Eosinophil extracellular vesicles and DNA traps in allergic inflammation.

Eosinophil granulocytes, a specialized subset of white blood cells, have traditionally been associated with allergic responses and parasitic infections. However, recent research has unveiled their versatile roles in immune regulation beyond these classical functions. This review highlights the emerging field of eosinophil biology, with a particular focus on their release of extracellular vesicles (EVs) and extracellular DNA traps (EETs). It further explores potential implications of eosinophil-derived EVs and EETs for immune responses during inflammatory diseases. The release of EVs/EETs from eosinophils, which also affects the eosinophils themselves, may influence both local and systemic immune reactions, affecting the pathophysiology of conditions such as airway inflammation, chronic rhinosinusitis and atopic dermatitis.

Eosinophils as Modulators of Host Defense during Parasitic, Fungal, Bacterial, and Viral Infections.

Eosinophils, traditionally associated as central innate effector cells with type-2 immunity during allergic and helminth parasitic diseases, have recently been revealed to have important roles in tissue homeostasis as well as host defense in a broader variety of infectious diseases. In a dedicated session at the 2023 biennial conference of the International Eosinophil Society titled "Eosinophils in Host Defense", the multifaceted roles eosinophils play against diverse pathogens ranging from parasites to fungi, bacteria, and viruses was presented. In this review, the session speakers offer a comprehensive summary of recent discoveries across pathogen classes, positioning eosinophils as pivotal leukocytes in both host defense and pathology. By unraveling the intricacies of eosinophil engagement in host resistance, this exploration may provide valuable insights not only to understand specific underpinnings of the eosinophil functions related to each class of pathogens, but also to develop novel therapeutics effective against a broad spectrum of infectious diseases.

CCR3-dependent eosinophil recruitment is regulated by sialyltransferase ST3Gal-IV.

Eosinophil recruitment is a pathological hallmark of many allergic and helminthic diseases. Here, we investigated chemokine receptor CCR3-induced eosinophil recruitment in sialyltransferase St3gal4-/- mice. We found a marked decrease in eosinophil extravasation into CCL11-stimulated cremaster muscles and into the inflamed peritoneal cavity of St3gal4-/- mice. Ex vivo flow chamber assays uncovered reduced adhesion of St3gal4-/- compared to wild type eosinophils. Using flow cytometry, we show reduced binding of CCL11 to St3gal4-/- eosinophils. Further, we noted reduced binding of CCL11 to its chemokine receptor CCR3 isolated from St3gal4-/- eosinophils. This was accompanied by almost absent CCR3 internalization of CCL11-stimulated St3gal4-/- eosinophils. Applying an ovalbumin-induced allergic airway disease model, we found a dramatic reduction in eosinophil numbers in bronchoalveolar lavage fluid following intratracheal challenge with ovalbumin in St3gal4-deficient mice. Finally, we also investigated tissue-resident eosinophils under homeostatic conditions and found reduced resident eosinophil numbers in the thymus and adipose tissue in the absence of ST3Gal-IV. Taken together, our results demonstrate an important role of ST3Gal-IV in CCR3-induced eosinophil recruitment in vivo rendering this enzyme an attractive target in reducing unwanted eosinophil infiltration in various disorders including allergic diseases.

Eosinophils preserve bone homeostasis by inhibiting excessive osteoclast formation and activity via eosinophil peroxidase.

Eosinophils are involved in tissue homeostasis. Herein, we unveiled eosinophils as important regulators of bone homeostasis. Eosinophils are localized in proximity to bone-resorbing osteoclasts in the bone marrow. The absence of eosinophils in ΔdblGATA mice results in lower bone mass under steady-state conditions and amplified bone loss upon sex hormone deprivation and inflammatory arthritis. Conversely, increased numbers of eosinophils in IL-5 transgenic mice enhance bone mass under steady-state conditions and protect from hormone- and inflammation- mediated bone loss. Eosinophils strongly inhibit the differentiation and demineralization activity of osteoclasts and lead to profound changes in the transcriptional profile of osteoclasts. This osteoclast-suppressive effect of eosinophils is based on the release of eosinophil peroxidase causing impaired reactive oxygen species and mitogen-activated protein kinase induction in osteoclast precursors. In humans, the number and the activity of eosinophils correlates with bone mass in healthy participants and rheumatoid arthritis patients. Taken together, experimental and human data indicate a regulatory function of eosinophils on bone.

STAT6-induced production of mucus and resistin-like molecules in lung Club cells does not protect against helminth or influenza A virus infection.

Airway epithelial cells contribute to a variety of lung diseases including allergic asthma, where IL-4 and IL-13 promote activation of the transcription factor STAT6. This leads to goblet cell hyperplasia and the secretion of effector molecules by epithelial cells. However, the specific effect of activated STAT6 in lung epithelial cells is only partially understood. Here, we created a mouse strain to selectively investigate the role of constitutively active STAT6 in Club cells, a subpopulation of airway epithelial cells. CCSP-Cre_STAT6vt mice and bronchiolar organoids derived from these show an enhanced expression of the chitinase-like protein Chil4 (Ym2) and resistin-like molecules (Relm-α, -ß, -γ). In addition, goblet cells of these mice spontaneously secrete mucus into the bronchi. However, the activated epithelium resulted neither in impaired lung function nor conferred a protective effect against the migrating helminth Nippostrongylus brasiliensis. Moreover, CCSP-Cre_STAT6vt mice showed similar allergic airway inflammation induced by live conidia of the fungus Aspergillus fumigatus and similar recovery after influenza A virus infection compared to control mice. Together these results highlight that STAT6 signaling in Club cells induces the secretion of Relm proteins and mucus without impairing lung function, but this is not sufficient to confer protection against helminth or viral infections.

Mast cells link immune sensing to antigen-avoidance behaviour.

The physiological functions of mast cells remain largely an enigma. In the context of barrier damage, mast cells are integrated in type 2 immunity and, together with immunoglobulin E (IgE), promote allergic diseases. Allergic symptoms may, however, facilitate expulsion of allergens, toxins and parasites and trigger future antigen avoidance1-3. Here, we show that antigen-specific avoidance behaviour in inbred mice4,5 is critically dependent on mast cells; hence, we identify the immunological sensor cell linking antigen recognition to avoidance behaviour. Avoidance prevented antigen-driven adaptive, innate and mucosal immune activation and inflammation in the stomach and small intestine. Avoidance was IgE dependent, promoted by Th2 cytokines in the immunization phase and by IgE in the execution phase. Mucosal mast cells lining the stomach and small intestine rapidly sensed antigen ingestion. We interrogated potential signalling routes between mast cells and the brain using mutant mice, pharmacological inhibition, neural activity recordings and vagotomy. Inhibition of leukotriene synthesis impaired avoidance, but overall no single pathway interruption completely abrogated avoidance, indicating complex regulation. Collectively, the stage for antigen avoidance is set when adaptive immunity equips mast cells with IgE as a telltale of past immune responses. On subsequent antigen ingestion, mast cells signal termination of antigen intake. Prevention of immunopathology-causing, continuous and futile responses against per se innocuous antigens or of repeated ingestion of toxins through mast-cell-mediated antigen-avoidance behaviour may be an important arm of immunity.

Th2-dependent disappearance and phenotypic conversion of mouse alveolar macrophages.

Alveolar macrophages (alvMs) play an important role for maintenance of lung function by constant removal of cellular debris in the alveolar space. They further contribute to defense against microbial or viral infections and limit tissue damage during acute lung injury. alvMs arise from embryonic progenitor cells, seed the alveoli before birth, and have life-long self-renewing capacity. However, recruited monocytes may also help to restore the alvM population after depletion caused by toxins or influenza virus infection. At present, the population dynamics and cellular plasticity of alvMs during allergic lung inflammation is poorly defined. To address this point, we used a mouse model of Aspergillus fumigatus-induced allergic lung inflammation and observed that Th2-derived IL-4 and IL-13 caused almost complete disappearance of alvMs. This effect required STAT6 expression in alvMs and also occurred in various other settings of type 2 immunity-mediated lung inflammation or administration of IL-4 complexes to the lung. In addition, Th2 cells promoted conversion of alvMs to alternatively activated macrophages and multinucleated giant cells. Given the well-established role of alvMs for maintenance of lung function, this process may have implications for resolution of inflammation and tissue homeostasis in allergic asthma.

Type 2 Immunity Regulates Dermal White Adipose Tissue Function.

Type 2 immune responses have been increasingly linked with tissue maintenance, regeneration, and metabolic homeostasis. The molecular basis of regulator and effector mechanisms of type 2 immunity in skin regeneration and homeostasis is still lacking. In this study, we analyzed the role of IL-4Rα signaling in the regeneration of diverse cellular compartments in the skin. Mutants with global IL-4Rα deficiency showed two major phenotypes: first, a pronounced atrophy of the interfollicular epidermis, and second, a significant increase in dermal white adipose tissue thickness in mice aged 3 weeks (postnatal day 21) compared with littermate controls. Notably, IL-4Rα deficiency decreased the activation of hormone-sensitive lipase, a rate-limiting step in lipolysis. Immunohistochemical and FACS analysis in IL-4/enhanced GFP reporter mice showed that IL-4 expression peaked on postnatal day 21 and that eosinophils are the predominant IL-4-expressing cells. Eosinophil-deficient mice recapitulated the lipolytic-defective dermal white adipose tissue phenotype of Il4ra-deficient mice, showing that eosinophils are necessary for dermal white adipose tissue lipolysis. Collectively, we provide mechanistic insights into the regulation of interfollicular epidermis and hormone-sensitive lipase-mediated lipolysis in dermal white adipose tissue in early life by IL-4Rα, and our findings show that eosinophils play a critical role in this process.

Th2-dependent STAT6-regulated genes in intestinal epithelial cells mediate larval trapping during secondary Heligmosomoides polygyrus bakeri infection.

Gastrointestinal helminths are a major health threat worldwide. Alternatively activated macrophages (AAMs) have been shown to contribute to host protection during secondary helminth infections. AAMs express effector molecules that depend on activation of the IL-4- or IL-13-induced transcription factor signal transducer and activator of transcription 6 (STAT6). However, the specific role of STAT6-regulated genes like Arginase-1 (Arg1) from AAMs or STAT6-regulated genes in other cell types for host protection remains unclear. To address this point, we generated mice expressing STAT6 only in macrophages (Mac-STAT6 mouse). In the model of Heligmosomoides polygyrus bakeri (Hpb) infection, Mac-STAT6 mice could not trap larvae in the submucosa of the small intestine after secondary infection. Further, mice lacking Arg1 in hematopoietic and endothelial cells were still protected from secondary Hpb infection. On the other hand, specific deletion of IL-4/IL-13 in T cells blunted AAM polarization, activation of intestinal epithelial cells (IECs) and protective immunity. Deletion of IL-4Rα on IEC also caused loss of larval trapping while AAM polarization remained intact. These results show that Th2-dependent and STAT6-regulated genes in IECs are required and AAMs are not sufficient for protection against secondary Hpb infection by mechanisms that remain to be investigated.

Granulocyte development, tissue recruitment, and function during allergic inflammation.

Granulocytes provide a fast innate response to pathogens and allergens. In allergy and anti-helminth immunity, epithelial cells of damaged barriers release alarmins like IL-25, IL-33, and thymic stromal lymphopoietin (TSLP) but also chemokines like CXCL1 or CCL11 to promote cell recruitment and inflammation. In addition, mast cells positioned at barrier tissue sites also quickly release mediators upon specifically sensing antigens through IgE bound to FcεR1 on their surface. Released mediators induce the recruitment of different granulocytes in a timely ordered manner. First, neutrophils extravasate from the blood vasculature to the side of alarmin release and promote a potent inflammatory response. Alarmins and activated mast cells further promote activation of ILC2s and recruitment of basophils and eosinophils, which inhibit neutrophil recruitment and enhance tissue type 2 immunity. In addition to their potent pro-inflammatory effector functions, granulocytes can also contribute to termination and resolution of inflammation. Here, we summarize the development and tissue recruitment of granulocyte subsets, and describe general effector functions and aspects of their increasingly appreciated role in limiting tissue damage. We further discuss targeting approaches for therapeutic interventions in allergic disorders.

IL-4 and helminth infection downregulate MINCLE-dependent macrophage response to mycobacteria and Th17 adjuvanticity.

The myeloid C-type lectin receptor (CLR) MINCLE senses the mycobacterial cell wall component trehalose-6,6'-dimycolate (TDM). Recently, we found that IL-4 downregulates MINCLE expression in macrophages. IL-4 is a hallmark cytokine in helminth infections, which appear to increase the risk for mycobacterial infection and active tuberculosis. Here, we investigated functional consequences of IL-4 and helminth infection on MINCLE-driven macrophage activation and Th1/Th17 adjuvanticity. IL-4 inhibited MINCLE and cytokine induction after macrophage infection with Mycobacterium bovis bacille Calmette-Guerin (BCG). Infection of mice with BCG upregulated MINCLE on myeloid cells, which was inhibited by IL-4 plasmid injection and by infection with the nematode Nippostrongylus brasiliensis in monocytes. To determine the impact of helminth infection on MINCLE-dependent immune responses, we vaccinated mice with a recombinant protein together with the MINCLE ligand trehalose-6,6-dibehenate (TDB) as adjuvant. Concurrent infection with N. brasiliensis or with Schistosoma mansoni promoted T cell-derived IL-4 production and suppressed Th1/Th17 differentiation in the spleen. In contrast, helminth infection did not reduce Th1/Th17 induction by TDB in draining peripheral lymph nodes, where IL-4 levels were unaltered. Upon use of the TLR4-dependent adjuvant G3D6A, N. brasiliensis infection impaired selectively the induction of splenic antigen-specific Th1 but not of Th17 cells. Inhibition of MINCLE-dependent Th1/Th17 responses in mice infected with N. brasiliensis was dependent on IL-4/IL-13. Thus, helminth infection attenuated the Th17 response to MINCLE-dependent immunization in an organ- and adjuvant-specific manner via the Th2 cytokines IL-4/IL-13. Taken together, our results demonstrate downregulation of MINCLE expression on monocytes and macrophages by IL-4 as a possible mechanism of thwarted Th17 vaccination responses by underlying helminth infection.

Interleukin-13 and its receptor are synaptic proteins involved in plasticity and neuroprotection.

Immune system molecules are expressed by neurons, yet their functions are often unknown. We have identified IL-13 and its receptor IL-13Ra1 as neuronal, synaptic proteins in mouse, rat, and human brains, whose engagement upregulates the phosphorylation of NMDAR and AMPAR subunits and, in turn, increases synaptic activity and CREB-mediated transcription. We demonstrate that increased IL-13 is a hallmark of traumatic brain injury (TBI) in male mice as well as in two distinct cohorts of human patients. We also provide evidence that IL-13 upregulation protects neurons from excitotoxic death. We show IL-13 upregulation occurring in several cohorts of human brain samples and in cerebrospinal fluid (CSF). Thus, IL-13 is a physiological modulator of synaptic physiology of neuronal origin, with implications for the establishment of synaptic plasticity and the survival of neurons under injury conditions. Furthermore, we suggest that the neuroprotection afforded through the upregulation of IL-13 represents an entry point for interventions in the pathophysiology of TBI.

Selective ablation of thymic and peripheral Foxp3+ regulatory T cell development.

Foxp3+ regulatory T (Treg) cells of thymic (tTreg) and peripheral (pTreg) developmental origin are thought to synergistically act to ensure immune homeostasis, with self-reactive tTreg cells primarily constraining autoimmune responses. Here we exploited a Foxp3-dependent reporter with thymus-specific GFP/Cre activity to selectively ablate either tTreg (ΔtTreg) or pTreg (ΔpTreg) cell development, while sparing the respective sister populations. We found that, in contrast to the tTreg cell behavior in ΔpTreg mice, pTreg cells acquired a highly activated suppressor phenotype and replenished the Treg cell pool of ΔtTreg mice on a non-autoimmune C57BL/6 background. Despite the absence of tTreg cells, pTreg cells prevented early mortality and fatal autoimmunity commonly observed in Foxp3-deficient models of complete Treg cell deficiency, and largely maintained immune tolerance even as the ΔtTreg mice aged. However, only two generations of backcrossing to the autoimmune-prone non-obese diabetic (NOD) background were sufficient to cause severe disease lethality associated with different, partially overlapping patterns of organ-specific autoimmunity. This included a particularly severe form of autoimmune diabetes characterized by an early onset and abrogation of the sex bias usually observed in the NOD mouse model of human type 1 diabetes. Genetic association studies further allowed us to define a small set of autoimmune risk loci sufficient to promote ß cell autoimmunity, including genes known to impinge on Treg cell biology. Overall, these studies show an unexpectedly high functional adaptability of pTreg cells, emphasizing their important role as mediators of bystander effects to ensure self-tolerance.

B cell fate mapping reveals their contribution to the memory immune response against helminths.

An estimated quarter of the human world population is infected with gastrointestinal helminths causing major socioeconomic problems in endemic countries. A better understanding of humoral immune responses against helminths is urgently needed to develop effective vaccination strategies. Here, we used a fate mapping (FM) approach to mark germinal center (GC) B cells and their developmental fates by induced expression of a fluorescent protein during infection of mice with the helminth Nippostrongylus brasiliensis. We could show that FM+ cells persist weeks after clearance of the primary infection mainly as CD80+CD73+PD-L2+ memory B cells. A secondary infection elicited expansion of helminth-specific memory B cells and plasma cells (PCs). Adoptive transfers and analysis of somatic mutations in immunoglobulin genes further revealed that FM+ B cells rapidly convert to PCs rather than participating again in a GC reaction. These results provide new insights in the population dynamics of the humoral immune response against helminths.

Neutrophil breaching of the blood vessel pericyte layer during diapedesis requires mast cell-derived IL-17A.

Neutrophil diapedesis is an immediate step following infections and injury and is driven by complex interactions between leukocytes and various components of the blood vessel wall. Here, we show that perivascular mast cells (MC) are key regulators of neutrophil behaviour within the sub-endothelial space of inflamed venules. Using confocal intravital microscopy, we observe directed abluminal neutrophil motility along pericyte processes towards perivascular MCs, a response that created neutrophil extravasation hotspots. Conversely, MC-deficiency and pharmacological or genetic blockade of IL-17A leads to impaired neutrophil sub-endothelial migration and breaching of the pericyte layer. Mechanistically, identifying MCs as a significant cellular source of IL-17A, we establish that MC-derived IL-17A regulates the enrichment of key effector molecules ICAM-1 and CXCL1 in nearby pericytes. Collectively, we identify a novel MC-IL-17A-pericyte axis as modulator of the final steps of neutrophil diapedesis, with potential translational implications for inflammatory disorders driven by increased neutrophil diapedesis.

IL-4/IL-13-producing ILC2s are required for timely control of intestinal helminth infection in mice.

Infection of mice with Nippostrongylus brasiliensis (Nb) serves as a model for human hookworm infection affecting about 600 million people world-wide. Expulsion of Nb from the intestine requires IL-13-mediated mucus secretion from goblet cells and activation of smooth muscles cells. Type 2 innate lymphoid cells (ILC2s) are a major cellular source of IL-13 but it remains unclear whether IL-13 secretion from ILC2s is required for Nb expulsion. Here, we compared the immune response to Nb infection in mixed bone marrow chimeras with wild-type or IL-4/IL-13-deficient ILC2s. ILC2-derived IL-4/IL-13 was required for recruitment of eosinophils to the lung but had no influence of systemic eosinophil levels. In the small intestine, goblet cell hyperplasia and tuft cell accumulation was largely dependent on IL-4/IL-13 secretion from ILC2s. This further translated to higher eggs counts and impaired worm expulsion in mice with IL-4/IL-13-deficient ILC2s. Overall, we demonstrate that ILC2s constitute a non-redundant source of IL-4/IL-13 required for protective immunity against primary Nb infection.

A newly identified secreted larval antigen elicits basophil-dependent protective immunity against N. brasiliensis infection.

Hookworms infect more that 400 million people and cause significant socio-economic burden on endemic countries. The lack of efficient vaccines and the emergence of anthelminthic drug resistance are of major concern. Free-living hookworm larvae infect their hosts via the skin and live as adult worms in the small intestine where they feed on host tissue and blood. Excretory/secretory (E/S) products, released by helminths as they migrate through their host, are thought to play a key role in facilitating infection and successful establishment of parasitism. However, E/S products can also elicit protective immune responses that might be harnessed for vaccine development. By performing Western blots with serum of Nippostrongylus brasiliensis (Nb) infected mice as a model for human hookworm infection, we identified a largely overlapping set of IgG1- and IgE-reactive antigens in E/S from infective L3 stage larvae. Mass spectrometry analysis led to the identification of a new protein family with 6 paralogues in the Nb genome which we termed Nb-LSA1 for "Nippostrongylus brasiliensis larval secreted protein 1". The recombinantly expressed 17 kDa family member Nb-LSA1a was recognized by antibodies in the serum of Nb immune mice. Immunization of mice with Nb-LSA1a in alum elicited a strong IgG1 response but no detectable antigen-specific IgE. Most importantly, immunized mice were largely protected against a challenge Nb infection. This effect was dependent on the presence of basophils and occurred before the parasites reached the intestine. Therefore, basophils appear to play a critical role for rapid control of infection with L3 stage larvae in mice immunized with a single secreted larval protein. A better understanding of basophil-mediated protective immunity and identification of potent larval antigens of human hookworms could help to develop promising vaccination strategies.

Th2 single-cell heterogeneity and clonal distribution at distant sites in helminth-infected mice.

Th2 cells provide effector functions in type 2 immune responses to helminths and allergens. Despite knowledge about molecular mechanisms of Th2 cell differentiation, there is little information on Th2 cell heterogeneity and clonal distribution between organs. To address this, we performed combined single-cell transcriptome and T-cell receptor (TCR) clonotype analysis on murine Th2 cells in mesenteric lymph nodes (MLNs) and lung after infection with Nippostrongylus brasiliensis (Nb) as a human hookworm infection model. We find organ-specific expression profiles, but also populations with conserved migration or effector/resident memory signatures that unexpectedly cluster with potentially regulatory Il10posFoxp3neg cells. A substantial MLN subpopulation with an interferon response signature suggests a role for interferon signaling in Th2 differentiation or diversification. Further RNA-inferred developmental directions indicate proliferation as a hub for differentiation decisions. Although the TCR repertoire is highly heterogeneous, we identified expanded clones and CDR3 motifs. Clonal relatedness between distant organs confirmed effective exchange of Th2 effector cells, although locally expanded clones dominated the response. We further cloned an Nb-specific TCR from an expanded clone in the lung effector cluster and describe surface markers that distinguish transcriptionally defined clusters. These results provide insights in Th2 cell subset diversity and clonal relatedness in distant organs.

Acetate, a metabolic product of Heligmosomoides polygyrus, facilitates intestinal epithelial barrier breakdown in a FFAR2-dependent manner.

Approximately 2 billion people worldwide and a significant part of the domestic livestock are infected with soil-transmitted helminths, of which many establish chronic infections causing substantial economic and welfare burdens. Beside intensive research on helminth-triggered mucosal and systemic immune responses, the local mechanism that enables infective larvae to cross the intestinal epithelial barrier and invade mucosal tissue remains poorly addressed. Here, we show that Heligmosomoides polygyrus infective L3s secrete acetate and that acetate potentially facilitates paracellular epithelial tissue invasion by changed epithelial tight junction claudin expression. In vitro, impedance-based real-time epithelial cell line barrier measurements together with ex vivo functional permeability assays in intestinal organoid cultures revealed that acetate decreased intercellular barrier function via the G-protein coupled free fatty acid receptor 2 (FFAR2, GPR43). In vivo validation experiments in FFAR2-/- mice showed lower H. polygyrus burdens, whereas oral acetate-treated C57BL/6 wild type mice showed higher burdens. These data suggest that locally secreted acetate - as a metabolic product of the energy metabolism of H. polygyrus L3s - provides a significant advantage to the parasite in crossing the intestinal epithelial barrier and invading mucosal tissues. This is the first and a rate-limiting step for helminths to establish chronic infections in their hosts and if modulated could have profound consequences for their life cycle.