Activation Of Innate Lymphoid Cells Research Articles

Central domain of IL-33 is cleaved by mast cell proteases for potent activation of group-2 innate lymphoid cells.

Interleukin-33 (IL-33) is an alarmin cytokine from the IL-1 family. IL-33 activates many immune cell types expressing the interleukin 1 receptor-like 1 (IL1RL1) receptor ST2, including group-2 innate lymphoid cells (ILC2s, natural helper cells, nuocytes), the major producers of IL-5 and IL-13 during type-2 innate immune responses and allergic airway inflammation. IL-33 is likely to play a critical role in asthma because the IL33 and ST2/IL1RL1 genes have been reproducibly identified as major susceptibility loci in large-scale genome-wide association studies. A better understanding of the mechanisms regulating IL-33 activity is thus urgently needed. Here, we investigated the role of mast cells, critical effector cells in allergic disorders, known to interact with ILC2s in vivo. We found that serine proteases secreted by activated mast cells (chymase and tryptase) generate mature forms of IL-33 with potent activity on ILC2s. The major forms produced by mast cell proteases, IL-33(95-270), IL-33(107-270), and IL-33(109-270), were 30-fold more potent than full-length human IL-33(1-270) for activation of ILC2s ex vivo. They induced a strong expansion of ILC2s and eosinophils in vivo, associated with elevated concentrations of IL-5 and IL-13. Murine IL-33 is also cleaved by mast cell tryptase, and a tryptase inhibitor reduced IL-33-dependent allergic airway inflammation in vivo. Our study identifies the central cleavage/activation domain of IL-33 (amino acids 66-111) as an important functional domain of the protein and suggests that interference with IL-33 cleavage and activation by mast cell and other inflammatory proteases could be useful to reduce IL-33-mediated responses in allergic asthma and other inflammatory diseases.

Rapid fucosylation of intestinal epithelium sustains host-commensal symbiosis in sickness.

Systemic infection induces conserved physiological responses that include both resistance and ‘tolerance of infection’ mechanisms1. Temporary anorexia associated with an infection is often beneficial2,3 reallocating energy from food foraging towards resistance to infection4 or depriving pathogens of nutrients 5. It imposes, however, a stress on intestinal commensals, as they also experience reduced substrate availability and impacting host fitness due to the loss of caloric intake and colonization resistance (protection from additional infections)6. We hypothesized that the host might utilize internal resources to support the gut microbiota during the acute phase of the disease. Here we show that systemic exposure to Toll-like receptor (TLR) ligands causes rapid α1,2-fucosylation of the small intestine epithelial cells (IEC), which requires sensing of TLR agonists and production of IL-23 by dendritic cells, activation of innate lymphoid cells and expression of α1,2-Fucosyltransferase-2 (Fut2) by IL-22-stimulated IECs. Fucosylated proteins are shed into the lumen and fucose is liberated and metabolized by the gut microbiota, as shown by reporter bacteria and community-wide analysis of microbial gene expression. Fucose affects the expression of microbial metabolic pathways and reduces the expression of bacterial virulence genes. It also improves host tolerance of the mild pathogen Citrobacter rodentium. Thus, rapid IEC fucosylation appears to be a protective mechanism that utilizes the host's resources to maintain host-microbial interactions during pathogen-induced stress.

Sa1769 TRIF Confers Expansion of Unique Innate Lymphoid Cell Populations During Enteric Infection With Gram-Negative Bacteria

Background: Increasing evidence has highlighted an important contribution of innate lymphoid cells (ILCs) to host defense against enteric pathogens. We have found that mice deficient in TIR-domain-containing adapter-inducing interferon-β (TRIF) (TrifLPS2 mice) have an increased susceptibility to Gram-negative enteropathogens due to defective induction of IFNs in macrophages and natural killer (NK) cells. Since other subsets of ILCs also express IFN-γ, this study examined the role of TRIF signaling in the regulation of IFN-γ expressing ILCs during enteric infection with a Gram-negative enteropathogen Yersinia enterocolitica. Method: WT and TrifLPS2 mice were orally infected with Y. enterocolitica. Four days post infection, IFN-γ expressing ILCs were analyzed in the mesenteric lymph nodes (MLN), Peyer's patches (PPs), and the small intestinal lamina propria by flow cytometry. The single cell suspensions of mononuclear cells were isolated from these organs and incubated with PMA and ionomycin for four hours in the presense of golgiplug. Specific surface markers and intracellular cytokines were stained. IFN-γ expressing ILCs were characterized by NKp46+IL-17-IL-22cells (include NK cells: ILC1), NKp46+IL-22+ cells (IL-22+ILC3), and NKp46-IL-17+ cells (IL-17+ILC3) within IFN-γ+CD3population. The expression of cytokines that are involved in the activation of ILCs (IL-12p35, IL-23p19, and IL-15) and IFN-β were analyzed in the MLN and PPs by real-time PCR. Results: WT mice had significantly greater population of IFN-γ+CD3cells in the MLN and PPs compared to TrifLPS2 mice: the population of these cells in the small intestine was similar between WT and TrifLPS2 mice. In the MLN, ILC1 was dominant in IFN-γ+CD3population, which was significantly greater in WT mice compared to TrifLPS2 mice. Whereas the number of ILC3 (both IL-22+ and IL-17+) in the MLN was similar between WT and TrifLPS2 mice. On the other hand, IL-22+ILC3 was more abundant in the small intestine than other ILCs, which was greater in TrifLPS2 mice compared to WT mice: there were no differences between WT and TrifLPS2 mice in the number of ILC1 or IL-17+ILC3. In the PPs, ILC1 was more abundant than ILC3. IL-22+ILC3 population in the PPs was slightly greater in TrifLPS2 mice than WT mice. Defective expression of IFN-γ, IL-12p35, and IL-15 in the MLN and PPs was found in TrifLPS2 mice suggesting the involvement of these cytokines in observed differences in ILC expansion. Conclusion: Taken together, TRIF signaling regulates IFN-γ expressing ILC1 expansion in the MLN and IL-22+ILC3 expansion in the small intestine and PPs during enteric infection with Y. enterocolitica. Establishing the role of TRIF signaling in intestinal ILC expansion may contribute to the managements of infectious as well as inflammatory diseases in the intestine.

Innate and adaptive type 2 immune cell responses in genetically controlled resistance to intestinal helminth infection.

The nematode Heligmosomoides polygyrus is an excellent model for intestinal helminth parasitism. Infection in mice persists for varying lengths of time in different inbred strains, with CBA and C57BL/6 mice being fully susceptible, BALB/c partially so and SJL able to expel worms within 2–3 weeks of infection. We find that resistance correlates not only with the adaptive Th2 response, including IL-10 but with activation of innate lymphoid cell and macrophage populations. In addition, the titer and specificity range of the serum antibody response is maximal in resistant mice. In susceptible strains, Th2 responses were found to be counterbalanced by IFN-γ-producing CD4+ and CD8+ cells, but these are not solely responsible for susceptibility as mice deficient in either CD8+ T cells or IFN-γ remain unable to expel the parasites. Foxp3+ Treg numbers were comparable in all strains, but in the most resistant SJL strain, this population does not upregulate CD103 in infection, and in the lamina propria the frequency of Foxp3+CD103+ T cells is significantly lower than in susceptible mice. The more resistant SJL and BALB/c mice develop macrophage-rich IL-4Rα-dependent Type 2 granulomas around intestinal sites of larval invasion, and expression of alternative activation markers Arginase-1, Ch3L3 (Ym1) and RELM-α within the intestine and the peritoneal lavage was also strongly correlated with helminth elimination in these strains. Clodronate depletion of phagocytic cells compromises resistance of BALB/c mice and slows expulsion in the SJL strain. Thus, Type 2 immunity involves IL-4Rα-dependent innate cells including but not limited to a phagocyte population, the latter likely involving the action of specific antibodies.

Interleukin-23 is sufficient to induce rapid de novo gut tumorigenesis, independent of carcinogens, through activation of innate lymphoid cells.

Chronic inflammation has been associated with increased risk for developing gastrointestinal cancer. Interleukin-23 (IL-23) receptor signaling has been correlated with inflammatory bowel disease pathogenesis, as well as promotion of tumor growth. However, little is known about the relative potential for IL-23-directed causality in gut tumorigenesis. We report that IL-23 transgene expression was sufficient to induce rapid (3-4 weeks) de novo development of intestinal adenomas with 100% incidence. Initiation of tumorigenesis was independent of exogenous carcinogens, Helicobacter colonization, or pre-existing tumor-suppressor gene mutations. Tumorigenesis was mediated by Thy1(+)IL-23R(+) innate lymphoid cells (ILC3), in part, through IL-17 responses as tumor development was inhibited in RAG(-/-) × IL-17(-/-) double knockout mice. Remarkably, IL-23 initiation of tumorigenesis by resident ILCs consistently occurred before recruitment of conspicuous inflammatory infiltrates. Our results reveal an explicit role for IL-23-mediated initiation of gut tumorigenesis and implicate a key role for IL-23R(+) ILC3 in the absence of overt cellular infiltrate recruitment.

Death Receptor 3 (DR3) Is Expressed By Innate Lymphoid Cells (ILC) and Ligation By Tumor Like Antigen-1 (TL1A) Leads To Costimulation and Significant ILC Expansion

ROR-gt expressing innate lymphoid cells (ILCs) are unique lymphocytes that play important roles in the immune system and have applications to hematopoietic cell transplantation. In fetal life, ILCs orchestrate lymph node organogenesis, while in adults they facilitate the repair of damaged lymphoid structures and mediate mucosal immunity. ILCs function through the production of IL-22 and the expression of TNF-superfamily molecules (lymphotoxin, BAFF, and OX40L) that act on stroma or other lymphocytes, respectively. ILCs are almost exclusively found in the secondary lymphoid tissues (i.e., lymph nodes) and are essentially absent from the peripheral blood, making the study of these cells difficult and clinical application nearly impossible. To overcome these limitations, we devised a method to generate ILCs from hematopoietic stem cells (HSCs) cultured on irradiated stroma in the presence of cytokines (IL-7, IL-15, SCF and FLT3L) [Blood 11:4052-5, 2011]. After ∼21 days of culture, functional ILCs and conventional NK cells differentiate. While this system robustly leads to the ILCs differentiation, GMP-compatible methods of expansion will be required for clinical translation. We sought to identify factors involved in ILC growth and proliferation. After screening gene expression arrays on purified ILCs, the TNF superfamily receptor, known as Death Receptor 3 (DR3, TNFRSF25) was selected for further study. DR3 expression was confirmed using quantitative PCR. Compared to cNK cells, ILCs expressed significant quantities of mRNA for DR3 (p<0.001). While DR3 contains death receptor signaling domains, under some conditions it can also mediate T cell growth. We assessed DR3 functionality in purified ILCs by stimulating them with recombinant TL1A (TNSF15), the only reported ligand for DR3. Consistent with known effect of TL1A on NF-kB activation in T cells, NK-kB phosphorylation was also observed in ILCs. To determine the impact of TL1A on ILC function, IL-22 production was tested. When TL1A was added to ILCs, there was surprisingly no IL-22 production by intracellular cytokine staining or ELISA. We reasoned that TL1a may costimulate ILC activation and combined TL1A with IL-1b and IL-23, known to activate IL-22 production in ILCs. Compared to IL-1b and IL-23, the addition of TL1A led to a significantly higher percentage of IL-22 producing ILCs (9.3% vs. 23.3%, n=8, p<0.001) and more IL-22 production as determined by ELISA (3399 pg/ml vs. 8757 pg/ml, n=3, p<0.001). Nearly identical results were obtained for IL-8 production (p<0.001). Prior studies in T cells show that DR3 signaling increases the expression of the high affinity IL-2 receptor (CD25). While TL1A alone did not increase CD25 on resting ILCs and IL1b + IL-23 only marginally increased CD25 expression (∼1.7x increase from baseline), the combination of TL1A and IL1b + IL-23 led to significantly higher amounts of CD25 on the surface of ILCs (∼3.8x induction). We then tested whether IL-2 could be used to expand ILCs in vitro. Purified ILCs were treated in the following conditions: media (control), TL1A alone, IL1b + IL-23, or the combination of TL1A + IL-1b + IL-23 for 16 hours. Cells were then washed and cultured in media containing IL-2 (1,000U/ml). In short term (5 day) cultures, there was significantly more proliferation with TL1A + IL-1b + IL-23 (14.4% vs. 21.2% vs. 17.4% vs. 42%, n=7, p<0.001) as measured by CSFE dilution. When ILCs were cultured for 14 days, TL1A + IL-1b + IL-23 resulted in significantly greater expansion than IL1-b + IL23 cultured cells (39.3x vs. 14x, n=7, p=0.007). Since TL1A has been associated with skewing of T cells to IL-17 production and the onset of inflammatory conditions (Crohn's disease), we assessed the expanded ILCs for the loss of IL-22 and/or the acquisition of IL-17. ILCs expanded in the presence of TLA1 + IL-1b + IL-23 had no change in their surface phenotype or capacity to produce IL-22. Importantly, neither IL-17 nor changes in RORgt or AHR mRNA expression were detected after expansion. Collectively, these studies identify a novel axis where DR3/TL1A signaling costimulates IL1b and IL-23 induced production of IL-22 and results in the expression of IL-2R (CD25) along with the associated proliferative response to IL-2. These studies significantly advance our ability to devise GMP-compliant methods to generate ILCs and pave the way for adoptive transfer experiments using ILCs in humans. Disclosures:Miller:Coronado Biosciences: Scientific Advisory Board Other.

Bee Venom Phospholipase A2 Induces a Primary Type 2 Response that Is Dependent on the Receptor ST2 and Confers Protective Immunity

Venoms consist of toxic components that are delivered to their victims via bites or stings. Venoms also represent a major class of allergens in humans. Phospholipase A2 (PLA2) is a conserved component of venoms from multiple species and is the major allergen in bee venom. Here we examined how bee venom PLA2 is sensed by the innate immune system and induces a type 2 immune response in mice. We found that bee venom PLA2 induced a T helper type 2 (Th2) cell-type response and group 2 innate lymphoid cell activation via the enzymatic cleavage of membrane phospholipids and release of interleukin-33. Furthermore, we showed that the IgE response to PLA2 could protect mice from future challenge with a near-lethal dose of PLA2. These data suggest that the innate immune system can detect the activity of a conserved component of venoms and induce a protective immune response against a venom toxin.

Interleukin-33-Dependent Innate Lymphoid Cells Mediate Hepatic Fibrosis

Liver fibrosis is a consequence of chronic liver diseases and thus a major cause of mortality and morbidity. Clinical evidence and animal studies suggest that local tissue homeostasis is disturbed due to immunological responses to chronic hepatocellular stress. Poorly defined stress-associated inflammatory networks are thought to mediate gradual accumulation of extracellular-matrix components, ultimately leading to fibrosis and liver failure. Here we have reported that hepatic expression of interleukin-33 (IL-33) was both required and sufficient for severe hepatic fibrosis invivo. We have demonstrated that IL-33's profibrotic effects related to activation and expansion of liver resident innate lymphoid cells (ILC2). We identified ILC2-derived IL-13, acting through type-II IL-4 receptor-dependent signaling via the transcription factor STAT6 and hepatic stellate-cell activation, as a critical downstream cytokine of IL-33-dependent pathologic tissue remodeling and fibrosis. Our data reveal key immunological networks implicated in hepatic fibrosis and support the concept of modulation of IL-33 bioactivity for therapeutic purposes.

Lipoxin A 4 Regulates Natural Killer Cell and Type 2 Innate Lymphoid Cell Activation in Asthma

Asthma is a prevalent disease of chronic inflammation in which endogenous counterregulatory signaling pathways are dysregulated. Recent evidence suggests that innate lymphoid cells (ILCs), including natural killer (NK) cells and type 2 ILCs (ILC2s), can participate in the regulation of allergic airway responses, in particular airway mucosal inflammation. We have identified both NK cells and ILC2s in human lung and peripheral blood in healthy and asthmatic subjects. NK cells were highly activated in severe asthma, were linked to eosinophilia, and interacted with autologous eosinophils to promote their apoptosis. ILC2s generated antigen-independent interleukin-13 (IL-13) in response to the mast cell product prostaglandin D2 alone and in a synergistic manner with the airway epithelial cytokines IL-25 and IL-33. Both NK cells and ILC2s expressed the pro-resolving ALX/FPR2 receptors. Lipoxin A4, a natural pro-resolving ligand for ALX/FPR2 receptors, significantly increased NK cell-mediated eosinophil apoptosis and decreased IL-13 release by ILC2s. Together, these findings indicate that ILCs are targets for lipoxin A4 to decrease airway inflammation and mediate the catabasis of eosinophilic inflammation. Because lipoxin A4 generation is decreased in severe asthma, these findings also implicate unrestrained ILC activation in asthma pathobiology.

IL-1β mediates chronic intestinal inflammation by promoting the accumulation of IL-17A secreting innate lymphoid cells and CD4+ Th17 cells

Although very high levels of interleukin (IL)-1β are present in the intestines of patients suffering from inflammatory bowel diseases (IBD), little is known about the contribution of IL-1β to intestinal pathology. Here, we used two complementary models of chronic intestinal inflammation to address the role of IL-1β in driving innate and adaptive pathology in the intestine. We show that IL-1β promotes innate immune pathology in Helicobacter hepaticus-triggered intestinal inflammation by augmenting the recruitment of granulocytes and the accumulation and activation of innate lymphoid cells (ILCs). Using a T cell transfer colitis model, we demonstrate a key role for T cell-specific IL-1 receptor (IL-1R) signals in the accumulation and survival of pathogenic CD4(+) T cells in the colon. Furthermore, we show that IL-1β promotes Th17 responses from CD4(+) T cells and ILCs in the intestine, and we describe synergistic interactions between IL-1β and IL-23 signals that sustain innate and adaptive inflammatory responses in the gut. These data identify multiple mechanisms through which IL-1β promotes intestinal pathology and suggest that targeting IL-1β may represent a useful therapeutic approach in IBD.

Gene expression signatures in BALB3T3 fibroblasts exposed to cobalt micro/nano-particles and cobalt ions

Innate lymphoid cells (ILCs) are a heterogeneous group of mucosal inflammatory cells that participate in chronic intestinal inflammation. We investigated the role of interleukin 6 (IL6) in inducing activation of ILCs in mice and in human beings with chronic intestinal inflammation.ILCs were isolated from colons of Tbx21-/- × Rag2-/- mice (TRUC), which develop colitis; patients with inflammatory bowel disease (IBD); and patients without colon inflammation (controls). ILCs were characterized by flow cytometry; cytokine production was measured by enzyme-linked immunosorbent assay and cytokine bead arrays. Mice were given intraperitoneal injections of depleting (CD4, CD90), neutralizing (IL6), or control antibodies. Isolated colon tissues were analyzed by histology, explant organ culture, and cell culture. Bacterial DNA was extracted from mouse fecal samples to assess the intestinal microbiota.IL17A- and IL22-producing, natural cytotoxicity receptor–negative, ILC3 were the major subset of ILCs detected in colons of TRUC mice. Combinations of IL23 and IL1α induced production of cytokines by these cells, which increased further after administration of IL6. Antibodies against IL6 reduced colitis in TRUC mice without significantly affecting the structure of their intestinal microbiota. Addition of IL6 increased production of IL17A, IL22, and interferon-γ by human intestinal CD3-negative, IL7-receptor–positive cells, in a dose-dependent manner.IL6 contributes to activation of colonic natural cytotoxicity receptor–negative, CD4-negative, ILC3s in mice with chronic intestinal inflammation (TRUC mice) by increasing IL23- and IL1α-induced production of IL17A and IL22. This pathway might be targeted to treat patients with IBD because IL6, which is highly produced in colonic tissue by some IBD patients, also increased the production of IL17A, IL22, and interferon-γ by cultured human colon CD3-negative, IL7-receptor–positive cells.

OR.75. Characterization of Il-17 Secreting T-Helper Cells (Thil-17)

Innate lymphoid cells (ILC) represent a novel group of lymphocytes that, different from T and B-lymphocytes lack recombinant activating genes (RAG-1 or RAG-2) and thus do not express rearranged antigen-specific receptors. Members of this family, i.e. NK cells, have been known since long time, while the other ILCs have been discovered only in recent years, possibly because of their predominant localization in tissues, primarily in mucosal tissues, skin and mucosa-associated lymphoid organs. ILC have been grouped in three major subsets on the basis of their phenotypic and functional features as well as of their dependency on given transcription factors (TF). Briefly, ILC-1 are dependent on T-bet TF and produce interferon (IFN)-γ. Group 2 ILC (ILC2) express GATA-3 TF and produce IL-5, IL-4 and IL-13 (Type 2) cytokines while group 3 ILC (ILC3) express RORγt TF and produce IL-17 and IL-22. ILC provide early defenses against pathogens and intervene in the repair of damaged tissues. ILC activation is mediated by cytokines (specifically acting on different ILC groups) and/or by activating receptors that are, at least in part, the same that had been previously identified in NK cells [1].