Potent Anti-Inflammatory Effects of Tetracyclines on Human Eosinophils

Eosinophils are potent pro-inflammatory cells. Not only in allergic diseases but also in other diseases there is a need for treatment strategies to induce resolution of eosinophil-mediated inflammation. During the last years beneficial non-antibiotic activities of tetracyclines (TCNs) have been shown in different diseases in which eosinophils play a role, for example, asthma and bullous pemphigoid. The working mechanism of these effects remains to be clarified. Aim of the present study was to investigate the effects of TCNs on eosinophils. Flow cytometry analysis of apoptosis, mitochondrial membrane potential, activation of caspases, intracellular H2O2 and calcium, surface expression of eosinophil activation markers was performed in highly purified peripheral blood eosinophils of non-atopic donors. Tetracycline hydrochloride, minocycline and doxycycline significantly induced eosinophil apoptosis. All TCNs were able to significantly overcome the strong survival enhancing effects of pro-eosinophilic cytokines and staphylococcus aureus enterotoxins. Tetracycline hydrochloride induced eosinophil apoptosis was accompanied by intracellular production of hydrogen peroxide, loss of mitochondrial membrane potential and activation of caspases. Moreover, tetracycline hydrochloride significantly down regulated eosinophil surface expression of CD9 and CD45, and of the activation markers CD11b and CD69, but not of CD54, CD63, or CD95. Our data, propably for the first time, point to a potent anti-inflammatory role of TCNs on eosinophils.

Novel mechanisms of action contributing to Benralizumab's potent anti-eosinophilic activity

Benralizumab is a humanised, anti-IL-5Rα monoclonal antibody with anti-eosinophilic activity. Lack of fucose (afucosylation) increases its affinity to CD16a and significantly enhances antibody-dependent cell-mediated cytotoxicity (ADCC) by NK cells. Although benralizumab proved clinically efficacious in clinical trials for patients with severe asthma and hypereosinophilic syndrome, in-depth characterisation of its anti-eosinophilic mechanisms of action remain elusive. Here, we further investigated the mechanisms involved in benralizumab's anti-eosinophilic activities. In the presence of NK cells benralizumab induced potent eosinophil apoptosis as demonstrated by the upstream induction of caspase 3/7 and upregulation of cytochrome C. In addition, we uncovered a previously unrecognised mechanism whereby benralizumab can induce eosinophil phagocytosis/efferocytosis by macrophages, a process called antibody-dependent cell phagocytosis (ADCP). Using live cell imaging we unravel the stepwise processes leading to eosinophil apoptosis and uptake by activated macrophages. Through careful observations of cellular co-culture assays we identified a novel role for macrophage derived TNF to further enhance benralizumab-mediated eosinophil apoptosis through activation of TNF-receptor 1 on eosinophils. TNF-induced eosinophil apoptosis was associated with Cytochrome C upregulation, mitochondrial membrane depolarisation, and increased caspase 3/7 activity. Moreover, activated NK cells were found to amplify this axis through the secretion of IFNγ, subsequently driving TNF expression by macrophages. Our data provide insights into the timely appearance of events leading to benralizumab-induced eosinophil apoptosis and suggest that additional mechanisms may contribute to the potent anti-eosinophilic activity of benralizumab in vivo. Importantly, afucosylation of benralizumab strongly enhanced its potency for all mechanisms investigated.

NK cells and Lipoxin A4 promote resolution of eosinophilic inflammation after nasal allergen challenge

Background: Resolution of inflammation is now recognized as a tightly regulated and active process. Lipoxins (LX) are lead members of a larger family of specialized pro-resolving mediators with unique anti-inflammatory and pro-resolving properties. Recent studies implicated natural killer (NK) cells in the resolution of allergic airway inflammation, notably in promoting eosinophil apoptosis. The aim of the study was to better understand the pro-resolving actions of NK cells and LXA4 during allergic eosinophilic airway inflammation. Methods: 20 subjects with grass pollen allergic rhinitis were included. A nasal provocation test with either a single grass pollen allergen threshold dose or diluent was used. Nasal lavage fluid and cells were collected at baseline and at different time points after challenge. For in vitro assays, eosinophils were incubated with NK cells. Results: We observed that NK cells were recruited to the nasal mucosa shortly after the initiation of the allergic inflammatory response. This recruitment correlated with eosinophilic inflammation. In vitro assays demonstrated that direct contact and a combined action of CD56bright and CD56dim NK cells were needed to promote autologous eosinophil apoptosis. We furthermore observed that local LXA4 production correlated with the peak of neutrophil nasal mucosal infiltration, suggesting a potential role of neutrophils in LXA4 biosynthesis during the early phase of the allergic inflammatory response. Last, LXA4 appeared as essential to inhibit the in vitro release of eosinophil superoxide triggered by NK cells. Conclusion: Together, these findings indicate a synergistic role for NK cells and LXA4 in the resolution of allergic eosinophilic inflammation.

Correction to: Assays of Eosinophil Apoptosis and Phagocytic Uptake

Chapter 10 was previously published non-open access. It has now been changed to open access under a CC BY 4.0 license and the copyright holder has been updated to ‘The Author(s)’. The book has been updated with these changes.

Benralizumab: eficacia y seguridad en pacientes con asma grave eosinofílica.

ResumenEl asma grave conlleva una carga de salud desproporcionadamente alta y cerca de la mitad de los adultos con esta patología tiene un fenotipo eosinofílico. En estos pacientes aunado a la producción de eosinófilos en médula ósea, se activan mecanismos de eosinopoyesis local en tejido pulmonar. Benralizumab es un anticuerpo monoclonal humanizado, que se une con alta afinidad y especificidad a la subunidad alfa del receptor de IL-5 (IL-5Rα) sobre la superficie de eosinófilos y otras células. El principal diferenciador de su mecanismo de acción se relaciona con la remoción de un residuo de fucosa en la Fc, lo cual incrementa hasta 50 veces la afinidad a células NK con apoptosis de eosinófilos mediante citotoxicidad celular dependiente de anticuerpos (CCDA), resultando en una reducción rápida y cercana al 100% tanto en suero como en médula ósea. Adicionalmente, benralizumab reduce >90% de los eosinófilos en tejido pulmonar y esputo. En diversos estudios clínicos controlados y en vida real se ha demostrado que esto se traduce en incremento actual del control del asma y disminución del riesgo futuro. El perfil de seguridad es adecuado sin haberse documentado infestaciones parasitarias ni efectos adversos a largo plazo relacionados con la reducción de los eosinófilos. Abstract Severe asthma carries a disproportionately high health burden and about half of adults with this pathology have an eosinophilic phenotype. In these patients, in addition to the production of eosinophils in bone marrow, local eosinopoiesis mechanisms are activated in lung tissue. Benralizumab is a humanized monoclonal antibody, which joins with high affinity and specificity to the alpha subunit of the IL-5 receptor (IL-5Rα) on the surface of eosinophils and other cells. The main differentiator of its mechanism of action is related to the removal of a fucose residue in Fc, which increases up to 50 times the affinity to NK cells with eosinophil apoptosis by antibody-dependent cell cytotoxicity (CCDA), that leads to a direct, rapid and nearly complete depletion in both peripheral blood and bone marrow. Additionally, benralizumab reduces >90% of eosinophils in lung tissue and sputum. Several controlled and real-life clinical studies have shown that this action over eosinophils is related to increased asthma control and decreased future risk. The safety profile is adequate without documenting parasitic infestations or long-term adverse effects related to the reduction of eosinophils.

Role of Leptin/Osteopontin Axis in the Function of Eosinophils in Allergic Rhinitis with Obesity

Background. Allergic rhinitis (AR) is characterized by tissue and blood eosinophilia. Previous studies showed enhanced eosinophilia in allergic rhinitis patients with obesity, suggesting an association between obesity and eosinophilia. However, the interaction and mechanism between obesity and eosinophilia is still unclear. Methods. We recruited thirty AR children and 30 controls in this study. Expression of leptin and osteopontin (OPN) proteins in serum was detected, and correlation analysis with eosinophilia was performed. The effect of leptin or OPN on eosinophil apoptosis, adhesion, migration, and activation of eosinophil was examined. Ovalbumin-sensitized mice were established to prove the role of obesity on eosinophil regulation by leptin and OPN. Results. We found that upregulated serum and nasal leptin and OPN expression in AR were positively correlated with eosinophilia and eosinophil cationic protein levels. Leptin or OPN inhibited eosinophil apoptosis, demonstrated as inhibited DNA fragmentation and phosphatidylserine (PS) redistribution (P<0.05). Leptin and OPN promote expression of cluster of differentiation 18 (CD-18) and intercellular adhesion molecule 1 (ICAM-1) and inhibit expression of ICAM-1 and L-selectin by eosinophils, which contribute to the adhesion of eosinophils. Leptin and OPN mediated migration and activation of eosinophil through phosphatidylinositol-3-OH kinase (PI3K) pathway. Obese AR mice presented with more severe eosinophilia and symptoms compared with nonobese AR mice or control mice. Immunochemistry staining of leptin and OPN of nasal turbinate in obese AR mice was also stronger than those in nonobese AR mice or control mice. Anti-OPN, anti-leptin, and anti-α4 treatments reduce nasal eosinophilia inflammation and clinical symptoms in model mice. Conclusion. Our results suggested that in an obese state, upregulation of leptin and OPN regulates apoptosis, adhesion, migration, and activation of eosinophils, and this process may be mediated by the PI3K and anti-α4 pathways.