Acidic Calcium-Independent Phospholipase A2 Regulates Eosinophil-Mediated Pathology during Filarial Manifestation of Tropical Pulmonary Eosinophilia

Abstract

Eosinophils mediate pathological manifestations during tropical pulmonary eosinophilia (TPE), a potentially fatal complication of lymphatic filariasis, by mechanisms that are incompletely understood. Using two-dimensional gel electrophoresis, mass spectrometry, flow cytometry, and pharmacological and functional studies, we identified acidic calcium-independent phospholipase A2 (aiPLA2) as the master regulator of TPE pathogenesis. FACS-sorted lung eosinophils from TPE mice exhibited aiPLA2-dependent activation characterized by heavy calcium influx, F-actin polymerization, increased degranulation, and heightened reactive oxygen species generation. Interestingly, aiPLA2 also promoted alternative activation in lung macrophages and regulated the release of inflammatory intermediates from them. Treatment of TPE mice with MJ33, a nontoxic pharmacological inhibitor of aiPLA2, lowered eosinophil counts in the bronchoalveolar lavage fluid, reduced eosinophil peroxidase and β-hexosaminidase activity, increased airway width, improved lung endothelial barrier, and lowered the production of inflammatory lipid intermediates, which significantly improved the pathological condition of the lungs. Importantly, ex vivo reconstitution of arachidonic acid to eosinophils from MJ33-treated TPE mice increased eosinophil degranulation and inflammatory lipid intermediates underlining the pivotal role of aiPLA2 in arachidonic acid metabolism. Mechanistically, phosphorylation of JNK-1 regulated phospholipase activity of aiPLA2, whereas IgG cross-linking mediated pathological activation of eosinophils. Taken together, ours is the first study, to our knowledge, to report hitherto undocumented role of aiPLA2 in regulating TPE pathogenesis.

Immunophenotypic and Functional Characterization of Eosinophil and Migratory Dendritic Cell Subsets during Filarial Manifestation of Tropical Pulmonary Eosinophilia

“…Interestingly, TPE iEos also significantly upregulated alarmins, viz., s100a8 and s100a9, which was an important observation as these molecules are released during injury or tissue damage and regulate inflammatory responses. − Also, since s100a8 and s100a9 regulate ROS, they might be involved in imprinting inflammatory signatures in TPE iEos. To test this, we looked at eosinophil’s intrinsic expression of genes responsible for NADPH oxidase assembly, an enzyme involved in ROS generation, and found significantly upregulated expression of Ncf2, Ncf4, Cyba, and Cybb in TPE iEos, which were in line with our recent findings wherein NADPH oxidase was found to be highly upregulated in TPE mice …”

“…To test this, we looked at eosinophil's intrinsic expression of genes responsible for NADPH oxidase assembly, an enzyme involved in ROS generation, and found significantly upregulated expression of Ncf2, Ncf4, Cyba, and Cybb in TPE iEos, which were in line with our recent findings wherein NADPH oxidase was found to be highly upregulated in TPE mice. 50 Next, we looked at the expression of crucial CD300 family molecules as they are associated with ITAM-containing adaptor FcRγ chain and can thus potentially modulate immunoglobulin receptor downstream signaling. 57−59 Of note, Cd300a, which regulates degranulation, activation, and chemotaxis of eosinophils and acts as an inhibitory receptor of the immune system, 58,59 was downregulated in TPE iEos, but CD300d, which stimulates the secretion of pro-inflammatory cytokines upon infection, was upregulated in TPE iEos, thus confirming the inflammatory nature of TPE iEos.…”

“…Thereafter, they were cut into small pieces and digested in incomplete RPMI medium containing 2 mg/mL collagenase and 80 U/mL DNase for 45 min at 37 °C. The digested tissue was passed through a 40 μm cell strainer to obtain single cell suspension, which was later used for immunophenotyping and cell sorting studies on FACS Aria Fusion Flow cytometer (BD Biosciences) fitted with a 70 μm nozzle. , Briefly, Fc- Block (CD16/32) was used before antibody staining to block the unspecific binding of antibodies to the Fc receptor of cells. The flow cytometry data was acquired on a 5-decade log scale dot plot displaying FSC area vs SSC area to exclude contaminating dead cells and debris.…”

“…Flow-sorted Cont. rEos, TPE rEos, and TPE iEos ( n = 50,000) were incubated in separate tubes in the presence of FITC-dextran (1 mg/mL) for 2 h in a 5% CO 2 incubator set at 37 °C, and the increase in FITC fluorescence was measured using flow cytometry, as described previously. ,, …”

Parasitic lung diseases

“…The levels of gGT-specific IgE/IgG4 ratio were 45 and 107 in patients with TES and those who live in the tropics without TES [ 13 ]. In addition, acidic calcium-independent phospholipase A2 was found to be a master regulator of TPE pathogenesis [ 14 ].…”