Increased CXCL10 (IP-10) associates with advanced myeloproliferative neoplasms and its loss dampens erythrocytosis in mouse models.

Key studies in pre-leukemic disorders have linked increases in pro-inflammatory cytokines with accelerated phases of disease, but the precise role of the cellular microenvironment in disease initiation and evolution remains poorly understood. In myeloproliferative neoplasms (MPNs), higher levels of specific cytokines have been previously correlated with increased disease severity (TNF-α, IP-10) and decreased survival (IL-8). Whereas TNF-α and IL-8 have been studied by numerous groups, there is a relative paucity of studies on IP-10 (CXCL10). Here we explore the relationship of IP-10 levels with detailed genomic and clinical data and undertake a complementary cytokine screen alongside functional assays in a wide range of MPN mouse models. Similar to patients, levels of IP-10 were increased in mice with more severe disease phenotypes (e.g., JAK2V617F/V617F TET2-/- double mutant mice) compared to those with less severe phenotypes (e.g., CALRdel52 or JAK2+/V617F mice) and WT littermate controls. While exposure to IP-10 did not directly alter proliferation or survival in single hematopoietic stem cells (HSCs) in vitro, IP-10-/- mice transplanted with disease initiating HSCs developed an MPN phenotype more slowly, suggesting that the effect of IP-10 loss was non-cell autonomous. To explore the broader effects of IP-10 loss, we crossed IP-10-/- mice into a series of MPN mouse models and show that its loss reduces the erythrocytosis observed in mice with the most severe phenotype. Together these data point to a potential role for blocking IP-10 activity in the management of MPNs.

Identification of aceNKPs, a committed common progenitor population of the ILC1 and NK cell continuum.

The development of innate lymphoid cell (ILC) transcription factor reporter mice has shown a previously unexpected complexity in ILC hematopoiesis. Using novel polychromic mice to achieve higher phenotypic resolution, we have characterized bone marrow progenitors that are committed to the group 1 ILC lineage. These common ILC1/NK cell progenitors (ILC1/NKP), which we call "aceNKPs", are defined as lineage-Id2+IL-7Rα+CD25-α4ß7-NKG2A/C/E+Bcl11b-. In vitro, aceNKPs differentiate into group 1 ILCs, including NK-like cells that express Eomes without the requirement for IL-15, and produce IFN-γ and perforin upon IL-15 stimulation. Following reconstitution of Rag2-/-Il2rg-/- hosts, aceNKPs give rise to a spectrum of mature ILC1/NK cells (regardless of their tissue location) that cannot be clearly segregated into the traditional ILC1 and NK subsets, suggesting that group 1 ILCs constitute a dynamic continuum of ILCs that can develop from a common progenitor. In addition, aceNKP-derived ILC1/NK cells effectively ameliorate tumor burden in a model of lung metastasis, where they acquired a cytotoxic NK cell phenotype. Our results identify the primary ILC1/NK progenitor that lacks ILC2 or ILC3 potential and is strictly committed to ILC1/NK cell production irrespective of tissue homing.

IL-6 effector function of group 2 innate lymphoid cells (ILC2) is NOD2 dependent.

Cutaneous group 2 innate lymphoid cells (ILC2) are spatially and epigenetically poised to respond to barrier compromise and associated immunological threats. ILC2, lacking rearranged antigen-specific receptors, are primarily activated by damage-associated cytokines and respond with type 2 cytokine production. To investigate ILC2 potential for direct sensing of skin pathogens and allergens, we performed RNA sequencing of ILC2 derived from in vivo challenged human skin or blood. We detected expression of NOD2 and TLR2 by skin and blood ILC2. Stimulation of ILC2 with TLR2 agonist alone not only induced interleukin-5 (IL-5) and IL-13 expression but also elicited IL-6 expression in combination with Staphylococcus aureus muramyl dipeptide (MDP). Heat-killed skin-resident bacteria provoked an IL-6 profile in ILC2 in vitro that was notably impaired in ILC2 derived from patients with nucleotide-binding oligomerization domain-containing protein 2 (NOD2) mutations. In addition, we show that NOD2 signaling can stimulate autophagy in ILC2, which was also impaired in patients with NOD2 mutations. Here, we have identified a role for ILC2 NOD2 signaling in the differential regulation of ILC2-derived IL-6 and have reported a previously unrecognized pathway of direct ILC2 bacterial sensing.

Mapping Rora expression in resting and activated CD4+ T cells.

The transcription factor Rora has been shown to be important for the development of ILC2 and the regulation of ILC3, macrophages and Treg cells. Here we investigate the role of Rora across CD4+ T cells in general, but with an emphasis on Th2 cells, both in vitro as well as in the context of several in vivo type 2 infection models. We dissect the function of Rora using overexpression and a CD4-conditional Rora-knockout mouse, as well as a RORA-reporter mouse. We establish the importance of Rora in CD4+ T cells for controlling lung inflammation induced by Nippostrongylus brasiliensis infection, and have measured the effect on downstream genes using RNA-seq. Using a systematic stimulation screen of CD4+ T cells, coupled with RNA-seq, we identify upstream regulators of Rora, most importantly IL-33 and CCL7. Our data suggest that Rora is a negative regulator of the immune system, possibly through several downstream pathways, and is under control of the local microenvironment.

RORα is a critical checkpoint for T cell and ILC2 commitment in the embryonic thymus.

Type 2 innate lymphoid cells (ILC2) contribute to immune homeostasis, protective immunity and tissue repair. Here we demonstrate that functional ILC2 cells can arise in the embryonic thymus from shared T cell precursors, preceding the emergence of CD4+CD8+ (double-positive) T cells. Thymic ILC2 cells migrated to mucosal tissues, with colonization of the intestinal lamina propria. Expression of the transcription factor RORα repressed T cell development while promoting ILC2 development in the thymus. From RNA-seq, assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation followed by sequencing (ChIP-seq) data, we propose a revised transcriptional circuit to explain the co-development of T cells and ILC2 cells from common progenitors in the thymus. When Notch signaling is present, BCL11B dampens Nfil3 and Id2 expression, permitting E protein-directed T cell commitment. However, concomitant expression of RORα overrides the repression of Nfil3 and Id2 repression, allowing ID2 to repress E proteins and promote ILC2 differentiation. Thus, we demonstrate that RORα expression represents a critical checkpoint at the bifurcation of the T cell and ILC2 lineages in the embryonic thymus.

Group-2 innate lymphoid cell-dependent regulation of tissue neutrophil migration by alternatively activated macrophage-secreted Ear11.

Type-2 immunity is characterised by interleukin (IL)-4, IL-5 and IL-13, eosinophilia, mucus production, IgE, and alternatively activated macrophages (AAM). However, despite the lack of neutrophil chemoattractants such as CXCL1, neutrophils, a feature of type-1 immunity, are observed in type-2 responses. Consequently, alternative mechanisms must exist to ensure that neutrophils can contribute to type-2 immune reactions without escalation of deleterious inflammation. We now demonstrate that type-2 immune-associated neutrophil infiltration is regulated by the mouse RNase A homologue, eosinophil-associated ribonuclease 11 (Ear11), which is secreted by AAM downstream of IL-25-stimulated ILC2. Transgenic overexpression of Ear11 resulted in tissue neutrophilia, whereas Ear11-deficient mice have fewer resting tissue neutrophils, whilst other type-2 immune responses are not impaired. Notably, administration of recombinant mouse Ear11 increases neutrophil motility and recruitment. Thus, Ear11 helps maintain tissue neutrophils at homoeostasis and during type-2 reactions when chemokine-producing classically activated macrophages are infrequently elicited.

Re-evaluation of human BDCA-2+ DC during acute sterile skin inflammation.

Plasmacytoid dendritic cells (pDCs) produce type I interferon (IFN-I) and are traditionally defined as being BDCA-2+CD123+. pDCs are not readily detectable in healthy human skin, but have been suggested to accumulate in wounds. Here, we describe a CD1a-bearing BDCA-2+CD123int DC subset that rapidly infiltrates human skin wounds and comprises a major DC population. Using single-cell RNA sequencing, we show that these cells are largely activated DCs acquiring features compatible with lymph node homing and antigen presentation, but unexpectedly express both BDCA-2 and CD123, potentially mimicking pDCs. Furthermore, a third BDCA-2-expressing population, Axl+Siglec-6+ DCs (ASDC), was also found to infiltrate human skin during wounding. These data demonstrate early skin infiltration of a previously unrecognized CD123intBDCA-2+CD1a+ DC subset during acute sterile inflammation, and prompt a re-evaluation of previously ascribed pDC involvement in skin disease.

A stromal cell niche sustains ILC2-mediated type-2 conditioning in adipose tissue.

Group-2 innate lymphoid cells (ILC2), type-2 cytokines, and eosinophils have all been implicated in sustaining adipose tissue homeostasis. However, the interplay between the stroma and adipose-resident immune cells is less well understood. We identify that white adipose tissue-resident multipotent stromal cells (WAT-MSCs) can act as a reservoir for IL-33, especially after cell stress, but also provide additional signals for sustaining ILC2. Indeed, we demonstrate that WAT-MSCs also support ICAM-1-mediated proliferation and activation of LFA-1-expressing ILC2s. Consequently, ILC2-derived IL-4 and IL-13 feed back to induce eotaxin secretion from WAT-MSCs, supporting eosinophil recruitment. Thus, MSCs provide a niche for multifaceted dialogue with ILC2 to sustain a type-2 immune environment in WAT.

Polychromic Reporter Mice Reveal Unappreciated Innate Lymphoid Cell Progenitor Heterogeneity and Elusive ILC3 Progenitors in Bone Marrow.

Innate lymphoid cells (ILCs) play strategic roles in tissue homeostasis and immunity. ILCs arise from lymphoid progenitors undergoing lineage restriction and the development of specialized ILC subsets. We generated "5x polychromILC" transcription factor reporter mice to delineate ILC precursor states by revealing the multifaceted expression of key ILC-associated transcription factors (Id2, Bcl11b, Gata3, RORγt, and RORα) during ILC development in the bone marrow. This approach allowed previously unattained enrichment of rare progenitor subsets and revealed hitherto unappreciated ILC precursor heterogeneity. In vivo and in vitro assays identified precursors with potential to generate all ILC subsets and natural killer (NK) cells, and also permitted discrimination of elusive ILC3 bone marrow antecedents. Single-cell gene expression analysis identified a discrete ILC2-committed population and delineated transition states between early progenitors and a highly heterogeneous ILC1, ILC3, and NK precursor cell cluster. This diversity might facilitate greater lineage potential upon progenitor recruitment to peripheral tissues.

BET Bromodomain Inhibitor iBET151 Impedes Human ILC2 Activation and Prevents Experimental Allergic Lung Inflammation.

Group 2 innate lymphoid cells (ILC2) increase in frequency in eczema and allergic asthma patients, and thus represent a new therapeutic target cell for type-2 immune-mediated disease. The bromodomain and extra-terminal (BET) protein family of epigenetic regulators are known to support the expression of cell cycle and pro-inflammatory genes during type-1 inflammation, but have not been evaluated in type-2 immune responses. We isolated human ILC2 and examined the capacity of the BET protein inhibitor, iBET151, to modulate human ILC2 activation following IL-33 stimulation. iBET151 profoundly blocked expression of genes critical for type-2 immunity, including type-2 cytokines, cell surface receptors and transcriptional regulators of ILC2 differentiation and activation. Furthermore, in vivo administration of iBET151 during experimental mouse models of allergic lung inflammation potently inhibited lung inflammation and airways resistance in response to cytokine or allergen exposure. Thus, iBET151 effectively prevents human ILC2 activation and dampens type-2 immune responses.

Innate Lymphoid Cells of the Lung.

Although, as the major organ of gas exchange, the lung is considered a nonlymphoid organ, an interconnected network of lung-resident innate cells, including epithelial cells, dendritic cells, macrophages, and natural killer cells is crucial for its protection. These cells provide defense against a daily assault by airborne bacteria, viruses, and fungi, as well as prevent the development of cancer, allergy, and the outgrowth of commensals. Our understanding of this innate immune environment has recently changed with the discovery of a family of innate lymphoid cells (ILCs): ILC1s, ILC2s, and ILC3s. All lack adaptive antigen receptors but can provide a substantial and rapid source of IFN-γ, IL-5 and IL-13, and IL-17A or IL-22, respectively. Their ability to afford immediate protection to the lung and to influence subsequent adaptive immune responses highlights the importance of understanding ILC-regulated immunity for the design of future therapeutic interventions.

Roles for T/B lymphocytes and ILC2s in experimental chronic obstructive pulmonary disease.

Pulmonary inflammation in chronic obstructive pulmonary disease (COPD) is characterized by both innate and adaptive immune responses; however, their specific roles in the pathogenesis of COPD are unclear. Therefore, we investigated the roles of T and B lymphocytes and group 2 innate lymphoid cells (ILC2s) in airway inflammation and remodelling, and lung function in an experimental model of COPD using mice that specifically lack these cells (Rag1-/- and Rorafl/fl Il7rCre [ILC2-deficient] mice). Wild-type (WT) C57BL/6 mice, Rag1-/- , and Rorafl/fl Il7rCre mice were exposed to cigarette smoke (CS; 12 cigarettes twice a day, 5 days a week) for up to 12 weeks, and airway inflammation, airway remodelling (collagen deposition and alveolar enlargement), and lung function were assessed. WT, Rag1-/- , and ILC2-deficient mice exposed to CS had similar levels of airway inflammation and impaired lung function. CS exposure increased small airway collagen deposition in WT mice. Rag1-/- normal air- and CS-exposed mice had significantly increased collagen deposition compared to similarly exposed WT mice, which was associated with increases in IL-33, IL-13, and ILC2 numbers. CS-exposed Rorafl/fl Il7rCre mice were protected from emphysema, but had increased IL-33/IL-13 expression and collagen deposition compared to WT CS-exposed mice. T/B lymphocytes and ILC2s play roles in airway collagen deposition/fibrosis, but not inflammation, in experimental COPD.

CD1a presentation of endogenous antigens by group 2 innate lymphoid cells.

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.

MHCII-mediated dialog between group 2 innate lymphoid cells and CD4(+) T cells potentiates type 2 immunity and promotes parasitic helminth expulsion.

Group 2 innate lymphoid cells (ILC2s) release interleukin-13 (IL-13) during protective immunity to helminth infection and detrimentally during allergy and asthma. Using two mouse models to deplete ILC2s in vivo, we demonstrate that T helper 2 (Th2) cell responses are impaired in the absence of ILC2s. We show that MHCII-expressing ILC2s interact with antigen-specific T cells to instigate a dialog in which IL-2 production from T cells promotes ILC2 proliferation and IL-13 production. Deletion of MHCII renders IL-13-expressing ILC2s incapable of efficiently inducing Nippostrongylus brasiliensis expulsion. Thus, during transition to adaptive T cell-mediated immunity, the ILC2 and T cell crosstalk contributes to their mutual maintenance, expansion and cytokine production. This interaction appears to augment dendritic-cell-induced T cell activation and identifies a previously unappreciated pathway in the regulation of type-2 immunity.

Type-2 innate lymphoid cells in human allergic disease.

PURPOSE OF REVIEW: Recent decades have seen allergic diseases become endemic in a number of developed countries. Understanding the inflammatory processes that dictate these allergic responses is therefore important. RECENT FINDINGS: Critical to many allergic responses is the inappropriate release of the type-2 immune-regulatory cytokines: interleukin-4, interleukin-5, interleukin-9, and interleukin-13. The study of these inflammatory mediators has led directly to the development of two new asthma treatments: anti-interleukin-5 and anti-interleukin-13. Until recently, T helper 2 cells were considered to be the major cellular source of type-2 cytokines; however, a paradigm shift occurred with the discovery of a novel population, type-2 innate lymphoid cells (ILC2s), that can produce huge levels of type-2 cytokines and are sufficient to induce allergy in mice. This discovery raises interesting questions about how innate and adaptive type-2 immunity might interact to induce relapsing and remitting episodes of allergy in patients. SUMMARY: It is essential that alongside the mechanistic investigation using model organisms, the roles of ILC2s in human disease be explored. Here, we discuss how ILC2 traits, discovered in mouse models, have informed research in humans and how newly identified human ILC2 pathways might provide potential therapeutic benefits in the future.

IL-25 and type 2 innate lymphoid cells induce pulmonary fibrosis.

Disease conditions associated with pulmonary fibrosis are progressive and have a poor long-term prognosis with irreversible changes in airway architecture leading to marked morbidity and mortalities. Using murine models we demonstrate a role for interleukin (IL)-25 in the generation of pulmonary fibrosis. Mechanistically, we identify IL-13 release from type 2 innate lymphoid cells (ILC2) as sufficient to drive collagen deposition in the lungs of challenged mice and suggest this as a potential mechanism through which IL-25 is acting. Additionally, we demonstrate that in human idiopathic pulmonary fibrosis there is increased pulmonary expression of IL-25 and also observe a population ILC2 in the lungs of idiopathic pulmonary fibrosis patients. Collectively, we present an innate mechanism for the generation of pulmonary fibrosis, via IL-25 and ILC2, that occurs independently of T-cell-mediated antigen-specific immune responses. These results suggest the potential of therapeutically targeting IL-25 and ILC2 for the treatment of human fibrotic diseases.

A role for IL-25 and IL-33-driven type-2 innate lymphoid cells in atopic dermatitis.

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.

A Trypanosoma brucei kinesin heavy chain promotes parasite growth by triggering host arginase activity.

BACKGROUND: In order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells. METHODOLOGY/PRINCIPAL FINDINGS: By screening a cDNA library of T. brucei with an antibody neutralizing the arginase-inducing activity of parasite released factors, we identified a Kinesin Heavy Chain isoform, termed TbKHC1, as responsible for this effect. Following interaction with mouse myeloid cells, natural or recombinant TbKHC1 triggered SIGN-R1 receptor-dependent induction of IL-10 production, resulting in arginase-1 activation concomitant with reduction of nitric oxide (NO) synthase activity. This TbKHC1 activity was IL-4Rα-independent and did not mirror M2 activation of myeloid cells. As compared to wild-type T. brucei, infection by TbKHC1 KO parasites was characterized by strongly reduced parasitaemia and prolonged host survival time. By treating infected mice with ornithine or with NO synthase inhibitor, we observed that during the first wave of parasitaemia the parasite growth-promoting effect of TbKHC1-mediated arginase activation resulted more from increased polyamine production than from reduction of NO synthesis. In late stage infection, TbKHC1-mediated reduction of NO synthesis appeared to contribute to liver damage linked to shortening of host survival time. CONCLUSION: A kinesin heavy chain released by T. brucei induces IL-10 and arginase-1 through SIGN-R1 signaling in myeloid cells, which promotes early trypanosome growth and favors parasite settlement in the host. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 contributes to liver pathogenicity.

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.

Innate lymphoid cells--how did we miss them?

Innate lymphoid cells (ILCs) are newly identified members of the lymphoid lineage that have emerging roles in mediating immune responses and in regulating tissue homeostasis and inflammation. Here, we review the developmental relationships between the various ILC lineages that have been identified to date and summarize their functions in protective immunity to infection and their pathological roles in allergic and autoimmune diseases.