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With the advent of biologicals, more and more therapeutics are available that specifically address specific switch points in the pathomechanism of immunologically dominated diseases. Thus, the focus of diagnostics and therapy (precision medicine) is more on the individual disease characteristics of the individual patient. Regarding the different phenotypes of atopic diseases, severe asthma was the first entity for which biologicals were approved, followed by urticaria, and finally atopic dermatitis and chronic rhinosinusitis with nasal polyps. Experience in the treatment of severe bronchial asthma has shown that the intensity of the response to biological therapy depends on the quality of clinical and immunological phenotyping of the patients. This also applies to different diseases of the atopic form, as patients can suffer from several atopic diseases at the same time, each with different characteristics. Biologics are already emerging that may represent a suitable therapy for allergic bronchial asthma, which often occurs together with severe neurodermatitis, and chronic rhinosinusitis with nasal polyps. In practice, however, the question of possible combinations of biologicals for the therapy of complex clinical pictures of individual patients is increasingly arising. In doing so, the side effect profile must be taken into account, including hypersensitivity reactions, whose diagnostic and logistical management must aim at a safe and efficient therapy of the underlying disease. Increased attention must also be paid to biological therapy in pregnancy and planned (predictable) vaccinations as well as existing infections, such as SARS-CoV-2 infection. Before starting a biological therapy, the immune status should be checked with regard to chronic viral and bacterial infections and, if necessary, the vaccination status should be refreshed or missing vaccinations should be made up for before starting therapy. Currently, reliable data on the effect of biologicals on the immunological situation of SARS-CoV-2 infection and COVID-19 are not available. Therefore, research and development of suitable diagnostic methods for detection of immunologically caused side effects as well as detection of potential therapy responders and non-responders is of great importance.

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.

Infection biology and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19), are incompletely understood. Here, we assessed the impact of airway epithelial cellular composition on infection in air-liquid interface (ALI) cultures of differentiated primary human tracheal (PTEC) and bronchial epithelial cells (PBEC). We first compared SARS-CoV-2 infection kinetics, related antiviral and inflammatory responses, and viral entry factors in PTEC and PBEC. Next, the contribution of differentiation time was investigated by differentiating ALI-PTEC/PBEC for 3-5 weeks and comparing dynamics of viral replication/spread, cellular composition and epithelial responses. We observed a gradual increase in viral load with prolonged culture duration. Ciliated and goblet cells were predominantly infected in both PTEC and PBEC. Immunofluorescence analysis and RT-qPCR showed that compared to other cell types mainly ciliated and goblet cell numbers were affected by increased culture duration. An increased proportion of these two target cell types was associated with increased viral load. Furthermore, modulation of cellular composition using IL-13 and the Notch signaling inhibitor DAPT, underlined the importance of both ciliated and goblet cells for infection. DAPT treatment resulted in a lower viral load and a relative increase in ciliated cells at the expense of goblet cells, compared to IL-13 treated cultures in which both cell types were present and viral load was higher.In conclusion, our results identify cellular composition as a contributing factor to airway epithelial susceptibility to SARS-CoV-2.IMPORTANCEIn this study, we determined an effect of culture duration and airway cellular composition of ALI-PBEC and ALI-PTEC cultures on SARS-CoV-2 infection. We found that SARS-CoV-2 infection was increased with prolonged cell culture time and the total percentage and proportion of ciliated and goblet cells played an important role in infection level, suggesting that airway epithelial differentiation/maturation levels may in part determine susceptibility of SARS-CoV-2 infection.The development of effective therapies either targeting virus replication or pathogenesis against SARS-CoV-2 requires robust cell culture-based infection models to test small molecules and biologicals. Therefore, it is important to identify factors that are essential for reliably modeling SARS-CoV-2-airway epithelial cell interactions. This study sheds light on virus-airway epithelial cell interactions and adds to the complexity of SARS-CoV-2 cell tropism in the airways. In addition, the effect of IL-13 on viral infection hints at a causal connection between SARS-CoV-2 infection and (allergic) asthma.

SummaryType I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies and have been successfully employed for the treatment of viral diseases. Humans express twelve IFN-alpha (α) subtypes, which activate downstream signalling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in type I IFN immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19, therefore, early administration of type I IFNs may be protective against life-threatening disease. Here we comprehensively analysed the antiviral activity of all IFNα subtypes against SARS-CoV-2 to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate and low antiviral IFNs. In particular IFNα5 showed superior antiviral activity against SARS-CoV-2 infection. Dose-dependency studies further displayed additive effects upon co-administered with the broad antiviral drug remdesivir in cell culture. Transcriptomics of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting and prototypical genes of individual IFNα subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in type I IFN signalling pathways, negative regulation of viral processes and immune effector processes for the potent antiviral IFNα5. Taken together, our data provide a systemic, multi-modular definition of antiviral host responses mediated by defined type I IFNs. This knowledge shall support the development of novel therapeutic approaches against SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) global pandemic, which so far has resulted in about 1 million deaths, is caused by severe acute respiratory coronavirus 2 (SARS-CoV-2). At present, infection dynamics and pathogenesis of the disease are incompletely understood. Here, we used well-differentiated primary human tracheal and bronchial epithelial cells (PTEC/PBEC) cultured at the air-liquid interface (ALI) to study the infection kinetics and subsequent epithelial response. ALI-PTEC and PBEC were differentiated for 3, 4 or 5 weeks, and next infected with SARS-CoV-2 to assess the impact of differentiation status on replication dynamics. We observed a gradual increase in viral load with prolonged differentiation time, and therefore focused on 5-weeks differentiated cultures. Next, we assessed the epithelial response to infection, identified the epithelial cell types that are predominantly infected and studied expression of SARS-CoV-2 entry-related host factors by using various methods (PCR, viral plaque assay and immunofluorescence [IF]). We found that ALI-PTEC cultures contained more goblet cells and less ciliated cells and showed higher expression of angiotensin-converting enzyme 2 (ACE2) compared to ALI-PBEC. Despite these differences, both cultures were efficiently infected by SARS-CoV-2. IF analysis showed that SARS-CoV-2 targets both ciliated and goblet cells. Expression of SARS-CoV-2 entry-related factors (ACE2 and TMPRSS2), IL-8 and IL-6 as well as type I and III interferons were increased after infection. These findings provide a foundation to further study virus-airway epithelial cell interactions in relation to host susceptibility to COVID-19. This study was supported by a grant from ZonMw and Proefdiervrij (# 114025007).

BACKGROUND: Malignant pleural mesothelioma (MPM) is a rare malignant tumor associated with asbestos exposure, with infaust prognosis and overall survival below 20 months in treated patients. Platinum is still the backbone of the chemotherapy protocols, and the reasons for the rather poor efficacy of platinum compounds in MPM remain largely unknown. Therefore, we aimed to analyze differences in key signaling pathways and biological mechanisms in therapy-naïve samples and samples after chemotherapy in order to evaluate the effect of platinum-based chemotherapy. METHODS: The study cohort comprised 24 MPM tumor specimens, 12 from therapy-naïve and 12 from patients after platinum-based therapy. Tumor samples were screened using the NanoString nCounter platform for digital gene expression analysis with an appurtenant custom-designed panel comprising a total of 366 mRNAs covering the most important tumor signaling pathways. Significant pathway associations were identified by gene set enrichment analysis using the WEB-based GEne SeT AnaLysis Toolkit (WebGestalt) RESULTS: We have found reduced activity of TNF (normalized enrichment score: 2.03), IL-17 (normalized enrichment score: 1.93), MAPK (normalized enrichment score: 1.51), and relaxin signaling pathways (normalized enrichment score: 1.42) in the samples obtained after platinum-based therapy. In contrast, AMPK (normalized enrichment score: –1.58), mTOR (normalized enrichment score: –1.50), Wnt (normalized enrichment score: –1.38), and longevity regulating pathway (normalized enrichment score: –1.31) showed significantly elevated expression in the same samples. CONCLUSIONS: We could identify deregulated signaling pathways due to a directed cellular response to platinum-induced cell stress. Our results are paving the ground for a better understanding of cellular responses and escape mechanisms, carrying a high potential for improved clinical management of patients with MPM.

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.

In dem Symposium "Adventure meets Experience", das auf dem 14. Deutschen Allergiekongress stattgefunden hat, beleuchteten die Redner die Probleme der Allergologie aus Sicht unterschiedlicher Generationen. Jung und Alt diskutierten uber …

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., Author summary From an epidemiological perspective, natural host-to-host transmission of human CMV mostly occurs in early childhood through saliva from virus-shedding intimate contact persons, such as family members or peers. The oronasal route of transmission involves also the airway mucosa and airway-draining lymph nodes. Almost unavoidably, CMV airway infection coincides with airway exposure to environmental antigens, which include potent classical allergens but also protein antigens that have low-to-no allergenic potential on their own. Ovalbumin (OVA), when administered as a purified protein, can serve as a well-studied model antigen for only poorly allergenic environmental antigens. In a murine model of airway exposure to OVA for allergenic sensitization, we have addressed the question if a simultaneous airway infection with murine CMV (mCMV) can promote allergic airway disease (AAD) elicited by challenge exposure to OVA. As anticipated by the model design, exposure to OVA alone did not sensitize for an allergic response to challenge exposure, nor did mCMV infection alone. Notably, based on viral activation of antigen uptake by DCs, both combined sensitized for a type 2 CD4+ T helper (Th2) cell-driven AAD histopathology. This is a novel aspect in CMV pathobiology with the medical relevance that CMV airway infection enlarges the spectrum of environmental allergens.

Simple Summary Malignant pleural mesothelioma (MPM) is a rare, biologically extremely aggressive tumor with an infaust prognosis. In this retrospective study, we aimed to assess the role of tumor-infiltrating immune cells and their activity in the respective histologic subtypes. We confirmed a substantial difference between epithelioid and sarcomatoid mesothelioma regarding the host’s anti-cancer immune reaction. Whereas antigen processing and presentation to resident cytotoxic T cells as well as phagocytosis is highly affected in sarcomatoid mesothelioma, cell–cell interaction via cytokines seems to be of greater importance in epithelioid cases. Our work reveals the specific role of the immune system within the different histologic subtypes of MPM, providing a more detailed background of their immunogenic potential. This is of great interest regarding therapeutic strategies addressing immunotherapy in mesothelioma. Abstract Malignant pleural mesothelioma (MPM) is an aggressive malignancy associated with asbestos exposure. Median survival ranges from 14 to 20 months after initial diagnosis. As of November 2020, the FDA approved a combination of immune checkpoint inhibitors after promising intermediate results. Nonetheless, responses remain unsatisfying. Adequate patient stratification to improve response rates is still lacking. This retrospective study analyzed formalin fixed paraffin embedded specimens from a cohort of 22 MPM. Twelve of those samples showed sarcomatoid, ten epithelioid differentiation. Complete follow-up, including radiological assessment of response by modRECIST and time to death, was available with reported deaths of all patients. RNA of all samples was isolated and subjected to digital gene expression pattern analysis. Our study revealed a notable difference between epithelioid and sarcomatoid mesothelioma, showing differential gene expression for 304/698 expressed genes. Whereas antigen processing and presentation to resident cytotoxic T cells as well as phagocytosis is highly affected in sarcomatoid mesothelioma, cell–cell interaction via cytokines seems to be of greater importance in epithelioid cases. Our work reveals the specific role of the immune system within the different histologic subtypes of MPM, providing a more detailed background of their immunogenic potential. This is of great interest regarding therapeutic strategies including immunotherapy in mesothelioma.

Background Regulatory T cells (Treg) represent a promising target for novel treatment strategies in patients with inflammatory/allergic diseases. A soluble derivate of the Treg surface molecule glycoprotein A repetitions predominant (sGARP) has strong anti-inflammatory and regulatory effects on human cells in vitro as well as in vivo through de novo induction of peripheral Treg. The aim of this study was to investigate the immunomodulatory function of sGARP and its possible role as a new therapeutic option in allergic diseases using a humanized mouse model. Methods To analyze the therapeutic effects of sGARP, adult NOD/Scidγc−/− (NSG) mice received peripheral blood mononuclear cells (PBMC) derived from allergic patients with sensitization against birch allergen. Subsequently, allergic inflammation was induced in the presence of Treg alone or in combination with sGARP. Results In comparison with mice that received Treg alone, additional treatment with sGARP reduced airway hyperresponsiveness (AHR), influx of neutrophils and macrophages into the bronchoalveolar lavage (BAL), and human CD45+ cells in the lungs. Furthermore, the numbers of mucus-producing goblet cells and inflammatory cell infiltrates were reduced. To elucidate whether the mechanism of action of sGARP involves the TGF-β receptor pathway, mice additionally received anti-TGF-β receptor II (TGF-βRII) antibodies. Blocking the signaling of TGF-β through TGF-βRII abrogated the anti-inflammatory effects of sGARP, confirming its essential role in inhibiting the allergic inflammation. Conclusion Induction of peripheral tolerance via sGARP is a promising potential approach to treat allergic airway diseases.

Background and Aims: In immunosuppressed individuals Aspergillus (A.) fumigatus is a frequent cause of invasive pulmonary aspergillosis (IPA) which is highly associated with relevant morbidity and mortality. Moreover, it often occurs in patients suffering from leukocyte-adhesion deficiency type 1 (LAD1) which is triggered by a functional loss of CD18 in ß2 integrin receptors as these receptors consist of an alpha subunit (CD11a-CD11d) and CD18 as the common beta subunit. ß2 integrin receptors are differentially expressed by leukocytes, and are required for cell-cell interaction, transendothelial migration, uptake of opsonized pathogens, and cell signaling processes. Here, we asked for the importance of CD11b/CD18 also termed MAC-1 which is required for phagocytosis of opsonized A. fumigatus conidia by polymorphonuclear neutrophils (PMN) for control of pulmonary A. fumigatus infection. Methods: We used a murine IPA model (C57BL/6) and challenged CD11b deficient (CD11b-/-) or wild type (WT) mice with A. fumigatus conidia intratracheally. Afterwards, some mice were sacrificed 24h after infection. In these mice PMN recruitment and cytokine patterns were examined by analyzing bronchoalveolar lavage fluid (BALF) and peripheral blood (PB) using flow cytometry, cytospin analysis, and cytometric bead array. Additionally, pulmonary fungal clearance and inflammation in murine lungs were analyzed by fungal culture assays and histopathologic examination using a scoring system. Furthermore, survival was studied with neutropenic animals serving as positive controls. To determine PMNs phagocytic activity and fungal killing capacity ex vivo, PMN were purified from bone marrow of CD11b-/- or WT mice by magnetic cell sorting using Ly6G specific antibodies. Afterwards, isolated PMN were stimulated with pacific blue-labeled tomato red-fluorescent modified A. fumigatus conidia and analyzed by flow cytometry. Results: We found that lung homogenates from CD11b-/- mice obtained 24h after infection showed an enhanced fungal burden as compared to lungs from WT mice. In contrast, lung tissue from infected CD11b-/- mice displayed impaired pulmonary inflammation as assessed by Hematoxilin-Eosin (HE) staining. Furthermore, the number of mucus-producing cells in bronchi of A. fumigatus infected CD11b-/- mice was decreased compared to cells in bronchi of WT mice. However, we observed markedly higher numbers of PMN in BALF of infected CD11b-/- mice compared to corresponding WT mice samples. In contrast, BALF derived from infected CD11b-/- mice contained lower levels of three different proinflammatory cytokines (TNF-α, IL-1α, IL-1β) compared to BALF from WT mice while levels of other cytokines and chemokines (IL-5, IL-6, IL-10, GM-CSF, CXCL1, CCL2) were comparable. In contrast, we measured higher levels of the chemokine CCL5 known as a relevant chemoattractant in innate and adaptive immune cells in BALF obtained from CD11b-/- mice. Interestingly, numbers of PMN, lymphocytes, and monocytes in the peripheral blood of A. fumigatus infected mice did not differ in a genotype-dependent manner. However, CD11b-/- mice PMN showed lower phagocytic activity than WT PMN, indicating an impaired capability to clear A. fumigatus. Nevertheless, CD11b-/- mice were finally characterized by similar long-term survival as WT mice after IPA induction while the PMN-depleted control mice died as expected. Conclusions: Our study demonstrates, that CD11b deficiency on myeloid cells affects the early course of IPA. This may be due to the importance of MAC-1 for PMN effector functions, and their interplay with DC and other leukocytes. Further work is necessary to elucidate the long-term course of IPA in CD11b-/- mice with regard to the interplay of PMN with dendritic cells, the efficacy of adaptive immune responses, and the potential pulmonary overexpression of CCL5. Beyond patients with LAD1 syndrome, we suggest our findings as being clinically relevant in other immunocompromised patients suffering from severe opportunistic infections. Disclosures Radsak: Celgene: Honoraria, Other: Travel grant; Jazz Pharmaceuticals: Other: Travel grant; TEVA: Consultancy; Gilead: Other: Travel grant; Takeda: Consultancy; Novartis: Consultancy, Honoraria; Daiichi Sankyo: Honoraria, Other: Travel grant.

There is a growing body of evidence for immunomodulatory side effects of antifungal agents on different immune cells, e.g., T cells. Therefore, the aim of our study was to clarify these interactions with regard to the effector functions of polymorphonuclear neutrophils (PMN). Human PMN were preincubated with fluconazole (FLC), voriconazole (VRC), posaconazole (POS), isavuconazole (ISA), caspofungin (CAS), micafungin (MFG), conventional amphotericin B (AMB), and liposomal amphotericin B (LAMB). PMN then were analyzed by flow cytometry for activation, degranulation, and phagocytosis and by dichlorofluorescein assay to detect reactive oxygen species (ROS). Additionally, interleukin-8 (IL-8) release was measured by enzyme-linked immunosorbent assay. POS led to enhanced activation, degranulation, and generation of ROS, whereas IL-8 release was reduced. In contrast, ISA-pretreated PMN showed decreased activation signaling, impaired degranulation, and lower generation of ROS. MFG caused enhanced expression of activation markers but impaired degranulation, phagocytosis, generation of ROS, and IL-8 release. CAS showed increased phagocytosis, whereas degranulation and generation of ROS were reduced. AMB led to activation of almost all effector functions besides impaired phagocytosis, whereas LAMB did not alter any effector functions. Independent from class, antifungal agents show variable influence on neutrophil effector functions in vitro. Whether this is clinically relevant needs to be clarified.

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.

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.

Neutrophil extracellular traps (NETs) are formed by polymorphonuclear neutrophils (PMN) and contribute to the innate host defense by binding and killing bacterial and fungal pathogens. Because NET formation depends on histone hypercitrullination by peptidylarginine deiminase 4 (PAD4), we used PAD4 gene deficient (Pad4-/-) mice in a mouse model of invasive pulmonary aspergillosis (IPA) to address the contribution of NETs to the innate host defense in vivo. After the induction (24 h) of IPA by i.t. infection with Aspergillus fumigatus conidia, Pad4-/- mice revealed lower fungal burden in the lungs, accompanied by less acute lung injury, TNFα and citH3 compared to wildtype controls. These findings suggest that release of NETs contributes to tissue damage and limits control of fungal outgrowth. Thus inhibition of NETosis might be a useful strategy to maintain neutrophil function and avoid lung damage in patients suffering from IPA, especially in those suffering from preexisting pulmonary disease.

Mit uber 28 eingereichten Posterbeitragen, dem 7. Junior-Member-Symposium und dem neuen Junior-Alumni-Symposium war der 14. Deutsche Allergiekongress in Hannover ein voller Erfolg fur die Junior Members der DGAKI. Die Sektion der Junior Members vertritt fast ein Viertel der gesamten DGAKI-Mitglieder und ist oft das Eingangstor fur junge, engagierte Arzte. Durch die vielfaltige Beteiligung konnte die Sichtbarkeit in diesem Jahr deutlich gesteigert werden.

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Epidemiological and basic research suggest that factors like genetic predisposition and environmental exposures contribute to the development of allergic disorder. There is increasing evidence that viral airway infections are also an important factor for the development of allergic airway diseases. In the present study the pulmonary interaction of murine CMV (mCMV) infection and concurrent exposure to a harmless inhaled antigen (Ovalbumin-OVA) was analyzed in mice. OVA or a fluorescent derivate was given intratracheally alone or in combination with a mCMV infection of the airways. At 24 hours later, migrating antigen loaded dendritic cells (DC) were analyzed. To assess the rechallenge response two weeks after this exposure, animals were challenged with OVA by nebulization on three consecutive days and developing inflammation was monitored. We can show, that 24 hours after application, number and activation status of OVA-positive DC in the draining lymph node in OVA/mCMV animals was enhanced compared to OVA only exposed animals. In OVA only animals these DC consisted mainly of CD11b-positive DC whereas in additionally infected animals equal numbers of CD103 and CD11b DC were detectable. After challenge with nebulized OVA, only animals which received OVA and mCMV in advance developed an increased inflammation in BAL and lung tissue and showed mucus-positive cells in the lung epithelium. Moreover only in OVA/mCMV pretreated mice specific immunoglobuline titers were detectable. These data demonstrate that mCMV can modulate the immune response towards harmless inhaled antigens by inducing sensitization and the development of allergic airway disease.

Introduction: Current observations reveal an anti-inflammatory effect of Wnt mediated β-catenin signaling during allergic airway disease. Indeed, lung specific overexpression of Wnt-1 attenuates experimental allergic airway disease (Reuter, S. et al. J Immunol 2014;193:485-95). The aim of the following study was to investigate the effect of recurrent Wnt-1 exposure in a therapeutic fashion in a murine model of allergic airway disease. Method: After the intraperitoneal sensitization with Ovalbumin(OVA)/Aluminium hydroxide on days 0 and 14, C57BL/6 mice were challenged via the airways by nebulization of a 1% OVA solution on days 28-30. Some animals received recombinant Wnt-1 intranasal one hour prior to each challenge. 48 hours after the last challenge the allergic airway disease was analyzed. Results: The application of Wnt-1 significant diminishes airway hyper responsiveness, goblet cell metaplasia and lung inflammation. Moreover, Wnt-1 treated animals demonstrated a reduced Interleukin (IL)-13 secretion of ex vivo lung and draining lymph node cells. Furthermore, the pattern of cDC (conventional dendritic cells) subpopulation in the lung differed between treated and untreated animals. Sensitized animals without Wnt-1 treatment demonstrated a CD11b dominated ratio of cDC in the lung whereas Wnt-1 treated animals revealed a balanced ratio of CD11b to CD103 positive cDC. The frequency of regulatory T cells or the secretion of IL-10 was unaffected. Conclusion: These data demonstrate the suppression of allergic airway disease by activating the canonical Wnt/β-catenin-pathway using Wnt-1 ligand.

CYLD is a negative regulator of NF-κB activity, yet in mice a short splice variant lacking exons 7 and 8 (or short CYLD (CYLD S )) with activating properties has also been described. In the present study we analyze the T cell-specific role of CYLD S in vitro and in vivo in a model of allergic airway disease. In addition we investigated if an equivalent is also expressed in humans. Using T cells derived from animals with a T cell specific expression of CYLD S we demonstrate that under Th9-polarizing conditions the production of IL-9 was enhanced, whereas under Th2-polarizing conditions production of IL-4 was not affected, compared to wild type (WT) cells. In contrast to WT animals, CyldFL*CD4cre animals developed an exacerbated asthma phenotype. Treatment with a neutralizing α-IL9 antibody during the challenge phase attenuates the asthma phenotype in CyldFL*CD4cre animals, but not in WT animals. By generation of specific primers we could demonstrate that humans express an undescribed splice variant of CYLD, lacking, as the mouse equivalent, the NEMO binding side. Activation of PBMC shifts the ratio of the full length CYLD (CYLD FL ) to CYLD S . Moreover, in comparison to healthy subjects asthmatics demonstrate by raising expression of CYLD FL a comparatively stronger expression of CYLD S . CYLD is an important regulator of NF-κB, driving the development of allergic airway disease by an IL-9 dependent pathway. Also a human homolog was discovered which was differently regulated in patients with asthma compared to healthy persons. Therefore, differential expression of CYLD splice variants could be an important determinant for the development of allergic airway disease.

Soluble derivate of Glycoprotein A repetitions predominant (sGARP) has strong anti-inflammatory and regulatory properties in vitro as well as in vivo , represses proliferation and cytokine production of CD4 + T helper cells and induces CD4 + regulatory T cells (Treg). Furthermore, sGARP prevents inflammation in a preclinical in vivo humanized mouse model of graft versus host disease (Hahn SA et al. Blood 2013). Modulation of Treg function could have therapeutic effects in many inflammatory diseases. Hence, the aim of the present study was to investigate the impact of sGARP in treatment of allergic airway diseases by using an established humanized mouse model (Martin H et al. 2012). To address this question, adult NSG mice received peripheral blood mononuclear cells (PBMC) from birch pollen asthmatic patients. To analyze the effects of sGARP on allergic inflammation in the humanized mouse model, mice received additionally Treg alone or in combination with sGARP. The allergic airway disease was induced by a three-day intranasal challenge with birch pollen allergen 20 days after PBMC transfer. 48 hours after last challenge allergic inflammation was assessed by measurement of airway hyperresponsiveness (AHR), quantification of cells in the bronchoalveolar lavage (BAL) and analysis of human immune cells in different tissues by flow cytometric and histological staining. Additional transfer with sGARP significantly reduced AHR, total human immune cell counts, neutrophils and macrophages in the BAL, human CD4 + T and mucus producing goblet cells in the lungs. Hence, amplifying of Treg cell function via sGARP seems to be an effective treatment option for the suppression of allergic airway diseases.

Signaling via the Wnt/β-catenin pathway plays crucial roles in embryogenesis and homeostasis of adult tissues. In the lung, the canonical Wnt/β-catenin pathway has been implicated in remodeling processes, development of emphysema, and fibrosis. However, its relevance for the modulation of allergic responses in the lung remains unclear. Using genetically modified mice with lung-specific inducible (doxycycline) Wnt-1 expression (CCSP-rtTA × tetO-Wnt1), the impact of Wnt on the development of allergic airway disease was analyzed. Overexpression of Wnt during the allergen challenge phase attenuated the development of airway inflammation in an acute model, as well as in a more therapeutic model of secondary challenge. These findings were further supported by treatment of allergen-sensitized mice with LiCl during challenge. Similar to Wnt, LiCl prevented the degradation of β-catenin and, thus, attenuated allergic airway inflammation and hyperresponsiveness. Migration studies revealed that lung-specific expression of Wnt reduced the migration of Ag-loaded dendritic cells (DCs) into the draining lymph nodes following allergen challenge. Administration of in vitro allergen-loaded DCs overcame Wnt-mediated suppression of airway inflammation. Furthermore, in vitro studies confirmed that DC-dependent T cell activation is impaired by blocking β-catenin degradation. These results demonstrate an important role for the canonical Wnt/β-catenin pathway in the DC-mediated regulation of allergic responses in the lung.