IL-5 antagonism reverses priming and activation of eosinophils in severe eosinophilic asthma.

Eosinophils are key effector cells mediating airway inflammation and exacerbation in patients with severe eosinophilic asthma. They are present in increased numbers and activation states in the airway mucosa and lumen. Interleukin-5 (IL-5) is the key eosinophil growth factor that is thought to play a role in eosinophil priming and activation. However, the mechanism of these effects is still not fully understood. The anti-IL-5 antibody mepolizumab reduces eosinophil counts in the airway modestly but has a large beneficial effect on the frequency of exacerbations of severe eosinophilic asthma, suggesting that reduction in eosinophil priming and activation is of central mechanistic importance. In this study, we used the therapeutic effect of mepolizumab and single-cell ribonucleic acid sequencing to investigate the mechanism of eosinophil priming and activation by IL-5. We demonstrated that IL-5 is a dominant driver of eosinophil priming and plays multifaceted roles in eosinophil function. It enhances eosinophil responses to other stimulators of migration, survival, and activation by activating phosphatidylinositol-3-kinases, extracellular signal-regulated kinases, and p38 mitogen-activated protein kinases signaling pathways. It also enhances the pro-fibrotic roles of eosinophils in airway remodeling via transforming growth factor-ß pathway. These findings provide a mechanistic understanding of eosinophil priming in severe eosinophilic asthma and the therapeutic effect of anti-IL-5 approaches in the disease.

Evaluation of perturbed iron-homeostasis in a prospective cohort of patients with COVID-19.

Background: Marked reductions in serum iron concentrations are commonly induced during the acute phase of infection. This phenomenon, termed hypoferremia of inflammation, leads to inflammatory anemia, but could also have broader pathophysiological implications. In patients with coronavirus disease 2019 (COVID-19), hypoferremia is associated with disease severity and poorer outcomes, although there are few reported cohorts. Methods: In this study, we leverage a well characterised prospective cohort of hospitalised COVID-19 patients and perform a set of analyses focussing on iron and related biomarkers and both acute severity of COVID-19 and longer-term symptomatology. Results: We observed no associations between acute serum iron and long-term outcomes (including fatigue, breathlessness or quality of life); however, lower haemoglobin was associated with poorer quality of life. We also quantified iron homeostasis associated parameters, demonstrating that among 50 circulating mediators of inflammation IL-6 concentrations were strongly associated with serum iron, consistent with its central role in inflammatory control of iron homeostasis. Surprisingly, we observed no association between serum hepcidin and serum iron concentrations. We also observed elevated erythroferrone concentrations in COVID-19 patients with anaemia of inflammation. Conclusions: These results enhance our understanding of the regulation and pathophysiological consequences of disturbed iron homeostasis during SARS-CoV-2 infection.

Neuromedin U promotes human type 2 immune responses.

Type 2 immunity mediates the immune responses against parasites and allergic stimuli. Evidence from studies of cell lines and animals implies that neuromedin U (NmU) acts as a pro-inflammatory mediator of type 2 inflammation. However, the role of NmU in human type 2 immunity remains unclear. Here we investigated the expression of NmU in human blood and airways, and the expression of NmU receptors by human immune cells in blood and lung tissue. We detected human NmU (hNmU-25) in blood and airways with higher concentrations in the latter. NmU receptor 1 (NmUR1) was expressed by most human immune cells with higher levels in type 2 cells including type 2 T helpers, type 2 cytotoxic T cells, group-2 innate lymphoid cells and eosinophils, and was upregulated in lung-resident and activated type 2 cells. We also assessed the effects of NmU in these cells. hNmU-25 elicited type 2 cytokine production by type 2 lymphocytes and induced cell migration, including eosinophils. hNmU-25 also enhanced the type 2 immune response to other stimuli, particularly prostaglandin D2. These results indicate that NmU could contribute to the pathogenic processes of type 2 immunity-mediated diseases in humans via its pro-inflammatory effects on type 2 lymphocytes and eosinophils.

Identification of immune correlates of fatal outcomes in critically ill COVID-19 patients.

Prior studies have demonstrated that immunologic dysfunction underpins severe illness in COVID-19 patients, but have lacked an in-depth analysis of the immunologic drivers of death in the most critically ill patients. We performed immunophenotyping of viral antigen-specific and unconventional T cell responses, neutralizing antibodies, and serum proteins in critically ill patients with SARS-CoV-2 infection, using influenza infection, SARS-CoV-2-convalescent health care workers, and healthy adults as controls. We identify mucosal-associated invariant T (MAIT) cell activation as an independent and significant predictor of death in COVID-19 (HR = 5.92, 95% CI = 2.49-14.1). MAIT cell activation correlates with several other mortality-associated immunologic measures including broad activation of CD8+ T cells and non-Vδ2 γδT cells, and elevated levels of cytokines and chemokines, including GM-CSF, CXCL10, CCL2, and IL-6. MAIT cell activation is also a predictor of disease severity in influenza (ECMO/death HR = 4.43, 95% CI = 1.08-18.2). Single-cell RNA-sequencing reveals a shift from focused IFNα-driven signals in COVID-19 ICU patients who survive to broad pro-inflammatory responses in fatal COVID-19 -a feature not observed in severe influenza. We conclude that fatal COVID-19 infection is driven by uncoordinated inflammatory responses that drive a hierarchy of T cell activation, elements of which can serve as prognostic indicators and potential targets for immune intervention.

Pre-existing asthma as a comorbidity does not modify cytokine responses and severity of COVID-19.

BACKGROUND: A significant portion of COVID-19 sufferers have asthma. The impacts of asthma on COVID-19 progression are still unclear but a modifying effect is plausible as respiratory viruses are acknowledged to be an important trigger for asthma exacerbations and a different, potentially type-2 biased, immune response might occur. In this study, we compared the blood circulating cytokine response to COVID-19 infection in patients with and without asthma. METHODS: Plasma samples and clinical information were collected from 80 patients with mild (25), severe (36) or critical (19) COVID-19 and 29 healthy subjects at the John Radcliffe Hospital, Oxford, UK. The concentrations of 51 circulating proteins in the plasma samples were measured with Luminex and compared between groups. RESULTS: Total 16 pre-existing asthma patients were found (3 in mild, 10 in severe, and 3 in critical COVID-19). The prevalence of asthma in COVID-19 severity groups did not suggest a clear correlation between asthma and COVID-19 severity. Within the same COVID-19 severity group, no differences were observed between patients with or without asthma on oxygen saturation, CRP, neutrophil counts, and length of hospital stay. The mortality in the COVID-19 patients with asthma (12.5%) was not higher than that in patients without asthma (17.2%). No significant difference was found between asthmatic and non-asthmatic in circulating cytokine response in different COVID-19 severity groups, including the cytokines strongly implicated in COVID-19 such as CXCL10, IL-6, CCL2, and IL-8. CONCLUSIONS: Pre-existing asthma was not associated with an enhanced cytokine response after COVID-19 infection, disease severity or mortality.

The Roles of Type 2 Cytotoxic T Cells in Inflammation, Tissue Remodeling, and Prostaglandin (PG) D2 Production Are Attenuated by PGD2 Receptor 2 Antagonism.

Human type 2 cytotoxic T (Tc2) cells are enriched in severe eosinophilic asthma and can contribute to airway eosinophilia. PGD2 and its receptor PGD2 receptor 2 (DP2) play important roles in Tc2 cell activation, including migration, cytokine production, and survival. In this study, we revealed novel, to our knowledge, functions of the PGD2/DP2 axis in Tc2 cells to induce tissue-remodeling effects and IgE-independent PGD2 autocrine production. PGD2 upregulated the expression of tissue-remodeling genes in Tc2 cells that enhanced the fibroblast proliferation and protein production required for tissue repair and myofibroblast differentiation. PGD2 stimulated Tc2 cells to produce PGD2 using the routine PGD2 synthesis pathway, which also contributed to TCR-dependent PGD2 production in Tc2 cells. Using fevipiprant, a specific DP2 antagonist, we demonstrated that competitive inhibition of DP2 not only completely blocked the cell migration, adhesion, proinflammatory cytokine production, and survival of Tc2 cells triggered by PGD2 but also attenuated the tissue-remodeling effects and autocrine/paracrine PGD2 production in Tc2 induced by PGD2 and other stimulators. These findings further confirmed the anti-inflammatory effect of fevipiprant and provided a better understanding of the role of Tc2 cells in the pathogenesis of asthma.

Neuromedin U: potential roles in immunity and inflammation.

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.

Correction: Synergistic activation of pro-inflammatory type-2 CD8 + T lymphocytes by lipid mediators in severe eosinophilic asthma.

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Synergistic activation of pro-inflammatory type-2 CD8+ T lymphocytes by lipid mediators in severe eosinophilic asthma.

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 CD8+CRTH2+ (Tc2) cells are enriched in blood and airways in severe eosinophilic asthma. Concentrations of prostaglandin D2 (PGD2) and cysteinyl leukotriene E4 (LTE4) are also increased in the airways of the same group of patients. In vitro PGD2 and LTE4 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.

Novel data analysis method for multicolour flow cytometry links variability of multiple markers on single cells to a clinical phenotype.

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.

Cysteinyl leukotriene E4 activates human group 2 innate lymphoid cells and enhances the effect of prostaglandin D2 and epithelial cytokines.

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.

Psoriatic T cells recognize neolipid antigens generated by mast cell phospholipase delivered by exosomes and presented by CD1a.

Psoriasis is a chronic inflammatory skin disease associated with a T helper 17 response. Yet, it has proved challenging to identify relevant peptide-based T cell antigens. Antigen-presenting Langerhans cells show a differential migration phenotype in psoriatic lesions and express constitutively high levels of CD1a, which presents lipid antigens to T cells. In addition, phospholipase A2 (PLA2) is highly expressed in psoriatic lesions and is known to generate neolipid skin antigens for recognition by CD1a-reactive T cells. In this study, we observed expression of a cytoplasmic PLA2 (PLA2G4D) in psoriatic mast cells but, unexpectedly, also found PLA2G4D activity to be extracellular. This was explained by IFN-α-induced mast cell release of exosomes, which transferred cytoplasmic PLA2 activity to neighboring CD1a-expressing cells. This led to the generation of neolipid antigens and subsequent recognition by lipid-specific CD1a-reactive T cells inducing production of IL-22 and IL-17A. Circulating and skin-derived T cells from patients with psoriasis showed elevated PLA2G4D responsiveness compared with healthy controls. Overall, these data present an alternative model of psoriasis pathogenesis in which lipid-specific CD1a-reactive T cells contribute to psoriatic inflammation. The findings suggest that PLA2 inhibition or CD1a blockade may have therapeutic potential for psoriasis.

Group 2 Innate Lymphoid Cells Express Functional NKp30 Receptor Inducing Type 2 Cytokine Production.

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.

Evidence for the efficacy and safety of anti-interleukin-5 treatment in the management of refractory eosinophilic asthma.

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.

Prostaglandin D2 and leukotriene E4 synergize to stimulate diverse TH2 functions and TH2 cell/neutrophil crosstalk.

BACKGROUND: Prostaglandin D2 (PGD2) and cysteinyl leukotrienes (cysLTs) are lipid mediators derived from mast cells, which activate TH2 cells. The combination of PGD2 and cysLTs (notably cysteinyl leukotriene E4 [LTE4]) enhances TH2 cytokine production. However, the synergistic interaction of cysLTs with PGD2 in promoting TH2 cell activation is still poorly understood. The receptors for these mediators are drug targets in the treatment of allergic diseases, and hence understanding their interaction is likely to have clinical implications. OBJECTIVE: We aimed to comprehensively define the roles of PGD2, LTE4, and their combination in activating human TH2 cells and how such activation might allow the TH2 cells to engage downstream effectors, such as neutrophils, which contribute to the pathology of allergic responses. METHODS: The effects of PGD2, LTE4, and their combination on human TH2 cell gene expression were defined by using a microarray, and changes in specific inflammatory pathways were confirmed by means of PCR array, quantitative RT-PCR, ELISA, Luminex, flow cytometry, and functional assays, including analysis of downstream neutrophil activation. Blockade of PGD2 and LTE4 was tested by using TM30089, an antagonist of chemoattractant receptor-homologous molecule expressed on TH2 cells, and montelukast, an antagonist of cysteinyl leukotriene receptor 1. RESULTS: PGD2 and LTE4 altered the transcription of a wide range of genes and induced diverse functional responses in TH2 cells, including cell adhesion, migration, and survival and cytokine production. The combination of these lipids synergistically or additively enhanced TH2 responses and, strikingly, induced marked production of diverse nonclassical TH2 inflammatory mediators, including IL-22, IL-8, and GM-CSF, at concentrations sufficient to affect neutrophil activation. CONCLUSIONS: PGD2 and LTE4 activate TH2 cells through different pathways but act synergistically to promote multiple downstream effector functions, including neutrophil migration and survival. Combined inhibition of both PGD2 and LTE4 pathways might provide an effective therapeutic strategy for allergic responses, particularly those involving interaction between TH2 cells and neutrophils, such as in patients with severe asthma.

Prostaglandin D2 activates group 2 innate lymphoid cells through chemoattractant receptor-homologous molecule expressed on TH2 cells.

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.