Neem leaf glycoprotein binding to Dectin-1 receptors on dendritic cell induces type-1 immunity through CARD9 mediated intracellular signal to NFκB. [Mass Spectrometry - Proteomics Facility]

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TitleNeem leaf glycoprotein binding to Dectin-1 receptors on dendritic cell induces type-1 immunity through CARD9 mediated intracellular signal to NFκB. [Mass Spectrometry - Proteomics Facility]
Publication TypeJournal Article
Year of Publication2024
AuthorsGanguly N, Das T, Bhuniya A, Guha I, Chakravarti M, Dhar S, Sarkar A, Bera S, Dhar J, Dasgupta S, Saha A, Ghosh T, Das J, Sk UHossain, Banerjee S, Laskar S, Bose A, Baral R
JournalCell Commun Signal
Date Published2024 Apr 23
KeywordsAnimals, Azadirachta, CARD Signaling Adaptor Proteins, Dendritic Cells, Lectins, C-Type, Mice, Mice, Inbred C57BL, NF-kappa B, Plant Leaves, Protein Binding, Signal Transduction

BACKGROUND: A water-soluble ingredient of mature leaves of the tropical mahogany 'Neem' (Azadirachta indica), was identified as glycoprotein, thus being named as 'Neem Leaf Glycoprotein' (NLGP). This non-toxic leaf-component regressed cancerous murine tumors (melanoma, carcinoma, sarcoma) recurrently in different experimental circumstances by boosting prime antitumor immune attributes. Such antitumor immunomodulation, aid cytotoxic T cell (T)-based annihilation of tumor cells. This study focused on identifying and characterizing the signaling gateway that initiate this systemic immunomodulation. In search of this gateway, antigen-presenting cells (APCs) were explored, which activate and induce the cytotoxic thrust in T cells.

METHODS: Six glycoprotein-binding C-type lectins found on APCs, namely, MBR, Dectin-1, Dectin-2, DC-SIGN, DEC205 and DNGR-1 were screened on bone marrow-derived dendritic cells from C57BL/6 J mice. Fluorescence microscopy, RT-PCR, flow cytometry and ELISA revealed Dectin-1 as the NLGP-binding receptor, followed by verifications through RNAi. Following detection of β-Glucans in NLGP, their interactions with Dectin-1 were explored in silico. Roles of second messengers and transcription factors in the downstream signal were studied by co-immunoprecipitation, western blotting, and chromatin-immunoprecipitation. Intracellularization of FITC-coupled NLGP was observed by processing confocal micrographs of DCs.

RESULTS: Considering extents of hindrance in NLGP-driven transcription rates of the cytokines IL-10 and IL-12p35 by receptor-neutralization, Dectin-1 receptors on dendritic cells were found to bind NLGP through the ligand's peripheral β-Glucan chains. The resulting signal phosphorylates PKCδ, forming a trimolecular complex of CARD9, Bcl10 and MALT1, which in turn activates the canonical NFκB-pathway of transcription-regulation. Consequently, the NFκB-heterodimer p65:p50 enhances Il12a transcription and the p50:p50 homodimer represses Il10 transcription, bringing about a cytokine-based systemic-bias towards type-1 immune environment. Further, NLGP gets engulfed within dendritic cells, possibly through endocytic activities of Dectin-1.

CONCLUSION: NLGP's binding to Dectin-1 receptors on murine dendritic cells, followed by the intracellular signal, lead to NFκB-mediated contrasting regulation of cytokine-transcriptions, initiating a pro-inflammatory immunopolarization, which amplifies further by the responding immune cells including T cells, alongside their enhanced cytotoxicity. These insights into the initiation of mammalian systemic immunomodulation by NLGP at cellular and molecular levels, may help uncovering its mode of action as a novel immunomodulator against human cancers, following clinical trials.

Alternate JournalCell Commun Signal
PubMed ID38649988
PubMed Central IDPMC11036628
Grant List09/030(0076)/2012-EMR-I / / Council of Scientific and Industrial Research /