House dust mite-driven asthma and allergen-specific T cells depend on B cells when the amount of inhaled allergen is limiting.

BACKGROUND: Allergic asthma is a CD4 TH2-lymphocyte driven disease characterized by airway hyperresponsiveness and eosinophilia. B cells can present antigens to CD4 T cells and produce IgE immunoglobulins that arm effector cells; however, mouse models are inconclusive on whether B cells are necessary for asthma development. OBJECTIVES: We sought to address the role of B cells in a house dust mite (HDM)-driven TH2-high asthma mouse model. METHODS: Wild-type and B cell-deficient muMT mice were sensitized and challenged through the airways with HDM extracts. The antigen-presenting capacities of B cells were studied by using new T-cell receptor transgenic 1-DER mice specific for the Der p 1 allergen. RESULTS: In vitro-activated B cells from HDM-exposed mice presented antigen to 1-DER T cells and induced a TH2 phenotype. In vivo B cells were dispensable for activation of naive 1-DER T cells but necessary for full expansion of primed 1-DER T cells. At high HDM challenge doses, B cells were not required for development of pulmonary asthmatic features yet contributed to TH2 expansion in the mediastinal lymph nodes but not in the lungs. When the amount of challenge allergen was decreased, muMT mice had reduced asthma features. Under these limiting conditions, B cells contributed also to expansion of TH2 effector cells in the lungs and central memory T cells in the mediastinal lymph nodes. CONCLUSION: B cells are a major part of the adaptive immune response to inhaled HDM allergen, particularly when the amount of inhaled allergen is low, by expanding allergen-specific T cells.

Perinatal Activation of the Interleukin-33 Pathway Promotes Type 2 Immunity in the Developing Lung.

Allergic disease originates in early life and polymorphisms in interleukin-33 gene (IL33) and IL1RL1, coding for IL-33R and decoy receptor sST2, confer allergy risk. Early life T helper 2 (Th2) cell skewing and allergy susceptibility are often seen as remnants of feto-maternal symbiosis. Here we report that shortly after birth, innate lymphoid type 2 cells (ILC2s), eosinophils, basophils, and mast cells spontaneously accumulated in developing lungs in an IL-33-dependent manner. During the phase of postnatal lung alveolarization, house dust mite exposure further increased IL-33, which boosted cytokine production in ILC2s and activated CD11b+ dendritic cells (DCs). IL-33 suppressed IL-12p35 and induced OX40L in neonatal DCs, thus promoting Th2 cell skewing. Decoy sST2 had a strong preventive effect on asthma in the neonatal period, less so in adulthood. Thus, enhanced neonatal Th2 cell skewing to inhaled allergens results from postnatal hyperactivity of the IL-33 axis during a period of maximal lung remodeling.

Cholesterol-sensing liver X receptors stimulate Th2-driven allergic eosinophilic asthma in mice.

INTRODUCTION: Liver X receptors (LXRs) are nuclear receptors that function as cholesterol sensors and regulate cholesterol homeostasis. High cholesterol has been recognized as a risk factor in asthma; however, the mechanism of this linkage is not known. METHODS: To explore the importance of cholesterol homeostasis for asthma, we investigated the contribution of LXR activity in an ovalbumin- and a house dust mite-driven eosinophilic asthma mouse model. RESULTS: In both models, airway inflammation, airway hyper-reactivity, and goblet cell hyperplasia were reduced in mice deficient for both LXRα and LXRß isoforms (LXRα(-/-)ß(-/-)) as compared to wild-type mice. Inversely, treatment with the LXR agonist GW3965 showed increased eosinophilic airway inflammation. LXR activity contributed to airway inflammation through promotion of type 2 cytokine production as LXRα(-/-)ß(-/-) mice showed strongly reduced protein levels of IL-5 and IL-13 in the lungs as well as reduced expression of these cytokines by CD4(+) lung cells and lung-draining lymph node cells. In line herewith, LXR activation resulted in increased type 2 cytokine production by the lung-draining lymph node cells. CONCLUSIONS: In conclusion, our study demonstrates that the cholesterol regulator LXR acts as a positive regulator of eosinophilic asthma in mice, contributing to airway inflammation through regulation of type 2 cytokine production.

A20 Deficiency in Lung Epithelial Cells Protects against Influenza A Virus Infection.

A20 negatively regulates multiple inflammatory signalling pathways. We here addressed the role of A20 in club cells (also known as Clara cells) of the bronchial epithelium in their response to influenza A virus infection. Club cells provide a niche for influenza virus replication, but little is known about the functions of these cells in antiviral immunity. Using airway epithelial cell-specific A20 knockout (A20AEC-KO) mice, we show that A20 in club cells critically controls innate immune responses upon TNF or double stranded RNA stimulation. Surprisingly, A20AEC-KO mice are better protected against influenza A virus challenge than their wild type littermates. This phenotype is not due to decreased viral replication. Instead host innate and adaptive immune responses and lung damage are reduced in A20AEC-KO mice. These attenuated responses correlate with a dampened cytotoxic T cell (CTL) response at later stages during infection, indicating that A20AEC-KO mice are better equipped to tolerate Influenza A virus infection. Expression of the chemokine CCL2 (also named MCP-1) is particularly suppressed in the lungs of A20AEC-KO mice during later stages of infection. When A20AEC-KO mice were treated with recombinant CCL2 the protective effect was abrogated demonstrating the crucial contribution of this chemokine to the protection of A20AEC-KO mice to Influenza A virus infection. Taken together, we propose a mechanism of action by which A20 expression in club cells controls inflammation and antiviral CTL responses in response to influenza virus infection.

GM-CSF treatment prevents respiratory syncytial virus-induced pulmonary exacerbation responses in postallergic mice by stimulating alveolar macrophage maturation.

BACKGROUND: Human respiratory syncytial virus (RSV) is a frequent cause of asthma exacerbations, yet the susceptibility of asthmatic patients to RSV is poorly understood. OBJECTIVE: We sought to address the contribution of resident alveolar macrophages (rAMs) to susceptibility to RSV infection in mice that recovered from allergic airway eosinophilia. METHODS: Mice were infected with RSV virus after clearance of allergic airway inflammation (AAI). The contribution of post-AAI rAMs was studied in vivo by means of clodronate liposome-mediated depletion, adoptive transfer, and treatment with recombinant cytokines before RSV infection. RESULTS: After clearing the allergic bronchial inflammation, post-AAI mice had bronchial hyperreactivity and increased inflammatory cell influx when infected with RSV compared with nonallergic mice, whereas viral clearance was comparable in both mouse groups. Post-AAI rAMs were necessary and sufficient for mediating these proinflammatory effects. In post-AAI mice the residing CD11c(hi) autofluorescent rAM population did not upregulate the terminal differentiation marker sialic acid-binding immunoglobulin-like lectin F and overproduced TNF and IL-6 through increased nuclear factor κB nuclear translocation. In line with these results, post-AAI lungs had reduced levels of the rAM maturation cytokine GM-CSF. Intratracheal administration of GM-CSF induced final rAM maturation in post-AAI mice and prevented the increased susceptibility to RSV-induced hyperreactivity and inflammation. CONCLUSION: Defective production of GM-CSF leads to insufficient post-AAI rAM maturation in mice that recovered from an AAI, causing increased susceptibility to RSV-induced immunopathology. Promoting the differentiation of post-AAI rAMs might be a therapeutic option for preventing RSV-induced exacerbations in human asthmatic patients.

Farm dust and endotoxin protect against allergy through A20 induction in lung epithelial cells.

Growing up on a dairy farm protects children from allergy, hay fever, and asthma. A mechanism linking exposure to this endotoxin (bacterial lipopolysaccharide)-rich environment with protection has remained elusive. Here we show that chronic exposure to low-dose endotoxin or farm dust protects mice from developing house dust mite (HDM)-induced asthma. Endotoxin reduced epithelial cell cytokines that activate dendritic cells (DCs), thus suppressing type 2 immunity to HDMs. Loss of the ubiquitin-modifying enzyme A20 in lung epithelium abolished the protective effect. A single-nucleotide polymorphism in the gene encoding A20 was associated with allergy and asthma risk in children growing up on farms. Thus, the farming environment protects from allergy by modifying the communication between barrier epithelial cells and DCs through A20 induction.

PAMPs and DAMPs in allergy exacerbation models.

Sensitization of mice to real-life allergens or harmless antigen with the use of adjuvants will lead to the induction of DAMPs in the immune system. We have shown that the Th2-inducing adjuvant aluminum hydroxide or exposure of the airways to house dust mite leads to the release of DAMPs: uric acid, ATP, and IL-1. Exposure to DAMPs or PAMPs present in allergens or added to harmless allergens, such as the experimental allergen ovalbumin, induces several immune responses, including cellular influx and activation. Cellular influx can be analyzed by flow cytometry. Likewise, cellular activation can be assessed by measuring increased expression and release of chemokines and cytokines. These inflammatory mediators can be analyzed by ELISA or confocal microscopy. Here, we describe the protocols for these assessments and a protocol that takes advantage of bone marrow chimeric mice to further elucidate mechanism.

Cytokine targets in airway inflammation.

Asthma is an inflammatory disease of the airway wall that leads to bronchial hyper-reactivity and airway obstruction, caused by inflammation, mucus hyper-production and airway wall remodelling. Central to pathogenesis, Th2 and Th17 lymphocytes of the adaptive immune system control many aspects of the disease by producing cytokines such as IL-4, IL-5, IL-13, and IL-17. In addition, many cells of the innate immune system such as mast cells, basophils, neutrophils, eosinophils, dendritic cells (DCs), and innate lymphoid cells (ILCs) play an important role in the initiation or maintenance of disease. Epithelial cells are ever more implicated in disease pathogenesis, as they are able to sense exposure to pathogens via pattern recognition receptors (PRRs) and can activate DCs. This review article will deal with the role of cytokines that are considered essential controllers of the inflammatory, immune and regenerative response to allergens, viruses and environmental pollutants. Emerging Th2 cytokines such as thymic stromal lymphopoietin, GM-CSF, IL-1, IL-33, IL-25 mediate the crosstalk between epithelial cells, DCs, and ILCs. Understanding the crosstalk between structural cells, innate and adaptive immune cells that is mediated by cytokines provides important mechanistic insights into how asthma develops and perpetuates itself. It could also provide the framework on which we will select new therapeutic strategies that prevent exacerbations and alter the natural course of the disease.

The mucosal adjuvant cholera toxin B instructs non-mucosal dendritic cells to promote IgA production via retinoic acid and TGF-ß.

It is currently unknown how mucosal adjuvants cause induction of secretory immunoglobulin A (IgA), and how T cell-dependent (TD) or -independent (TI) pathways might be involved. Mucosal dendritic cells (DCs) are the primary antigen presenting cells driving TI IgA synthesis, by producing a proliferation-inducing ligand (APRIL), B cell activating factor (BAFF), Retinoic Acid (RA), TGF-ß or nitric oxide (NO). We hypothesized that the mucosal adjuvant Cholera Toxin subunit B (CTB) could imprint non-mucosal DCs to induce IgA synthesis, and studied the mechanism of its induction. In vitro, CTB-treated bone marrow derived DCs primed for IgA production by B cells without the help of T cells, yet required co-signaling by different Toll-like receptor (TLR) ligands acting via the MyD88 pathway. CTB-DC induced IgA production was blocked in vitro or in vivo when RA receptor antagonist, TGF-ß signaling inhibitor or neutralizing anti-TGF-ß was added, demonstrating the involvement of RA and TGF-ß in promoting IgA responses. There was no major involvement for BAFF, APRIL or NO. This study highlights that synergism between CTB and MyD88-dependent TLR signals selectively imprints a TI IgA-inducing capacity in non-mucosal DCs, explaining how CTB acts as an IgA promoting adjuvant.

Interleukin-1α controls allergic sensitization to inhaled house dust mite via the epithelial release of GM-CSF and IL-33.

House dust mite (HDM) is one of the most common allergens worldwide. In this study, we have addressed the involvement of IL-1 in the interaction between HDM and the innate immune response driven by lung epithelial cells (ECs) and dendritic cells (DCs) that leads to asthma. Mice lacking IL-1R on radioresistant cells, but not hematopoietic cells, failed to mount a Th2 immune response and did not develop asthma to HDM. Experiments performed in vivo and in isolated air-liquid interface cultures of bronchial ECs showed that TLR4 signals induced the release of IL-1α, which then acted in an autocrine manner to trigger the release of DC-attracting chemokines, GM-CSF, and IL-33. Consequently, allergic sensitization to HDM was abolished in vivo when IL-1α, GM-CSF, or IL-33 was neutralized. Thymic stromal lymphopoietin (TSLP) became important only when high doses of allergen were administered. These findings put IL-1α upstream in the cytokine cascade leading to epithelial and DC activation in response to inhaled HDM allergen.

Surface-engineered polyelectrolyte multilayer capsules: synthetic vaccines mimicking microbial structure and function.

Immunizing: to evoke highly potent immune responses against recombinant antigens, hollow capsules consisting of layers of dextran sulphate and poly-L-arginine that encapsulate the antigen ovalbumin (orange circles) were coated with immune-activating CpG-containing oligonucleotides (green). These capsules were readily internalized by dendritic cells and showed activity in further immunization experiments.

Polymeric multilayer capsule-mediated vaccination induces protective immunity against cancer and viral infection.

Recombinant antigens hold high potential to develop vaccines against lethal intracellular pathogens and cancer. However, they are poorly immunogenic and fail to induce potent cellular immunity. In this paper, we demonstrate that polymeric multilayer capsules (PMLC) strongly increase antigen delivery toward professional antigen-presenting cells in vivo, including dendritic cells (DCs), macrophages, and B cells, thereby enforcing antigen presentation and stimulating T cell proliferation. A thorough analysis of the T cell response demonstrated their capacity to induce IFN-γ secreting CD4 and CD8 T cells, in addition to follicular T-helper cells, a recently identified CD4 T cell subset supporting antibody responses. On the B cell level, PMLC-mediated antigen delivery promoted the formation of germinal centers, resulting in increased numbers of antibody-secreting plasma cells and elevated antibody titers. The functional relevance of the induced immune responses was validated in murine models of influenza and melanoma. On a mechanistic level, we have demonstrated the capacity of PMLC to activate the NALP3 inflammasome and trigger the release of the potent pro-inflammatory cytokine IL-1ß. Finally, using DC-depleted mice, we have identified DCs as the key mediators of the immunogenic properties of PMLC.

Lung dendritic cell-epithelial cell crosstalk in Th2 responses to allergens.

Dendritic cells (DC) have been shown to be responsible for the initiation and maintenance of adaptive Th2 responses in asthma. It is increasingly clear that DC functions are strongly influenced by crosstalk with neighboring cells like epithelial cells, which can release a number of innate cytokines promoting Th2 responses. Clinically relevant allergens often interfere directly or indirectly with the innate immune functions of airway epithelial cells and DC. A better understanding of these interactions might lead to a better prevention and ultimately to new treatments for asthma.

Persistent activation of dendritic cells after resolution of allergic airway inflammation breaks tolerance to inhaled allergens in mice.

RATIONALE: Polysensitization of patients who are allergic is a common feature. The underlying immunologic mechanism is not clear. The maturation status of dendritic cells (DCs) is considered to be important for priming naive T cells in the draining lymph nodes. We hypothesized that chronic airway inflammation can induce an enhanced maturation of airway DCs and facilitate subsequent priming to neoallergens. OBJECTIVES: To investigate whether chronic airway inflammation could induce an altered activation of airway DCs in mice and whether this influences the development of allergic sensitization. METHODS: Balb/c mice were repeatedly challenged with DCs to induce a chronic airway inflammation. We evaluated (1) the induction of the main characteristic features of human asthma including persistent remodeling, (2) the maturation status of airway DCs 1 month after inflammation resolved, (3) whether this influences tolerance to inhaled neoallergen, and (4) what type of T helper response would be induced by DCs. MEASUREMENTS AND MAIN RESULTS: Airway DCs displayed a mature phenotype after complete resolution of airway eosinophilia. Inhalation of a neoallergen without any adjuvant was able to induce airway inflammation in postinflammation lungs but not in control lungs. One month after inflammation, airway DCs were able to induce Th2 polarization in naive T cells consistent with the up-regulation of the Th2 skewing molecules Ym1/2 and OX-40L compared with DCs of control airways. CONCLUSIONS: This study provides evidence that sustained maturation of DCs after resolution of Th2-mediated inflammation can contribute to polysensitization.

An unexpected role for uric acid as an inducer of T helper 2 cell immunity to inhaled antigens and inflammatory mediator of allergic asthma.

Although deposition of uric acid (UA) crystals is known as the cause of gout, it is unclear whether UA plays a role in other inflammatory diseases. We here have shown that UA is released in the airways of allergen-challenged asthmatic patients and mice, where it was necessary for mounting T helper 2 (Th2) cell immunity, airway eosinophilia, and bronchial hyperreactivity to inhaled harmless proteins and clinically relevant house dust mite allergen. Conversely, administration of UA crystals together with protein antigen was sufficient to promote Th2 cell immunity and features of asthma. The adjuvant effects of UA did not require the inflammasome (Nlrp3, Pycard) or the interleukin-1 (Myd88, IL-1r) axis. UA crystals promoted Th2 cell immunity by activating dendritic cells through spleen tyrosine kinase and PI3-kinase δ signaling. These findings provide further molecular insight into Th2 cell development and identify UA as an essential initiator and amplifier of allergic inflammation.

Dual Role of IL-22 in allergic airway inflammation and its cross-talk with IL-17A.

RATIONALE: IL-22 has both proinflammatory and antiinflammatory properties. Its role in allergic lung inflammation has not been explored. OBJECTIVES: To investigate the expression and roles of IL-22 in the onset and resolution of experimental allergic asthma and its cross-talk with IL-17A. METHODS: IL-22 expression was assessed in patient samples and in the lung of mice immunized and challenged with ovalbumin. IL-22 functions in allergic airway inflammation were evaluated using mice deficient in IL-22 or anti-IL-22 neutralizing antibodies. Moreover, the effects of recombinant IL-22 and IL-17A neutralizing antibodies were investigated. MEASUREMENTS AND MAIN RESULTS: Increased pulmonary IL-22 expression is found in the serum of patients with asthma and mice immunized and challenged with ovalbumin. Allergic lung inflammation is IL-22 dependent because eosinophil recruitment, Th2 cytokine including IL-13 and IL-33, chemokine production, airway hyperreactivity, and mucus production are drastically reduced in mice deficient in IL-22 or by IL-22 antibody neutralization during immunization of wild-type mice. By contrast, IL-22 neutralization during antigen challenge enhanced allergic lung inflammation with increased Th2 cytokines. Consistent with this, recombinant IL-22 given with allergen challenge protects mice from lung inflammation. Finally, IL-22 may regulate the expression and proinflammatory properties of IL-17A in allergic lung inflammation. CONCLUSIONS: IL-22 is required for the onset of allergic asthma, but functions as a negative regulator of established allergic inflammation. Our study reveals that IL-22 contributes to the proinflammatory properties of IL-17A in experimental allergic asthma.

Disruption of the SapM locus in Mycobacterium bovis BCG improves its protective efficacy as a vaccine against M. tuberculosis.

Mycobacterium bovis bacille Calmette-Guerin (BCG) provides only limited protection against pulmonary tuberculosis. We tested the hypothesis that BCG might have retained immunomodulatory properties from its pathogenic parent that limit its protective immunogenicity. Mutation of the molecules involved in immunomodulation might then improve its vaccine potential. We studied the vaccine potential of BCG mutants deficient in the secreted acid phosphatase, SapM, or in the capping of the immunomodulatory ManLAM cell wall component with α-1,2-oligomannoside. Both systemic and intratracheal challenge of mice with Mycobacterium tuberculosis following vaccination showed that the SapM mutant, compared to the parental BCG vaccine, provided better protection: it led to longer-term survival. Persistence of the SapM-mutated BCG in vivo resembled that of the parental BCG indicating that this mutation will likely not compromise the safety of the BCG vaccine. The SapM mutant BCG vaccine was more effective than the parental vaccine in inducing recruitment and activation of CD11c(+) MHC-II(int) CD40(int) dendritic cells (DCs) to the draining lymph nodes. Thus, SapM acts by inhibiting recruitment of DCs and their activation at the site of vaccination.

Designing polymeric particles for antigen delivery.

By targeting dendritic cells, polymeric carriers in the nano to lower micron range constitute very interesting tools for antigen delivery. In this critical review, we review how new immunological insights can be exploited to design new carriers allowing one to tune immune responses and to further increase vaccine potency (137 references).

Inflammatory dendritic cells--not basophils--are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen.

It is unclear how Th2 immunity is induced in response to allergens like house dust mite (HDM). Here, we show that HDM inhalation leads to the TLR4/MyD88-dependent recruitment of IL-4 competent basophils and eosinophils, and of inflammatory DCs to the draining mediastinal nodes. Depletion of basophils only partially reduced Th2 immunity, and depletion of eosinophils had no effect on the Th2 response. Basophils did not take up inhaled antigen, present it to T cells, or express antigen presentation machinery, whereas a population of FceRI(+) DCs readily did. Inflammatory DCs were necessary and sufficient for induction of Th2 immunity and features of asthma, whereas basophils were not required. We favor a model whereby DCs initiate and basophils amplify Th2 immunity to HDM allergen.

Lentiviral gene therapy of murine hematopoietic stem cells ameliorates the Pompe disease phenotype.

Pompe disease (acid alpha-glucosidase deficiency) is a lysosomal glycogen storage disorder characterized in its most severe early-onset form by rapidly progressive muscle weakness and mortality within the first year of life due to cardiac and respiratory failure. Enzyme replacement therapy prolongs the life of affected infants and supports the condition of older children and adults but entails lifelong treatment and can be counteracted by immune responses to the recombinant enzyme. We have explored the potential of lentiviral vector-mediated expression of human acid alpha-glucosidase in hematopoietic stem cells (HSCs) in a Pompe mouse model. After mild conditioning, transplantation of genetically engineered HSCs resulted in stable chimerism of approximately 35% hematopoietic cells that overexpress acid alpha-glucosidase and in major clearance of glycogen in heart, diaphragm, spleen, and liver. Cardiac remodeling was reversed, and respiratory function, skeletal muscle strength, and motor performance improved. Overexpression of acid alpha-glucosidase did not affect overall hematopoietic cell function and led to immune tolerance as shown by challenge with the human recombinant protein. On the basis of the prominent and sustained therapeutic efficacy without adverse events in mice we conclude that ex vivo HSC gene therapy is a treatment option worthwhile to pursue.