The overriding goal of this project is to provide an understanding of how the immunologic responses to filarial and related parasites are controlled. The major aspects of this project involve the parasite-specific responses in lymphatic filariasis (LF), loiasis, onchocerciasis and most recently strongyloidiasis in terms of regulation, pathogenesis protective immunity, and the genetic underpinnings of these host responses. The major objectives are to identify the mechanisms by which the modulation/regulation of immune responsiveness works in filarial and related parasitic infections; 2) to identify factors involved in the pathogenesis of disease in filarial infections; 3) to identify the role of host and parasite factors underlying the differential responsiveness to parasite antigens and the subsequent clinical and immunologic outcome; and 4) to understand the immunologic correlates of immunity in human filarial infection. IL-10 has been shown to be the primary immunoregulatory cytokine driving the modulation of the host response in many filarial infections. To determine whether parasite-derived molecules could modulate the host IL-10/IL-10R pathways, we performed solid-phase immobilization of human IL-10Ra followed by binding assays with a Brugia malayi (Bm) adult antigen extracts. Bm proteins that bound to human IL-10Ra were eluted and analyzed by liquid chromatography-tandem mass spectrometry. Our proteomic analysis identified 5 Bm molecules that bound human IL-10Ra. Using a structural alignment program, we identified a 164 amino acid sequence from Bm5539 (the top hit) that shared high structural homology with the human IL-10 functional dimer. Using a baculovirus-expressed truncated form of Bm5539, we examined its ability to signal through the human IL-10R, reading out phosphorylation of STAT3 (pSTAT3) in human monocytes by flow cytometry, and showed that Bm5539 appears to be an IL-10 antagonist. Furthermore, as IL-10 belongs to a superfamily of cytokines and 2 of the IL-10 superfamily members IL-24 and IL-19 are increased in active (antigen positive) W. bancrofti infections and to understand the role played by microfilaria (mf) in their induction, peripheral blood mononuclear cells from patients with filarial infections were stimulated with or without filarial antigen they were shown to produce significant levels of IL-10, IL-13, IL-5. IL-4, IL-9 IL-2, and IL-27. When comparing mf positive (mf+) and mf negative (mf-) patients, there were no significant differences in most of the cytokines; in marked contrast, mf+ individuals had significantly increased filarial antigen-driven IL-24 and IL-19 (compared to mf- subjects. These data provide an important link between IL-10 and its related family members IL-19 and IL-24 in the modulation of the immune response in human filarial infections that appears to be driven by microfilariae. Although IL-10 plays a major role in the altering the response to both both filarial antigen and bystander antigens/diseases (e.g. allergic diseases, Mtb, malaria), we developed methods to understand more fully this issue. Using a murine model of house dust mite (HDM)-induced allergic asthma inflammation followed by Ascaris infection, we demonstrated that pre-existing allergen sensitization markedly limits Ascaris larval development and reduces up to 70% the parasite burden in the lung. This reduction in parasite burden is driven by an eosinophil-dependent pulmonary type-2-immune response. To explore further the role played by IL-13/IL-13R signaling in mediating this eosinophil-dependent phenomenon, we show that in HDM-sensitized-IL-13Ra1 deficient mice, there was a significant reduction in tissue eosinophil number (a reduction similar to that found in HDM-sensitized eosinophil-deficient mice) that, as a consequence. failed to limit parasite development or numbers. RNA-seq analyses of Ascaris larvae isolated from the lungs of allergen pre-sensitized mice compared to larvae from non-allergic mice showed a transcriptomic-signature of the L3-liver stage. Our data suggest that HDM-induced allergic sensitization drives a response that mimics a primary Ascaris infection, such that lung-specific IL-13Ra1 signaling driven by allergen sensitization mediates eosinophil-dependent helminth larval killing in the tissue occurs. In related human studies, in which we examined how helminth infections are known to modulate T cell and cytokine responses in latent Mycobacterium tuberculosis infection (LTBI) and because chemokines play a vital role in the protective immune responses in LTBI, we demonstrated that in LTBI with concomitant Strongyloides infection there was a marked decrease in the production of CCL1, CCL2, CCL4, CCL11, CXCL9, CXCL10, and CXCL11 that was reversible with definitive treatment of the Strongyloides infection. We have previously shown that the microfilarial (mf) stage of Brugia malayi can inhibit the mammalian target of rapamycin (mTOR; a conserved serine/threonine kinase critical for immune regulation and cellular growth) in human dendritic cells (DC) and we have proposed that this mTOR inhibition is associated with the DC dysfunction seen in filarial infections. Because exosome-like vesicles (ELV) contain many proteins and nucleic acids such as microRNAs (miRNAs) that affect a variety of cellular pathways, we hypothesized that ELV secreted from mf are enriched in miRNAs that target and downregulate the mTOR pathway in human DC. Thus, ELV, purified from mf of Brugia malayi and confirmed by morphology and size through electron microscopy and Nanosight analysis, were shown to be enriched for miR100, miR71, miR34, miR7, and let-7. After confirming their presence in ELVs using qPCR for these miRNA targets, target predictions suggested that mir100 and let-7 targeted mTOR and its downstream regulatory protein 4-EBP1 respectively. We then exposed human DC and monocytes to these ELVs. After confirming the internalization of mf-derived ELV, we were able to demonstrate through western blotting and flow cytometry that mf ELV downregulate the phosphorylation of 4E-BP1 and mTOR to the same degree that rapamycin (a known mTOR inhibitor) does. Our data collectively suggest that mf ELV are enriched in miRNAs that regulate mTOR.
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