched in blood and airways in severe eosinophilic asthma. Concentrations of prostaglandin D 2 (PGD 2 ) and cysteinyl leukotriene E 4 (LTE 4 ) are also increased in the airways of the same group of patients. In vitro PGD 2 and LTE 4 function synergistically to trigger Tc2 cell recruitment and activation in a TCR-independent manner. These lipids regulate diverse genes in Tc2 cells inducing type-2 cytokines and many other pro-inflammatory cytokines and chemokines which could contribute to eosinophilia. These findings are consistent with an important innate-like role for human Tc2 cells in severe eosinophilic asthma and suggest a potential target for therapeutic intervention in this and other diseases.

No abstract