Olfactory receptors impact pathophysiological processes of lung diseases in bronchial epithelial cells.

BACKGROUND: Therapeutic options for steroid-resistant non-type 2 inflammation in obstructive lung diseases are limited. Bronchial epithelial cells are key in the pathogenesis by releasing the central proinflammatory cytokine interleukine-8 (IL-8). Olfactory receptors (ORs) are expressed in various cell types. This study examined the drug target potential of ORs by investigating their impact on associated pathophysiological processes in lung epithelial cells. METHODS: Experiments were performed in the A549 cell line and in primary human bronchial epithelial cells. OR expression was investigated using RT-PCR, Western blot, and immunocytochemical staining. OR-mediated effects were analyzed by measuring 1) intracellular calcium concentration via calcium imaging, 2) cAMP concentration by luminescence-based assays, 3) wound healing by scratch assays, 4) proliferation by MTS-based assays, 5) cellular vitality by Annexin V/PI-based FACS staining, and 6) the secretion of IL-8 in culture supernatants by ELISA. RESULTS: By screening 100 potential OR agonists, we identified two, Brahmanol and Cinnamaldehyde, that increased intracellular calcium concentrations. The mRNA and proteins of the corresponding receptors OR2AT4 and OR2J3 were detected. Stimulation of OR2J3 with Cinnamaldehyde reduced 1) IL-8 in the absence and presence of bacterial and viral pathogen-associated molecular patterns (PAMPs), 2) proliferation, and 3) wound healing but increased cAMP. In contrast, stimulation of OR2AT4 by Brahmanol increased wound healing but did not affect cAMP and proliferation. Both ORs did not influence cell vitality. CONCLUSION: ORs might be promising drug target candidates for lung diseases with non-type 2 inflammation. Their stimulation might reduce inflammation or prevent tissue remodeling by promoting wound healing.

Plasma levels of chemokines decrease during elexacaftor/tezacaftor/ivacaftor therapy in adults with cystic fibrosis.

Background: Cystic fibrosis (CF) is associated with dysregulated immune responses, exaggerated inflammation and chronic infection. CF transmembrane conductance regulator (CFTR) modulator therapies directly target the underlying protein defects and resulted in significant clinical benefits for people with CF (pwCF). This study analysed the effects of triple CFTR modulator therapy elexacaftor/tezacaftor/ivacaftor (ETI) on CF-associated inflammation, especially systemic chemokines. Methods: A bead-based immunoassay was used to quantify proinflammatory chemokines (IL-8, IP-10, Eotaxin, TARC, RANTES, MIP-1α, MIP-1ß, MIP-3α, MIG, ENA-78, GROα, I-TAC) in plasma samples from pwCF collected before, at three, and at six months after starting ETI therapy. Results: Fifty-one pwCF (47 % female; mean age 32 ± 10.4 years) were included. At baseline, 67 % were already receiving CFTR modulator therapy with tezacaftor/ivacaftor or lumacaftor/ivacaftor. After initiation of ETI therapy there was a significant improvement in percent predicted forced expiratory volume in 1 s (+12.7 points, p < 0.001) and a significant decrease in sweat chloride levels (-53.6 %, p < 0.001). After 6 months' treatment with ETI therapy there were significant decreases in plasma levels of MIP-3α (-68.2 %, p = 0.018), GROα (-17.7 %, p = 0.013), ENA-78 (-16.3 %, p = 0.034) and I-TAC (-3.4 %, p = 0.032). IL-8 exhibited a reduction that did not reach statistical significance (-17.8 %, p = 0.057); levels of other assessed cytokines did not change significantly from baseline. Conclusions: ETI appears to affect a distinct group of chemokines that are predominately associated with neutrophilic inflammation, demonstrating the anti-inflammatory properties of ETI therapy.

Therapeutic properties of Helicobacter pylori-derived vacuolating cytotoxin A in an animal model of chronic allergic airway disease.

BACKGROUND: It has previously been shown that the Helicobacter pylori (H. pylori)-derived molecule vacuolating cytotoxin A (VacA) could be suitable for the treatment of allergic airway disease. The therapeutic activity of the protein, which acts through modulation of dendritic cells (DC) and regulatory T cells (Tregs), was demonstrated in murine short-term acute models. The aim of this study is to further evaluate the therapeutic potential of VacA by determining the effectiveness of different application routes and the suitability of the protein for treating the chronic phase of allergic airway disease. METHODS: VacA was administered by the intraperitoneal (i.p.), oral (p.o.) or intratracheal (i.t.) routes, and long-term therapeutic effectiveness, allergic airway disease hallmarks, and immune phenotype were analyzed in murine models of acute and chronic allergic airway disease. RESULTS: Administration of VacA via the i.p., p.o or i.t. routes was associated with a reduction in airway inflammation. The i.p. route showed the most consistent effect in reducing airway inflammation and i.p. treatment with VacA was the only treatment that significantly reduced mucus cell hyperplasia. In a murine model of chronic allergic airway disease, both short- and long-term treatment with VacA showed a therapeutic effect, with a reduction in a variety of asthma hallmarks, including bronchoalveolar lavage eosinophilia, lung inflammation and goblet cell metaplasia. Short-term treatment was associated with induction of Tregs, while repetitive long-term administration of VacA influenced immunological memory in the lung. CONCLUSIONS: In addition to showing therapeutic efficacy in short-term models, treatment with VacA also appeared to be effective in suppressing inflammation in a chronic airway disease model. The observation that treatment was effective after administration via several different routes highlights the potential of VacA as a therapeutic agent with different routes of administration in humans.

TROP2 expression and SN38 antitumor activity in malignant pleural mesothelioma cells provide a rationale for antibody-drug conjugate therapy.

OBJECTIVES: Malignant pleural mesothelioma (MPM) is an aggressive cancer which at large is not amenable to curative surgery. Despite the recent approval of immune checkpoint inhibitor therapy, the response rates and survival following systemic therapy is still limited. Sacituzumab govitecan is an antibody-drug conjugate targeting the topoisomerase I inhibitor SN38 to trophoblast cell-surface antigen 2 (TROP-2)-positive cells. Here we have explored the therapeutic potential of sacituzumab govitecan in MPM models. MATERIALS AND METHODS: TROP2 expression was analyzed in a panel of two well established and 15 pleural effusion derived novel lines by RT-QPCR and immunoblotting, TROP2 membrane-localization was studied by flow cytometry and immunohistochemistry. Cultured mesothelial cells and pneumothorax pleura served as controls. The sensitivity of MPM cell lines to irinotecan and SN38 was studied using cell viability, cell cycle, apoptosis and DNA damage assays. Drug sensitivity of cell lines was correlated with RNA expression of DNA repair genes. Drug sensitivity was defined as an IC50 below 5 nM in the cell viability assay. RESULTS: TROP2 expression was detected at RNA and protein level in 6 of the 17 MPM cell lines, but not in in cultured mesothelial control cells or in the mesothelial layer of the pleura. TROP2 was detectable on the cell membrane in 5 MPM lines and was present in the nucleus in 6 cell models. Ten of 17 MPM cell lines showed sensitivity to SN38 treatment, among those 4 expressed TROP2. High AURKA RNA expression and high proliferation rate correlated with sensitivity to SN38-induced cell death, DNA damage response, cell cycle arrest and cell death. Sacituzumab govitecan treatment effectively induced cell cycle arrest and cell death in TROP2-positive MPM cells. CONCLUSION: TROP2 expression and sensitivity to SN38 in MPM cell lines support biomarker-selected clinical exploration of sacituzumab govitecan in patients with MPM.

Regulatory T cell enhancement in adults with cystic fibrosis receiving Elexacaftor/Tezacaftor/Ivacaftor therapy.

Introduction: Cystic fibrosis (CF), especially CF lung disease, is characterized by chronic infection, immune dysfunction including impairment of regulatory T cells (Tregs) and an exaggerated inflammatory response. CF transmembrane conductance regulator (CFTR) modulators have shown to improve clinical outcomes in people with CF (PwCF) with a wide range of CFTR mutations. However, it remains unclear whether CFTR modulator therapy also affects CF-associated inflammation. We aimed to examine the effect of elexacaftor/tezacaftor/ivacaftor therapy on lymphocyte subsets and systemic cytokines in PwCF. Methods: Peripheral blood mononuclear cells and plasma were collected before and at three and six months after the initiation of elexacaftor/tezacaftor/ivacaftor therapy; lymphocyte subsets and systemic cytokines were determined using flow cytometry. Results: Elexacaftor/tezacaftor/ivacaftor treatment was initiated in 77 PwCF and improved percent predicted FEV1 by 12.5 points (p<0.001) at 3 months. During elexacaftor/tezacaftor/ivacaftor therapy, percentages of Tregs were enhanced (+18.7%, p<0.001), with an increased proportion of Tregs expressing CD39 as a marker of stability (+14.4%, p<0.001). Treg enhancement was more pronounced in PwCF clearing Pseudomonas aeruginosa infection. Only minor, non-significant shifts were observed among Th1-, Th2- and Th17-expressing effector T helper cells. These results were stable at 3- and 6-month follow-up. Cytokine measurements showed a significant decrease in interleukin-6 levels during treatment with elexacaftor/tezacaftor/ivacaftor (-50.2%, p<0.001). Conclusion: Treatment with elexacaftor/tezacaftor/ivacaftor was associated with an increased percentage of Tregs, especially in PwCF clearing Pseudomonas aeruginosa infection. Targeting Treg homeostasis is a therapeutic option for PwCF with persistent Treg impairment.

Treatment with Helicobacter pylori-derived VacA attenuates allergic airway disease.

Background: Asthma is an incurable heterogeneous disease with variations in clinical and underlying immunological phenotype. New approaches could help to support existing therapy concepts. Neonatal infection of mice with Helicobacter pylori or administration of H. pylori-derived extracts or molecules after birth have been shown to prevent the development of allergic airway disease later in life. This study evaluated the potential therapeutic efficacy of H. pylori vacuolating cytotoxin A (VacA) in allergic airway inflammation and investigated the underlying immunological mechanisms for its actions. Methods: Murine models of allergic airway diseases, and murine and human in vitro models were used. Results: In both an acute model and a therapeutic house dust mite model of allergic airway disease, treatment with H. pylori-derived VacA reduced several asthma hallmarks, including airway hyperresponsiveness, inflammation and goblet cell metaplasia. Flow cytometry and ELISA analyses revealed induction of tolerogenic dendritic cells (DC) and FoxP3 positive regulatory T cells (Tregs), and a shift in the composition of allergen-specific immunoglobulins. Depletion of Tregs during treatment with VacA reversed treatment-mediated suppression of allergic airway disease. Human monocyte derived DCs (moDC) that were exposed to VacA induced Tregs in co-cultured naïve autologous T cells, replicating key observations made in vivo. Conclusion: H. pylori-derived VacA suppressed allergic airway inflammation via induction of Tregs in both allergic airway disease models. These data suggest that the immunomodulatory activity of VacA could potentially be exploited for the prevention and treatment of allergic airway disease.

The molecular mechanisms of remodeling in asthma, COPD and IPF with a special emphasis on the complex role of Wnt5A.

INTRODUCTION: Chronic inflammatory lung diseases are a common cause of suffering and death. Chronic obstructive pulmonary disease (COPD) is the reason for 6% of all deaths worldwide. A total of 262 million people are affected by asthma and 461,000 people died in 2019. Idiopathic pulmonary fibrosis (IPF) is diagnosed in 3 million people worldwide, with an onset over the age of 50 with a mean survival of only 24-30 months. These three diseases have in common that remodeling of the lung tissue takes place, which is responsible for an irreversible decline of lung function. Pathological lung remodeling is mediated by a complex interaction of different, often misguided, repair processes regulated by a variety of mediators. One group of these, as has recently become known, are the Wnt ligands. In addition to their well-characterized role in embryogenesis, this group of glycoproteins is also involved in immunological and structural repair processes. Depending on the combination of the Wnt ligand with its receptors and co-receptors, canonical and noncanonical signaling cascades can be induced. Wnt5A is a mediator that is described mainly in noncanonical Wnt signaling and has been shown to play an important role in different inflammatory diseases and malignancies. OBJECTIVES: In this review, we summarize the literature available regarding the role of Wnt5A as an immune modulator and its role in the development of asthma, COPD and IPF. We will focus specifically on what is known about Wnt5A concerning its role in the remodeling processes involved in the chronification of the diseases. CONCLUSION: Wnt5A has been shown to be involved in all three inflammatory lung diseases. Since the ligand affects both structural and immunological processes, it is an interesting target for the treatment of lung diseases whose pathology involves a restructuring of the lung tissue triggered in part by an inflammatory immune response.

CFTR modulator therapy alters plasma sphingolipid profiles in people with cystic fibrosis.

BACKGROUND: Sphingolipids, in particular ceramides, play an important role in the pathogenesis of cystic fibrosis (CF) lung disease. Ceramides seem to be dysregulated in people with CF (PWCF): An elevated ratio of ceramides C16Cer/ C24Cer has been linked to inflammation and disease severity. CFTR modulators might influence sphingolipid dysregulation in PWCF. METHODS: Sphingolipid profiles were retrospectively analyzed in serum from 112 PWCF and 96 healthy controls as well as in plasma from 25 PWCF before and after treatment with the CFTR modulator elexacaftor/tezacaftor/ivacaftor (ELX/TEZ/IVA) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Lipid data were correlated with clinical parameters. RESULTS: There were significantly higher levels of long-chain ceramides C18Cer and C20Cer and of the very long-chain ceramide C24:1Cer in PWCF versus healthy controls. Sphingosine levels were significantly reduced and accurately distinguished PWCF from healthy controls. Treatment with ELX/TEZ/IVA was associated with a decrease in levels of long-chain ceramides C16Cer, C18Cer and C20Cer and very long-chain ceramide C24:1Cer. Plasma levels of the most abundant very long-chain ceramide C24Cer as well as sphingosine-1-phosphate increased. Consequently, the ratio of ceramides C16Cer/ C24Cer decreased. Sphingolipid levels showed weak correlations with clinical parameters. CONCLUSIONS: These findings highlight the existence of a distinctive sphingolipid profile in blood from PWCF, which appears to be altered by ELX/TEZ/IVA therapy. Thus, strategies for sphingolipid remodeling need to be reassessed and adjusted in the light of highly effective CFTR modulator therapies.

Pseudomonas aeruginosa infection, but not mono or dual-combination CFTR modulator therapy affects circulating regulatory T cells in an adult population with cystic fibrosis.

BACKGROUND: Chronic infection and an exaggerated inflammatory response are key drivers of the pathogenesis of cystic fibrosis (CF), especially CF lung disease. An imbalance of pro- and anti-inflammatory mediators, including dysregulated Th2/Th17 cells and impairment of regulatory T cells (Tregs), maintain CF inflammation. CF transmembrane conductance regulator (CFTR) modulator therapy might influence these immune cell abnormalities. METHODS: Peripheral blood mononuclear cells and serum samples were collected from 108 patients with CF (PWCF) and 40 patients with non-CF bronchiectasis. Samples were analysed for peripheral blood lymphocytes subsets (Tregs; Th1-, Th1/17-, Th17- and Th2-effector cells) and systemic T helper cell-associated cytokines (interleukin [IL]-5, IL-13, IL-2, IL-6, IL-9, IL-10, IL-17A, IL-17F, IL-4, IL-22, interferon-γ, tumour necrosis factor-α) using flow cytometry. RESULTS: 51% of PWCF received CFTR modulators (ivacaftor, ivacaftor/ lumacaftor or tezacaftor/ ivacaftor). There were no differences in proportions of analysed T cell subsets or cytokines between PWCF who were versus were not receiving CFTR modulators. Additional analysis revealed lower percentages of Tregs in PWCF and chronic pulmonary Pseudomonas aeruginosa infection; this difference was also present in PWCF treated with CFTR modulators. Patients with non-CF bronchiectasis tended to have higher percentages of Th2- and Th17-cells and higher levels of peripheral cytokines versus PWCF. CONCLUSIONS: Chronic P. aeruginosa lung infection appears to impair Tregs in PWCF (independent of CFTR modulator therapy) but not those with non-CF bronchiectasis. Moreover, our data showed no statistically significant differences in major subsets of peripheral lymphocytes and cytokines among PWCF who were versus were not receiving CFTR modulators.

Preconceptional smoking alters spermatozoal miRNAs of murine fathers and affects offspring's body weight.

BACKGROUND: Active smoking has been reported among 7% of teenagers worldwide, with ages ranging from 13 to 15 years. An epidemiological study suggested that preconceptional paternal smoking is associated with adolescent obesity in boys. We developed a murine adolescent smoking model before conception to investigate the paternal molecular causes of changes in offspring's phenotype. METHOD: Male and female C57BL/6J mice were exposed to increasing doses of mainstream cigarette smoke (CS) from onset of puberty for 6 weeks and mated with room air (RA) controls. RESULTS: Thirteen miRNAs were upregulated and 32 downregulated in the spermatozoa of CS-exposed fathers, while there were no significant differences in the count and morphological integrity of spermatozoa, as well as the proliferation of spermatogonia between CS- and RA-exposed fathers. Offspring from preconceptional CS-exposed mothers had lower body weights (p = 0.007). Moreover, data from offspring from CS-exposed fathers suggested a potential increase in body weight (p = 0.062). CONCLUSION: We showed that preconceptional paternal CS exposure regulates spermatozoal miRNAs, and possibly influences the body weight of F1 progeny in early life. The regulated miRNAs may modulate transmittable epigenetic changes to offspring, thus influence the development of respiratory- and metabolic-related diseases such as obesity, a mechanism that warrants further studies for elaborate explanations.

Cigarette Smoke Affects Dendritic Cell Populations, Epithelial Barrier Function, and the Immune Response to Viral Infection With H1N1.

Smokers with apparently "healthy" lungs suffer from more severe and frequent viral respiratory infections, but the mechanisms underlying this observation are still unclear. Epithelial cells and dendritic cells (DC) form the first line of defense against inhaled noxes such as smoke or viruses. We therefore aimed to obtain insight into how cigarette smoke affects DCs and epithelial cells and how this influences the response to viral infection. Female C57BL/6J mice were exposed to cigarette smoke (CS) for 1 h daily for 24 days and then challenged i.n. with the viral mimic and Toll-like receptor 3 (TLR3) ligand poly (I:C) after the last exposure. DC subpopulations were analyzed 24 h later in whole lung homogenates by flow cytometry. Calu-3 cells or human precision-cut lung slices (PCLS) cultured at air-liquid interface were exposed to CS or air and subsequently inoculated with influenza H1N1. At 48 h post infection cytokines were analyzed by multiplex technology. Cytotoxic effects were measured by release of lactate dehydrogenase (LDH) and confocal imaging. In Calu-3 cells the trans-epithelial electrical resistance (TEER) was assessed. Smoke exposure of mice increased numbers of inflammatory and plasmacytoid DCs in lung tissue. Additional poly (I:C) challenge further increased the population of inflammatory DCs and conventional DCs, especially CD11b+ cDCs. Smoke exposure led to a loss of the barrier function in Calu-3 cells, which was further exaggerated by additional influenza H1N1 infection. Influenza H1N1-induced secretion of antiviral cytokines (IFN-α2a, IFN-λ, interferon-γ-induced protein 10 [IP-10]), pro-inflammatory cytokine IL-6, as well as T cell-associated cytokines (e.g., I-TAC) were completely suppressed in both Calu-3 cells and human PCLS after smoke exposure. In summary, cigarette smoke exposure increased the number of inflammatory DCs in the lung and disrupted epithelial barrier functions, both of which was further enhanced by viral stimulation. Additionally, the antiviral immune response to influenza H1N1 was strongly suppressed by smoke. These data suggest that smoke impairs protective innate mechanisms in the lung, which could be responsible for the increased susceptibility to viral infections in "healthy" smokers.

IRF4 Expression Is Required for the Immunoregulatory Activity of Conventional Type 2 Dendritic Cells in Settings of Chronic Bacterial Infection and Cancer.

The lamina propria of the gastrointestinal tract and other mucosal surfaces of humans and mice host a network of mononuclear phagocytes that differ in their ontogeny, surface marker and transcription factor expression, and functional specialization. Conventional dendritic cells (DCs) in particular exist as two major subpopulations in both lymphoid and nonlymphoid organs that can be distinguished based on their surface marker and transcription factor expression. In this study, we show in various Th1- and/or Th17-polarized settings of acute and chronic bacterial infection and of tumor growth that the conditional ablation of Irf4 in CD11c+ DCs results in more efficient immune control of Helicobacter pylori, Mycobacterium bovis bacillus Calmette-Guérin, and Citrobacter rodentium and of tumor growth in a syngeneic tumor model. We attribute the phenotype of IRF4ΔDC mice to unrestricted Th1 responses and in particular to IFN-γ- and TNF-α-expressing CD4+ T cells. This activity of IRF4-expressing DCs is linked to a DC-specific immunoregulatory transcriptional program. In contrast, in Th2-polarized settings such as house dust mite-induced allergic airway inflammation, the lack of IRF4 expression in the DC compartment alleviates inflammation and goblet cell metaplasia. The combined data provide evidence for immunoregulatory properties of this versatile DC population in Th1-polarized infection settings.

Single and Synergistic Effects of Type 2 Cytokines on Eosinophils and Asthma Hallmarks.

The type 2 cytokines IL-5, IL-13, and IL-4 play an important role in the induction and progression of asthma. According to the Global Initiative for Asthma guidelines, blood eosinophil numbers are one marker that helps to guide treatment decisions in patients suffering from severe forms of asthma. Effects of type 2 cytokines were analyzed, alone or in combination, on eosinophils in blood and other compartments and on the development of asthma symptoms. C57BL/6 mice received a single intranasal application of equimolar amounts of IL-5, IL-13, and IL-4, alone or in combination. Numbers, activation state, and migratory behavior of eosinophils in bone marrow (BM), blood, lung, and bronchoalveolar lavage as well as airway hyperresponsiveness and goblet cell metaplasia were evaluated. Only IL-13 was associated with airway eosinophilia, development of airway hyperresponsiveness, and goblet cell metaplasia, without any synergistic effects. IL-5 increased the number of eosinophils in BM and lung tissue but failed to affect structural changes. IL-4 had similar, but weaker, effects to IL-13. Cytokine combinations synergistically affected eosinophils but failed to enhance IL-13-driven effects on lung function or goblet cell metaplasia. IL-5 and IL-13 markedly increased eosinophil numbers locally in lung and airways and distally in blood and BM, whereas IL-5 and IL-4 only increased eosinophils in lung and BM. IL-13 together with IL-4 failed to demonstrate any synergistic effect. These insights into single and combined effects of type 2 cytokines on disease-driving mechanisms could improve understanding of the impact and effectiveness of new therapies in asthma.

Coincident airway exposure to low-potency allergen and cytomegalovirus sensitizes for allergic airway disease by viral activation of migratory dendritic cells.

Despite a broad cell-type tropism, cytomegalovirus (CMV) is an evidentially pulmonary pathogen. Predilection for the lungs is of medical relevance in immunocompromised recipients of hematopoietic cell transplantation, in whom interstitial CMV pneumonia is a frequent and, if left untreated, fatal clinical manifestation of human CMV infection. A conceivable contribution of CMV to airway diseases of other etiology is an issue that so far attracted little medical attention. As the route of primary CMV infection upon host-to-host transmission in early childhood involves airway mucosa, coincidence of CMV airway infection and exposure to airborne environmental antigens is almost unavoidable. For investigating possible consequences of such a coincidence, we established a mouse model of airway co-exposure to CMV and ovalbumin (OVA) representing a protein antigen of an inherently low allergenic potential. Accordingly, intratracheal OVA exposure alone failed to sensitize for allergic airway disease (AAD) upon OVA aerosol challenge. In contrast, airway infection at the time of OVA sensitization predisposed for AAD that was characterized by airway inflammation, IgE secretion, thickening of airway epithelia, and goblet cell hyperplasia. This AAD histopathology was associated with a T helper type 2 (Th2) transcription profile in the lungs, including IL-4, IL-5, IL-9, and IL-25, known inducers of Th2-driven AAD. These symptoms were all prevented by a pre-challenge depletion of CD4+ T cells, but not of CD8+ T cells. As to the underlying mechanism, murine CMV activated migratory CD11b+ as well as CD103+ conventional dendritic cells (cDCs), which have been associated with Th2 cytokine-driven AAD and with antigen cross-presentation, respectively. This resulted in an enhanced OVA uptake and recruitment of the OVA-laden cDCs selectively to the draining tracheal lymph nodes for antigen presentation. We thus propose that CMV, through activation of migratory cDCs in the airway mucosa, can enhance the allergenic potential of otherwise poorly allergenic environmental protein antigens.

In utero exposure to cigarette smoke and effects across generations: A conference of animals on asthma.

BACKGROUND: The prevalence of asthma and chronic obstructive pulmonary disease (COPD) has risen markedly over the last decades and is reaching epidemic proportions. However, underlying molecular mechanisms are not fully understood, hampering the urgently needed development of approaches to prevent these diseases. It is well established from epidemiological studies that prenatal exposure to cigarette smoke is one of the main risk factors for aberrant lung function development or reduced fetal growth, but also for the development of asthma and possibly COPD later in life. Of note, recent evidence suggests that the disease risk can be transferred across generations, that is, from grandparents to their grandchildren. While initial studies in mouse models on in utero smoke exposure have provided important mechanistic insights, there are still knowledge gaps that need to be filled. OBJECTIVE: Thus, in this review, we summarize current knowledge on this topic derived from mouse models, while also introducing two other relevant animal models: the fruit fly Drosophila melanogaster and the zebrafish Danio rerio. METHODS: This review is based on an intensive review of PubMed-listed transgenerational animal studies from 1902 to 2018 and focuses in detail on selected literature due to space limitations. RESULTS: This review gives a comprehensive overview of mechanistic insights obtained in studies with the three species, while highlighting the remaining knowledge gaps. We will further discuss potential (dis)advantages of all three animal models. CONCLUSION/CLINICAL RELEVANCE: Many studies have already addressed transgenerational inheritance of disease risk in mouse, zebrafish or fly models. We here propose a novel strategy for how these three model organisms can be synergistically combined to achieve a more detailed understanding of in utero cigarette smoke-induced transgenerational inheritance of disease risk.

The Canonical but Not the Noncanonical Wnt Pathway Inhibits the Development of Allergic Airway Disease.

Asthma is a syndrome with multifactorial causes, resulting in a variety of different phenotypes. Current treatment options are not curative and are sometimes ineffective in certain disease phenotypes. Therefore, novel therapeutic approaches are required. Recent findings have shown that activation of the canonical Wnt signaling pathway suppresses the development of allergic airway disease. In contrast, the effect of the noncanonical Wnt signaling pathway activation on allergic airway disease is not well described. The aim of this study was to validate the therapeutic effectiveness of Wnt-1-driven canonical Wnt signaling compared with Wnt-5a-driven noncanonical signaling in murine models. In vitro, both ligands were capable of attenuating allergen-specific T cell activation in a dendritic cell-dependent manner. In addition, the therapeutic effects of Wnt ligands were assessed in two different models of allergic airway disease. Application of Wnt-1 resulted in suppression of airway inflammation as well as airway hyperresponsiveness and mucus production. In contrast, administration of Wnt-5a was less effective in reducing airway inflammation or goblet cell metaplasia. These results suggest an immune modulating function for canonical as well as noncanonical Wnt signaling, but canonical Wnt pathway activation appears to be more effective in suppressing allergic airway disease than noncanonical Wnt activation.

Cylindromatosis (Cyld) gene mutation in T cells promotes the development of an IL-9-dependent allergic phenotype in experimental asthma.

Cylindromatosis (CYLD) is a ubiquitously expressed deubiquitinating enzyme which removes activating ubiquitin residues from important signaling molecules of the NF-κB pathway. In CYLDex7/8 transgenic mice, a naturally occurring short isoform (sCYLD) is overexpressed in the absence of full length CYLD, leading to excessive NF-κB activity. Herein, we investigated the impact of the CYLDex7/8 mutation selectively in T cells on the development of experimental allergic airway disease induced by sensitization and challenge with ovalbumin. Compared with their wildtype littermates, mice bearing the T cell-specific mutation (CD4+CYLDex7/8) display stronger eosinophilia and mucus production in the lungs and higher IgE serum levels. The reason for these observations is excessive production of T cell-derived IL-9, a cytokine to whom allergy-promoting properties were ascribed. Consequently, blockade of IL-9 in CD4+CYLDex7/8 mice alleviates the development of disease symptoms. Thus, by polarization of the T cell cytokine response, sCYLD can favor the development of allergic airway disease.

Take the Wnt out of the inflammatory sails: modulatory effects of Wnt in airway diseases.

Bronchial asthma and chronic obstructive pulmonary disease (COPD) are chronic diseases that are associated with inflammation and structural changes in the airways and lungs. Recent findings have implicated Wnt pathways in critically regulating inflammatory responses, especially in asthma. Furthermore, canonical and noncanonical Wnt pathways are involved in structural changes such as airway remodeling, goblet cell metaplasia, and airway smooth muscle (ASM) proliferation. In COPD, Wnt pathways are not only associated with structural changes in the airways but also involved in the development of emphysema. The present review summarizes the role and function of the canonical and noncanonical Wnt pathway with regard to airway inflammation and structural changes in asthma and COPD. Further identification of the role and function of different Wnt molecules and pathways could help to develop novel therapeutic options for these diseases.

Tick Salivary Sialostatin L Represses the Initiation of Immune Responses by Targeting IRF4-Dependent Transcription in Murine Mast Cells.

Coevolution of ticks and the vertebrate immune system has led to the development of immunosuppressive molecules that prevent immediate response of skin-resident immune cells to quickly fend off the parasite. In this article, we demonstrate that the tick-derived immunosuppressor sialostatin L restrains IL-9 production by mast cells, whereas degranulation and IL-6 expression are both unaffected. In addition, the expression of IL-1ß and IRF4 is strongly reduced in the presence of sialostatin L. Correspondingly, IRF4- or IL-1R-deficient mast cells exhibit a strong impairment in IL-9 production, demonstrating the importance of IRF4 and IL-1 in the regulation of the Il9 locus in mast cells. Furthermore, IRF4 binds to the promoters of Il1b and Il9, suggesting that sialostatin L suppresses mast cell-derived IL-9 preferentially by inhibiting IRF4. In an experimental asthma model, mast cell-specific deficiency in IRF4 or administration of sialostatin L results in a strong reduction in asthma symptoms, demonstrating the immunosuppressive potency of tick-derived molecules.

IL-10 and regulatory T cells cooperate in allergen-specific immunotherapy to ameliorate allergic asthma.

Human studies demonstrated that allergen-specific immunotherapy (IT) represents an effective treatment for allergic diseases. IT involves repeated administration of the sensitizing allergen, indicating a crucial contribution of T cells to its medicinal benefit. However, the underlying mechanisms of IT, especially in a chronic disease, are far from being definitive. In the current study, we sought to elucidate the suppressive mechanisms of IT in a mouse model of chronic allergic asthma. OVA-sensitized mice were challenged with OVA or PBS for 4 wk. After development of chronic airway inflammation, mice received OVA-specific IT or placebo alternately to airway challenge for 3 wk. To analyze the T cell-mediated mechanisms underlying IT in vivo, we elaborated the role of T-bet-expressing Th1 cells, T cell-derived IL-10, and Ag-specific thymic as well as peripherally induced Foxp3(+) regulatory T (Treg) cells. IT ameliorated airway hyperresponsiveness and airway inflammation in a chronic asthma model. Of note, IT even resulted in a regression of structural changes in the airways following chronic inhaled allergen exposure. Concomitantly, IT induced Th1 cells, Foxp3(+), and IL-10-producing Treg cells. Detailed analyses revealed that thymic Treg cells crucially contribute to the effectiveness of IT by promoting IL-10 production in Foxp3-negative T cells. Together with the peripherally induced Ag-specific Foxp3(+) Treg cells, thymic Foxp3(+) Treg cells orchestrate the curative mechanisms of IT. Taken together, we demonstrate that IT is effective in a chronic allergic disease and dependent on IL-10 and thymic as well as peripherally induced Ag-specific Treg cells.