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

The role of eosinophil and migratory dendritic cell (migDC) subsets during tropical pulmonary eosinophilia (TPE), a potentially fatal complication of lymphatic filariasis, has not been explored. We show that the onset of TPE is characterized by the accumulation of ROS and anaphylatoxins and a rapid influx of morphologically distinct Siglec-Fint resident eosinophils (rEos) and Siglec-Fhi inflammatory eosinophils (iEos) in the lungs, BAL fluid, and blood of TPE mice. While rEos display regulatory behavior, iEos are highly inflammatory cells, as evident in upregulated expression of activation markers CD69 and CD101, anaphylatoxin receptor C5AR1, alarmins s100a8 and s100a9, components of NADPH oxidase, and copious secretion of TNF-α, IFN-γ, IL-6, IL-1ß, IL-4, IL-10, IL-12, and TGF-ß. Importantly, iEos exhibited heightened ROS generation, higher phagocytic and increased antigen presentation capacity, elevated Ca2+ influx, and increased F-actin polymerization but downregulated negative regulators of the immune response, i.e., Cd300a, Anaxa1, Runx3, Lilrb3, and Serpinb1a, underlining their essential role in promoting lung damage during TPE. Interestingly, TPE mice also showed significant expansion of CD24+CD11b+ migDCs, which showed upregulated expression of maturation and costimulatory markers CD40, CD80, CD83, CD86, and MHCII, increased antigen presentation capacity, and higher migratory potential as evidenced by increased expression of cytokine receptors CCR4, CCR5, CXCR4, and CXCR5. CD24+CD11b+ migDCs also upregulated the expression of immunoregulators PD-L1 and PD-L2 and secreted proinflammatory cytokines, suggesting their significant involvement during TPE. Taken together, we document important morphological, immunophenotypic, and functional characteristics of eosinophil and migDC subsets in the lungs of TPE mice and suggest that they contribute to worsening lung histopathological conditions during TPE.

Immunization with Brugia malayi Calreticulin Protein Generates Robust Antiparasitic Immunity and Offers Protection during Experimental Lymphatic Filariasis.

Lymphatic filariasis causes permanent and long-term disability worldwide. Lack of potent adulticidal drugs, the emergence of drug resistance, and the nonavailability of effective vaccines are the major drawbacks toward LF elimination. However, immunomodulatory proteins present in the parasite secretome are capable of providing good protection against LF and thus offer hope in designing new vaccines against LF. Here, we evaluated the immunogenicity and protective efficacy of B. malayi calreticulin protein (BmCRT) using in vitro and in vivo approaches. Stimulation with recombinant BmCRT (rBmCRT) significantly upregulated Th1 cytokine production in mouse splenocytes, mesenteric lymph nodes (mLNs), and splenic and peritoneal macrophages (PMΦs). Heightened NO release, ROS generation, increased lymphocyte proliferation, and increased antigen uptake were also observed after rBmCRT exposure. Mice immunized with rBmCRT responded with increased Th1 and Th2 cytokine secretion and exhibited highly elevated titers of anti-BmCRT specific IgG at day 14 and day 28 postimmunization while splenocytes and mLNs from immunized mice showed a robust recall response on restimulation with rBmCRT. Infective larvae (L3) challenge and protection studies undertaken in Mastomys coucha, a permissive model for LF, showed that rBmCRT-immunized animals mounted a robust humoral immune response as evident by elevated levels of total IgG, IgG1, IgG2a, IgG2b, and IgG3 in their serum even 150 days after L3 challenge, which led to significantly reduced microfilariae and worm burden in infected animals. BmCRT is highly immunogenic and generates robust antiparasitic immunity in immunized animals and should therefore be explored further as a putative vaccine candidate against LF.

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

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 ß-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.

Infective Larvae of Brugia malayi Induce Polarization of Host Macrophages that Helps in Immune Evasion.

Filarial parasites suppress, divert, or polarize the host immune response to aid their survival. However, mechanisms that govern the polarization of host MΦs during early filarial infection are not completely understood. In this study, we infected BALB/c mice with infective larvae stage-3 of Brugia malayi (Bm-L3) and studied their effect on the polarization of splenic MΦs. Results showed that MΦs displayed M2-phenotype by day 3 p.i. characterized by upregulated IL-4, but reduced IL-12 and Prostaglandin-D2 secretion. Increased arginase activity, higher arginase-1 but reduced NOS2 expression and poor phagocytic and antigen processing capacity was also observed. M2 MΦs supported T-cell proliferation and characteristically upregulated p-ERK but downregulated NF-κB-p65 and NF-κB-p50/105. Notably, Bm-L3 synergized with host regulatory T-cells (Tregs) and polarized M2 MΦs to regulatory MΦs (Mregs) by day 7 p.i., which secreted copious amounts of IL-10 and prostaglandin-E2. Mregs also showed upregulated expression levels of MHC-II, CD80, and CD86 and exhibited increased antigen-processing capacity but displayed impaired activation of NF-κB-p65 and NF-κB-p50/105. Neutralization of Tregs by anti-GITR + anti-CD25 antibodies checked the polarization of M2 MΦs to Mregs, decreased accumulation of regulatory B cells and inflammatory monocytes, and reduced secretion of IL-10, but enhanced IL-4 production and percentages of eosinophils, which led to Bm-L3 killing. In summary, we report hitherto undocumented effects of early Bm-L3 infection on the polarization of splenic MΦs and show how infective larvae deftly utilize the functional plasticity of host MΦs to establish themselves inside the host.