Clare S. Hardman

Transcription factor ROR[alpha] is critical for nuocyte development

Nuocytes are essential in innate type 2 immunity and contribute to the exacerbation of asthma responses. Here we found that nuocytes arose in the bone marrow and differentiated from common lymphoid progenitors, which indicates they are distinct, previously unknown members of the lymphoid lineage. Nuocytes required interleukin 7 (IL-7), IL-33 and Notch signaling for development in vitro. Pro-T cell progenitors at double-negative stage 1 (DN1) and DN2 maintained nuocyte potential in vitro, although the thymus was not essential for nuocyte development. Notably, the transcription factor RORα was critical for the development of nuocytes and their role in the expulsion of parasitic worms.Nuocytes are essential in innate type-2 immunity and contribute to the exacerbation of asthma responses. Here we show that nuocytes arise in the bone marrow and differentiate from common lymphoid progenitors, which makes them distinct new members of the lymphoid lineage. Nuocytes required interleukin 7 (IL-7), IL-33 and Notch signalling for development in vitro. Double negative 1 (DN1) and DN2 pro-T-cell progenitors maintained nuocyte potential in vitro, although the thymus was not essential for nuocyte development. Notably, the transcription factor Rorα was critical for nuocyte development and their role in parasitic worm expulsion.

Innate IL-13–producing nuocytes arise during allergic lung inflammation and contribute to airways hyperreactivity

BACKGROUND IL-4, IL-5, and IL-13 are thought to be central to the allergic asthmatic response. Previous work supposed that the essential source of these cytokines was CD4(+) T(H)2 cells. However, more recent studies have suggested that other innate production of type 2 cytokines might be as important. OBJECTIVES Nuocytes are a novel population of IL-13-producing innate cells, which are critical for protective immunity in Nippostrongylus brasiliensis infection. Given this, we investigated the potential existence and functional importance of nuocytes in experimental allergic asthma. METHODS We generated Il4(+/eGFP)Il13(+/Tomato) dual-reporter mice to study cytokine-producing cells during allergic inflammation. We adoptively transferred innate IL-13-producing cells to investigate their role in airways hyperreactivity (AHR). RESULTS We show that allergen-induced nuocytes infiltrate the lung and are a major innate source of IL-13. CD4(+) T cells in the lung almost exclusively express only IL-13, whereas IL-4-producing T cells were restricted to the draining lymph nodes. Intranasal administration of IL-25 or IL-33 induced IL-13-producing nuocytes in the BAL fluid. Strikingly, adoptive transfer of wild-type nuocytes, but not Il13(-/-) nuocytes, into Il13(-/-) mice, which are normally resistant to IL-25-induced AHR, restored airways resistance and lung cell infiltration. CONCLUSIONS These findings identify nuocytes as a novel cell type in allergic lung inflammation and an innate source of IL-13 that can directly induce AHR in the absence of IL-13-producing CD4(+) T cells. These data highlight nuocytes as an important new consideration in the development of future allergic asthma therapy.

IL-33 is more potent than IL-25 in provoking IL-13–producing nuocytes (type 2 innate lymphoid cells) and airway contraction

BACKGROUND IL-25 and IL-33 belong to distinct cytokine families, but experimental mouse studies suggest their immunologic functions in type 2 immunity are almost entirely overlapping. However, only polymorphisms in the IL-33 pathway (IL1RL1 and IL33) have been significantly associated with asthma in large-cohort genome-wide association studies. OBJECTIVE We sought to identify distinct pathways for IL-25 and IL-33 in the lung that might provide insight into their roles in asthma pathogenesis and potential for therapeutic intervention. METHODS IL-25 receptor-deficient (Il17rb(-/-)), IL-33 receptor-deficient (ST2, Il1rl1(-/-)), and double-deficient (Il17rb(-/-)Il1rl1(-/-)) mice were analyzed in models of allergic asthma. Microarrays, an ex vivo lung slice airway contraction model, and Il13(+/eGFP) mice were then used to identify specific effects of IL-25 and IL-33 administration. RESULTS Comparison of IL-25 and IL-33 pathway-deficient mice demonstrates that IL-33 signaling plays a more important in vivo role in airways hyperreactivity than IL-25. Furthermore, methacholine-induced airway contraction ex vivo increases after treatment with IL-33 but not IL-25. This is dependent on expression of the IL-33 receptor and type 2 cytokines. Confocal studies with Il13(+/eGFP) mice show that IL-33 more potently induces expansion of IL-13-producing type 2 innate lymphoid cells, correlating with airway contraction. This predominance of IL-33 activity is enforced in vivo because IL-33 is more rapidly expressed and released in comparison with IL-25. CONCLUSION Our data demonstrate that IL-33 plays a critical role in the rapid induction of airway contraction by stimulating the prompt expansion of IL-13-producing type 2 innate lymphoid cells, whereas IL-25-induced responses are slower and less potent.

IL-33 citrine reporter mice reveal the temporal and spatial expression of IL-33 during allergic lung inflammation

Interleukin‐33 (IL‐33) is an IL‐1 family cytokine that signals via its receptor T1/ST2, and is a key regulator of inflammation, notably the type‐2 response implicated in allergic asthma. Critical to our understanding of the role of IL‐33 is the identification of the cellular sources of IL‐33. Although progress has been made in this area, the development of a robust live cell reporter of expression would allow the localisation of IL‐33 during ongoing immune responses. We have generated a fluorescent reporter mouse line, Il33Cit/+, to define the expression profile of IL‐33 in vivo and demonstrate its temporal and spatial expression during experimental allergic asthma responses. We found that type‐2 pneumocytes constitute the major source of IL‐33 upon allergic lung inflammation following exposure to OVA, fungal extract or ragweed pollen. Using Il33Cit/Cit mice (IL‐33‐deficient), we establish a role for IL‐33 early in the initiation of type‐2 responses and the induction of nuocytes (ILC2). We also demonstrate a potential mechanism of action by which IL‐33 rapidly initiates type‐2 immune responses. Il33Cit/+ mice have enabled new insights into the initiation of type‐2 responses and will provide an important tool for further dissection of this important inflammatory pathway in vivo.

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

In psoriasis, IFN-α–stimulated mast cells release exosomes containing cytoplasmic PLA2 that are transferred to CD1a-expressing cells and generate neolipid antigens which induce the production of IL-22 and IL-17A by CD1a-reactive T cells.

Filaggrin inhibits generation of CD1a neolipid antigens by house dust mite–derived phospholipase

Lack of the skin barrier protein filaggrin worsens atopic dermatitis by allergenic activation of CD1a-reactive T cells. Bringing atopic dermatitis up to scratch Targeted therapies are transforming medicine, but complex diseases such as atopic dermatitis are difficult to target. Now, Jarrett et al. report a mechanism that links two contributors to atopic dermatitis pathogenesis—cutaneous inflammation and barrier dysfunction. They found that house dust mite allergen phospholipase (PLA2) can induce neolipid antigens in human skin. These antigens can then be presented by the nonclassical MHC family member CD1a to CD1a-restricted T cells, which contribute to inflammation. The skin barrier protein filaggrin can inhibit PLA2 and decrease this inflammation. Indeed, individuals with filaggrin mutations experience severe atopic dermatitis. These data suggest that barrier dysfunction and inflammation may be linked, and support PLA2 as a target for atopic dermatitis. Atopic dermatitis is a common pruritic skin disease in which barrier dysfunction and cutaneous inflammation contribute to pathogenesis. Mechanisms underlying the associated inflammation are not fully understood, and although Langerhans cells expressing the nonclassical major histocompatibility complex (MHC) family member CD1a are known to be enriched within lesions, their role in clinical disease pathogenesis has not been studied. We observed that house dust mite (HDM) allergen generates neolipid antigens presented by CD1a to T cells in the blood and skin lesions of affected individuals. HDM-responsive CD1a-reactive T cells increased in frequency after birth in individuals with atopic dermatitis and showed rapid effector function, consistent with antigen-driven maturation. In HDM-challenged human skin, we observed phospholipase A2 (PLA2) activity in vivo. CD1a-reactive T cell activation was dependent on HDM-derived PLA2, and such cells infiltrated the skin after allergen challenge. Moreover, we observed that the skin barrier protein filaggrin, insufficiency of which is associated with atopic skin disease, inhibited PLA2 activity and decreased CD1a-reactive PLA2-generated neolipid-specific T cell activity from skin and blood. The most widely used classification schemes of hypersensitivity suggest that nonpeptide stimulants of T cells act as haptens that modify peptides or proteins; however, our results show that HDM proteins may also generate neolipid antigens that directly activate T cells. These data define PLA2 inhibition as a function of filaggrin, supporting PLA2 inhibition as a therapeutic approach.

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.

Interleukin-33, friend and foe in type-2 immune responses.

IL-33 is the most recent addition to the IL-1 cytokine family, identified in 2005 as the ligand of T1/ST2 and inducer of type-2 immune responses. IL-33 has been implicated in a wide range of disease settings, in anti-inflammatory responses and homeostasis, and thus signalling must be strictly regulated. Altered gene expression, post-translational modification, decoy receptor, and receptor signalling are all modulatory mechanisms used to control the IL-33 pathway. Understanding both the genetic and post-translational factors influencing IL-33 activity will be critical for provision of safe effective treatment of type-2 disorders.

CD1a presentation of endogenous antigens by group 2 innate lymphoid cells

Human skin–derived ILC2 express CD1a and present endogenous PLA2G4A-dependent antigens to T cells. A new look at lipid surveillance Human group 2 innate lymphoid cells (ILC2) play roles in maintaining homeostasis and defending against pathogens, but dysregulated ILC2 responses have been linked to asthma and allergic responses. Hardman et al. now use an in vivo human skin challenge model to show that ILC2 express CD1a, which is regulated by TSLP, and that CD1a+ILC2 can present endogenous lipid antigens to CD1a-reactive T cells and induce inflammatory responses. CD1a+ILC2 expressed the phospholipase PLA2G4A, which contributed to CD1a-mediated T cell activation, and this pathway was involved in sensing Staphylococcus aureus–associated skin inflammation. These results provide insight into lipid sensing by skin-resident ILC2 and how this pathway may contribute to atopic skin inflammation and pathogen surveillance. Group 2 innate lymphoid cells (ILC2) are effectors of barrier immunity, with roles in infection, wound healing, and allergy. A proportion of ILC2 express MHCII (major histocompatibility complex II) and are capable of presenting peptide antigens to T cells and amplifying the subsequent adaptive immune response. Recent studies have highlighted the importance of CD1a-reactive T cells in allergy and infection, activated by the presentation of endogenous neolipid antigens and bacterial components. Using a human skin challenge model, we unexpectedly show that human skin–derived ILC2 can express CD1a and are capable of presenting endogenous antigens to T cells. CD1a expression is up-regulated by TSLP (thymic stromal lymphopoietin) at levels observed in the skin of patients with atopic dermatitis, and the response is dependent on PLA2G4A. Furthermore, this pathway is used to sense Staphylococcus aureus by promoting Toll-like receptor–dependent CD1a-reactive T cell responses to endogenous ligands. These findings define a previously unrecognized role for ILC2 in lipid surveillance and identify shared pathways of CD1a- and PLA2G4A-dependent ILC2 inflammation amenable to therapeutic intervention.

Role of NS1 antibodies in the pathogenesis of acute dengue infection

The role of NS1-specific antibodies in the pathogenesis of dengue virus infection is of particular interest to the dengue field, yet remains poorly understood. We therefore investigated the immunoglobulin responses of patients with dengue fever (DF) and dengue hemorrhagic fever (DHF) to NS1. Antibody responses to recombinant-NS1 were assessed in serum samples obtained throughout illness of patients with acute secondary DENV1 and DENV2 infection by ELISA. NS1 antibody titres were significantly higher in patients with DHF compared to those with DF for both serotypes, during the critical phase of illness. Antibody responses were further assessed to NS1 peptides and showed that in both acute secondary DENV1 and DENV2 infection, the antibody repertoire of DF and DHF patients is directed towards distinct regions of the NS1 protein. Further experiments in healthy individuals, with either past severe dengue or past asymptomatic dengue infection revealed that individuals with past inapparent disease mounted antibody responses directed to the same NS1 epitope regions as those with mild acute infection (DF). Our results suggest that the specific epitope target of NS1-antibodies generated by patients could predict disease severity and be of potential therapeutic benefit in aiding vaccine and treatment design.