Neuromedin U promotes immune cell activation and cytokine production, and contributes to several inflammatory disorders. The inflammatory roles of NmU have been indicated to be neurogenic or ...neuron‐independent. Understanding the potential roles of NmU in immunity and inflammation could be beneficial for the development of novel therapies for NmU‐involving diseases.
Summary
Since the discovery of neuromedin U (NmU) from porcine spinal cord in 1985, this neuropeptide has been subsequently identified in many other species with multiple physiological and pathophysiological roles detected, ranging from smooth muscle contraction, feeding, energy balance to tumorigenesis. Intriguingly, NmU is also emerging to play pro‐inflammatory roles involving immune cell activation and cytokine release in a neuron‐dependent or neuron‐independent manner. The NmU‐mediated inflammatory responses have already been observed in worm infection, sepsis, autoimmune arthritis and allergic animal models. In this review, we focus on the roles of NmU in immunity and inflammation by highlighting the interactions between NmU and immune cells, summarizing the signalling mechanism involved in their reactions and discussing its potential contributions to inflammatory diseases.
Activation of the group 2 innate lymphoid cell (ILC2) population leads to production of the classical type 2 cytokines, thus promoting type 2 immunity. Chemoattractant receptor-homologous molecule ...expressed on TH2 cells (CRTH2), a receptor for prostaglandin D2 (PGD2), is expressed by human ILC2s. However, the function of CRTH2 in these cells is unclear.
We sought to determine the role of PGD2 and CRTH2 in human ILC2s and compare it with that of the established ILC2 activators IL-25 and IL-33.
The effects of PGD2, IL-25, and IL-33 on the cell migration, cytokine production, gene regulation, and receptor expression of ILC2s were measured with chemotaxis, ELISA, Luminex, flow cytometry, quantitative RT-PCR, and QuantiGene assays. The effects of PGD2 under physiologic conditions were evaluated by using the supernatant from activated mast cells.
PGD2 binding to CRTH2 induced ILC2 migration and production of type 2 cytokines and many other cytokines. ILC2 activation through CRTH2 also upregulated the expression of IL-33 and IL-25 receptor subunits (ST2 and IL-17RA). The effects of PGD2 on ILC2s could be mimicked by the supernatant from activated human mast cells and inhibited by a CRTH2 antagonist.
PGD2 is an important and potent activator of ILC2s through CRTH2 mediating strong proallergic inflammatory responses. Through IgE-mediated mast cell degranulation, these innate cells can also contribute to adaptive type 2 immunity; thus CRTH2 bridges the innate and adaptive pathways in human ILC2s.
Type 2 innate lymphoid cells (ILC2s, nuocytes, NHC) require RORA and GATA3 for their development. We show that human ILC2s express skin homing receptors and infiltrate the skin after allergen ...challenge, where they produce the type 2 cytokines IL-5 and IL-13. Skin-derived ILC2s express the IL-33 receptor ST2, which is up-regulated during activation, and are enriched in lesional skin biopsies from atopic patients. Signaling via IL-33 induces type 2 cytokine and amphiregulin expression, and increases ILC2 migration. Furthermore, we demonstrate that E-cadherin ligation on human ILC2 dramatically inhibits IL-5 and IL-13 production. Interestingly, down-regulation of E-cadherin is characteristic of filaggrin insufficiency, a cardinal feature of atopic dermatitis (AD). ILC2 may contribute to increases in type 2 cytokine production in the absence of the suppressive E-cadherin ligation through this novel mechanism of barrier sensing. Using Rag1(-/-) and RORα-deficient mice, we confirm that ILC2s are present in mouse skin and promote AD-like inflammation. IL-25 and IL-33 are the predominant ILC2-inducing cytokines in this model. The presence of ILC2s in skin, and their production of type 2 cytokines in response to IL-33, identifies a role for ILC2s in the pathogenesis of cutaneous atopic disease.
Background Group 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. Objectives We sought to determine the role of cysLTs and their relationship with other ILC2 stimulators in the activation of human ILC2s. Methods For 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. Results Human 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. Conclusion CysLTs, particularly LTE4 , are important contributors to the triggering of human ILC2s in inflammatory responses, particularly when combined with other ILC2 activators.
Group 2 innate lymphoid cells (ILC2) are important in effector functions for eliciting allergic inflammation, parasite defense, epithelial repair, and lipid homeostasis. ILC2 lack rearranged ...Ag-specific receptors, and although many soluble factors such as cytokines and lipid mediators can influence ILC2, direct interaction of these cells with the microenvironment and other cells has been less explored. Natural cytotoxicity receptors are expressed by subsets of group 1 ILC and group 3 ILC and thought to be important for their effector function, but they have not been shown to be expressed by ILC2. Therefore, we sought to investigate the expression and functional properties of the natural cytotoxicity receptor NKp30 on human ILC2. A subset of ex vivo and cultured ILC2 express NKp30 that upon interaction with its cognate activatory ligand B7-H6 induces rapid production of type 2 cytokines. This interaction can be blocked by NKp30 blocking Ab and an inhibitory ligand, galectin-3. Higher expression of B7-H6 was observed in lesional skin biopsies of patients with atopic dermatitis, and incubation of keratinocytes with proinflammatory and type 2 cytokines upregulated B7-H6, leading to increased ILC2 cytokine production. NKp30-B7-H6 interaction is a novel cell contact mechanism that mediates activation of ILC2 and identifies a potential target for the development of novel therapeutics for atopic dermatitis and other atopic diseases.
It is now well established that interaction of PGD(2) with chemoattractant receptor- homologous molecule expressed on Th2 cells (CRTH2) promotes chemotaxis and proinflammatory cytokine production by ...Th2 lymphocytes. In this study we show a novel function of CRTH2 in mediating an inhibitory effect of PGD(2) on the apoptosis of human Th2 cells induced by cytokine deprivation. This effect was mimicked by the selective CRTH2 agonist 13,14-dihydro-15-keto-PGD(2), inhibited by the CRTH2 antagonists ramatroban and TM30089, and not observed in CRTH2-negative T cells. D prostanoid receptor 1 (DP(1)) or the thromboxane-like prostanoid (TP) receptor did not play a role in mediating the effects of PGD(2) on the apoptosis of Th2 cells because neither the DP(1) antagonist BW868C nor the TP antagonist SQ29548 had any effect on the antiapoptotic effect of PGD(2). Apoptosis of Th2 cells induced by Fas ligation was not suppressed by treatment with PGD(2), illustrating that activation of CRTH2 only inhibits apoptosis induced by cytokine deprivation. Treatment with PGD(2) induced phosphorylation of Akt and BAD, prevented release of cytochrome c from mitochondria, and suppressed cleavage of caspase-3 and poly(ADP-ribose) polymerase in Th2 cells deprived of IL-2. The PI3K inhibitor LY294002 blocked the effect of PGD(2) both on the signaling events and on the apoptotic death of Th2 cells. These data suggest that in addition to promoting the recruitment and activation of Th2 cells, PGD(2) may also impede the resolution of allergic inflammation through inhibiting apoptosis of Th2 cells.
Two recent phase III trials in patients with severe eosinophilic asthma have shown that anti-interleukin 5 (IL-5) therapy with mepolizumab reduces the frequency of asthma attacks, improves symptoms ...and allows patients to reduce oral glucocorticoid use without loss of control of asthma. An earlier large 616 patient Dose Ranging Efficacy And safety with Mepolizumab in severe asthma (DREAM) study had shown that the only variables associated with treatment efficacy were a prior history of asthma attacks and the peripheral blood eosinophil count. The link between blood eosinophil counts and treatment efficacy is biologically obvious given that IL-5 has a pivotal role in eosinophil production, proliferation and chemotaxis. It is also clinically relevant as the blood eosinophil count is routinely measured and thus readily available in patients with asthma. Recognition of the link between airway or blood eosinophilia and treatment response was also important in the clinical testing of the alternative IL-5 blocker, such as reslizumab, which is currently being evaluated in a phase III randomized controlled trial (RCT) after having shown to improve lung function, improve symptom score and reduce sputum eosinophilia in a smaller phase IIb study. In addition, benralizumab, an IL-5α receptor blocker, has shown good effects in a phase IIb RCT with patients with severe asthma that had sputum eosinophilia and more recently in a phase IIa trial with patients with eosinophilic chronic obstructive pulmonary disease. Therefore anti-IL-5 treatment seems generally effective in eosinophilic asthma, either assessed by blood or airway eosinophilia. This factor together with the impressive clinical efficacy and good safety profile make anti-IL-5 (mepolizumab, reslizumab) and benralizumab (anti-IL-5 receptor α) very promising drugs for the treatment of patients with severe eosinophilic asthma, a subgroup that is in desperate need of better treatments.
PGD2, produced by mast cells, has been detected in high concentrations at sites of allergic inflammation. It can stimulate vascular and other inflammatory responses by interaction with D prostanoid ...receptor (DP) and chemoattractant receptor-like molecule expressed on Th2 cells (CRTH2) receptors. A significant role for PGD2 in mediating allergic responses has been suggested based on the observation that enhanced eosinophilic lung inflammation and cytokine production is apparent in the allergen-challenged airways of transgenic mice overexpressing human PGD2 synthase, and PGD2 can enhance Th2 cytokine production in vitro from CD3/CD28-costimulated Th2 cells. In the present study, we investigated whether PGD2 has the ability to stimulate Th2 cytokine production in the absence of costimulation. At concentrations found at sites of allergic inflammation, PGD2 preferentially elicited the production of IL-4, IL-5, and IL-13 by human Th2 cells in a dose-dependent manner without affecting the level of the anti-inflammatory cytokine IL-10. Gene transcription peaked within 2 h, and protein release peaked approximately 8 h after stimulation. The effect of PGD2 was mimicked by the selective CRTH2 agonist 13,14-dihydro-15-keto-PGD2 but not by the selective DP agonist BW245C, suggesting that the stimulation is mediated by CRTH2 and not DP. Ramatroban, a dual CRTH2/thromboxane-like prostanoid receptor antagonist, markedly inhibited Th2 cytokine production induced by PGD2, while the selective thromboxane-like prostanoid receptor antagonist SQ29548 was without effect. These data suggest that PGD2 preferentially up-regulates proinflammatory cytokine production in human Th2 cells through a CRTH2-dependent mechanism in the absence of any other costimulation and highlight the potential utility of CRTH2 antagonists in the treatment of allergic diseases.
Multicolour Flow Cytometry (MFC) produces multidimensional analytical data on the quantitative expression of multiple markers on single cells. This data contains invaluable biomedical information on ...(1) the marker expressions per cell, (2) the variation in such expression across cells, (3) the variability of cell marker expression across samples that (4) may vary systematically between cells collected from donors and patients. Current conventional and even advanced data analysis methods for MFC data explore only a subset of these levels. The Discriminant Analysis of MultiAspect CYtometry (DAMACY) we present here provides a comprehensive view on health and disease responses by integrating all four levels. We validate DAMACY by using three distinct datasets: in vivo response of neutrophils evoked by systemic endotoxin challenge, the clonal response of leukocytes in bone marrow of acute myeloid leukaemia (AML) patients, and the complex immune response in blood of asthmatics. DAMACY provided good accuracy 91-100% in the discrimination between health and disease, on par with literature values. Additionally, the method provides figures that give insight into the marker expression and cell variability for more in-depth interpretation, that can benefit both physicians and biomedical researchers to better diagnose and monitor diseases that are reflected by changes in blood leukocytes.
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