Sublingual allergen immunotherapy prevents house dust mite inhalant type 2 immunity through dendritic cell-mediated induction of Foxp3+ regulatory T cells.

Sublingual allergen immunotherapy (SLIT) is an emerging treatment option for allergic asthma and a potential disease-modifying strategy for asthma prevention. The key cellular events leading to such long-term tolerance remain to be fully elucidated. We administered prophylactic SLIT in a mouse model of house dust mite (HDM)-driven allergic asthma. HDM extract was sublingually administered over 3 weeks followed by intratracheal sensitization and intranasal challenges with HDM. Prophylactic SLIT prevented allergic airway inflammation and hyperreactivity with a low lab-to-lab variation. The HDM-specific T helper (Th)2 (cluster of differentiation 4 Th) response was shifted by SLIT toward a regulatory and Th17 response in the lung and mediastinal lymph node. By using Derp1-specific cluster of differentiation 4+ T cells (1-DER), we found that SLIT blocked 1-DER T cell recruitment to the mediastinal lymph node and dampened IL-4 secretion following intratracheal HDM sensitization. Sublingually administered Derp1 protein activated 1-DER T cells in the cervical lymph node via chemokine receptor7+ migratory dendritic cells (DC). DCs migrating from the oral submucosa to the cervical lymph node after SLIT-induced Foxp3+ regulatory T cells. When mice were sensitized with HDM, prior prophylactic SLIT increased Derp1 specific regulatory T cells (Tregs) and lowered Th2 recruitment in the lung. By using Foxp3-diphtheria toxin receptor mice, Tregs were found to contribute to the immunoregulatory prophylactic effect of SLIT on type 2 immunity. These findings in a mouse model suggest that DC-mediated functional Treg induction in oral mucosa draining lymph nodes is one of the driving mechanisms behind the disease-modifying effect of prophylactic SLIT.

Airway remodelling in asthma and the epithelium: on the edge of a new era.

Asthma is a chronic, heterogeneous disease of the airways, often characterised by structural changes known collectively as airway remodelling. In response to environmental insults, including pathogens, allergens and pollutants, the epithelium can initiate remodelling via an inflammatory cascade involving a variety of mediators that have downstream effects on both structural and immune cells. These mediators include the epithelial cytokines thymic stromal lymphopoietin, interleukin (IL)-33 and IL-25, which facilitate airway remodelling through cross-talk between epithelial cells and fibroblasts, and between mast cells and airway smooth muscle cells, as well as through signalling with immune cells such as macrophages. The epithelium can also initiate airway remodelling independently of inflammation in response to the mechanical stress present during bronchoconstriction. Furthermore, genetic and epigenetic alterations to epithelial components are believed to influence remodelling. Here, we review recent advances in our understanding of the roles of the epithelium and epithelial cytokines in driving airway remodelling, facilitated by developments in genetic sequencing and imaging techniques. We also explore how new and existing therapeutics that target the epithelium and epithelial cytokines could modify airway remodelling.

Ym1 protein crystals promote type 2 immunity.

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 Chil3 and Chil4 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.

Physicochemical and biological stability of diluted vedolizumab in intravenous infusion bags.

INTRODUCTION: Intravenous vedolizumab is a widely used monoclonal antibody for outpatients with inflammatory bowel disease. Drug preparation is performed on the day of administration, but is time consuming, causing unnecessary in-hospital patient delay and inefficient logistics for preparation and distribution. Storage of vedolizumab ready-to-administer infusions and distribution via pneumatic air tubes could streamline logistics in the outpatient setting. The aim of this study was to test the shelf life and stability of ready-to-administer intravenous infusion bags containing vedolizumab. METHODS: For assessing in-use shelf life, the reconstituted product (300 mg fixed dose) was diluted to a concentration of 1.2 mg/mL in 0.9% NaCl under aseptic conditions, and stored in polyolefin infusion bags at 2-8°C prior to analysis. On replicate samples, we measured concentration, physical and chemical stability using sodium dodecyl sulphate polyacrylamide gel electrophoresis, size exclusion chromatography, and multi-angle laser light scattering, as well as biological activity using a biolayer interferometry assay to study target engagement, and endotoxin content to assess microbiological stability. Stability of ready-to-use vedolizumab was assessed also after transportation via pneumatic tube system. Samples were taken at different time points over an observation period of 30 days on four replicate samples. RESULTS: For all parameters assessed, the ready-to-use solution of vedolizumab remained stable over a period of at least 30 days. There were no signs of protein aggregation, chemical instability, or loss of binding of the antibody to the α4ß7 integrin target. There was no increase in endotoxin concentration over time. No significant difference was seen in antibody structural stability and protein aggregation between samples before and after transportation via pneumatic tube system. CONCLUSION: When prepared under aseptic conditions, dissolved ready-to-administer vedolizumab infusion bags can be stored long term at 2-8°C and transported via pneumatic air tube, without observable loss of antibody stability or binding activity.

Aspergillus and the Lung.

The filamentous fungus Aspergillus causes a wide spectrum of diseases in the human lung, with Aspergillus fumigatus being the most pathogenic and allergenic subspecies. The broad range of clinical syndromes that can develop from the presence of Aspergillus in the respiratory tract is determined by the interaction between host and pathogen. In this review, an oversight of the different clinical entities of pulmonary aspergillosis is given, categorized by their main pathophysiological mechanisms. The underlying immune processes are discussed, and the main clinical, radiological, biochemical, microbiological, and histopathological findings are summarized.

Lipid Nanoparticle Encapsulation Empowers Poly(I:C) to Activate Cytoplasmic RLRs and Thereby Increases Its Adjuvanticity.

Poly(I:C) is a synthetic analogue of dsRNA capable of activating both TLR3 and RLRs, such as MDA-5 and RIG-I, as pathogen recognition receptors. While poly(I:C) is known to provoke a robust type I IFN, type III IFN, and Th1 cytokine response, its therapeutic use as a vaccine adjuvant is limited due to its vulnerability to nucleases and poor uptake by immune cells. is encapsulated poly(I:C) into lipid nanoparticles (LNPs) containing an ionizable cationic lipid that can electrostatically interact with poly(I:C). LNP-formulated poly(I:C) triggered both lysosomal TLR3 and cytoplasmic RLRs, in vitro and in vivo, whereas poly(I:C) in an unformulated soluble form only triggered endosomal-localized TLR3. Administration of LNP-formulated poly(I:C) in mouse models led to efficient translocation to lymphoid tissue and concurrent innate immune activation following intramuscular (IM) administration, resulting in a significant increase in innate immune activation compared to unformulated soluble poly(I:C). When used as an adjuvant for recombinant full-length SARS-CoV-2 spike protein, LNP-formulated poly(I:C) elicited potent anti-spike antibody titers, surpassing those of unformulated soluble poly(I:C) by orders of magnitude and offered complete protection against a SARS-CoV-2 viral challenge in vivo, and serum from these mice are capable of significantly reducing viral infection in vitro.

Protein citrullination and NET formation do not contribute to the pathology of A20/TNFAIP3 mutant mice.

A20 serves as a critical brake on NF-κB-dependent inflammation. In humans, polymorphisms in or near the TNFAIP3/A20 gene have been linked to various inflammatory disorders, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Experimental gene knockout studies in mice have confirmed A20 as a susceptibility gene for SLE and RA. Here, we examine the significance of protein citrullination and NET formation in the autoimmune pathology of A20 mutant mice because autoimmunity directed against citrullinated antigens released by neutrophil extracellular traps (NETs) is central to the pathogenesis of RA and SLE. Furthermore, genetic variants impairing the deubiquitinase (DUB) function of A20 have been shown to contribute to autoimmune susceptibility. Our findings demonstrate that genetic disruption of A20 DUB function in A20 C103R knockin mice does not result in autoimmune pathology. Moreover, we show that PAD4 deficiency, which abolishes protein citrullination and NET formation, does not prevent the development of autoimmunity in A20 deficient mice. Collectively, these findings provide experimental confirmation that PAD4-dependent protein citrullination and NET formation do not serve as pathogenic mechanisms in the development of RA and SLE pathology in mice with A20 mutations.

Lipid Nanoparticle Delivery Alters the Adjuvanticity of the TLR9 Agonist CpG by Innate Immune Activation in Lymphoid Tissue.

Pharmacological strategies to activate innate immune cells are of great relevance in the context of vaccine design and anticancer immune therapy, to mount broad immune responses able to clear infection and malignant cells. Synthetic CpG oligodeoxynucleotides (CpG-ODNs) are short single-stranded DNA molecules containing unmethylated CpG dinucleotides and a phosphorothioate backbone. Class B CpG ODNs activate robust innate immune responses through a TLR9-dependent NF-κB signaling pathway. This feature is attractive to exploit in the context of vaccine design and cancer immunotherapy. Soluble CpG-ODNs cause hepatic toxicity, which reduces its therapeutic applicability. The formulation of class B CpG ODN1826 in lipid nanoparticles (LNPs) containing an ionizable cationic lipid that complexes CpG through electrostatic interaction is reported. Upon local administration, LNP-formulated CpG drains to lymph nodes and triggers robust innate immune activation. Unformulated, soluble, CpG, by contrast, is unable to induce robust innate activation in draining lymph nodes and is distributed systemically. In a vaccination setting, LNP-formulated CpG, admixed with a protein antigen, induces higher antigen-specific antibody titers and T cell responses than antigen admixed with unformulated soluble CpG.

A conversation on allergy: recognizing the past and looking to the future.

Allergy is an ever-evolving group of disorders, which includes asthma, atopic dermatitis, rhinitis and food allergies and that currently affects over 1 billion people worldwide. This group of disorders has exploded in incidence since around the start of the 20th century, implying that genetics is not solely responsible for its development but that environmental factors have an important role. Here, Fabio Luciani and Jonathan Coquet, in their role as editors at Immunology & Cell Biology, asked nine prominent researchers in the field of allergy to define the term 'allergy', discuss the role of genetics and the environment, nominate the most important discoveries of the past decade and describe the best strategies to combat allergy at the population level going forward.

A complement atlas identifies interleukin-6-dependent alternative pathway dysregulation as a key druggable feature of COVID-19.

Improvements in COVID-19 treatments, especially for the critically ill, require deeper understanding of the mechanisms driving disease pathology. The complement system is not only a crucial component of innate host defense but can also contribute to tissue injury. Although all complement pathways have been implicated in COVID-19 pathogenesis, the upstream drivers and downstream effects on tissue injury remain poorly defined. We demonstrate that complement activation is primarily mediated by the alternative pathway, and we provide a comprehensive atlas of the complement alterations around the time of respiratory deterioration. Proteomic and single-cell sequencing mapping across cell types and tissues reveals a division of labor between lung epithelial, stromal, and myeloid cells in complement production, in addition to liver-derived factors. We identify IL-6 and STAT1/3 signaling as an upstream driver of complement responses, linking complement dysregulation to approved COVID-19 therapies. Furthermore, an exploratory proteomic study indicates that inhibition of complement C5 decreases epithelial damage and markers of disease severity. Collectively, these results support complement dysregulation as a key druggable feature of COVID-19.

STE20 kinase TAOK3 regulates type 2 immunity and metabolism in obesity.

Healthy adipose tissue (AT) contains ST2+ Tregs, ILC2s, and alternatively activated macrophages that are lost in mice or humans on high caloric diet. Understanding how this form of type 2 immunity is regulated could improve treatment of obesity. The STE20 kinase Thousand And One amino acid Kinase-3 (TAOK3) has been linked to obesity in mice and humans, but its precise function is unknown. We found that ST2+ Tregs are upregulated in visceral epididymal white AT (eWAT) of Taok3-/- mice, dependent on IL-33 and the kinase activity of TAOK3. Upon high fat diet feeding, metabolic dysfunction was attenuated in Taok3-/- mice. ST2+ Tregs disappeared from eWAT in obese wild-type mice, but this was not the case in Taok3-/- mice. Mechanistically, AT Taok3-/- Tregs were intrinsically more responsive to IL-33, through higher expression of ST2, and expressed more PPARγ and type 2 cytokines. Thus, TAOK3 inhibits adipose tissue Tregs and regulates immunometabolism under excessive caloric intake.

LXR signaling controls homeostatic dendritic cell maturation.

Dendritic cells (DCs) mature in an immunogenic or tolerogenic manner depending on the context in which an antigen is perceived, preserving the balance between immunity and tolerance. Whereas the pathways driving immunogenic maturation in response to infectious insults are well-characterized, the signals that drive tolerogenic maturation during homeostasis are still poorly understood. We found that the engulfment of apoptotic cells triggered homeostatic maturation of type 1 conventional DCs (cDC1s) within the spleen. This maturation process could be mimicked by engulfment of empty, nonadjuvanted lipid nanoparticles (LNPs), was marked by intracellular accumulation of cholesterol, and was highly specific to cDC1s. Engulfment of either apoptotic cells or cholesterol-rich LNPs led to the activation of the liver X receptor (LXR) pathway, which promotes the efflux of cellular cholesterol, and repressed genes associated with immunogenic maturation. In contrast, simultaneous engagement of TLR3 to mimic viral infection via administration of poly(I:C)-adjuvanted LNPs repressed the LXR pathway, thus delaying cellular cholesterol efflux and inducing genes that promote T cell-mediated immunity. These data demonstrate that conserved cellular cholesterol efflux pathways are differentially regulated in tolerogenic versus immunogenic cDC1s and suggest that administration of nonadjuvanted cholesterol-rich LNPs may be an approach for inducing tolerogenic DC maturation.

PD-L1 checkpoint blockade promotes regulatory T cell activity that underlies therapy resistance.

Despite the clinical success of immune checkpoint blockade (ICB), in certain cancer types, most patients with cancer do not respond well. Furthermore, in patients for whom ICB is initially successful, this is often short-lived because of the development of resistance to ICB. The mechanisms underlying primary or secondary ICB resistance are incompletely understood. Here, we identified preferential activation and enhanced suppressive capacity of regulatory T cells (Treg cells) in αPD-L1 therapy-resistant solid tumor-bearing mice. Treg cell depletion reversed resistance to αPD-L1 with concomitant expansion of effector T cells. Moreover, we found that tumor-infiltrating Treg cells in human patients with skin cancer, and in patients with non-small cell lung cancer, up-regulated a suppressive transcriptional gene program after ICB treatment, which correlated with lack of treatment response. αPD-1/PD-L1-induced PD-1+ Treg cell activation was also seen in peripheral blood of patients with lung cancer and mesothelioma, especially in nonresponders. Together, these data reveal that treatment with αPD-1 and αPD-L1 unleashes the immunosuppressive role of Treg cells, resulting in therapy resistance, suggesting that Treg cell targeting is an important adjunct strategy to enhance therapeutic efficacy.

Chitinases and chitinase-like proteins in asthma.

Despite the lack of endogenous chitin synthesis, mammalian genomes encode two enzymatically active true chitinases (chitotriosidase and acidic mammalian chitinase) and a variable number of chitinase-like proteins (CLPs) that have no enzyme activity but bind chitin. Chitinases and CLPs are prominent components of type-2 immune response-mediated respiratory diseases. However, despite extensive research into their role in allergic airway disease, there is still no agreement on whether they are mere biomarkers of disease or actual disease drivers. Functions ascribed to chitinases and CLPs include, but are not limited to host defense against chitin-containing pathogens, directly promoting inflammation, and modulating tissue remodeling and fibrosis. Here, we discuss in detail the chitin-dependent and -independent roles of chitinases and CLPs in the context of allergic airway disease, and recent advances and emerging concepts in the field that might identify opportunities for new therapies.

CCL5-producing migratory dendritic cells guide CCR5+ monocytes into the draining lymph nodes.

Dendritic cells (DCs) and monocytes capture, transport, and present antigen to cognate T cells in the draining lymph nodes (LNs) in a CCR7-dependent manner. Since only migratory DCs express this chemokine receptor, it is unclear how monocytes reach the LN. In steady-state and following inhalation of several PAMPs, scRNA-seq identified LN mononuclear phagocytes as monocytes, resident, or migratory type 1 and type 2 conventional (c)DCs, despite the downregulation of Xcr1, Clec9a, H2-Ab1, Sirpa, and Clec10a transcripts on migratory cDCs. Migratory cDCs, however, upregulated Ccr7, Ccl17, Ccl22, and Ccl5. Migratory monocytes expressed Ccr5, a high-affinity receptor for Ccl5. Using two tracking methods, we observed that both CD88hiCD26lomonocytes and CD88-CD26hi cDCs captured inhaled antigens in the lung and migrated to LNs. Antigen exposure in mixed-chimeric Ccl5-, Ccr2-, Ccr5-, Ccr7-, and Batf3-deficient mice demonstrated that while antigen-bearing DCs use CCR7 to reach the LN, monocytes use CCR5 to follow CCL5-secreting migratory cDCs into the LN, where they regulate DC-mediated immunity.

The Pathology of Asthma: What Is Obstructing Our View?

Despite the advent of sophisticated and efficient new biologics to treat inflammation in asthma, the disease persists. Even following treatment, many patients still experience the well-known symptoms of wheezing, shortness of breath, and coughing. What are we missing? Here we examine the evidence that mucus plugs contribute to a substantial portion of disease, not only by physically obstructing the airways but also by perpetuating inflammation. In this way, mucus plugs may act as an immunogenic stimulus even in the absence of allergen or with the use of current therapeutics. The alterations of several parameters of mucus biology, driven by type 2 inflammation, result in sticky and tenacious sputum, which represents a potent threat, first due to the difficulties in expectoration and second by acting as a platform for viral, bacterial, or fungal colonization that allows exacerbations. Therefore, in this way, mucus plugs are an overlooked but critical feature of asthmatic airway disease.

RNA viruses alter house dust mite physiology and allergen production with no detected consequences for allergenicity.

RNA viruses have recently been detected in association with house dust mites, including laboratory cultures, dust samples, and mite-derived pharmaceuticals used for allergy diagnosis. This study aimed to assess the incidence of viral infection on Dermatophagoides pteronyssinus physiology and on the allergenic performance of extracts derived from its culture. Transcriptional changes between genetically identical control and virus-infected mite colonies were analysed by RNAseq with the support of a new D. pteronyssinus high-quality annotated genome (56.8 Mb, 108 scaffolds, N50 = 2.73 Mb, 96.7% BUSCO-completeness). Extracts of cultures and bodies from both colonies were compared by inspecting major allergen accumulation by enzyme-linked immunosorbent assay (ELISA), allergen-related enzymatic activities by specific assays, airway inflammation in a mouse model of allergic asthma, and binding to allergic patient's sera IgE by ImmunoCAP. Viral infection induced a significant transcriptional response, including several immunity and stress-response genes, and affected the expression of seven allergens, putative isoallergens and allergen orthologs. Major allergens were unaffected except for Der p 23 that was upregulated, increasing ELISA titers up to 29% in infected-mite extracts. By contrast, serine protease allergens Der p 3, 6 and 9 were downregulated, being trypsin and chymotrypsin enzymatic activities reduced up to 21% in extracts. None of the parameters analysed in our mouse model, nor binding to human IgE were significantly different when comparing control and infected-mite extracts. Despite the described physiological impact of viral infection on the mites, no significant consequences for the allergenicity of derived extracts or their practical use in allergy diagnosis have been detected.

Canonical IRE1 function needed to sustain vigorous natural killer cell proliferation during viral infection.

The unfolded protein response (UPR) aims to restore ER homeostasis under conditions of high protein folding load, a function primarily serving secretory cells. Additional, non-canonical UPR functions have recently been unraveled in immune cells. We addressed the function of the inositol-requiring enzyme 1 (IRE1) signaling branch of the UPR in NK cells in homeostasis and microbial challenge. Cell-intrinsic compound deficiency of IRE1 and its downstream transcription factor XBP1 in NKp46+ NK cells, did not affect basal NK cell homeostasis, or overall outcome of viral MCMV infection. However, mixed bone marrow chimeras revealed a competitive advantage in the proliferation of IRE1-sufficient Ly49H+ NK cells after viral infection. CITE-Seq analysis confirmed strong induction of IRE1 early upon infection, concomitant with the activation of a canonical UPR signature. Therefore, we conclude that IRE1/XBP1 activation is required during vigorous NK cell proliferation early upon viral infection, as part of a canonical UPR response.

Loss of GM-CSF-dependent instruction of alveolar macrophages in COVID-19 provides a rationale for inhaled GM-CSF treatment.

GM-CSF promotes myelopoiesis and inflammation, and GM-CSF blockade is being evaluated as a treatment for COVID-19-associated hyperinflammation. Alveolar GM-CSF is, however, required for monocytes to differentiate into alveolar macrophages (AMs) that control alveolar homeostasis. By mapping cross-species AM development to clinical lung samples, we discovered that COVID-19 is marked by defective GM-CSF-dependent AM instruction and accumulation of pro-inflammatory macrophages. In a multi-center, open-label RCT in 81 non-ventilated COVID-19 patients with respiratory failure, we found that inhalation of rhu-GM-CSF did not improve mean oxygenation parameters compared with standard treatment. However, more patients on GM-CSF had a clinical response, and GM-CSF inhalation induced higher numbers of virus-specific CD8 effector lymphocytes and class-switched B cells, without exacerbating systemic hyperinflammation. This translational proof-of-concept study provides a rationale for further testing of inhaled GM-CSF as a non-invasive treatment to improve alveolar gas exchange and simultaneously boost antiviral immunity in COVID-19. This study is registered at ClinicalTrials.gov (NCT04326920) and EudraCT (2020-001254-22).

GTF3A mutations predispose to herpes simplex encephalitis by disrupting biogenesis of the host-derived RIG-I ligand RNA5SP141.

Herpes simplex virus 1 (HSV-1) infects several billion people worldwide and can cause life-threatening herpes simplex encephalitis (HSE) in some patients. Monogenic defects in components of the type I interferon system have been identified in patients with HSE, emphasizing the role of inborn errors of immunity underlying HSE pathogenesis. Here, we identify compound heterozygous loss-of-function mutations in the gene GTF3A encoding for transcription factor IIIA (TFIIIA), a component of the RNA polymerase III complex, in a patient with common variable immunodeficiency and HSE. Patient fibroblasts and GTF3A gene-edited cells displayed impaired HSV-1-induced innate immune responses and enhanced HSV-1 replication. Chromatin immunoprecipitation sequencing analysis identified the 5S ribosomal RNA pseudogene 141 (RNA5SP141), an endogenous ligand of the RNA sensor RIG-I, as a transcriptional target of TFIIIA. GTF3A mutant cells exhibited diminished RNA5SP141 expression and abrogated RIG-I activation upon HSV-1 infection. Our work unveils a crucial role for TFIIIA in transcriptional regulation of a cellular RIG-I agonist and shows that GTF3A genetic defects lead to impaired cell-intrinsic anti-HSV-1 responses and can predispose to HSE.