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The recently published new European guidelines for diagnosis and treatment of pulmonary hypertension now offer the so far most extensive description of genetic testing and counselling for pulmonary arterial hypertension patients. In addition, the importance of a clinical screening of healthy mutation carriers is highlighted as well as the genetic testing of patients with a suspicion of pulmonary veno-occlusive disease. We frame the respective parts of the guidelines on genetic testing and counselling in the context of recent data and provide comments. Finally, we give an outlook on novel molecular approaches starting from Sotatercept, addressing ion channels and novel therapeutic developments., Competing Interests: C.A.E. und K.H. sind Erfinderinnen des europäischen Patentens (EP3507380) “Gene panel specific for pulmonary hypertension and its uses”. CAE hat von MSD Vortragshonorare erhalten, unabhängig von dieser Arbeit.N.S. hat Berater- und Vortragstätigkeiten für MSD und Janssen.R.T.S. erhielt Forschungsförderung von Gossamer.S.S.P. erhielt Forschungsgelder und Honorare von Gossamer Bio, Inc.N.W. ist Besitzer des Patentes: L-NIL als Inhibitor zur Regeneration der Lunge von an COPD leidenden Patienten (EP2591777B1).L.H. erhielt Vortragshonorare von Janssen-Cilag und AOP Health sowie Forschungsmittel von MSD.C.T. erhielt finanzielle Förderung für Forschung von der Deutschen Gesellschaft für Pneumologie und Beatmungsmedizin e. V., Bayer HealthCare, Boehringer Ingelheim, und für Vorträge und Beratungstätigkeit von Actelion Pharmaceuticals, AstraZeneca, Berlin-Chemie, Boehringer Ingelheim, GlaxoSmithKline, und nicht-finanzielle Förderung von Actelion, ALK-Abelló, Bayer HealthCare, Boehringer Ingelheim und GlaxoSmithKline.M.W. erhielt Vortragshonorare von Janssen.V.F. erhielt Reisekosten und Vortragstätigkeiten von Boehringer Ingelheim, BMS, Chiesi, Janssen, MSD, alle nicht im Zusammenhang mit der aktuellen Übersichtsarbeit.A.O. ist Erfinderin des Patentes (WO2017153472A1 priority date 09.03.2016, erteilt in US, KR, JP, pending in CA, EP, AU) “Biomarker for the diagnosis of pulmonary hypertension (PH)”, ohne daraus persönlichen Gewinn zu erhalten. A.O. hat Honorare für Präsentationen und Reiseförderung von MSD erhalten, unabhängig von dieser Arbeit.O.B., W.M.K., G.K. haben keinen Interessenkonflikt., (Thieme. All rights reserved.)

Background: Systemic sclerosis (SSc) belongs to the group of connective tissue diseases and is associated with the occurrence of disease-specific autoantibodies. Although it is still controversial whether these antibodies contribute to pathogenesis, there are new insights into the development of these specific antibodies and their possible pathophysiological properties. Interestingly, they are associated with specific clinical manifestations, but for some rarer antibodies this association is not fully clarified. The aim of this study is a comprehensive analysis of the serum autoantibody status in patients with SSc followed by correlation analyses of autoantibodies with the clinical course of the disease., Methods: Serum from SSc patients was analyzed using a line blot (EUROLINE, EUROIMMUN AG) for SSc-related autoantibodies. Autoantibodies to centromere, Topo-1, antimitochondrial antibodies (AMA) M2 subunit, angiotensin II type 1 receptors (AT 1 R) and endothelin-1 type-A-receptors (ET A R) were also determined by ELISA. We formed immunological clusters and used principal components analysis (PCA) to assign specific clinical characteristics to these clusters., Results: A total of 372 SSc patients were included. 95.3% of the patients were antinuclear antibody positive and in 333 patients at least one SSc specific antibody could be detected. Four immunological clusters could be found by PCA. Centromere, Topo-1 and RP3 all formed own clusters, which are associated with distinct clinical phenotypes. We found that patients with an inverted phenotype, such as limited cutaneous SSc patients within the Topo-1 cluster show an increased risk for interstital lung disease compared to ACA positive patients. Anti-AT 1 R and anti-ET A R autoantibodies were measured in 176 SSc patients; no association with SSc disease manifestation was found. SSc patients with AMA-M2 antibodies showed an increased risk of cardiovascular events., Conclusion: In our in large cluster analysis, which included an extended autoantibody profile, we were able to show that serologic status of SSc patients provides important clues to disease manifestation, co-morbidities and complications. Line blot was a reliable technique to detect autoantibodies in SSc and detected rarer autoantibodies in 42% of our patients., Competing Interests: CT received funding for research from Deutsche Gesellschaft für Pneumologie, Bayer HealthCare, Boehringer Ingelheim, and for lectures and advisory from Actelion Pharmaceuticals, Boehringer Ingelheim, GlaxoSmithKline, and for non-financial support from Actelion, ALK-Abelló, Bayer HealthCare, Boehringer Ingelheim and GlaxoSmithKline. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest., (Copyright © 2023 Höppner, Tabeling, Casteleyn, Kedor, Windisch, Burmester, Huscher and Siegert.)

Purpose: Six to 19% of critically ill COVID-19 patients display circulating auto-antibodies against type I interferons (IFN-AABs). Here, we establish a clinically applicable strategy for early identification of IFN-AAB-positive patients for potential subsequent clinical interventions., Methods: We analyzed sera of 430 COVID-19 patients from four hospitals for presence of IFN-AABs by ELISA. Binding specificity and neutralizing activity were evaluated via competition assay and virus-infection-based neutralization assay. We defined clinical parameters associated with IFN-AAB positivity. In a subgroup of critically ill patients, we analyzed effects of therapeutic plasma exchange (TPE) on the levels of IFN-AABs, SARS-CoV-2 antibodies and clinical outcome., Results: The prevalence of neutralizing AABs to IFN-α and IFN-ω in COVID-19 patients from all cohorts was 4.2% (18/430), while being undetectable in an uninfected control cohort. Neutralizing IFN-AABs were detectable exclusively in critically affected (max. WHO score 6-8), predominantly male (83%) patients (7.6%, 18/237 for IFN-α-AABs and 4.6%, 11/237 for IFN-ω-AABs in 237 patients with critical COVID-19). IFN-AABs were present early post-symptom onset and at the peak of disease. Fever and oxygen requirement at hospital admission co-presented with neutralizing IFN-AAB positivity. IFN-AABs were associated with lower probability of survival (7.7% versus 80.9% in patients without IFN-AABs). TPE reduced levels of IFN-AABs in three of five patients and may increase survival of IFN-AAB-positive patients compared to those not undergoing TPE., Conclusion: IFN-AABs may serve as early biomarker for the development of severe COVID-19. We propose to implement routine screening of hospitalized COVID-19 patients for rapid identification of patients with IFN-AABs who most likely benefit from specific therapies., (© 2022. The Author(s).)

Introduction: Inflammation is a major pathological feature of pulmonary arterial hypertension (PAH), particularly in the context of inflammatory conditions such as systemic sclerosis (SSc). The endothelin system and anti-endothelin A receptor (ET A ) autoantibodies have been implicated in the pathogenesis of PAH, and endothelin receptor antagonists are routinely used treatments for PAH. However, immunological functions of the endothelin B receptor (ET B ) remain obscure., Methods: Serum levels of anti-ET B receptor autoantibodies were quantified in healthy donors and SSc patients with or without PAH. Age-dependent effects of overexpression of prepro-endothelin-1 or ET B deficiency on pulmonary inflammation and the cardiovascular system were studied in mice. Rescued ET B -deficient mice (ET B -/- ) were used to prevent congenital Hirschsprung disease. The effects of pulmonary T-helper type 2 (Th2) inflammation on PAH-associated pathologies were analyzed in ET B -/- mice. Pulmonary vascular hemodynamics were investigated in isolated perfused mouse lungs. Hearts were assessed for right ventricular hypertrophy. Pulmonary inflammation and collagen deposition were assessed via lung microscopy and bronchoalveolar lavage fluid analyses., Results: Anti-ET B autoantibody levels were elevated in patients with PAH secondary to SSc. Both overexpression of prepro-endothelin-1 and rescued ET B deficiency led to pulmonary hypertension, pulmonary vascular hyperresponsiveness, and right ventricular hypertrophy with accompanying lymphocytic alveolitis. Marked perivascular lymphocytic infiltrates were exclusively found in ET B -/- mice. Following induction of pulmonary Th2 inflammation, PAH-associated pathologies and perivascular collagen deposition were aggravated in ET B -/- mice., Conclusion: This study provides evidence for an anti-inflammatory role of ET B . ET B seems to have protective effects on Th2-evoked pathologies of the cardiovascular system. Anti-ET B autoantibodies may modulate ET B -mediated immune homeostasis., Competing Interests: CT received funding for research from Deutsche Gesellschaft für Pneumologie, Bayer HealthCare, Boehringer Ingelheim, and for lectures from Actelion Pharmaceuticals, Boehringer Ingelheim. HH is CEO of CellTrend GmbH, Luckenwalde, Germany. MW received funding for research from Deutsche Forschungsgemeinschaft, Bundesministerium für Bildung und Forschung, Deutsche Gesellschaft für Pneumologie, European Respiratory Society, Marie Curie Foundation, Else Kröner Fresenius Stiftung, CAPNETZ STIFTUNG, International Max Planck Research School, Actelion, Bayer Health Care, Biotest AG, Boehringer Ingelheim, NOXXON Pharma, Pantherna, Quark Pharma, Silence Therapeutics, Vaxxilon, and for lectures and advisory from Actelion, Alexion, Aptarion, Astra Zeneca, Bayer Health Care, Berlin Chemie, Biotest, Boehringer Ingelheim, Chiesi, Glaxo Smith Kline, Insmed, Novartis, Teva and Vaxxilon. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tabeling, González Calera, Lienau, Höppner, Tschernig, Kershaw, Gutbier, Naujoks, Herbert, Opitz, Gruber, Hocher, Suttorp, Heidecke, Burmester, Riemekasten, Siegert, Kuebler and Witzenrath.)

Coronavirus disease 2019 (COVID-19) is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Type I interferons are important in the defense of viral infections. Recently, neutralizing IgG auto-antibodies against type I interferons were found in patients with severe COVID-19 infection. Here, we analyzed expression of CD169/SIGLEC1, a well described downstream molecule in interferon signaling, and found increased monocytic CD169/SIGLEC1 expression levels in patients with mild, acute COVID-19, compared to patients with severe disease. We recommend further clinical studies to evaluate the value of CD169/SIGLEC1 expression in patients with COVID-19 with or without auto-antibodies against type I interferons., (© 2021. The Author(s).)

Idiopathic pulmonary fibrosis (IPF) is a deadly condition characterized by progressive respiratory dysfunction. Exacerbations due to airway infections are believed to promote disease progression, and presence of Streptococcus in the lung microbiome has been associated with the progression of IPF and mortality. The aim of this study was to analyze the effect of lung fibrosis on susceptibility to pneumococcal pneumonia and bacteremia. The effects of subclinical (low dose) infection with Streptococcus pneumoniae were studied in a well characterized fos-related antigen-2 (Fra-2) transgenic (TG) mouse model of spontaneous, progressive pulmonary fibrosis. Forty-eight hours after transnasal infection with S. pneumoniae , bacterial load was assessed in lung tissue, bronchoalveolar lavage (BAL), blood, and spleen. Leukocyte subsets and cytokine levels were analyzed in BAL and blood. Lung compliance and arterial blood gases were assessed. In contrast to wildtype mice, low dose lung infection with S. pneumoniae in Fra-2 TG mice resulted in substantial pneumonia including weight loss, increased lung bacterial load, and bacteremia. BAL alveolar macrophages were reduced in Fra-2 TG mice compared to the corresponding WT mice. Proinflammatory cytokines and chemokines (IL-1β, IL-6, TNF-α, and CXCL1) were elevated upon infection in BAL supernatant and plasma of Fra-2 TG mice. Lung compliance was decreased in Fra-2 TG mice following low dose infection with S. pneumoniae . Pulmonary fibrosis increases susceptibility to pneumococcal pneumonia and bacteremia possibly via impaired alveolar bacterial clearance.

Background: Maternal asthma during pregnancy is considered an environmental risk factor for asthma development in children. Immunoglobulin G (IgG) antibodies that are transferred from the mother to the fetus are known to act in a pro- or anti-inflammatory manner depending on their glycosylation status., Objective: Using a mouse model, we examined how maternal allergic airway inflammation during pregnancy influenced offspring experimental asthma severity, as well as maternal and offspring serum IgG antibody glycosylation patterns. Additionally, the effects of maternal and offspring exposure to the same or different allergens were investigated., Methods: Female mice were either sham sensitized or sensitized to casein (CAS) or ovalbumin (OVA) before mating. Subsequently, allergic lung inflammation was induced in pregnant dams via aerosol allergen challenge (sham, CAS or OVA). After weaning, pups were subjected to an experimental asthma protocol using OVA. Asn-297 IgG glycosylation was analysed in maternal and offspring serum., Results: When mothers and offspring were sensitized to the same allergen (OVA-OVA), offspring had more severe experimental asthma. This was evidenced by altered antibody concentrations, increased bronchoalveolar lavage inflammatory cell influx and decreased lung tissue and lung draining lymph node regulatory T cell percentages. When mothers and offspring were sensitized to different allergens (CAS-OVA), this phenotype was no longer observed. Additionally, maternal serum from allergic mothers had significantly higher levels of pro-inflammatory IgG1, shown by decreased galactosylation and sialylation at the Asn-297 glycosylation site. Similar glycosylation patterns were observed in the serum of adult allergic offspring from allergic mothers., Conclusions and Clinical Relevance: We observed a strong association between maternal experimental asthma during pregnancy, increased offspring airway inflammation and pro-inflammatory IgG glycosylation patterns in mothers and offspring. IgG glycosylation is not a standard measurement in the clinical setting, and we argue that it may be an important parameter to include in future clinical studies., (© 2020 The Authors. Clinical & Experimental Allergy published by John Wiley & Sons Ltd.)

Background Hypertension is the major risk factor for cardiovascular disease, the most common cause of death worldwide. Resistance arteries are capable of adapting their diameter independently in response to pressure and flow-associated shear stress. Ryanodine receptors (RyRs) are major Ca 2+ -release channels in the sarcoplasmic reticulum membrane of myocytes that contribute to the regulation of contractility. Vascular smooth muscle cells exhibit 3 different RyR isoforms (RyR1, RyR2, and RyR3), but the impact of individual RyR isoforms on adaptive vascular responses is largely unknown. Herein, we generated tamoxifen-inducible smooth muscle cell-specific RyR2-deficient mice and tested the hypothesis that vascular smooth muscle cell RyR2s play a specific role in elementary Ca 2+ signaling and adaptive vascular responses to vascular pressure and/or flow. Methods and Results Targeted deletion of the Ryr2 gene resulted in a complete loss of sarcoplasmic reticulum-mediated Ca 2+ -release events and associated Ca 2+ -activated, large-conductance K + channel currents in peripheral arteries, leading to increased myogenic tone and systemic blood pressure. In the absence of RyR2, the pulmonary artery pressure response to sustained hypoxia was enhanced, but flow-dependent effects, including blood flow recovery in ischemic hind limbs, were unaffected. Conclusions Our results establish that RyR2-mediated Ca 2+ -release events in VSCM s specifically regulate myogenic tone (systemic circulation) and arterial adaptation in response to changes in pressure (hypoxic lung model), but not flow. They further suggest that vascular smooth muscle cell-expressed RyR2 deserves scrutiny as a therapeutic target for the treatment of vascular responses in hypertension and chronic vascular diseases.

Th17 cells provide protection at barrier tissues but may also contribute to immune pathology. The relevance and induction mechanisms of pathologic Th17 responses in humans are poorly understood. Here, we identify the mucocutaneous pathobiont Candida albicans as the major direct inducer of human anti-fungal Th17 cells. Th17 cells directed against other fungi are induced by cross-reactivity to C. albicans. Intestinal inflammation expands total C. albicans and cross-reactive Th17 cells. Strikingly, Th17 cells cross-reactive to the airborne fungus Aspergillus fumigatus are selectively activated and expanded in patients with airway inflammation, especially during acute allergic bronchopulmonary aspergillosis. This indicates a direct link between protective intestinal Th17 responses against C. albicans and lung inflammation caused by airborne fungi. We identify heterologous immunity to a single, ubiquitous member of the microbiota as a central mechanism for systemic induction of human anti-fungal Th17 responses and as a potential risk factor for pulmonary inflammatory diseases., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Hydrogen sulfide (H 2 S) and NO are important gasotransmitters, but how endogenous H 2 S affects the circulatory system has remained incompletely understood. Here, we show that CTH or CSE (cystathionine γ-lyase)-produced H 2 S scavenges vascular NO and controls its endogenous levels in peripheral arteries, which contribute to blood pressure regulation. Furthermore, eNOS (endothelial NO synthase) and phospho-eNOS protein levels were unaffected, but levels of nitroxyl were low in CTH-deficient arteries, demonstrating reduced direct chemical interaction between H 2 S and NO. Pretreatment of arterial rings from CTH-deficient mice with exogenous H 2 S donor rescued the endothelial vasorelaxant response and decreased tissue NO levels. Our discovery that CTH-produced H 2 S inhibits endogenous endothelial NO bioavailability and vascular tone is novel and fundamentally important for understanding how regulation of vascular tone is tailored for endogenous H 2 S to contribute to systemic blood pressure function., (© 2018 American Heart Association, Inc.)

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease in which the amount of emphysema and airway disease may be very different between individuals, even in end-stage disease. Emphysema formation may be linked to the involvement of the small pulmonary vessels. The NAPDH oxidase (Nox) family is emerging as a key disease-related factor in vascular diseases, but currently its role in hypoxia-induced pulmonary remodelling in COPD remains unclear.Here we investigate the role of p22phox, a regulatory subunit of Nox, in COPD lungs, hypoxic pulmonary vasoconstriction (HPV), hypoxia-induced pulmonary vascular remodelling and pulmonary hypertension.In COPD, compared to control lungs, p22phox expression was significantly reduced. The expression was correlated positively with mean pulmonary arterial pressure and oxygenation index and negatively with the diffusing capacity of the lung for carbon monoxide (p<0.02). This suggests a role of p22phox in ventilation/perfusion ratio matching, vascular remodelling and loss of perfused lung area. In p22phox -/- mice, HPV was significantly impaired. In the chronic hypoxic setting, lack of p22phox was associated with improved right ventricular function and decreased pulmonary vascular remodelling.p22phox-dependent Nox plays an important role in the COPD phenotype, by its action on phase II HPV and chronic vascular remodelling., Competing Interests: Conflict of interest: Disclosures can be found alongside this article at erj.ersjournals.com, (Copyright ©ERS 2017.)

Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria.

Hypoxic pulmonary vasoconstriction (HPV) optimizes pulmonary ventilation-perfusion matching in regional hypoxia, but promotes pulmonary hypertension in global hypoxia. Ventilation-perfusion mismatch is a major cause of hypoxemia in cystic fibrosis. We hypothesized that cystic fibrosis transmembrane conductance regulator (CFTR) may be critical in HPV, potentially by modulating the response to sphingolipids as mediators of HPV. HPV and ventilation-perfusion mismatch were analyzed in isolated mouse lungs or in vivo. Ca(2+) mobilization and transient receptor potential canonical 6 (TRPC6) translocation were studied in human pulmonary (PASMCs) or coronary (CASMCs) artery smooth muscle cells. CFTR inhibition or deficiency diminished HPV and aggravated ventilation-perfusion mismatch. In PASMCs, hypoxia caused CFTR to interact with TRPC6, whereas CFTR inhibition attenuated hypoxia-induced TRPC6 translocation to caveolae and Ca(2+) mobilization. Ca(2+) mobilization by sphingosine-1-phosphate (S1P) was also attenuated by CFTR inhibition in PASMCs, but amplified in CASMCs. Inhibition of neutral sphingomyelinase (nSMase) blocked HPV, whereas exogenous nSMase caused TRPC6 translocation and vasoconstriction that were blocked by CFTR inhibition. nSMase- and hypoxia-induced vasoconstriction, yet not TRPC6 translocation, were blocked by inhibition or deficiency of sphingosine kinase 1 (SphK1) or antagonism of S1P receptors 2 and 4 (S1P2/4). S1P and nSMase had synergistic effects on pulmonary vasoconstriction that involved TRPC6, phospholipase C, and rho kinase. Our findings demonstrate a central role of CFTR and sphingolipids in HPV. Upon hypoxia, nSMase triggers TRPC6 translocation, which requires its interaction with CFTR. Concomitant SphK1-dependent formation of S1P and activation of S1P2/4 result in phospholipase C-mediated TRPC6 and rho kinase activation, which conjointly trigger vasoconstriction.

Pulmonary vascular hyperresponsiveness is a main characteristic of pulmonary arterial hypertension (PAH). In PAH patients, elevated levels of the vasoconstrictors thromboxane A2 (TXA2), endothelin (ET)-1 and serotonin further contribute to pulmonary hypertension. Protein kinase C (PKC) isozyme alpha (PKCα) is a known modulator of smooth muscle cell contraction. However, the effects of PKCα deficiency on pulmonary vasoconstriction have not yet been investigated. Thus, the role of PKCα in pulmonary vascular responsiveness to the TXA2 analog U46619, ET-1, serotonin and acute hypoxia was investigated in isolated lungs of PKCα-/- mice and corresponding wild-type mice, with or without prior administration of the PKC inhibitor bisindolylmaleimide I or Gö6976. mRNA was quantified from microdissected intrapulmonary arteries. We found that broad-spectrum PKC inhibition reduced pulmonary vascular responsiveness to ET-1 and acute hypoxia and, by trend, to U46619. Analogously, selective inhibition of conventional PKC isozymes or PKCα deficiency reduced ET-1-evoked pulmonary vasoconstriction. The pulmonary vasopressor response to serotonin was unaffected by either broad PKC inhibition or PKCα deficiency. Surprisingly, PKCα-/- mice showed pulmonary vascular hyperresponsiveness to U46619 and increased TXA2 receptor (TP receptor) expression in the intrapulmonary arteries. To conclude, PKCα regulates ET-1-induced pulmonary vasoconstriction. However, PKCα deficiency leads to pulmonary vascular hyperresponsiveness to TXA2, possibly via increased pulmonary arterial TP receptor expression., (© 2016 S. Karger AG, Basel.)

Lung diseases, including pneumonia and asthma, are among the most prevalent human disorders, and murine models have been established to investigate their pathobiology and develop novel treatment approaches. Whereas bronchoscopy is valuable for diagnostic and therapeutic procedures in patients, no equivalent for small rodents has been established. Here, we introduce a miniaturized video-bronchoscopy system offering new opportunities in experimental lung research. With an outer diameter of 0.75 mm, it is possible to advance the optics into the main bronchi of mice. An irrigation channel allows bronchoalveolar lavage and unilateral application of substances to one lung. Even a unilateral infection is possible, enabling researchers to use the contralateral lung as internal control.

Rationale: Systemic sclerosis (SSc)-associated pulmonary arterial hypertension (PAH) portends worse outcome than other forms of PAH. Vasoconstrictive and vascular remodeling actions of endothelin (ET) 1 and angiotensin (Ang) II via endothelin receptor type A (ETAR) and Ang receptor type-1 (AT1R) activation are implicated in PAH pathogenesis., Objectives: We hypothesized that stimulating autoantibodies (Abs) targeting and activating AT1R and ETAR may contribute to SSc-PAH pathogenesis, and tested their functional and biomarker relevance., Methods: Anti-AT1R and -ETAR Abs were detected by ELISA in different cohorts of patients and tested in vitro and in an animal model for their pathophysiological effects., Measurements and Main Results: The Abs were significantly higher and more prevalent in patients with SSc-PAH (n = 81) and connective tissue disease-associated PAH (n = 110) compared with other forms of PAH/pulmonary hypertension (n = 106). High anti-AT1R and anti-ETAR Abs predicted development of SSc-PAH and SSc-PAH-related mortality in a prospective analysis. Both Abs increased endothelial cytosolic Ca(2+) concentrations in isolated perfused rat lungs, which could be blocked by respective specific receptor antagonists. Ab-mediated stimulation of intralobar pulmonary rat artery ring segments increased vasoconstrictive responses to Ang II and ET-1, and implicated cross-talk between both pathways demonstrated by reciprocal blockade with respective antagonists. Transfer of SSc-IgG containing both autoantibodies into healthy C57BL/6J mice led to more abundant vascular and airway α-smooth muscle actin expression and inflammatory pulmonary vasculopathy., Conclusions: Anti-AT1R and -ETAR Abs are more frequent in SSc-PAH/connective tissue disease-PAH compared with other forms of pulmonary hypertension, and serve as predictive and prognostic biomarkers in SSc-PAH. Both antibodies may contribute to SSc-PAH via increased vascular endothelial reactivity and induction of pulmonary vasculopathy.

Megaesophagus in mice has been associated with several genetic defects. In the present study we expand the range of genes associated with esophageal function and morphology by protein kinase C alpha (PKCα). PKCα-deficient mice showed a six times increased prevalence of megaesophagus at the age of 9-10 weeks compared to wild-type animals. In contrast, in a restricted number of 14-month-old animals of both genotypes a similar prevalence of megaesophagus was found. Megaesophagus was associated with an increased portion of the distal esophagus lined by smooth muscle cells. Achalasia-like degeneration or loss of neuronal cells, inflammation or fibrosis was not present in any of the animals. The results of the study therefore suggest that PKCα expression is associated with a delayed replacement of embryonic smooth muscle by skeletal muscle at the distal esophagus and consecutive megaesophagus in young mice, which, however, is not present at the same prevalence at an advanced age., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Calcitriol (1α,25-dihydroxyvitamin D3) is the active vitamin D metabolite and mediates immunological functions, which are relevant in allergy. Its therapeutic use is limited by hypercalcaemic toxicity. We have previously shown that the activation of the vitamin D receptor inhibits IgE production and that B cells can synthesize calcitriol from its precursor 25-hydroxyvitamin D3 (inactive precursor) [25(OH)D] upon antigenic stimulation. In this study, we address the impact of 25(OH)D on the development of type I sensitization and determine its role in allergen-specific immunotherapy. BALB/c mice were sensitized to OVA, under 25(OH)D-deficient or sufficient conditions. The humoral immune response over time was measured by ELISA. OVA-specific immunotherapy was established and studied in a murine model of allergic airway inflammation using lung histology, pulmonary cytokine expression analysis, and functional parameters in isolated and perfused mouse lungs. In 25(OH)D-deficient mice, OVA-specific IgE and IgG1 serum concentrations were increased compared with control mice. OVA-specific immunotherapy reduced the humoral immune reaction after OVA recall dose-dependently. Coadministration of 25(OH)D in the context of OVA-specific immunotherapy reduced the allergic airway inflammation and responsiveness upon OVA challenge. These findings were paralleled by reduced Th2 cytokine expression in the lungs. In conclusion, 25(OH)D deficiency promotes the development of type I sensitization and correction of its serum concentrations enhances the benefit of specific immunotherapy., (Copyright © 2014 by The American Association of Immunologists, Inc.)

The cytosolic nucleotide oligomerization domain (NOD)-like receptors NOD1 and NOD2 are important contributors to the intracellular recognition of pathogens including Chlamydophila pneumoniae, but little is known about their influence on allergen-induced airway inflammation. In BALB/c mice, we observed that infection with C. pneumoniae before systemic sensitization with ovalbumin (OVA) and local OVA airway exposure diminished airway hyperresponsiveness (AHR). Thus, the impact of the NOD1 agonist FK156 and the NOD2 agonist muramyl dipeptide given 6 hours before each sensitization or airway challenge was evaluated regarding AHR, OVA-specific plasma immunoglobulins, bronchoalveolar lavage fluid differentials, and cytokines. Spleen dendritic cells of FK156-treated mice were isolated and cocultured with OVA-specific T cells isolated from DO11.10 mice, and T-cell proliferation was quantified after OVA restimulation. T-cell proliferation was investigated in vivo in lungs and lymph nodes of FK156-treated and OVA-exposed DO11.10 mice. FK156, but not muramyl dipeptide, reduced AHR and pulmonary eosinophilic infiltration if given before OVA sensitization or challenge, whereas T-helper (Th)2 cytokines were not diminished. Dendritic cells from FK156-treated mice evoked less OVA-specific T-cell proliferation as compared with solvent-treated controls. Similarly, antigen-specific T-cell activation in lung tissue was diminished after FK156 treatment. We conclude that NOD1 activation reduced AHR in allergen-induced lung inflammation, which was accompanied by a reduction of allergen-specific T-cell proliferation.

Introduction: Vasculopathy, inflammatory fibrosis and functional autoantibodies (Abs) are major manifestations of systemic sclerosis (SSc). Abs directed against the angiotensin II type 1 receptor (AT₁R) and endothelin-1 type A receptor (ETAR) are associated with characteristic disease features including vascular, inflammatory, and fibrotic complications indicating their role in SSc pathogenesis. Therefore, the impact of anti-AT₁R and anti-ETAR Abs on initiation of inflammation and fibrosis was analyzed., Methods: Anti-AT₁R and anti-ETAR Ab-positive immunoglobulin G (IgG) from SSc patients (SSc-IgG) was used for experiments. Healthy donor IgG served as a normal control, and AT₁R and ETAR activation was inhibited by antagonists. Protein expression was measured with ELISA, mRNA expression with real time-PCR, endothelial repair with a scratch assay, and collagen expression with immunocytochemistry. Transendothelial neutrophil migration was measured with a culture insert system, and neutrophil ROS activation with immunofluorescence. Neutrophils in bronchoalveolar lavage fluids (BALFs) were analyzed microscopically after passive transfer of SSc-IgG or NC-IgG into naïve C57BL/6J mice. KC plasma levels were quantified by a suspension array system. Histologic analyses were performed by using light microscopy., Results: Anti-AT₁R and anti-ETAR Ab-positive SSc-IgG induced activation of human microvascular endothelial cells (HMEC-1). Elevated protein and mRNA levels of the proinflammatory chemokine interleukin-8 (IL-8, CXCL8) and elevated mRNA levels of the vascular cell adhesion molecule-1 (VCAM-1) were induced in HMEC-1. Furthermore, activation of HMEC-1 with SSc-IgG increased neutrophil migration through an endothelial cell layer and activation of reactive oxygen species (ROS). SSc-IgG decreased HMEC-1 wound repair and induced type I collagen production in healthy donor skin fibroblasts. Effects of migration, wound repair, and collagen expression were dependent on the Ab-levels. Passive transfer of anti-AT1R and anti-ETAR Ab-positive SSc-IgG into naïve C57BL/6J mice increased neutrophil BALF counts. In parallel, increased levels of the murine functional IL-8 homologue, chemokine KC, were found in the plasma of SSc-IgG-treated mice as well as structural alterations of the lungs., Conclusions: We conclude that angiotensin and endothelin-receptor activation via anti-AT₁R and anti-ETAR Abs mediate pathogenic effects, indicating their contribution to pathogenesis of SSc. Therefore, anti-AT₁R and anti-ETAR Abs could provide novel targets for therapeutic intervention in the treatment of SSc.

Hypoxic pulmonary vasoconstriction (HPV) is a physiological mechanism by which pulmonary arteries constrict in hypoxic lung areas in order to redirect blood flow to areas with greater oxygen supply. Both oxygen sensing and the contractile response are thought to be intrinsic to pulmonary arterial smooth muscle cells. Here we speculated that the ideal site for oxygen sensing might instead be at the alveolocapillary level, with subsequent retrograde propagation to upstream arterioles via connexin 40 (Cx40) endothelial gap junctions. HPV was largely attenuated by Cx40-specific and nonspecific gap junction uncouplers in the lungs of wild-type mice and in lungs from mice lacking Cx40 (Cx40-/-). In vivo, hypoxemia was more severe in Cx40-/- mice than in wild-type mice. Real-time fluorescence imaging revealed that hypoxia caused endothelial membrane depolarization in alveolar capillaries that propagated to upstream arterioles in wild-type, but not Cx40-/-, mice. Transformation of endothelial depolarization into vasoconstriction involved endothelial voltage-dependent α1G subtype Ca2+ channels, cytosolic phospholipase A2, and epoxyeicosatrienoic acids. Based on these data, we propose that HPV originates at the alveolocapillary level, from which the hypoxic signal is propagated as endothelial membrane depolarization to upstream arterioles in a Cx40-dependent manner.

Background: Even protective ventilation may aggravate or induce lung failure, particularly in preinjured lungs. Thus, new adjuvant pharmacologic strategies are needed to minimize ventilator-induced lung injury (VILI). Intermedin/Adrenomedullin-2 (IMD) stabilized pulmonary endothelial barrier function in vitro. We hypothesized that IMD may attenuate VILI-associated lung permeability in vivo., Methodology/principal Findings: Human pulmonary microvascular endothelial cell (HPMVEC) monolayers were incubated with IMD, and transcellular electrical resistance was measured to quantify endothelial barrier function. Expression and localization of endogenous pulmonary IMD, and its receptor complexes composed of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMPs) 1-3 were analyzed by qRT-PCR and immunofluorescence in non ventilated mouse lungs and in lungs ventilated for 6 h. In untreated and IMD treated mice, lung permeability, pulmonary leukocyte recruitment and cytokine levels were assessed after mechanical ventilation. Further, the impact of IMD on pulmonary vasoconstriction was investigated in precision cut lung slices (PCLS) and in isolated perfused and ventilated mouse lungs. IMD stabilized endothelial barrier function in HPMVECs. Mechanical ventilation reduced the expression of RAMP3, but not of IMD, CRLR, and RAMP1 and 2. Mechanical ventilation induced lung hyperpermeability, which was ameliorated by IMD treatment. Oxygenation was not improved by IMD, which may be attributed to impaired hypoxic vasoconstriction due to IMD treatment. IMD had minor impact on pulmonary leukocyte recruitment and did not reduce cytokine levels in VILI., Conclusions/significance: IMD may possibly provide a new approach to attenuate VILI.

Aims: Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide. Despite its importance, treatment methods are limited and restricted to symptomatic care, highlighting the urgent need for new treatment options. Tissue damage in COPD is thought to result from an inability of the normal repair processes with accumulation of apoptotic material and impaired clearance of this material by macrophages in the airways. Lung inflammation involves the bioactive sphingolipid sphingosine 1-phosphate (S1P)., Main Methods: We investigated lung tissue samples from 55 patients (25 with COPD) undergoing lobectomies for management of cancer. We analysed the sphingosine-kinase (SphK) mRNA expression profile, SphK enzyme activity as well as the localisation and expression of individual proteins related to the SphK-signalling system., Key Findings: We show in this study for the first time a comprehensive expression profile of all synthesising enzymes, receptors and degrading enzymes of the SphK-signalling system in the human lung. Multivariate ANOVA showed that the relative mRNA expression of S1P receptor (S1PR) subtype 5 was reduced in COPD. There were strong positive correlations between the mRNA expression of S1PR5 and S1PR1 and S1PR3, and between S1PR3 and S1PR2. A significant negative correlation was found between S1PR1 and SphK protein activity., Significance: The correlations between expression levels of receptors and enzymes involved in the sphingosine kinase signalling system in the lung suggest common regulatory mechanisms. Our findings of reduced S1PR5 in COPD and the correlation with other S1P receptors in COPD identify S1PR5 as a possible novel target for pharmacotherapy., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Although predominantly expressed on lymphocytic and hematopoietic cells, the role of sphingosine-1-phospate receptor 4 (S1P(4)) in immune homeostasis is still poorly understood. In this report, we used a S1P(4)-deficient murine model to characterize the biological role of S1P(4)-mediated S1P signaling in the immune system. S1p(4)(-/-) animals showed normal peripheral lymphocyte numbers and a regular architecture of secondary lymphoid organs. Interestingly, S1P(4) only marginally affects T-cell function in vivo. In contrast, dendritic cell (DC) migration and cytokine secretion are profoundly affected by S1P(4) deficiency. Lack of S1P(4) expression on DCs significantly reduces T(H)17 differentiation of T(H) cells. Furthermore, in various in vivo models of T(H)1- or T(H)2-dominated immune reactions, S1P(4) deficiency consistently increased the amplitude of T(H)2-dominated immune responses, while those depending on T(H)1-dominated mechanisms were diminished. Finally, S1p(4)(-/-) mice showed decreased pathology in a model of dextran sulfate sodium-induced colitis. In summary, for the first time, we show that S1P(4) signaling is involved in the regulation of DC function and T(H)17 T-cell differentiation. S1P(4)-mediated S1P signaling also modifies the course of various immune diseases in a murine model. We propose that S1P(4) may constitute an interesting target to influence the course of various autoimmune pathologies.

Defence mechanisms against intracellular bacterial pathogens are incompletely understood. Our study characterizes a type I IFN-dependent cell-autonomous defence pathway directed against Legionella pneumophila, an intracellular model organism and frequent cause of pneumonia. We show that macrophages infected with L. pneumophila produced IFNβ in a STING- and IRF3- dependent manner. Paracrine type I IFNs stimulated upregulation of IFN-stimulated genes and a cell-autonomous defence pathway acting on replicating and non-replicating Legionella within their specialized vacuole. Our infection experiments in mice lacking receptors for type I and/or II IFNs show that type I IFNs contribute to expression of IFN-stimulated genes and to bacterial clearance as well as resistance in L. pneumophila pneumonia in addition to type II IFN. Overall, our study shows that paracrine type I IFNs mediate defence against L. pneumophila, and demonstrates a protective role of type I IFNs in in vivo infections with intracellular bacteria., (© 2011 Blackwell Publishing Ltd.)

Streptococcus pneumoniae is a leading cause of pneumonia, meningitis, and sepsis. Pneumococci can be divided into >90 serotypes that show differences in the pathogenicity and invasiveness. We tested the hypotheses that the innate immune inflammasome pathway is involved in fighting pneumococcal pneumonia and that some invasive pneumococcal types are not recognized by this pathway. We show that human and murine mononuclear cells responded to S. pneumoniae expressing hemolytic pneumolysin by producing IL-1β. This IL-1β production depended on the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Some serotype 1, serotype 8, and serotype 7F bacteria, which have previously been associated with increased invasiveness and with production of toxins with reduced hemolytic activity, or bacterial mutants lacking pneumolysin did not stimulate notable IL-1β production. We further found that NLRP3 was beneficial for mice during pneumonia caused by pneumococci expressing hemolytic pneumolysin and was involved in cytokine production and maintenance of the pulmonary microvascular barrier. Overall, the inflammasome pathway is protective in pneumonia caused by pneumococci expressing hemolytic toxin but is not activated by clinically important pneumococcal sequence types causing invasive disease. The study indicates that a virulence factor polymorphism may substantially affect the recognition of bacteria by the innate immune system.

Background: Immunologic processes might contribute to the pathogenesis of pulmonary arterial hypertension (PAH), a fatal condition characterized by progressive pulmonary arterial remodeling, increased pulmonary vascular resistance, and right ventricular failure. Experimental allergen-driven lung inflammation evoked morphologic and functional vascular changes that resembled those observed in patients with PAH. Sphingosine kinase 1 (SphK1) is the main pulmonary contributor to sphingosine-1-phosphate (S1P) synthesis, a modulator of immune and vascular functions., Objective: We sought to investigate the role of SphK1 in allergen-induced lung inflammation., Methods: SphK1-deficient mice and C57Bl/6 littermates (wild-type [WT] animals) were subjected to acute or chronic allergen exposure., Results: After 4 weeks of systemic ovalbumin sensitization and local airway challenge, airway responsiveness increased less in SphK1(-/-) compared with WT mice, whereas pulmonary vascular responsiveness was greatly increased and did not differ between strains. Acute lung inflammation led to an increase in eosinophils and mRNA expression for S1P phosphatase 2 and S1P lyase in lungs of WT but not SphK1(-/-) mice. After repetitive allergen exposure for 8 weeks, airway responsiveness was not augmented in SphK1(-/-) or WT mice, but pulmonary vascular responsiveness was increased in both strains, with significantly higher vascular responsiveness in SphK1(-/-) mice compared with that seen in WT mice. Increased vascular responsiveness was accompanied by remodeling of the small and intra-acinar arteries., Conclusion: : The data support a role for SphK1 and S1P in allergen-induced airway inflammation. However, SphK1 deficiency increased pulmonary vascular hyperresponsiveness, which is a component of PAH pathobiology. Moreover, we show for the first time the dissociation between inflammation-induced remodeling of the airways and pulmonary vasculature.