HpARI Protein Secreted by a Helminth Parasite Suppresses Interleukin-33.

Infection by helminth parasites is associated with amelioration of allergic reactivity, but mechanistic insights into this association are lacking. Products secreted by the mouse parasite Heligmosomoides polygyrus suppress type 2 (allergic) immune responses through interference in the interleukin-33 (IL-33) pathway. Here, we identified H. polygyrus Alarmin Release Inhibitor (HpARI), an IL-33-suppressive 26-kDa protein, containing three predicted complement control protein (CCP) modules. In vivo, recombinant HpARI abrogated IL-33, group 2 innate lymphoid cell (ILC2) and eosinophilic responses to Alternaria allergen administration, and diminished eosinophilic responses to Nippostrongylus brasiliensis, increasing parasite burden. HpARI bound directly to both mouse and human IL-33 (in the cytokine's activated state) and also to nuclear DNA via its N-terminal CCP module pair (CCP1/2), tethering active IL-33 within necrotic cells, preventing its release, and forestalling initiation of type 2 allergic responses. Thus, HpARI employs a novel molecular strategy to suppress type 2 immunity in both infection and allergy.

Basophils promote barrier dysfunction and resolution in the atopic skin.

BACKGROUND: The type 2 cytokines IL-4 and IL-13 promote not only atopic dermatitis (AD) but also the resolution of inflammation. How type 2 cytokines participate in the resolution of AD is poorly known. OBJECTIVE: Our aim was to determine the mechanisms and cell types governing skin inflammation, barrier dysfunction, and resolution of inflammation in a model of AD. METHODS: Mice that exhibit expression of IL-4, IL-13, and MCPT8 or that could be depleted of basophils or eosinophils, be deficient in IL-4 or MHC class II molecules, or have basophils lacking macrophage colony-stimulating factor (M-CSF) were treated with calcipotriol (MC903) as an acute model of AD. Kinetics of the disease; keratinocyte differentiation; and leukocyte accumulation, phenotype, function, and cytokine production were measured by transepidermal water loss, histopathology, molecular biology, or unbiased analysis of spectral flow cytometry. RESULTS: In this model of AD, basophils were activated systemically and were the initial and main source of IL-4 in the skin. Basophils and IL-4 promoted epidermal hyperplasia and skin barrier dysfunction by acting on keratinocyte differentiation during inflammation. Basophils, IL-4, and basophil-derived M-CSF inhibited the accumulation of proinflammatory cells in the skin while promoting the expansion and function of proresolution M2-like macrophages and the expression of probarrier genes. Basophils kept their proresolution properties during AD resolution. CONCLUSION: Basophils can display both beneficial and detrimental type 2 functions simultaneously during atopic inflammation.

Cell surface IL-1α trafficking is specifically inhibited by interferon-γ, and associates with the membrane via IL-1R2 and GPI anchors.

IL-1 is a powerful cytokine that drives inflammation and modulates adaptive immunity. Both IL-1α and IL-1ß are translated as proforms that require cleavage for full cytokine activity and release, while IL-1α is reported to occur as an alternative plasma membrane-associated form on many cell types. However, the existence of cell surface IL-1α (csIL-1α) is contested, how IL-1α tethers to the membrane is unknown, and signaling pathways controlling trafficking are not specified. Using a robust and fully validated system, we show that macrophages present bona fide csIL-1α after ligation of TLRs. Pro-IL-1α tethers to the plasma membrane in part through IL-1R2 or via association with a glycosylphosphatidylinositol-anchored protein, and can be cleaved, activated, and released by proteases. csIL-1α requires de novo protein synthesis and its trafficking to the plasma membrane is exquisitely sensitive to inhibition by IFN-γ, independent of expression level. We also reveal how prior csIL-1α detection could occur through inadvertent cell permeabilisation, and that senescent cells do not drive the senescent-associated secretory phenotype via csIL-1α, but rather via soluble IL-1α. We believe these data are important for determining the local or systemic context in which IL-1α can contribute to disease and/or physiological processes.

A novel blood-feeding detoxification pathway in Nippostrongylus brasiliensis L3 reveals a potential checkpoint for arresting hookworm development.

As part of on-going efforts to control hookworm infection, the "human hookworm vaccine initiative" has recognised blood feeding as a feasible therapeutic target for inducing immunity against hookworm infection. To this end, molecular approaches have been used to identify candidate targets, such as Necator americanus (Na) haemoglobinase aspartic protease-1 (APR-1), with immunogenicity profiled in canine and hamster models. We sought to accelerate the immune analysis of these identified therapeutic targets by developing an appropriate mouse model. Here we demonstrate that Nippostrongylus brasiliensis (Nb), a phylogenetically distant strongylid nematode of rodents, begins blood feeding early in its development and that immunisation with Na-APR-1 can block its growth and completion of its life cycle. Furthermore, we identify a new haem detoxification pathway in Nb required for blood feeding that can be blocked by drugs of the quinolone family, reducing both infection burden and the associated anaemia in rodents. Collectively, our findings show that haem metabolism has potential as a checkpoint for interrupting hookworm development in early stages of the hookworm life cycle and that the Nippostrongylus brasiliensis rodent model is relevant for identifying novel therapeutic targets against human hookworm.

Concerted activity of IgG1 antibodies and IL-4/IL-25-dependent effector cells trap helminth larvae in the tissues following vaccination with defined secreted antigens, providing sterile immunity to challenge infection.

Over 25% of the world's population are infected with helminth parasites, the majority of which colonise the gastrointestinal tract. However, no vaccine is yet available for human use, and mechanisms of protective immunity remain unclear. In the mouse model of Heligmosomoides polygyrus infection, vaccination with excretory-secretory (HES) antigens from adult parasites elicits sterilising immunity. Notably, three purified HES antigens (VAL-1, -2 and -3) are sufficient for effective vaccination. Protection is fully dependent upon specific IgG1 antibodies, but passive transfer confers only partial immunity to infection, indicating that cellular components are also required. Moreover, immune mice show greater cellular infiltration associated with trapping of larvae in the gut wall prior to their maturation. Intra-vital imaging of infected intestinal tissue revealed a four-fold increase in extravasation by LysM+GFP+ myeloid cells in vaccinated mice, and the massing of these cells around immature larvae. Mice deficient in FcRγ chain or C3 complement component remain fully immune, suggesting that in the presence of antibodies that directly neutralise parasite molecules, the myeloid compartment may attack larvae more quickly and effectively. Immunity to challenge infection was compromised in IL-4Rα- and IL-25-deficient mice, despite levels of specific antibody comparable to immune wild-type controls, while deficiencies in basophils, eosinophils or mast cells or CCR2-dependent inflammatory monocytes did not diminish immunity. Finally, we identify a suite of previously uncharacterised heat-labile vaccine antigens with homologs in human and veterinary parasites that together promote full immunity. Taken together, these data indicate that vaccine-induced immunity to intestinal helminths involves IgG1 antibodies directed against secreted proteins acting in concert with IL-25-dependent Type 2 myeloid effector populations.

Secretion of protective antigens by tissue-stage nematode larvae revealed by proteomic analysis and vaccination-induced sterile immunity.

Gastrointestinal nematode parasites infect over 1 billion humans, with little evidence for generation of sterilising immunity. These helminths are highly adapted to their mammalian host, following a developmental program through successive niches, while effectively down-modulating host immune responsiveness. Larvae of Heligmosomoides polygyrus, for example, encyst in the intestinal submucosa, before emerging as adult worms into the duodenal lumen. Adults release immunomodulatory excretory-secretory (ES) products, but mice immunised with adult H. polygyrus ES become fully immune to challenge infection. ES products of the intestinal wall 4th stage (L4) larvae are similarly important in host-parasite interactions, as they readily generate sterile immunity against infection, while released material from the egg stage is ineffective. Proteomic analyses of L4 ES identifies protective antigen targets as well as potential tissue-phase immunomodulatory molecules, using as comparators the adult ES proteome and a profile of H. polygyrus egg-released material. While 135 proteins are shared between L4 and adult ES, 72 are L4 ES-specific; L4-specific proteins correspond to those whose transcription is restricted to larval stages, while shared proteins are generally transcribed by all life cycle forms. Two protein families are more heavily represented in the L4 secretome, the Sushi domain, associated with complement regulation, and the ShK/SXC domain related to a toxin interfering with T cell signalling. Both adult and L4 ES contain extensive but distinct arrays of Venom allergen/Ancylostoma secreted protein-Like (VAL) members, with acetylcholinesterases (ACEs) and apyrase APY-3 particularly abundant in L4 ES. Serum antibodies from mice vaccinated with L4 and adult ES react strongly to the VAL-1 protein and to ACE-1, indicating that these two antigens represent major vaccine targets for this intestinal nematode. We have thus defined an extensive and novel repertoire of H. polygyrus proteins closely implicated in immune modulation and protective immunity.

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.

Suppression of type 2 immunity and allergic airway inflammation by secreted products of the helminth Heligmosomoides polygyrus.

Allergic asthma is less prevalent in countries with parasitic helminth infections, and mice infected with parasites such as Heligmosomoides polygyrus are protected from allergic airway inflammation. To establish whether suppression of allergy could be mediated by soluble products of this helminth, we tested H. polygyrus excretory-secretory (HES) material for its ability to impair allergic inflammation. When HES was added to sensitising doses of ovalbumin, the subsequent allergic airway response was suppressed, with ablated cell infiltration, a lower ratio of effector (CD4(+) CD25(+) Foxp3(-) ) to regulatory (CD4(+) Foxp3(+) ) T (Treg) cells, and reduced Th1, Th2 and Th17 cytokine production. HES exposure reduced IL-5 responses and eosinophilia, abolished IgE production and inhibited the type 2 innate molecules arginase-1 and RELM-α (resistin-like molecule-α). Although HES contains a TGF-ß-like activity, similar effects in modulating allergy were not observed when administering mammalian TGF-ß alone. HES also protected previously sensitised mice, suppressing recruitment of eosinophils to the airways when given at challenge, but no change in Th or Treg cell populations was apparent. Because heat-treatment of HES did not impair suppression at sensitisation, but compromised its ability to suppress at challenge, we propose that HES contains distinct heat-stable and heat-labile immunomodulatory molecules, which modulate pro-allergic adaptive and innate cell populations.

Heligmosomoides polygyrus elicits a dominant nonprotective antibody response directed against restricted glycan and peptide epitopes.

Heligmosomoides polygyrus is a widely used gastrointestinal helminth model of long-term chronic infection in mice, which has not been well-characterized at the antigenic level. We now identify the major targets of the murine primary Ab response as a subset of the secreted products in H. polygyrus excretory-secretory (HES) Ag. An immunodominant epitope is an O-linked glycan (named glycan A) carried on three highly expressed HES glycoproteins (venom allergen Ancylostoma-secreted protein-like [VAL]-1, -2, and -5), which stimulates only IgM Abs, is exposed on the adult worm surface, and is poorly represented in somatic parasite extracts. A second carbohydrate epitope (glycan B), present on both a non-protein high molecular mass component and a 65-kDa molecule, is widely distributed in adult somatic tissues. Whereas the high molecular mass component and 65-kDa molecules bear phosphorylcholine, the glycan B epitope itself is not phosphorylcholine. Class-switched IgG1 Abs are found to glycan B, but the dominant primary IgG1 response is to the polypeptides of VAL proteins, including also VAL-3 and VAL-4. Secondary Ab responses include the same specificities while also recognizing VAL-7. Although vaccination with HES conferred complete protection against challenge H. polygyrus infection, mAbs raised against each of the glycan epitopes and against VAL-1, VAL-2, and VAL-4 proteins were unable to do so, even though these specificities (with the exception of VAL-2) are also secreted by tissue-phase L4 larvae. The primary immune response in susceptible mice is, therefore, dominated by nonprotective Abs against a small subset of antigenic epitopes, raising the possibility that these act as decoy specificities that generate ineffective humoral immunity.

Immune modulation and modulators in Heligmosomoides polygyrus infection.

The intestinal nematode parasite Heligmosomoides polygyrus bakeri exerts widespread immunomodulatory effects on both the innate and adaptive immune system of the host. Infected mice adopt an immunoregulated phenotype, with abated allergic and autoimmune reactions. At the cellular level, infection is accompanied by expanded regulatory T cell populations, skewed dendritic cell and macrophage phenotypes, B cell hyperstimulation and multiple localised changes within the intestinal environment. In most mouse strains, these act to block protective Th2 immunity. The molecular basis of parasite interactions with the host immune system centres upon secreted products termed HES (H. polygyrus excretory-secretory antigen), which include a TGF-ß-like ligand that induces de novo regulatory T cells, factors that modify innate inflammatory responses, and molecules that block allergy in vivo. Proteomic and transcriptomic definition of parasite proteins, combined with biochemical identification of immunogenic molecules in resistant mice, will provide new candidate immunomodulators and vaccine antigens for future research.

The IL-25-dependent tuft cell circuit driven by intestinal helminths requires macrophage migration inhibitory factor (MIF).

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 RELMß, 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-ß, 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.

Integrated miRNA/cytokine/chemokine profiling reveals severity-associated step changes and principal correlates of fatality in COVID-19.

Inflammatory cytokines and chemokines (CC) drive COVID-19 pathology. Yet, patients with similar circulating CC levels present with different disease severity. Here, we determined 171 microRNAomes from 58 hospitalized COVID-19 patients (Cohort 1) and levels of 25 cytokines and chemokines (CC) in the same samples. Combining microRNA (miRNA) and CC measurements allowed for discrimination of severe cases with greater accuracy than using miRNA or CC levels alone. Severity group-specific associations between miRNAs and COVID-19-associated CC (e.g., IL6, CCL20) or clinical hallmarks of COVID-19 (e.g., neutrophilia, hypoalbuminemia) separated patients with similar CC levels but different disease severity. Analysis of an independent cohort of 108 patients from a different center (Cohort 2) demonstrated feasibility of CC/miRNA profiling in leftover hospital blood samples with similar severe disease CC and miRNA profiles, and revealed CCL20, IL6, IL10, and miR-451a as key correlates of fatal COVID-19. These findings highlight that systemic miRNA/CC networks underpin severe COVID-19.

Helminth-induced CD19+CD23hi B cells modulate experimental allergic and autoimmune inflammation.

Numerous population studies and experimental models suggest that helminth infections can ameliorate immuno-inflammatory disorders such as asthma and autoimmunity. Immunosuppressive cell populations associated with helminth infections include Treg and alternatively-activated macrophages. In previous studies, we showed that both CD4(+)CD25(+) Treg, and CD4(-) MLN cells from Heligmosomoides polygyus-infected C57BL/6 mice were able to transfer protection against allergic airway inflammation to sensitized but uninfected animals. We now show that CD4(-)CD19(+) MLN B cells from infected, but not naïve, mice are able to transfer a down-modulatory effect on allergy, significantly suppressing airway eosinophilia, IL-5 secretion and pathology following allergen challenge. We further demonstrate that the same cell population can alleviate autoimmune-mediated inflammatory events in the CNS, when transferred to uninfected mice undergoing myelin oligodendrocyte glycoprotein((p35-55))-induced EAE. In both allergic and autoimmune models, reduction of disease was achieved with B cells from helminth-infected IL-10(-/-) donors, indicating that donor cell-derived IL-10 is not required. Phenotypically, MLN B cells from helminth-infected mice expressed uniformly high levels of CD23, with follicular (B2) cell surface markers. These data expand previous observations and highlight the broad regulatory environment that develops during helminth infections that can abate diverse inflammatory disorders in vivo.

The Gastrointestinal Helminth Heligmosomoides bakeri Suppresses Inflammation in a Model of Contact Hypersensitivity.

Helminths regulate host immune responses to ensure their own long-term survival. Numerous studies have demonstrated that these helminth-induced regulatory mechanisms can also limit host inflammatory responses in several disease models. We used the Heligmosomoides bakeri (Hb) infection model (also known as H. polygyrus or H. polygyrus bakeri in the literature) to test whether such immune regulation affects skin inflammatory responses induced by the model contact sensitiser dibutyl phthalate fluorescein isothiocynate (DBP-FITC). Skin lysates from DBP-FITC-sensitized, Hb-infected mice produced less neutrophil specific chemokines and had significantly reduced levels of skin thickening and cellular inflammatory responses in tissue and draining lymph nodes (LNs) compared to uninfected mice. Hb-induced suppression did not appear to be mediated by regulatory T cells, nor was it due to impaired dendritic cell (DC) activity. Mice cleared of infection remained unresponsive to DBP-FITC sensitization indicating that suppression was not via the secretion of Hb-derived short-lived regulatory molecules, although long-term effects on cells cannot be ruled out. Importantly, similar helminth-induced suppression of inflammation was also seen in the draining LN after intradermal injection of the ubiquitous allergen house dust mite (HDM). These findings demonstrate that Hb infection attenuates skin inflammatory responses by suppressing chemokine production and recruitment of innate cells. These findings further contribute to the growing body of evidence that helminth infection can modulate inflammatory and allergic responses via a number of mechanisms with potential to be exploited in therapeutic and preventative strategies in the future.

Intestinal helminth infection promotes IL-5- and CD4+ T cell-dependent immunity in the lung against migrating parasites.

The ability of helminths to manipulate the immune system of their hosts to ensure their own survival is often credited with affecting responses to other pathogens. We undertook co-infection experiments in mice to determine how infection with the intestinal helminth Heligmosomoides polygyrus affected the parasitological, immunological and physiological outcomes of a primary infection with a distinct species of helminth; the lung migratory parasite Nippostrongylus brasiliensis. We found that migrating N. brasiliensis larvae were killed in the lungs of H. polygyrus-infected mice by a process involving IL-33-activated CD4+ T cells that released IL-5 and recruited activated eosinophils. The lung pathology normally associated with N. brasiliensis larval migration was also reduced. Importantly, lung immunity remained intact in mice cleared of prior H. polygyrus infection and also occurred during infection with another entirely enteric helminth, Trichuris muris. This study identifies a cross-mucosal immune mechanism by which intestinal helminths may protect their hosts against co-infection by a different parasite at a distal site, via circulation of activated CD4+ T cells that can be triggered to release effector cytokines and mount inflammatory responses by tissue damage-associated alarmins, such as IL-33.

The Basoph8 Mice Enable an Unbiased Detection and a Conditional Depletion of Basophils.

Basophils are granulocytes involved in parasite immunity and allergic diseases, known for their potent secretion of type 2 cytokines. Identifying their functions has proven to be controversial due to their relative rarity and their complex lineage phenotype. Here, we show that the expression of basophils lineage markers CD200R3 and FcεRIα is highly variable in inflammatory settings and hinders basophils identification by flow cytometry across multiple disease states or tissues. Fluorophore-conjugated antibody staining of these lineage markers strongly activates basophil type 2 cytokine expression, and represents a potential bias for coculture or in vivo transfer experiments. The Basoph8 is a mouse model where basophils specifically express a strong fluorescent reporter and the Cre recombinase. Basophils can be identified and FACS sorted unambiguously by their expression of the enhanced yellow fluorescent protein (eYFP) in these mice. We show that the expression of the eYFP is robust in vivo during inflammation, and in vitro on living basophils for at least 72 h, including during the induction of anaphylactoid degranulation. We bred and characterized the Basoph8xiDTR mice, in which basophils specifically express eYFP and the simian diphtheria toxin receptor (DTR). This model enables basophils conditional depletion relatively specifically ex vivo and in vivo during allergic inflammation and their detection as eYFP+ cells. In conclusion, we report underappreciated benefits of the commercially available Basoph8 mice to study basophils function.

Macrophage Migration Inhibitory Factor (MIF) Is Essential for Type 2 Effector Cell Immunity to an Intestinal Helminth Parasite.

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.

A structurally distinct TGF-ß mimic from an intestinal helminth parasite potently induces regulatory T cells.

Helminth parasites defy immune exclusion through sophisticated evasion mechanisms, including activation of host immunosuppressive regulatory T (Treg) cells. The mouse parasite Heligmosomoides polygyrus can expand the host Treg population by secreting products that activate TGF-ß signalling, but the identity of the active molecule is unknown. Here we identify an H. polygyrus TGF-ß mimic (Hp-TGM) that replicates the biological and functional properties of TGF-ß, including binding to mammalian TGF-ß receptors and inducing mouse and human Foxp3+ Treg cells. Hp-TGM has no homology with mammalian TGF-ß or other members of the TGF-ß family, but is a member of the complement control protein superfamily. Thus, our data indicate that through convergent evolution, the parasite has acquired a protein with cytokine-like function that is able to exploit an endogenous pathway of immunoregulation in the host.