Although spontaneous protein crystallization is a rare event in vivo, Charcot-Leyden crystals (CLCs) consisting of galectin-10 (Gal10) protein are frequently observed in eosinophilic diseases, such ...as asthma. We found that CLCs derived from patients showed crystal packing and Gal10 structure identical to those of Gal10 crystals grown in vitro. When administered to the airways, crystalline Gal10 stimulated innate and adaptive immunity and acted as a type 2 adjuvant. By contrast, a soluble Gal10 mutein was inert. Antibodies directed against key epitopes of the CLC crystallization interface dissolved preexisting CLCs in patient-derived mucus within hours and reversed crystal-driven inflammation, goblet-cell metaplasia, immunoglobulin E (IgE) synthesis, and bronchial hyperreactivity (BHR) in a humanized mouse model of asthma. Thus, protein crystals may promote hallmark features of asthma and are targetable by crystal-dissolving antibodies.
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.
Localized therapeutic modalities that subvert the tumor microenvironment from immune‐suppressive to pro‐immunogenic can elicit systemic antitumor immune responses that induce regression of directly ...treated as well as nontreated distal tumors. A key toward generating robust antitumor T cell responses is the activation of dendritic cells (DCs) in the tumor microenvironment. Treatment with agonists triggering various pattern recognition receptors is very efficient to activate DCs, yet suffers from the induction of serious immune‐related adverse effects, which is closely linked to their unfavorable PK/PD profile causing systemic immune activation and cytokine release. Here, it is reported that nanoparticle conjugation of a highly potent TLR7/8 agonist restricts immune activation to the tumor bed and its sentinel lymph nodes without hampering therapeutic antitumor efficacy. On a mechanistic level, it is confirmed that localized treatment with a nanoparticle‐conjugated TLR7/8 agonist leads to potent activation of DCs in the sentinel lymph nodes and promotes proliferation of tumor antigen‐specific CD8 T cells. Furthermore, therapeutic improvement upon combination with anti‐PDL1 checkpoint inhibition and Flt3L, a growth factor that expands and mobilizes DCs from the bone marrow, is demonstrated. The findings provide a rational base for localized tumor engineering by nanomedicine strategies that provide spatial control over immune‐activation.
Conjugation of TLR7/8 agonist provides spatial control of immune activation to avoid systemic inflammation. Local immune stimulation allows the generation of tumor‐specific immune responses that show therapeutic improvement in combination with checkpoint inhibitor treatment to combat immune suppression.
Asthma is a chronic inflammatory airway disease in which innate and adaptive immune cells act together to cause eosinophilic inflammation, goblet cell metaplasia (GCM), and bronchial hyperreactivity ...(BHR). In clinical trials using biologicals against IL-4 receptor (IL-4R) α or IL-5, only a subset of patients with moderate-to-severe asthma responded favorably, suggesting that distinct pathophysiologic mechanisms are at play in subgroups of patients called endotypes. However, the effect of multiple cytokine blockade using bispecific antibodies has not been tested.
We sought to target simultaneously the IL-4, IL-13, and IL-5 signaling pathways with a novel IL-4Rα/IL-5–bispecific antibody in a murine house dust mite (HDM) model of asthma.
Two mAbs neutralizing IL-4Rα and IL-5 were generated by using a llama-based antibody platform. Their heavy and light chains were then cotransfected in mammalian cells, resulting in a heterogeneous antibody mixture from which the bispecific antibody was isolated by using a dual anti-idiotypic purification process. C57BL/6J mice were finally sensitized and challenged to HDM extracts and treated during challenge with the antibodies.
We successfully generated and characterized the monospecific and bispecific antibodies targeting IL-4Rα and IL-5. The monospecific antibodies could suppress eosinophilia, IgE synthesis, or both, whereas only the IL-4Rα/IL-5–bispecific antibody and the combination of monospecific antibodies additionally inhibited GCM and BHR.
Type 2 cytokines act synergistically to cause GCM and BHR in HDM-exposed mice. These preclinical results show the feasibility of generating bispecific antibodies that target multiple cytokine signaling pathways as superior inhibitors of asthma features, including the difficult-to-treat GCM.
Spontaneous protein crystallization is a rare event, yet protein crystals are frequently found in eosinophil-rich inflammation. In humans, Charcot-Leyden crystals (CLCs) are made from galectin-10 ...(Gal10) protein, an abundant protein in eosinophils. Although mice do not encode Gal10 in their genome, they do form pseudo-CLCs, made from the chitinase-like proteins Ym1 and/or Ym2, encoded by
and
and made by myeloid and epithelial cells respectively. Here, we investigated the biological effects of pseudo-CLCs since their function is currently unknown. We produced recombinant Ym1 crystals which were shown to have identical crystal packing and structure by X-ray crystallography as in vivo native crystals derived from murine lung. When administered to the airways of mice, crystalline but not soluble Ym1 stimulated innate and adaptive immunity and acted as a type 2 immune adjuvant for eosinophilic inflammation via triggering of dendritic cells (DCs). Murine Ym1 protein crystals found at sites of eosinophilic inflammation reinforce type 2 immunity and could serve as a surrogate model for studying the biology of human CLCs.
Genome-wide association studies in asthma have repeatedly identified single nucleotide polymorphisms in the ORM (yeast)–like protein isoform 3 (ORMDL3) gene across different populations. Although the ...ORM homologues in yeast are well-known inhibitors of sphingolipid synthesis, it is still unclear whether and how mammalian ORMDL3 regulates sphingolipid metabolism and whether altered sphingolipid synthesis would be causally related to asthma risk.
We sought to examine the in vivo role of ORMDL3 in sphingolipid metabolism and allergic asthma.
Ormdl3-LacZ reporter mice, gene-deficient Ormdl3−/− mice, and overexpressing Ormdl3Tg/wt mice were exposed to physiologically relevant aeroallergens, such as house dust mite (HDM) or Alternaria alternata, to induce experimental asthma. Mass spectrometry–based sphingolipidomics were performed, and airway eosinophilia, TH2 cytokine production, immunoglobulin synthesis, airway remodeling, and bronchial hyperreactivity were measured.
HDM challenge significantly increased levels of total sphingolipids in the lungs of HDM-sensitized mice compared with those in control mice. In Ormdl3Tg/wt mice the allergen-induced increase in lung ceramide levels was significantly reduced, whereas total sphingolipid levels were not affected. Conversely, in liver and serum, levels of total sphingolipids, including ceramides, were increased in Ormdl3−/− mice, whereas they were decreased in Ormdl3Tg/wt mice. This difference was independent of allergen exposure. Despite these changes, all features of asthma were identical between wild-type, Ormdl3Tg/wt, and Ormdl3−/− mice across several models of experimental asthma.
ORMDL3 regulates systemic ceramide levels, but genetically interfering with Ormdl3 expression does not result in altered experimental asthma.
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Small-molecular Toll-like receptor 7/8 (TLR7/8) agonists hold promise as immune modulators for a variety of immune therapeutic purposes including cancer therapy or vaccination. However, due to their ...rapid systemic distribution causing difficult-to-control inflammatory off-target effects, their application is still problematic, in particular systemically. To address this problem, we designed and robustly fabricated pH-responsive nanogels serving as versatile immunodrug nanocarriers for safe delivery of TLR7/8-stimulating imidazoquinolines after intravenous administration. To this aim, a primary amine-reactive methacrylamide monomer bearing a pendant squaric ester amide is introduced, which is polymerized under controlled RAFT polymerization conditions. Corresponding PEG-derived squaric ester amide block copolymers self-assemble into precursor micelles in polar protic solvents. Their cores are amine-reactive and can sequentially be transformed by acid-sensitive cross-linkers, dyes, and imidazoquinolines. Remaining squaric ester amides are hydrophilized affording fully hydrophilic nanogels with profound stability in human plasma but stimuli-responsive degradation upon exposure to endolysosomal pH conditions. The immunomodulatory behavior of the imidazoquinolines alone or conjugated to the nanogels was demonstrated by macrophages in vitro. In vivo, however, we observed a remarkable impact of the nanogel: After intravenous injection, a spatially controlled immunostimulatory activity was evident in the spleen, whereas systemic off-target inflammatory responses triggered by the small-molecular imidazoquinoline analogue were absent. These findings underline the potential of squaric ester-based, pH-degradable nanogels as a promising platform to permit intravenous administration routes of small-molecular TLR7/8 agonists and, thus, the opportunity to explore their adjuvant potency for systemic vaccination or cancer immunotherapy purposes.
Several studies have suggested that the natural route of allergen exposure (eg, ingestion of peanut allergen in infants, bee stings in beekeepers, and inhalation of high doses of cat allergens in pet ...owners) can be very successful in preventing the onset of clinical allergies.3-5 Exposure to immunodominant allergens in these studies, such as Ara h 6 peanut allergen in breast milk, phospholipase A2 in bee venom, and Fel d 1 cat allergen in house dust, suggests that the same allergens that can cause disease are also best at inducing tolerance through the natural route of exposure. In mice treated with sham PBS AIT, HDM sensitization and challenge induced robust asthma features, including airway and tissue eosinophilia (Fig 1, B and C), goblet cell metaplasia (Fig 1, B), T- and B-cell influx in bronchoalveolar lavage (BAL) fluid (Fig 1, C), TH2 cytokine production by lung-draining lymph node (LDLN) cells (Fig 1, D), IgE synthesis (Fig 1, E), and bronchial hyperreactivity (BHR) to methacholine (Fig 1, F). ...we addressed the mechanism of the reduced cDC2-mediated antigen presentation in mice treated with prophylactic Der p 2 AIT. ...our findings in mice show that prophylactic exposure to the single immunodominant HDM allergen Der p 2 through the airways offers an alternative way to prevent respiratory allergy to the complex allergen HDM by suppressing GM-CSF–driven activation of lung cDC2s.Methods Mice Mb1iCre mice were obtained from Michael Reth,E1 ROSADTA mice were from the Jackson laboratory (Bar Harbor, Me), and female C57Bl/6J mice were from Janvier Labs (Le Genest St Isle, France).
Inducible bronchus-associated lymphoid tissue (iBALT) is a long lasting tertiary lymphoid tissue that can be induced following influenza A virus (IAV) infection. Previous studies have shown that ...iBALT structures containing germinal center (GC) B cells protect against repeated infection by contributing locally to the cellular and humoral immune response. If we are to exploit this in vaccination strategies, we need a better understanding on how iBALT structures are induced. One hypothesis is that the strength of the initial innate response dictates induction of iBALT. In the present study, we investigated the role of interleukin (IL)-1 and IL-1R signaling on iBALT formation. Mice lacking the IL-1R had a delayed viral clearance and, thus, a prolonged exposure to viral replication, leading to increased disease severity, compared to wild-type mice. Contradictorily, iBALT formation following clearance of the virus was heavily compromised in Il1r1 (-/-) mice. Quantification of gene induction after IAV infection demonstrated induction of IL-1α and to a much lesser extent of IL-1β. Administration of recombinant IL-1α to the lungs of wild-type mice, early but not late, after IAV infection led to more pronounced iBALT formation and an increased amount of GC B cells in the lungs. Bone marrow chimeric mice identified the stromal compartment as the crucial IL-1 responsive cell for iBALT induction. Mechanistically, Q-PCR analysis of lung homogenates revealed a strongly diminished production of CXCL13, a B cell-attracting chemokine, in Il1r (-/-) mice during the early innate phase of IAV infection. These experiments demonstrate that appropriate innate IL-1α-IL-1R signaling is necessary for IAV clearance and at the same time instructs the formation of organized tertiary lymphoid tissues through induction of CXCL13 early after infection. These findings are discussed in the light of recent insights on the pathogenesis of tertiary lymphoid organ formation in the lung in various diseases where the IL-1 axis is hyperactive, such as rheumatoid arthritis and COPD.
House dust mite (HDM)‐allergic asthma is driven by T helper 2 (Th2) lymphocytes, but also innate immune cells control key aspects of the disease. The precise function of innate natural killer (NK) ...cells during the initiation and propagation of asthma has been very confusing, in part because different, not entirely specific, strategies were used to target these cells. We show that HDM inhalation rapidly led to the accumulation of NK cells in the lung‐draining lymph nodes and of activated CD69+ NK cells in the bronchoalveolar lumen. However, genetically engineered Ncr1‐DTA or Ncr1‐DTR mice that constitutively or temporarily lack NK cells, still developed all key features of acute or chronic HDM‐driven asthma, such as bronchial hyperreactivity, Th2 cytokine production, eosinophilia, mucus overproduction, and Th2‐dependent immunoglobulin serum titers. The same results were obtained by administration of conventional NK1.1 or asialo‐GM1 NK cell‐depleting antibodies, antibody‐mediated blocking of the NKG2D receptor, or genetic NKG2D deficiency. Thus, although NK cells accumulate in allergen‐challenged lungs, our findings comprehensively demonstrate that these cells are not required for HDM‐driven asthma in the mouse.
Synopsis
The function of NK cells in allergic asthma development has been confusing, as targeting strategies with different specificities have generated conflicting results. Here, conventional NK cells are genetically and specifically depleted and shown to be dispensable for asthma development in mice.
Allergic asthma to house dust mite or ovalbumin can be induced in genetically engineered Ncr1‐DTA mice that constitutively lack all NKp46+ cells.
The asthma phenotype is neither affected by temporarily depleting NK cells in Ncr1‐DTR mice, nor by antibody‐mediated depletion of NK1.1+ or asialoGM1+ cells.
Antibody‐mediated blocking or genetic depletion of the NKG2D receptor does not influence allergic asthma development.
The function of NK cells in allergic asthma development has been confusing, as targeting strategies with different specificities have generated conflicting results. Here, conventional NK cells are genetically and specifically depleted and shown to be dispensable for asthma development in mice.
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