Lysophosphatidylcholines inhibit human eosinophil activation and suppress eosinophil migration in vivo.

Eosinophils are important multifaceted effector cells involved in allergic inflammation. Following allergen challenge, eosinophils and other immune cells release secreted phospholipases, generating lysophosphatidylcholines (LPCs). LPCs are potent lipid mediators, and serum levels of LPCs associate with asthma severity, suggesting a regulatory activity of LPCs in asthma development. As of yet, the direct effects of LPCs on eosinophils remain unclear. In the present study, we tested the effects of the major LPC species (16:0, 18:0 and 18:1) on eosinophils isolated from healthy human donors. Addition of saturated LPCs in the presence of albumin rapidly disrupted cholesterol-rich nanodomains on eosinophil cell membranes and suppressed multiple eosinophil effector responses, such as CD11b upregulation, degranulation, chemotaxis, and downstream signaling. Furthermore, we demonstrate in a mouse model of allergic cell recruitment, that LPC treatment markedly reduces immune cell infiltration into the lungs. Our observations suggest a strong modulatory activity of LPCs in the regulation of eosinophilic inflammation in vitro and in vivo.

Apolipoprotein A-IV acts as an endogenous anti-inflammatory protein and is reduced in treatment-naïve allergic patients and allergen-challenged mice.

BACKGROUND: Recent studies pointed to a crucial role for apolipoproteins in the pathogenesis of inflammatory diseases. However, the role of apolipoprotein-IV (ApoA-IV) in allergic inflammation has not been addressed thoroughly thus far. OBJECTIVE: Here, we explored the anti-inflammatory effects and underlying signaling pathways of ApoA-IV on eosinophil effector function in vitro and in vivo. METHODS: Migratory responsiveness, Ca2+ -flux and apoptosis of human peripheral blood eosinophils were assessed in vitro. Allergen-driven airway inflammation was assessed in a mouse model of acute house dust mite-induced asthma. ApoA-IV serum levels were determined by ELISA. RESULTS: Recombinant ApoA-IV potently inhibited eosinophil responsiveness in vitro as measured by Ca2+ -flux, shape change, integrin (CD11b) expression, and chemotaxis. The underlying molecular mechanism involved the activation of Rev-ErbA-α and induced a PI3K/PDK1/PKA-dependent signaling cascade. Systemic application of ApoA-IV prevented airway hyperresponsiveness (AHR) and airway eosinophilia in mice following allergen challenge. ApoA-IV levels were decreased in serum from allergic patients compared to healthy controls. CONCLUSION: Our data suggest that ApoA-IV is an endogenous anti-inflammatory protein that potently suppresses effector cell functions in eosinophils. Thus, exogenously applied ApoA-IV may represent a novel pharmacological approach for the treatment of allergic inflammation and other eosinophil-driven disorders.

Inhibiting eicosanoid degradation exerts antifibrotic effects in a pulmonary fibrosis mouse model and human tissue.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a disease with high 5-year mortality and few therapeutic options. Prostaglandin (PG) E2 exhibits antifibrotic properties and is reduced in bronchoalveolar lavage from patients with IPF. 15-Prostaglandin dehydrogenase (15-PGDH) is the key enzyme in PGE2 metabolism under the control of TGF-ß and microRNA 218. OBJECTIVE: We sought to investigate the expression of 15-PGDH in IPF and the therapeutic potential of a specific inhibitor of this enzyme in a mouse model and human tissue. METHODS: In vitro studies, including fibrocyte differentiation, regulation of 15-PGDH, RT-PCR, and Western blot, were performed using peripheral blood from healthy donors and patients with IPF and A549 cells. Immunohistochemistry, immunofluorescence, 15-PGDH activity assays, and in situ hybridization as well as ex vivo IPF tissue culture experiments were done using healthy donor and IPF lungs. Therapeutic effects of 15-PGDH inhibition were studied in the bleomycin mouse model of pulmonary fibrosis. RESULTS: We demonstrate that 15-PGDH shows areas of increased expression in patients with IPF. Inhibition of this enzyme increases PGE2 levels and reduces collagen production in IPF precision cut lung slices and in the bleomycin model. Inhibitor-treated mice show amelioration of lung function, decreased alveolar epithelial cell apoptosis, and fibroblast proliferation. Pulmonary fibrocyte accumulation is also decreased by inhibitor treatment in mice, similar to PGE2 that inhibits fibrocyte differentiation from blood of healthy donors and patients with IPF. Finally, microRNA 218-5p, which is downregulated in patients with IPF, suppressed 15-PGDH expression in vivo and in vitro. CONCLUSIONS: These findings highlight the role of 15-PGDH in IPF and suggest 15-PGDH inhibition as a promising therapeutic approach.

Butyrate ameliorates allergic airway inflammation by limiting eosinophil trafficking and survival.

BACKGROUND: Lung eosinophilia is a hallmark of asthma, and eosinophils are believed to play a crucial role in the pathogenesis of allergic inflammatory diseases. Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are produced in high amounts in the gastrointestinal tract by commensal bacteria and can be absorbed into the bloodstream. Although there is recent evidence that SCFAs are beneficial in allergic asthma models, the effect on eosinophils has remained elusive. OBJECTIVE: The role of SCFAs was investigated in human eosinophil function and a mouse model of allergic asthma. METHODS: Eosinophils were purified from self-reported allergic or healthy donors. Migration, adhesion to the endothelium, and eosinophil survival were studied in vitro. Ca2+ flux, apoptosis, mitochondrial membrane potential, and expression of surface markers were determined by using flow cytometry and in part by using real-time PCR. Allergic airway inflammation was assessed in vivo in an ovalbumin-induced asthma model by using invasive spirometry. RESULTS: For the first time, we observed that SCFAs were able to attenuate human eosinophils at several functional levels, including (1) adhesion to the endothelium, (2) migration, and (3) survival. These effects were independent from GPR41 and GPR43 but were accompanied by histone acetylation and mimicked by trichostatin A, a pan-histone deacetylase inhibitor. In vivo butyrate ameliorated allergen-induced airway and lung eosinophilia, reduced type 2 cytokine levels in bronchial fluid, and improved airway hyperresponsiveness in mice. CONCLUSION: These in vitro and in vivo findings highlight the importance of SCFAs, especially butyrate as a promising therapeutic agent in allergic inflammatory diseases.

DP1 receptor signaling prevents the onset of intrinsic apoptosis in eosinophils and functions as a transcriptional modulator.

Prostaglandin (PG) D2 is the ligand for the G-protein coupled receptors DP1 (D-type prostanoid receptor 1) and DP2 (also known as chemoattractant receptor homologous molecule, expressed on Th2 cells; CRTH2). Both, DP1 and DP2 are expressed on the cellular surface of eosinophils; although it has become quite clear that PGD2 induces eosinophil migration mainly via DP2 receptors, the role of DP1 in eosinophil responses has remained elusive. In this study, we addressed how DP1 receptor signaling complements the pro-inflammatory effects of DP2. We found that PGD2 prolongs the survival of eosinophils via a DP1 receptor-mediated mechanism that inhibits the onset of the intrinsic apoptotic cascade. The DP1 agonist BW245c prevented the activation of effector caspases in eosinophils and protected mitochondrial membranes from depolarization which-as a consequence-sustained viability of eosinophils. DP1 activation in eosinophils enhanced the expression of the anti-apoptotic gene BCL-XL , but also induced pro-inflammatory genes, such as VLA-4 and CCR3. In HEK293 cells that overexpress recombinant DP1 and/or DP2 receptors, activation of DP1, but not DP2, delayed cell death and stimulated proliferation, along with induction of serum response element (SRE), a regulator of anti-apoptotic, early-response genes. We conclude that DP1 receptors promote the survival via SRE induction and induction of pro-inflammatory genes. Therefore, targeting DP1 receptors, along with DP2, may contribute to anti-inflammatory therapy in eosinophilic diseases.

The Role of PGE2 in Alveolar Epithelial and Lung Microvascular Endothelial Crosstalk.

Disruption of the blood-air barrier, which is formed by lung microvascular endothelial and alveolar epithelial cells, is a hallmark of acute lung injury. It was shown that alveolar epithelial cells release an unidentified soluble factor that enhances the barrier function of lung microvascular endothelial cells. In this study we reveal that primarily prostaglandin (PG) E2 accounts for this endothelial barrier-promoting activity. Conditioned media from alveolar epithelial cells (primary ATI-like cells) collected from BALB/c mice and A549 cells increased the electrical resistance of pulmonary human microvascular endothelial cells, respectively. This effect was reversed by pretreating alveolar epithelial cells with a cyclooxygenase-2 inhibitor or by blockade of EP4 receptors on endothelial cells, and in A549 cells also by blocking the sphingosine-1-phosphate1 receptor. Cyclooxygenase-2 was constitutively expressed in A549 cells and in primary ATI-like cells, and was upregulated by lipopolysaccharide treatment. This was accompanied by enhanced PGE2 secretion into conditioned media. Therefore, we conclude that epithelium-derived PGE2 is a key regulator of endothelial barrier integrity via EP4 receptors under physiologic and inflammatory conditions. Given that pharmacologic treatment options are still unavailable for diseases with compromised air-blood barrier, like acute lung injury, our data thus support the therapeutic potential of selective EP4 receptor agonists.

The EP1/EP3 receptor agonist 17-pt-PGE2 acts as an EP4 receptor agonist on endothelial barrier function and in a model of LPS-induced pulmonary inflammation.

Endothelial dysfunction is a hallmark of inflammatory conditions. We recently demonstrated that prostaglandin (PG)E2 enhances the resistance of pulmonary endothelium in vitro and counteracts lipopolysaccharide (LPS)-induced pulmonary inflammation in vivo via EP4 receptors. The aim of this study was to investigate the role of the EP1/EP3 receptor agonist 17-phenyl-trinor-(pt)-PGE2 on acute lung inflammation in a mouse model. In LPS-induced pulmonary inflammation in mice, 17-pt-PGE2 reduced neutrophil infiltration and inhibited vascular leakage. These effects were unaltered by an EP1 antagonist, but reversed by EP4 receptor antagonists. 17-pt-PGE2 increased the resistance of pulmonary microvascular endothelial cells and prevented thrombin-induced disruption of endothelial junctions. Again, these effects were not mediated via EP1 or EP3 but through activation of the EP4 receptor, as demonstrated by the lack of effect of more selective EP1 and EP3 receptor agonists, prevention of these effects by EP4 antagonists and EP4 receptor knock-down by siRNA. In contrast, the aggregation enhancing effect of 17-pt-PGE2 in human platelets was mediated via EP3 receptors. Our results demonstrate that 17-pt-PGE2 enhances the endothelial barrier in vitro on pulmonary microvascular endothelial cells, and accordingly ameliorates the recruitment of neutrophils, via EP4 receptors in vivo. This suggests a beneficial effect of 17-pt-PGE2 on pulmonary inflammatory diseases.

Presence of human polyomavirus 6 in mutation-specific BRAF inhibitor-induced epithelial proliferations.

IMPORTANCE: A frequent adverse effect of mutation-specific BRAF inhibitor therapy is the induction of epithelial proliferations including cutaneous squamous cell carcinomas. To date, the only factor identified contributing to their development is the activation of the mitogen-activated signal transduction cascade by mutations in the RAS genes. However, these mutations explain only 60% of the tumors; hence, it is important to identify what is causing the remaining tumors. OBJECTIVE: To test for the presence of human papillomaviruses (HPVs) and the recently identified human polyomaviruses (HPyVs), Merkel cell polyomavirus (MCPyV), and trichodysplasia spinulosa-associated polyomavirus (TSPyV), as well as HPyV-6, HPyV-7, HPyV-9, and HPyV-10, in epithelial proliferations occurring after BRAF inhibitor therapy to determine whether these oncogenic viruses may contribute to BRAF inhibitor-induced skin tumors. DESIGN, SETTING, AND PARTICIPANTS: Retrospective study at a university hospital in Austria of epithelial proliferations that developed in patients with melanoma after initiation of treatment with the BRAF inhibitor vemurafenib. Samples were analyzed for (1) presence of the most frequently observed RAS mutations by SNaPshot technology, (2) detection of the viruses by real-time polymerase chain reaction, and (3) presence of capsid proteins of the most abundantly detected virus by immunohistochemical analysis. MAIN OUTCOMES AND MEASURES: RAS mutational status, as well as HPV and HPyV presence, in BRAF inhibitor-induced epithelial proliferations. RESULTS: Eighteen biopsy samples from 6 patients were retrieved from our hospital's archive. We identified RAS mutations in 10 (62%) of the 16 samples with clear results. DNA of HPyV-9, HPyV-10, and TSPyV were virtually absent in the samples. MCPyV DNA was present in 13 of 18 samples, and HPV, HPyV-6, and HPyV-7 DNA were present in all samples. In general, the amount of DNA encoding the latter viruses was rather low, with the exception of HPyV-6 in several samples of 1 individual patient. Notably, the relevance of the presence of HPyV-6 in the epithelial proliferation was underlined by immunohistochemical detection of the core protein VP1 of HPyV-6. CONCLUSIONS AND RELEVANCE: The presence of both high HPyV-6 DNA load and VP1 protein suggests that polyomaviruses may contribute to the epithelial proliferations observed in patients receiving BRAF inhibitor therapy, albeit the relative impact as compared with that of RAS mutations appears circumstantial.

Endothelial E-type prostanoid 4 receptors promote barrier function and inhibit neutrophil trafficking.

BACKGROUND: Increased vascular permeability is a fundamental characteristic of inflammation. Substances that are released during inflammation, such as prostaglandin (PG) E(2), can counteract vascular leakage, thereby hampering tissue damage. OBJECTIVE: In this study we investigated the role of PGE(2) and its receptors in the barrier function of human pulmonary microvascular endothelial cells and in neutrophil trafficking. METHODS: Endothelial barrier function was determined based on electrical impedance measurements. Neutrophil recruitment was assessed based on adhesion and transendothelial migration. Morphologic alterations are shown by using immunofluorescence microscopy. RESULTS: We observed that activation of E-type prostanoid (EP) 4 receptor by PGE(2) or an EP4-selective agonist (ONO AE1-329) enhanced the barrier function of human microvascular lung endothelial cells. EP4 receptor activation prompted similar responses in pulmonary artery and coronary artery endothelial cells. These effects were reversed by an EP4 antagonist (ONO AE3-208), as well as by blocking actin polymerization with cytochalasin B. The EP4 receptor-induced increase in barrier function was independent of the classical cyclic AMP/protein kinase A signaling machinery, endothelial nitric oxide synthase, and Rac1. Most importantly, EP4 receptor stimulation showed potent anti-inflammatory activities by (1) facilitating wound healing of pulmonary microvascular endothelial monolayers, (2) preventing junctional and cytoskeletal reorganization of activated endothelial cells, and (3) impairing neutrophil adhesion to endothelial cells and transendothelial migration. The latter effects could be partially attributed to reduced E-selectin expression after EP4 receptor stimulation. CONCLUSION: These data indicate that EP4 agonists as anti-inflammatory agents represent a potential therapy for diseases with increased vascular permeability and neutrophil extravasation.