BackgroundGenome-wide association studies have identified the ORM (yeast)-like protein isoform 3 (ORMDL3) gene locus on human chromosome 17q to be a highly significant risk factor for childhood-onset asthma.ObjectiveWe sought to investigate in vivo the functional role of ORMDL3 in disease inception.MethodsAn Ormdl3-deficient mouse was generated and the role of ORMDL3 in the generation of allergic airways disease to the fungal aeroallergen Alternaria alternata was determined. An adeno-associated viral vector was also used to reconstitute ORMDL3 expression in airway epithelial cells of Ormdl3 knockout mice.ResultsOrmdl3 knockout mice were found to be protected from developing allergic airways disease and showed a marked decrease in pathophysiology, including lung function and airway eosinophilia induced by Alternaria. Alternaria is a potent inducer of cellular stress and the unfolded protein response, and ORMDL3 was found to play a critical role in driving the activating transcription factor 6–mediated arm of this response through Xbp1 and downstream activation of the endoplasmic reticulum–associated degradation pathway. In addition, ORMDL3 mediated uric acid release, another marker of cellular stress. In the knockout mice, reconstitution of Ormdl3 transcript levels specifically in the bronchial epithelium resulted in reinstatement of susceptibility to fungal allergen–induced allergic airways disease.ConclusionsThis study demonstrates that ORMDL3, an asthma susceptibility gene identified by genome-wide association studies, contributes to key pathways that promote changes in airway physiology during allergic immune responses.

Helminth parasites are adept manipulators of the immune system, using multiple strategies to evade the host type 2 response. In the intestinal niche, the epithelium is crucial for initiating type 2 immunity via tuft cells, which together with goblet cells expand dramatically in response to the type 2 cytokines IL-4 and IL-13. However, it is not known whether helminths modulate these epithelial cell populations. In vitro, using small intestinal organoids, we found that excretory/secretory products (HpES) from Heligmosomoides polygyrus blocked the effects of IL-4/13, inhibiting tuft and goblet cell gene expression and expansion, and inducing spheroid growth characteristic of fetal epithelium and homeostatic repair. Similar outcomes were seen in organoids exposed to parasite larvae. In vivo, H. polygyrus infection inhibited tuft cell responses to heterologous Nippostrongylus brasiliensis infection or succinate, and HpES also reduced succinate-stimulated tuft cell expansion. Our results demonstrate that helminth parasites reshape their intestinal environment in a novel strategy for undermining the host protective response.

Interleukin 25 (IL-25) is a major 'alarmin' cytokine, capable of initiating and amplifying the type immune response to helminth parasites. However, its role in the later effector phase of clearing chronic infection remains unclear. The helminth Heligmosomoides polygyrus establishes long-term infections in susceptible C57BL/6 mice, but is slowly expelled in BALB/c mice from day 14 onwards. We noted that IL-25R (Il17rb)-deficient BALB/c mice were unable to expel parasites despite type 2 immune activation comparable to the wild-type. We then established that in C57BL/6 mice, IL-25 adminstered late in infection (days 14–17) drove immunity. Moreover, when IL-25 and IL-4 were delivered to Rag1-deficient mice, the combination resulted in near complete expulsion of the parasite, even following administration of an anti-CD90 antibody to deplete innate lymphoid cells (ILCs). Hence, effective anti-helminth immunity during chronic infection requires an innate effector cell population that is synergistically activated by the combination of IL-4Rα and IL-25R signaling.

Immunity to intestinal helminths is known to require both innate and adaptive components of the immune system activated along the Type 2 IL-4R/STAT6-dependent pathway. We have found that macrophage migration inhibitory factor (MIF) is essential for the development of effective immunity to the intestinal helminth Heligmosomoides polygyrus, even following vaccination which induces sterile immunity in wild-type mice. A chemical inhibitor of MIF, 4-IPP, was similarly found to compromise anti-parasite immunity. Cellular analyses found that the adaptive arm of the immune response, including IgG1 antibody responses and Th2-derived cytokines, was intact and that Foxp3+ T regulatory cell responses were unaltered in the absence of MIF. However, MIF was found to be an essential cytokine for innate cells, with ablated eosinophilia and ILC2 responses, and delayed recruitment and activation of macrophages to the M2 phenotype (expressing Arginase 1, Chil3, and RELM-α) upon infection of MIF-deficient mice; a macrophage deficit was also seen in wild-type BALB/c mice exposed to 4-IPP. Gene expression analysis of intestinal and lymph node tissues from MIF-deficient and -sufficient infected mice indicated significantly reduced levels of Arl2bp, encoding a factor involved in nuclear localization of STAT3. We further found that STAT3-deficient macrophages expressed less Arginase-1, and that mice lacking STAT3 in the myeloid compartment (LysMCrexSTAT3fl/fl) were unable to reject a secondary infection with H. polygyrus. We thus conclude that in the context of a Type 2 infection, MIF plays a critical role in polarizing macrophages into the protective alternatively-activated phenotype, and that STAT3 signaling may make a previously unrecognized contribution to immunity to helminths.

Macrophage migration inhibitory factor (MIF) is a key innate immune mediator with chemokine- and cytokine-like properties in the inflammatory pathway. While its actions on macrophages are well-studied, its effects on other cell types are less understood. Here we report that MIF is required for expansion of intestinal tuft cells during infection with the helminth Nippostrongylus brasiliensis. MIF-deficient mice show defective innate responses following infection, lacking intestinal epithelial tuft cell hyperplasia or upregulation of goblet cell RELMβ, and fail to expand eosinophil, type 2 innate lymphoid cell (ILC2) and macrophage (M2) populations. Similar effects were observed in MIF-sufficient wild-type mice given the MIF inhibitor 4-IPP. MIF had no direct effect on epithelial cells in organoid cultures, and MIF-deficient intestinal stem cells could generate tuft cells in vitro in the presence of type 2 cytokines. In vivo the lack of MIF could be fully compensated by administration of IL-25, restoring tuft cell differentiation and goblet cell expression of RELM-β, demonstrating its requirement upstream of the ILC2-tuft cell circuit. Both ILC2s and macrophages expressed the MIF receptor CXCR4, indicating that MIF may act as an essential co-factor on both cell types to activate responses to IL-25 in helminth infection.

The immunoproteasome subunit β5i has been shown to play an important role in Th1/Th17 driven models of colitis and arthritis. However, the function of β5i in Th2 dependent diseases remains enigmatic. To study the role of β5i in Th2-driven pathology, β5i knockout (KO) and control mice were tested in different models of experimental allergic asthma. β5i-deficient mice showed reduced OVA/Alum- and subcutaneous/OVA-induced acute asthma with decreased eosinophilia in the bronchoalveolar lavage (BAL), low OVA-specific IgG1 and reduced local and systemic Th2 cytokines. While Th2 cells in the lungs were reduced, Tregs and Th1 cells were not affected. Attenuated asthma in β5i KO mice could not be attributed to defects in OVA uptake or maturation of dendritic cells in the lung. Surprisingly, β5i deficient mice developed HDM asthma which was comparable to control mice. Here, we present novel evidence for the requirement of the β5i immunosubunit to generate a strong Th2 response during OVA- but not HDM-induced acute asthma. The unexpected role of β5i in OVA asthma remains to be clarified.

ILCs burst onto the immunological scene with their involvement in bacterial and helminth infections. As their influence has emerged, it has become clear that they play a fundamental role in regulating barrier tissue homeostasis and the immune response during inflammation. A subset of ILCs, ILC2s, has become the focus of attention for many helminth biologists—stepping into the limelight as both the elusive initiator and amplifier of the type-2 response. In many of the early reports, conclusions as to their function were based on experiments using unadapted parasites or immune-compromised hosts. In this review we re-examine the generation and function of type-2 ILCs in helminth infection and the extent to which their roles may be essential or redundant, in both primary and challenge infections. ILC2s will be discussed in terms of a broader innate network, which when in dialogue with adaptive immunity, allows the generation of the anti-parasite response. Finally, we will review how helminths manipulate ILC2 populations to benefit their survival, as well as dampen systemic inflammation in the host, and how this understanding may be used to improve strategies to control disease.

Type 2-high asthma is a chronic inflammatory disease of the airways which is increasingly prevalent in countries where helminth parasite infections are rare, and characterized by T helper 2 (Th2)-dependent accumulation of eosinophils in the lungs. Regulatory cytokines such as TGF-β can restrain inflammatory reactions, dampen allergic Th2 responses, and control eosinophil activation. The murine helminth parasite Heligmosomoides polygyrus releases a TGF-β mimic (Hp-TGM) that replicates the biological and functional properties of TGF-β despite bearing no structural similarity to the mammalian protein. Here, we investigated if Hp-TGM could alleviate allergic airway inflammation in mice exposed to Alternaria alternata allergen, house dust mite (HDM) extract or alumadjuvanted ovalbumin protein (OVA). Intranasal administration of Hp-TGM during Alternaria exposure sharply reduced airway and lung tissue eosinophilia along with bronchoalveolar lavage fluid IL-5 and lung IL-33 cytokine levels at 24 h. The protective effect of Hp-TGM on airway eosinophilia was also obtained in the longer T-cell mediated models of HDM or OVA sensitisation with significant inhibition of eotaxin-1, IL-4 and IL-13 responses depending on the model and timepoint. Hp-TGM was also protective when administered parenterally either when given at the time of allergic sensitisation or during airway allergen challenge. This project has taken the first steps in identifying the role of Hp-TGM in allergic asthma and highlighted its ability to control lung inflammation and allergic pathology. Future research will investigate the mode of action of Hp-TGM against airway allergic eosinophilia, and further explore its potential to be developed as a biotherapeutic in allergic asthma.