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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.

The signals driving the adaptation of type 2 dendritic cells (DC2s) to diverse peripheral environments remain mostly undefined. We show that differentiation of CD11blo migratory DC2s-a DC2 population unique to the dermis-required IL-13 signaling dependent on the transcription factors STAT6 and KLF4, whereas DC2s in lung and small intestine were STAT6-independent. Similarly, human DC2s in skin expressed an IL-4 and IL-13 gene signature that was not found in blood, spleen and lung DCs. In mice, IL-13 was secreted homeostatically by dermal innate lymphoid cells and was independent of microbiota, TSLP or IL-33. In the absence of IL-13 signaling, dermal DC2s were stable in number but remained CD11bhi and showed defective activation in response to allergens, with diminished ability to support the development of IL-4+GATA3+ helper T cells (TH), whereas antifungal IL-17+RORγt+ TH cells were increased. Therefore, homeostatic IL-13 fosters a noninflammatory skin environment that supports allergic sensitization.

In the version of this article initially published, there were labeling errors in Fig. 8. In Fig. 8a, the top row, now reading “GSE43184:CD1c+ lung versus blood,” originally appeared as “GSE35457….” The bottom row, now reading “GSE35457: CD1c+ skin versus blood,” originally appeared as “GSE43184: CD1c+ lung versus blood.” In Fig. 8b, the top-left plot, now reading “skin versus blood,” originally appeared as “lung versus blood.” The errors have been corrected in the HTML and PDF versions of the article.

A characteristic feature of host responses to helminth infections is the development of profound systemic and tissue-localised Type 2 immune responses that play critical roles in immunity, tissue repair and tolerance of the parasite at tissue sites. These same Type 2 responses are also seen in the tissue-associated immune-pathologies seen in asthma, atopic dermatitis and many forms of allergies. The recent identification of new subtypes of immune cells and cytokine pathways that influence both immune and non-immune cells and tissues creates the opportunity for reviewing helminth parasite-host responses in the context of tissue specific immunity. This review focuses on the new discoveries of the cells and cytokines involved in tissue specific immune responses to helminths and how these contribute to host immunity against helminth infection and allow the host to accommodate the presence of parasites when they cannot be eliminated.

Recent advances in the field of host immunity against parasitic nematodes have revealed the importance of macrophages in trapping tissue migratory larvae. Protective immune mechanisms against the rodent hookworm Nippostrongylus brasiliensis (Nb) are mediated, at least in part, by IL-4-activated macrophages that bind and trap larvae in the lung. However, it is still not clear how host macrophages recognize the parasite. An in vitro co-culture system of bone marrow-derived macrophages and Nb infective larvae was utilized to screen for the possible ligand-receptor pair involved in macrophage attack of larvae. Competitive binding assays revealed an important role for β-glucan recognition in the process. We further identified a role for CD11b and the non-classical pattern recognition receptor ephrin-A2 (EphA2), but not the highly expressed β-glucan dectin-1 receptor, in this process of recognition. This work raises the possibility that parasitic nematodes synthesize β-glucans and it identifies CD11b and ephrin-A2 as important pattern recognition receptors involved in the host recognition of these evolutionary old pathogens. To our knowledge, this is the first time that EphA2 has been implicated in immune responses to a helminth.

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.

Background: Neural pathways may play a role in non-eosinophilic asthma, but this has rarely been studied. Aims: To assess whether non-eosinophilic asthma is characterised by increased sensory nerve sensitivity compared to eosinophilic asthmatics and controls. Methods: We recruited 38 asthmatics (18 eosinophilic and 20 non-eosinophilic) and 19 non-asthmatics (14-21 years) who underwent a capsaicin challenge, combined sputum induction and airway hyperreactivity (AHR), exhaled nitric oxide (FENO), atopy and spirometry tests. Sensory nerve sensitivity was defined as the lowest capsaicin concentration that evoked 2 (C2) or 5 (C5) coughs. Results: Non-eosinophilic asthmatics (NEA; eosinophil cut-point Conclusion: Heightened airway sensory sensitivity may play a role in non-eosinophilic asthma.

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.

Antibody‐mediated immunity is highly protective against disease. The majority of current vaccines confer protection through humoral immunity, but there is high variability in responsiveness across populations. Identifying immune mechanisms that mediate low antibody responsiveness may provide potential strategies to boost vaccine efficacy. Here, we report diverse antibody responsiveness to unadjuvanted as well as adjuvanted immunization in substrains of BALB/c mice, resulting in high and low antibody response phenotypes. Furthermore, these antibody phenotypes were not affected by changes in environmental factors such as the gut microbiota composition. Antigen‐specific B cells following immunization had a marked difference in capability to class switch, resulting in perturbed IgG isotype antibody production. In vitro, a B‐cell intrinsic defect in the regulation of class‐switch recombination was identified in mice with low IgG antibody production. Whole genome sequencing identified polymorphisms associated with the magnitude of antibody produced, and we propose candidate genes that may regulate isotype class‐switching capability. This study highlights that mice sourced from different vendors can have significantly altered humoral immune response profiles, and provides a resource to interrogate genetic regulators of antibody responsiveness. Together these results further our understanding of immune heterogeneity and suggest additional research on the genetic influences of adjuvanted vaccine strategies is warranted for enhancing vaccine efficacy.

Significance T helper 2 (Th2) cells are defined by their ability to produce the hallmark cytokine IL-4. However, to mediate allergic inflammation in tissues, Th2 cells must secrete additional cytokines including IL-13 and IL-5. We used IL-4 and IL-13 dual-reporter mice to show that naive CD4+ T cells cultured in the presence of IL-4 and thymic stromal lymphopoietin (TSLP) generate a population of IL-4negIL-13pos Th2 cells that develop from IL-4neg precursors and express the Th2 effector cytokines IL-5 and IL-9. In vivo, high TSLP levels promote the development of a similar population of IL-4negIL-13pos T cells that also express Gata3, Il5, and Il3 transcripts. Thus, TSLP drives the early differentiation of a distinct population of effector Th2 cells with pro-inflammatory properties., T helper 2 (Th2) cells are pivotal in the development of allergy. Allergen exposure primes IL-4+ Th2 cells in lymph node, but production of effector cytokines including IL-5 and IL-13 is thought to require additional signals from antigen and the environment. Here we report that a substantial proportion of naive CD4+ T cells in spleen and lymph node express receptors for the epithelium-derived inflammatory cytokine thymic stromal lymphopoietin (TSLP). Culture of naive CD4+ T cells in anti-(a)CD3, aCD28, and TSLP-supplemented Th2 conditions enabled the development of a unique population of IL-13-single positive (IL-13-SP) CD4+ T cells; TSLP and Th2 conditions were both required for their development. Sorting experiments revealed that IL-13-SP Th2 cells originated from IL-4-negative precursors and coexpressed transcripts for the Th2 cytokines IL-5 and IL-9. In vivo, high TSLP levels acted directly on CD4+ T cells to induce the development of IL-13-SP and IL-4+IL-13+ double-positive populations in lymph node. These cells were phenotypically similar to Th2 effector cells and were CXCR5lowPD1low and expressed low levels of Bcl6 and Il21 transcripts and high levels of Gata3, Il3, and Il5. Our findings suggest a role of TSLP in directly promoting Th2 cell effector function and support the notion of TSLP as a key driver of Th2 inflammation.

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.

New Zealand has avoided the major health impacts of the SARS-CoV-2 pandemic due to a strict country-wide lockdown, the end-goal of which was elimination rather than mitigation and suppression. The New Zealand government’s use of scientific expertise, spanning public health, infectious diseases, genomics, modeling and immunology, has been one of the keys to the success of its SARS-CoV-2 elimination and control strategy.

Single cell isolation from helminth-infected murine intestines has been notoriously difficult, due to the strong anti-parasite type 2 immune responses that drive mucus production, tissue remodeling and immune cell infiltration. Through the systematic optimization of a standard intestinal digestion protocol, we were able to successfully isolate millions of immune cells from the heavily infected duodenum. To validate that these cells gave an accurate representation of intestinal immune responses, we analyzed them using a high-dimensional spectral flow cytometry panel and confirmed our findings by confocal microscopy. Our cell isolation protocol and high-dimensional analysis allowed us to identify many known hallmarks of anti-parasite immune responses throughout the entire course of helminth infection and has the potential to accelerate single-cell discoveries of local helminth immune responses that have previously been unfeasible., eLife digest Parasitic worms known as helminths represent an important health problem in large parts of Africa, South America and Asia. Once their larvae enter the body, they head to the gut where they mature into adults and start laying eggs. In areas with poor sanitation, these may then get passed on to other individuals. To defend the body, the immune system sends large numbers of immune cells to the gut, but it usually struggles to eliminate the parasites. Without deworming medication, the infection can last for many years. Scientists study helminth infections in the laboratory by using worms that naturally infect mice. Understanding exactly how the immune system responds to the infection is essential to grasp why it fails to clear the worms. However, it is difficult to extract immune cells from an infected gut, as the infection creates strong local responses – such as an intense ‘slime’ production to try to flush out the worms. The standard procedure to obtain immune cells from the gut consists of three steps: collecting a gut segment and washing it, stripping away the surface layers with chemicals, and finally using enzymes to digest the tissues, which are then filtered to obtain individual cells. However, this protocol is not able to extract cells during infection. Ferrer-Font et al. therefore methodically refined every step of this method, and finally succeeded in obtaining millions of immune cells from infected guts. For the first time, these cells could then be studied and identified using a new technology called spectral flow cytometry. Over 40 immune cell types were followed throughout the course of infection, revealing that many ‘first responders’ immune cells were recruited to the gut early on, when the worms were still larvae. However, these cells disappeared once the worms developed into adults. These findings were confirmed by microscopy, which also showed that the first responder cells were found around the developing larvae, likely attacking them. When the adult worms developed, these cells were replaced by other immune cells, which also decreased the longer the worms were present in the gut. This new extraction process established by Ferrer-Font et al. can also be paired with other technologies that can, for example, reveal which genes are turned on in individual cells. This could help map out exactly how the body fights helminth infections, and how to improve this response. The method could also be useful to extract immune cells from the gut in other challenging scenarios, such food allergies or inflammatory bowel disorders.

Single cell isolation from helminth infected intestines has been notoriously difficult, due to the strong anti-parasite type 2 immune responses that drive mucus production, tissue remodeling and immune cell infiltration. Through the systematic optimization of a standard intestinal digestion protocol, we were able to isolate millions of immune cells from heavily infected tissues. Using this protocol, we validated many hallmarks of anti-parasite immunity and analyzed immune cells from the lamina propria and granulomas during helminth development, as well as acute and chronic worm infection.

Group 2 innate lymphoid cells (ILC2s) were first discovered in experimental studies of intestinal helminth infection - and much of our current knowledge of ILC2 activation and function is based on the use of these models. It is perhaps not surprising therefore that these cells have also been found to play a key role in mediating protection against these large multicellular parasites. ILC2s have been intensively studied over the last decade, and are known to respond quickly and robustly to the presence of helminths – both by increasing in number and producing type 2 cytokines. These mediators function to activate and repair epithelial barriers, to recruit other innate cells such as eosinophils, and to help activate T helper 2 cells. More recent investigations have focused on the mechanisms by which the host senses helminth parasites to activate ILC2s. Such studies have identified novel stromal cell types as being involved in this process - including intestinal tuft cells and enteric neurons, which respond to the presence of helminths and activate ILC2s by producing IL-25 and Neuromedin, respectively. In the current review, we will outline the latest insights into ILC2 activation and discuss the requirement for – or redundancy of – ILC2s in providing protective immunity against intestinal helminth parasites.

Although IL-4 is long associated with CD4 Th2 immune responses, its role in Th2 subset development in non-lymphoid tissues is less clear. We sought to better define IL-4's role in CD4 Th2 responses by using transgenic mice that express a dual IL-4 AmCyan/IL-13 DsRed (IL-4AC/IL-13DR) fluorescent reporter on an IL-4-sufficient or IL-4-deficient background. Using primary Th2 immune response models against house dust mite or Nippostrongylus brasiliensis (Nb) allergens, we examined the requirement for IL-4 by each of the defined Th2 subsets in the antigen draining lymph node, skin, and lung tissues. In the lymph node, a CXCR5+PD-1+ T follicular helper (Tfh) and a CXCR5loPD-1lo Th2 subset could be detected that expressed only IL-4AC but no IL-13DR. The number of IL-4AC+ Tfh cells was not affected by IL-4 deficiency whereas the number of IL-4AC+ Th2 cells was significantly reduced. In the non-lymphoid dermal or lung tissues of allergen primed or Nb-infected mice, three strikingly distinct T cell subsets could be detected that were IL-4AC, or IL-4AC/IL-13DR, or IL-13DR CD4. The IL-4- and IL-4/IL-13-expressing subsets were significantly reduced in IL-4-deficient mice, while the numbers of IL-13-expressing CD4 T cells were not affected by IL-4 deficiency indicating that other factors can play a role in directing the development of this Th2 subtype. Taken together, these data indicate that the appearance of IL-4-expressing Tfh cells in the lymph node is not dependent on IL-4 while the appearance of IL-4-expressing Th2 subsets in the lymph node and IL-4, IL-4/IL-13-expressing Th2 subsets in skin and lung tissues of antigen primed mice is significantly IL-4 dependent.

Background and objective Neutrophilic inflammation has been implicated in non-eosinophilic asthma (NEA) in adults, but little is known about NEA in children/adolescents. We assessed clinical and inflammatory characteristics of NEA in adolescent asthma. Methods Airway inflammation, sputum endotoxin, airway hyper-reactivity, atopy and lung function were assessed in 77 adolescents with asthma and 68 without asthma (12–17 years). Asthma was identified on the basis of wheeze and asthma history. Results The proportion of NEA (sputum eosinophils

Atopic dermatitis (AD) is a highly debilitating disease with significant health impacts worldwide. It has been a difficult disease to treat because of the wide spectrum of clinical manifestations. Therefore, the current clinical management strategies are nonspecific. Previous studies have documented that AD disease progression is precipitated by a combination of skin barrier dysfunction, itch, and immune dysregulation. However, the precise roles played by effector cells and cytokines have not been fully elucidated. To address this, we established a prolonged model of AD, using MC903. The phenotype of this MC903 model closely resembles the one observed in AD patients, including inflammatory parameters, barrier dysfunction, itch, and histopathological characteristics, thereby providing a platform to evaluate targets for the treatment of AD. This model exposed cells and cytokines that are critically associated with disease severity, including eosinophils, TSLP, and IL-4/IL-13. Indeed, eosinophil depletion significantly ameliorated AD pathology, most notably barrier dysfunction, to a similar extent as blocking of the IL-4/IL-13 axis by genetic deletion of STAT6. Thus, this study has identified eosinophils to be critical for the development and maintenance of AD, thereby proposing these effector cells as therapeutic targets for the treatment of AD.

The induction of Th2 responses is thought to be multifactorial, and emerge from specific pathways distinct from those associated with antagonistic antibacterial or antiviral Th1 responses. Here, we show that the recognition of non-viable Nippostrongylus brasiliensis (Nb) in the skin induces a strong recruitment of monocytes and neutrophils and the release of neutrophil extracellular traps (NETs). Nb also activates toll-like receptor 4 (TLR4) signaling with expression of Ifnb transcripts in the skin and the development of an IFN type I signature on helminth antigen-bearing dendritic cells in draining lymph nodes. Co-injection of Nb together with about 10,000 Gram-negative bacteria amplified this TLR4-dependent but NET-independent IFN type I response and enhanced the development of Th2 responses. Thus, a limited activation of antibacterial signaling pathways is able to boost antihelminthic responses, suggesting a role for bacterial sensing in the optimal induction of Th2 immunity.

Type 2 immune responses in the skin cause a variety of pathologies, including urticaria and atopic dermatitis. Traditionally, CD4(+) helper T cells have been implicated in the pathogenesis of these conditions. However, recently a new player, the group 2 innate lymphoid (ILC2) cell, has emerged as an important contributor to skin allergies. In this review, we summarize our current knowledge of the role ILC2 cells play in the physiology and pathology of mouse and human skin.

Dendritic cells (DC) are critical for the initiation of immune responses; however, their role in priming IL-4–producing Th2 cells in vivo is not fully understood. We used a model of intradermal injection with fluorescent-labeled, nonviable larvae from the helminth parasite nonviable Nippostrongylus brasiliensis L3 larvae (Nb), a strong inducer of Th2 responses, together with IL-4–GFP reporter mice that enable a sensitive detection of IL-4 production to examine the contribution of DC to the priming of IL-4–producing CD4+ T cells in vivo. We found that parasite material is taken up by two distinct DC populations in draining lymph nodes: a mostly CD11cintMHC class II (MHCII)hiCD11b+Ly6C− dermal DC population and a CD11chiMHCIIintCD11b+Ly6C+ monocyte-derived DC population. After Nb treatment, these two DC populations appeared in the draining lymph nodes in comparable numbers and with similar kinetics; however, treatment with pertussis toxin blocked the migration of dermal DC and the priming of IL-4–producing T cells, but only partially affected monocyte-derived DC numbers. In line with this observation, transfer of OVA-loaded CD11cintMHCIIhi DC from Nb-treated mice into naive hosts could sensitize OVA-specific CD4+ T cells to IL-4 production, whereas transfer of CD11cintMHCIIhi DC from naive mice, or CD11chiMHCIIint DC from Nb-treated or naive mice, induced CD4+ T cell expansion but no IL-4 production. Phenotypic analysis of Nb-loaded CD11cintMHCIIhi DC revealed expression of programmed death ligand 2, CD301b, IFN regulatory factor 4, and moderate upregulation of OX40 ligand. However, thymic stromal lymphopoietin and OX40 ligand were not required for Th2 priming. Thus, our data suggest that appropriate stimuli can induce DC to express the unique signals sufficient to direct CD4+ T cells to Th2 differentiation.

A keenly sought therapeutic approach for the treatment of allergic disease is the identification and neutralization of the cytokine that regulates the differentiation of T helper 2 (Th2) cells. Th2 cells are exciting targets for asthma therapies. Recently, the cytokine IL-25 has been shown to enhance Th2-type immune activity and play important roles in mediating allergic inflammatory responses. To investigate this further, we crossed IL-25(-/-) C57BL/6 mice with G4 IL-4 C57BL/6 reporter mice and developed an assay for in vitro and in vivo IL-4-independent Th2-cell differentiation. These assays were used to determine whether IL-25 was critical for the formation of Th2 cells. We found there was no physiological role for IL-25 in either the differentiation of Th2 cells or their development to effector or memory Th2-cell subsets. Importantly, this data challenges the newly found and growing status of the cytokine IL-25 and its proposed role in promoting Th2-cell responses.

How Th2 immunity develops in vivo remains obscure. Basophils have been considered key innate cells producing IL-4, a cytokine essential for Th2 immunity. Increasing evidence suggests that basophils are dispensable for the initiation of Th2 immunity. In this study, we revisited the role of basophils in Th2 immune responses induced by various types of adjuvants. Mice deficient in IL-3 or IL-3 receptor, in which basophil lymph node recruitment is completely abolished, fully developed wild type level Th2 CD4 T cell responses in response to parasite antigen or papain immunization. Similar finding was also observed in mice where basophils are inducibly ablated. Interestingly, IL-4-derived from non-T cells appeared to be critical for the generation of IL-4-producing CD4 T cells. Other Th2 promoting factors including IL-25 and thymic stromal lymphopoietin (TSLP) were dispensable. Therefore, our results suggest that IL-3- and basophil-independent in vivo Th2 immunity develops with the help of non-T cell-derived IL-4, offering an additional mechanism by which Th2 type immune responses arise in vivo.

Exposure to allergens, both man-made and from our environment is increasingly associated with the development of significant human health issues such as allergy and asthma. Allergen induced production of the cytokine interleukin (IL-)4 by Th2 cells is central to the pathogenesis of allergic disease (Gavett et al., 1994). The development of the G4 mouse, that expresses green fluorescent protein (GFP) as a surrogate for IL-4 protein expression has made it possible to directly track the immune cells that produce IL-4. By combining a reliable intradermal immunisation technique with the transgenic G4 mouse we have been able to develop a novel & unique in vivo primary Th2 immune response model (PTh2). When allergens relevant to human disease are evaluated using the PTh2 assay a dose dependent hierarchy of allergenicity is revealed with environmental allergens (cockroach, house dust mite) the most potent and food allergens being the least. In addition, the PTh2 assay is extremely sensitive to the immunoregulatory effects of Mycobacterial extracts and immunosuppressive drugs on primary Th2 cell development. Taken together, this assay provides a standardised method for the identification of the structural and functional properties of proteins relevant to allergenicity, and is a powerful screening tool for novel lead compounds that are effective at inhibiting the primary Th2 response in allergic diseases.

Polymorphisms in genes involved in IL-4 responses segregate with allergic disease risk and correlate with IgE levels in humans, and IL-4 promotes IgE and IgG1 Ab production against allergens in mice. We report that mice with only one intact Il4 gene copy are significantly impaired in their ability to make specific IgE responses against allergens, whereas IgG1 responses to allergens remain unaffected. Il4-hemizygosity also resulted in a modest but detectable drop in IL-4 production by CD4+ T cells isolated from lymph nodes and prevented IgE-dependent oral allergen–induced diarrhea. We conclude that a state of haploinsufficiency for the Il4 gene locus is specifically relevant for IL-4–dependent IgE responses to allergens with the amount of IL-4 produced in the hemizygous condition falling close to the threshold required for switching to IgE production. These results may be relevant for how polymorphisms in genes affecting IL-4 responses influence the risk of IgE-mediated allergic disease in humans.

The induction of Th2 responses is thought to be multifactorial, and emerge from specific pathways distinct from those associated with antagonistic antibacterial or antiviral Th1 responses. Here, we show that the recognition of non-viable Nippostrongylus brasiliensis (Nb) in the skin induces a strong recruitment of monocytes and neutrophils and the release of neutrophil extracellular traps (NETs). Nb also activates toll-like receptor 4 (TLR4) signaling with expression of Ifnb transcripts in the skin and the development of an IFN type I signature on helminth antigen-bearing dendritic cells in draining lymph nodes. Co-injection of Nb together with about 10,000 Gram-negative bacteria amplified this TLR4-dependent but NET-independent IFN type I response and enhanced the development of Th2 responses. Thus, a limited activation of antibacterial signaling pathways is able to boost antihelminthic responses, suggesting a role for bacterial sensing in the optimal induction of Th2 immunity. Published version

Traditional jet-in-air cell sorters have been designed and optimized to isolate small particles such as mammalian lymphocytes with an average diameter of 10 μm. We discuss the practical considerations of setting up a conventional jet-in-air cell sorter, using a 200-μm nozzle, to isolate the large parasitic nematode eggs of Nippostrongylus brasiliensis, with a maximum size of 60 μm. The eggs were separated based on light scattering properties, no fluorescent dye or molecule was required.

The rodent hookwormNippostrongylus brasiliensistypically infects its host by penetrating the skin and rapidly migrating to the lungs and gut. Following primary infection, immunocompetent mice become highly protected from reinfection withN. brasiliensis, with the numbers of worms gaining access to the lungs and gut being reduced by up to 90%. We used green fluorescent protein/interleukin-4 (IL-4) reporter mice and truncated infection studies to identify both the tissue site and mechanism(s) by which the host protects itself from reinfection withN. brasiliensis. Strikingly, we demonstrated that the lung is an important site for priming immune protection. Furthermore, a lung-initiated, CD4 T-cell-dependent, and IL-4- and STAT6-dependent response was sufficient to confer protection against reinfection. In conclusion, vaccination strategies which seek to break the cycle of reinfection and egg production by helminths such as hookworms can include strategies which directly stimulate Th2 responses in the lung.

The immune mechanisms that orchestrate protection against tuberculosis as a result of BCG vaccination are not fully understood. We used the immunomodulatory properties of fingolimod (FTY720) treatment to test whether the lung-resident memory T lymphocytes generated by BCG vaccination were sufficient to maintain immunity against challenge infection with mycobacteria (BCG). Mice were given daily fingolimod treatment, starting either immediately before s.c. BCG vaccination or during subsequent BCG i.n. challenge, to prevent LN effector and memory lymphocytes from entering the periphery either during priming or challenge, respectively. Treatment with fingolimod during vaccination reduced vaccine-mediated protection against subsequent infection. By contrast, BCG-vaccinated mice were protected when fingolimod was given during the infectious challenge, suggesting that memory lymphocytes that migrate to the lung following vaccination are sufficient for protection. Notably, the antigen-reactive IFN-gamma or multicytokine-producing CD4(+) T cells present in the lung when fingolimod was given during BCG challenge did not correlate with protection; however, expression of MHC class II on macrophages isolated from the lungs post BCG challenge was increased in the protected mice. We conclude that protection conferred by BCG vaccination is dependent on memory lymphocytes retained in the lung, although IFN-gamma production by this population is not correlated with vaccine-mediated protection.

Improved analytical tools have revealed that the development and expression of a Th2 immune response can be broken down into distinct stages with respect to the cytokine microenvironment that is required. Although IL-4 and its STAT6-signalling pathway are critical for the expression of Th2 effector immune responses in peripheral tissues such as the skin, lung and gut, IL-4 and STAT6 signalling are not required for the initial generation of IL-4-producing Th2 cells in the lymph node. This finding reveals that we have yet to identify the key cytokine or microenvironment that stimulates the development of this most intriguing CD4(+) T-helper subset and emphasises the tissue specificity and timing of IL-4/STAT6-dependent Th2 effector responses.

The expression of interleukin-4 (IL-4) is viewed as the hallmark of a Th2 lymphocyte, whereas the subsequent action of IL-4 and IL-13, mediated through the STAT6 signaling pathway, is seen as a prerequisite for the full development of Th2 immune responses to parasites and allergens. G4 mice, whose IL-4 gene locus contains the fluorescent reporter eGFP, were used to quantify the number of Th2 cells that develop during Nippostrongylus brasiliensis - or allergen-induced immune responses under conditions where IL-4 or STAT6 was absent. Here, we show that deletion of IL-4 or STAT6 had little impact on the number or timing of appearance of IL-4-producing Th2 cells. These data indicate that in vivo differentiation of naïve CD4 T cells to Th2 status often occurs independently of IL-4 and STAT6 and that recently described pathways of Th2 cell differentiation may explain how allergens and parasites selectively induce Th2-mediated immunity.

The antigen-induced activation and differentiation of CD4+ T cells into the specialized subtypes Th2 and Th9 is controlled by a distinct stimuli resulting in the expression of subtype-specific transcription factors that further define and reinforce the specific functional roles each has in an immune response. The Th2 phenotype is regulated by the transcription factor GATA3, which alongside phosphorylated STAT5 promotes the expression of interleukin-4 (IL-4), IL-5, and IL-13. These three cytokines mediate the Type 2 immune responses that mediate atopic disease and the immunity and pathology associated with helminth infections. The development of the Th9 phenotype is governed by a set of transcription factors that includes Pu.1, IRF4, and BATF and through their expression of IL-9 contribute to a range of proinflammatory conditions including atopy and intestinal inflammation and also antitumor immunity.

Defining the immune mechanisms underlying protective immunity to helminth infection remains an important challenge. Here we report that lung CD4(+) T cells and Group 2 innate lymphoid cells (ILC2s) work in concert to block Nippostrongylus brasiliensis (Nb) development in the parenchyma within 48 h in mice. Immune-damaged larvae have a striking morphological defect that is dependent on the expansion of IL-13-producing ILC2 and CD4(+) T cells, and the activation of M2 macrophages. This T-cell requirement can be bypassed by administration of IL-2 or IL-33, resulting in expansion of IL-13-producing ILC2s and larval killing. Depletion of ILC2s inhibits larval killing in IL-2-treated mice. Our results broaden understanding of ILC2's role in immunity to helminths by demonstrating that they not only act as alarmin sensors, but can also be sustained by CD4(+) T cells, ensuring both the prompt activation and the maintenance of IL-13-dependent M2 macrophage immunity in the lung.

Previous activation of effector Th2 cells is central to the development of allergic inflammatory responses. We have observed that priming of allergen-specific Th2 cells in C57BL/6 or B10.A mice with allergen delivered via the i.p. or s.c. routes results in very different outcomes following subsequent airway exposure to the same allergen. Systemic allergen immunization (via the i.p. route) resulted in the formation of a lung-resident population of allergen-specific T cells, and mice developed severe allergic airway inflammation in response to inhaled allergen. The localization of cells to the lung did not require the presence of antigen at this site, but reflected a large pool of circulating activated allergen-specific T cells. In contrast, localized immunization (via the s.c. route) resulted in a small T-cell response restricted to the draining lymph node, and mice were not responsive to inhaled allergen. These data indicate that prior sensitization to an allergen alone was not sufficient for the induction of allergic inflammation; rather, responsiveness was largely determined by precursor frequency and tissue localization of the allergen-specific effector Th2 cells.

It is often argued that T cell-mediated immunity to secondary infection is dependent on the 'accelerated' responses of memory T cells in lymph nodes. However, new evidence points to a crucial role for effector memory T cells, which are resident in peripheral tissues, in immune protection. These T cells, which reside in peripheral tissues, are not necessarily bound by an anatomical structure and can be present at many sites. Collectively, they represent a third functional tissue of the immune system, uniquely specialized to mediate protective immunity. We propose that the paradigm 'effector lymphoid tissue' needs to be articulated and developed as a focus of new research to describe and understand the unique role this tissue has in protective immunity.

Allergic asthma is responsible for widespread morbidity and mortality and its incidence has increased dramatically in industrialized countries over the past two decades. Here, we describe a new murine model of allergic asthma utilizing a novel allergen with intrinsic enzymatic activity similar to that reported for many environmental allergens. The allergen, NES, is excreted and secreted from the nematode Nippostrongylus brasiliensis, and can readily be isolated from in vitro parasite cultures. When NES is administered intranasally to presensitized mice, allergic airway disease develops, including airway hyper-responsiveness, airway eosinophilia, IgE antibody production and Th2 cytokine production. This disease is characteristic of atopic asthma and can be induced within 11 days, thus providing a platform for the rapid analysis of allergic disease and high throughput testing of immunomodulatory factors.

Administration of heat-killed mycobacteria can suppress allergic disease in mice and humans. The active components of mycobacteria mediating these effects remain unresolved.We sought to identify the active components of mycobacteria mediating suppression of allergic disease and to determine structural features important for function.Using a murine model of allergic airway disease, we tested the ability of the lipoglycan fractions of the mycobacterial cell wall to suppress airway eosinophilia. Lipoglycans isolated from different strains of mycobacteria and chemical modifications were used to explore structure-function relationships. Markers of allergic disease including bronchoalvealor lavage cytokines, spleen and lymph node T-cell cytokine production, and serum specific immunoglobulin (Ig) E/IgG1 were examined.We identified the mycobacterial cell wall lipoglycans lipoarabinomannan and phosphatidylinositol mannan as components of mycobacteria capable of suppressing airway disease (70% reduction in airway eosinophilia; P.03). Structure-function analysis identified the acyl chains and mannose groups of the molecules as having a role in mediating this effect. Mechanistic studies provided no evidence for a T-helper cell (Th) 1-mediated suppression of an ongoing Th2 response. An increased capacity of T cells to secrete interleukin 10 in the spleen and lymph node of treated animals was identified, suggesting a potential T-cell-mediated suppression mechanism.We have identified immunomodulatory component(s) of mycobacteria responsible for the protective effects observed in allergic disease; these findings will lead to the generation of synthetic compounds or agonists devoid of the unwanted characteristics of whole mycobacteria for evaluation in a human clinical setting.

CD8+ memory T cells have recently been recognized as playing a key role in natural immunity against unrelated viral infections, a phenomenon referred to as “heterologous antiviral immunity.” We now provide data that the cellular immunological interactions that underlie such heterologous immunity can play an equally important role in regulating T helper 2 immune responses and protecting mucosal surfaces from allergen-induced inflammation. Our data show that CD8+ T cells, either retained in the lung after infection with influenza virus, or adoptively transferred via the intranasal route can suppress allergic airway inflammation. The suppression is mediated by IFN-γ, which acts to reduce the activation level, T helper 2 cytokine production, airways hyperresponsiveness, and migration of allergen-specific CD4+ T cells into the lung, whereas the systemic and draining lymph node responses remain unchanged. Of note, adoptive transfer of previously activated transgenic CD8+ T cells conferred protection against allergic airway inflammation, even in the absence of specific-antigen. Airway resident CD8+ T cells produced IFN-γ when directly exposed to conditioned media from activated dendritic cells or the proinflammatory cytokines IL-12 and IL-18. Taken together these data indicate that effector/memory CD8+ T cells present in the airways produce IFN-γ after inflammatory stimuli, independent of specific-antigen, and as a consequence play a key role in modifying the degree and frequency of allergic responses in the lung.

A cohort of immunodeficient children lead near-normal lives after bone marrow transplantation, despite a profound deficiency of innate lymphoid cells (ILCs).

Certain forms of the neuroendocrine hormone preproenkephalin (PPNK) are produced by T cells, B cells and macrophages. This hormone has been shown to be important in regulating a variety of immune responses; however, the basic mechanisms of this regulation are unknown. Here we examine the ability of CD8 and CD4 PPNK-deficient T lymphocytes to proliferate to antigenic and mitogenic stimuli. We found that lymphocyte activation and proliferation to suboptimal concentrations of both anti-CD3 and antigen was reduced in the absence of PPNK. Proliferation could be rescued by increasing antigen or by co-incubation of PPNK-deficient cells with wild-type cells. These data confirm the importance of neuroendocrine hormones such as PPNK in T cell activation and proliferation and provides a potential mechanism for the regulation of T cell responses by PPNK or its peptide derivatives.

Parasitic helminth infections can be associated with lifelong morbidity such as immune-mediated organ failure. A better understanding of the host immune response to helminths could provide new avenues to promote parasite clearance and/or alleviate infection-associated morbidity. Murine resistin-like molecules (RELM) exhibit pleiotropic functions following helminth infection including modulating the host immune response; however, the relevance of human RELM proteins in helminth infection is unknown. To examine the function of human resistin (hResistin), we utilized transgenic mice expressing the human resistin gene (hRetnTg+). Following infection with the helminth Nippostrongylus brasiliensis (Nb), hResistin expression was significantly upregulated in infected tissue. Compared to control hRetnTg− mice, hRetnTg+ mice suffered from exacerbated Nb-induced inflammation characterized by weight loss and increased infiltration of inflammatory monocytes in the lung, along with elevated Nb egg burdens and delayed parasite expulsion. Genome-wide transcriptional profiling of the infected tissue revealed that hResistin promoted expression of proinflammatory cytokines and genes downstream of toll-like receptor signaling. Moreover, hResistin preferentially bound lung monocytes, and exogenous treatment of mice with recombinant hResistin promoted monocyte recruitment and proinflammatory cytokine expression. In human studies, increased serum resistin was associated with higher parasite load in individuals infected with soil-transmitted helminths or filarial nematode Wuchereria bancrofti, and was positively correlated with proinflammatory cytokines. Together, these studies identify human resistin as a detrimental factor induced by multiple helminth infections, where it promotes proinflammatory cytokines and impedes parasite clearance. Targeting the resistin/proinflammatory cytokine immune axis may provide new diagnostic or treatment strategies for helminth infection and associated immune-mediated pathology., Author Summary Parasitic helminths, which infect an estimated two billion people worldwide, represent a significant global public health problem. Infection is associated with life-long morbidity including growth retardation and organ failure. Despite these debilitating conditions, there are currently no successful vaccines against helminths. Further, great variability in the host immune response to helminths exists, with the ability of some individuals to develop immunity, while others are susceptible when re-exposed or maintain life-long chronic infections. Identifying new factors that are differentially expressed in immune versus susceptible individuals could provide new targeting strategies for diagnosis or treatment of helminth infection. Here, we identify an important immunoregulatory function for human resistin in helminth infection. Employing transgenic mice in which the human resistin gene was inserted, we show that human resistin is induced by infection with the helminth Nippostrongylus brasiliensis, where it promotes excessive inflammation and impedes parasite killing. Moreover, analysis of clinical samples from two cohorts of individuals infected with filarial nematodes or soil-transmitted helminths revealed increased resistin and serum proinflammatory cytokines compared to putatively immune individuals. Together, these studies suggest that human resistin is a detrimental cytokine that is expressed in multiple helminth infections, mediates pathogenic inflammation, and delays parasite clearance.

The c-Jun NH(2)-terminal kinase (JNK) signaling pathway is induced by cytokines and stress stimuli and is implicated in cell death and differentiation, but the specific function of this pathway depends on the cell type. Here we examined the role of JNK1 and JNK2 in CD8(+) T cells. Unlike CD4(+) T cells, the absence of JNK2 causes increased interleukin (IL)-2 production and proliferation of CD8(+) T cells. In contrast, JNK1-deficient CD8(+) T cells are unable to undergo antigen-stimulated expansion in vitro, even in the presence of exogenous IL-2. The hypoproliferation of these cells is associated with impaired IL-2 receptor alpha chain (CD25) gene and cell surface expression. The reduced level of nuclear activating protein 1 (AP-1) complexes in activated JNK1-deficient CD8(+) T cells can account for the impaired IL-2 receptor alpha chain gene expression. Thus, JNK1 and JNK2 play different roles during CD8(+) T cell activation and these roles differ from those in CD4(+) T cells.

Helminths are credited with being the major selective force driving the evolution of the so-called "type 2" immune responses in vertebrate animals, with their size and infection strategies presenting unique challenges to the immune system. Originally, type 2 immune responses were defined by the presence and activities of the CD4(+) T-helper 2 subset producing the canonical cytokines IL-4, IL-5, and IL-13. This picture is now being challenged by the discovery of a more complex pattern of CD4(+) T-helper cell subsets that appear during infection, including Tregs, Th17, Tfh, and more recently, Th22, Th9, and ThGM. In addition, a clearer view of the mechanisms by which helminths and their products selectively prime the CD4(+) T-cell subsets is emerging. In this review, we have focused on recent data concerning the selective priming, differentiation, and functional role of CD4(+) T-helper cell subsets in the context of helminth infection. We argue for a re-evaluation of the original Th2 paradigm and discuss how the observed plasticity of the T-helper subsets may enable the parasitized host to achieve an appropriate compromise between elimination, tissue repair, containment, and pathology.

The cytokine thymic stromal lymphopoietin (TSLP) is produced by epithelia exposed to the contact sensitizer dibutyl phthalate (DBP), and it is critical for the induction of Th2 immune responses by DBP-FITC. TSLP is thought to act on dendritic cells (DC), but the precise DC subsets involved in the response to TSLP remain to be fully characterized. In this study we show that a subset of CD326loCD103loCD11blo dermal DC, which we termed “triple-negative (TN) DC,” is highly responsive to TSLP. In DBP-FITC–treated mice, TN DC upregulated expression of CD86 and rapidly migrated to the draining lymph node to become the most abundant skin-derived DC subset at 24 and 48 h after sensitization. None of these responses was observed in TSLPR-deficient mice. In contrast, TN DC numbers were not increased after treatment with the allergen house dust mite or the bacteria Escherichia coli and bacillus Calmette–Guérin, which increased other DC subsets. In vivo, treatment with rTSLP preferentially increased the numbers of TN DC in lymph nodes. In vitro, TN DC responded to rTSLP treatment with a higher level of STAT5 phosphorylation compared with other skin-derived DC subsets. The TN DC subset shared the morphology, phenotype, and developmental requirements of conventional DC, depending on FLT3 expression for their optimal development from bone marrow precursors, and CCR7 for migration to the draining lymph node. Thus, TN DC represent a dermal DC subset that should be considered in future studies of TSLP-dependent contact sensitization and skin immune responses.

We have used preproenkephalin (PPNK)-deficient mice to study the role of PPNK in the development of airway inflammation. Airway eosinophilia was established by either sensitization followed by airway challenge with OVA or by infection with Nippostrongylus brasiliensis. Both models induce a strong Th2 immune response, characterized by an IL-5-dependent airway eosinophilia. We observed that although the accumulation of lymphocytes in the airways of PPNK-deficient mice was similar to that induced in control mice, IL-5 production and eosinophil infiltration were reduced. We conclude from this work that PPNK has a role in enhancing Th2 cell function and that this molecule may be an important target in asthma immunotherapy.

Asthma is an atopic disorder characterised by the activation and recruitment of eosinophils to the lung resulting in chronic swelling and inflammation of the airways. Allergic disorders such as atopic asthma and dermatitis have been increasingly prevalent in developed countries, and the inverse correlation between exposure to major diseases such as tuberculosis and atopy prevalence has been reported. Intranasal administration of Mycobacterium bovis-Bacillus Calmette-Guerin (BCG) has been demonstrated to suppress airway eosinophilia in a model of atopic asthma. This immunomodulation is attributed to the ability of interferon (IFN)-gamma produced by BCG-specific T(H)1 lymphocytes to inhibit the development of lung T(H)2 responses such as airway eosinophilia. The mechanism of IFNgamma-induced inhibition is yet to be defined, but could involve activation of macrophages, direct suppression of developing T(H)2 lymphocytes, or altered dendritic cell activation and antigen presentation. Mycobacteria such as BCG and certain mycobacterial fractions are strong inducers of a T(H)1 immune response. The effectiveness of BCG in inhibiting atopic airway eosinophilia suggests its potential as a useful therapeutic agent in the treatment of atopic asthma.