RESUMO
Immune modulation is a hallmark of patent filarial infection, including suppression of antigen-presenting cell function and downmodulation of filarial antigen-specific T cell responses. The mammalian target of rapamycin (mTOR) signaling pathway has been implicated in immune regulation, not only by suppressing T cell responses but also by regulating autophagy (through mTOR sensing amino acid availability). Global proteomic analysis (liquid chromatography-tandem mass spectrometry) of microfilaria (mf)-exposed monocyte-derived dendritic cells (DC) indicated that multiple components of the mTOR signaling pathway, including mTOR, eIF4A, and eIF4E, are downregulated by mf, suggesting that mf target this pathway for immune modulation in DC. Utilizing Western blot analysis, we demonstrate that similar to rapamycin (a known mTOR inhibitor), mf downregulate the phosphorylation of mTOR and its regulatory proteins, p70S6K1 and 4E-BP1, a process essential for DC protein synthesis. As active mTOR signaling regulates autophagy, we examined whether mf exposure alters autophagy-associated processes. mf-induced autophagy was reflected in marked upregulation of phosphorylated Beclin 1, known to play an important role in both autophagosome formation and autolysosome fusion, in induction of LC3II, a marker of autophagosome formation, and in induced degradation of p62, a ubiquitin-binding protein that aggregates protein in autophagosomes and is degraded upon autophagy that was reduced significantly by mf exposure and by rapamycin. Together, these results suggest that Brugia malayi mf employ mechanisms of metabolic modulation in DC to influence the regulation of the host immune response by downregulating mTOR signaling, resulting in increased autophagy. Whether this is a result of the parasite-secreted rapamycin homolog is currently under study.
Assuntos
Autofagia/fisiologia , Brugia Malayi/parasitologia , Células Dendríticas/parasitologia , Microfilárias/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Autofagossomos/metabolismo , Autofagossomos/parasitologia , Proteína Beclina-1/metabolismo , Proteínas de Ciclo Celular , Células Dendríticas/metabolismo , Regulação para Baixo/fisiologia , Fator de Iniciação 4A em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Lisossomos/metabolismo , Lisossomos/parasitologia , Monócitos/metabolismo , Monócitos/parasitologia , Fosfoproteínas/metabolismo , Fosforilação/fisiologia , Proteômica/métodos , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/fisiologia , Ubiquitina/metabolismo , Regulação para Cima/fisiologiaRESUMO
To characterize the function and plasticity of the major human circulating monocyte populations and to explore their role in systemic helminth infection, highly purified (by flow-based sorting) human monocyte subsets (CD14(hi)/CD16(neg) [classical], CD14(+ or hi)/CD16(med) [intermediate], and CD14(neg)/CD16(hi) [nonclassical]) were examined at homeostasis and after activation. Among these three subsets the classical and intermediate subsets were found to be the major sources of inflammatory and regulatory cytokines, as well as cytokines/chemokines associated with alternative activation, whereas the nonclassical and classical populations demonstrated an ability to transmigrate through endothelial monolayers. Moreover, it was primarily the classical subset that was the most efficient in promoting autologous T cell proliferation. The distribution of these subsets changed in the context of a systemic helminth (Wuchereria bancrofti) infection such that patent infection altered the frequency and distribution of these monocyte subsets with the nonclassical monocytes being expanded (almost 2-fold) in filarial infection. To understand further the filarial/monocyte interface, in vitro modeling demonstrated that the classical subset internalized filarial antigens more efficiently than the other two subsets but that the parasite-driven regulatory cytokine interleukin-10 was exclusively coming from the intermediate subset. Our data suggest that monocyte subsets have a differential function at homeostasis and in response to helminth parasites.
Assuntos
Filariose/imunologia , Homeostase/fisiologia , Monócitos/classificação , Monócitos/fisiologia , Adulto , Animais , Brugia Malayi , Estudos de Casos e Controles , Movimento Celular , Células Cultivadas , Feminino , Gerbillinae , Humanos , Interferon gama/farmacologia , Interleucina-4 , Lipopolissacarídeos/farmacologia , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
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. Extracellular vesicles (EVs) contain many proteins and nucleic acids including microRNAs (miRNAs) that might affect a variety of intracellular pathways. Thus, EVs secreted from mf may elucidate the mechanism by which the parasite is able to modulate the host immune response during infection. EVs, purified from mf of Brugia malayi and confirmed by size through nanoparticle tracking analysis, were assessed by miRNA microarrays (accession number GSE157226) and shown to be enriched (>2-fold, p-value<0.05, FDR = 0.05) for miR100, miR71, miR34, and miR7. The microarray analysis compared mf-derived EVs and mf supernatant. After confirming their presence in EVs using qPCR for these miRNA targets, web-based target predictions (using MIRPathv3, TarBAse and MicroT-CD) predicted that miR100 targeted mTOR and its downstream regulatory protein 4E-BP1. Our previous data with live parasites demonstrated that mf downregulate the phosphorylation of mTOR and its downstream effectors. Additionally, our proteomic analysis of the mf-derived EVs revealed the presence of proteins commonly found in these vesicles (data are available via ProteomeXchange with identifier PXD021844). We confirmed internalization of mf-derived EVs by human DCs and monocytes using confocal microscopy and flow cytometry, and further demonstrated through flow cytometry, that mf-derived EVs downregulate the phosphorylation of mTOR in human monocytes (THP-1 cells) to the same degree that rapamycin (a known mTOR inhibitor) does. Our data collectively suggest that mf release EVs that interact with host cells, such as DC, to modulate host responses.
Assuntos
Brugia Malayi/metabolismo , Regulação para Baixo , Vesículas Extracelulares/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Brugia Malayi/imunologia , Proteínas de Ciclo Celular/metabolismo , Células Dendríticas/imunologia , Filariose/imunologia , Humanos , MicroRNAs/metabolismo , Microfilárias/imunologia , Monócitos/metabolismo , Fosforilação , Proteômica , Células THP-1 , Serina-Treonina Quinases TOR/genéticaRESUMO
A number of features at the host-parasite interface are reminiscent of those that are also observed at the host-tumor interface. Both cancer cells and parasites establish a tissue microenvironment that allows for immune evasion and may reflect functional alterations of various innate cells. Here, we investigated how the phenotype and function of human monocytes is altered by exposure to cancer cell lines and if these functional and phenotypic alterations parallel those induced by exposure to helminth parasites. Thus, human monocytes were exposed to three different cancer cell lines (breast, ovarian, or glioblastoma) or to live microfilariae (mf) of Brugia malayi-a causative agent of lymphatic filariasis. After 2 days of co-culture, monocytes exposed to cancer cell lines showed markedly upregulated expression of M1-associated (TNF-α, IL-1ß), M2-associated (CCL13, CD206), Mreg-associated (IL-10, TGF-ß), and angiogenesis associated (MMP9, VEGF) genes. Similar to cancer cell lines, but less dramatically, mf altered the mRNA expression of IL-1ß, CCL13, TGM2 and MMP9. When surface expression of the inhibitory ligands PDL1 and PDL2 was assessed, monocytes exposed to both cancer cell lines and to live mf significantly upregulated PDL1 and PDL2 expression. In contrast to exposure to mf, exposure to cancer cell lines increased the phagocytic ability of monocytes and reduced their ability to induce T cell proliferation and to expand Granzyme A+ CD8+ T cells. Our data suggest that despite the fact that helminth parasites and cancer cell lines are extraordinarily disparate, they share the ability to alter the phenotype of human monocytes.
Assuntos
Brugia Malayi/imunologia , Filariose/imunologia , Evasão da Resposta Imune , Monócitos/imunologia , Monócitos/parasitologia , Neoplasias/imunologia , Animais , Brugia Malayi/genética , Brugia Malayi/fisiologia , Linhagem Celular Tumoral , Filariose/parasitologia , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Ativação Linfocitária , Fagocitose , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
T-cell interaction with antigen-presenting cells (APCs) results in activation and clonal expansion of naive T cells. CD80 expression/acquisition in T cells has been implicated in disease processes in patients with rheumatoid arthritis and multiple myeloma and patients infected with HIV. Our previous data indicate that antigen-specific activation of naive T cells results in T-cell acquisition of CD80 molecules from APCs. However, the functional relevance of the acquired CD80 by T cells in signal pathways has remained unresolved. This study aims to define for the first time the role of acquired CD80 in T-cell clonal expansion. We demonstrate the following: (1) T cells, upon CD80 acquisition, sustain their proliferative response in the absence of APCs; (2) T cells that acquire CD80 sustain the activity of transcriptional factors such as nuclear factor-kappaB (NFkappaB) and activator protein-1 (AP1) for 24 hours after separation from APCs and up-regulate signal transducer and activator of transcription-5 (Stat5) in the absence of APCs or exogenous signal 1; and (3) maintenance of these signals results in unique cytokine production. Collectively, our data support the unique concept that naive T cells sustain their activation by removing "antigen presentasome" (APS; eg, antigen-presenting complex) from APCs, thereby releasing the constraint of APC requirement for further activation.