Date of Award

11-12-2023

Document Type

Thesis

School

School of Chemical & Biotechnology

Programme

Ph.D.-Doctoral of Philosophy

First Advisor

Dr.Santanu Kar Mahapatra

Keywords

Nut Shell Glycoprotein, Macrophage Activation, Toll Like Receptor-4, Leishmania Donovani, Immune Modulators

Abstract

Glycoproteins are among several immunomodulatory agents known to activate the innate immune system. Plant derived glycoproteins are a generous source of glycoproteins that could be utilized as cost effective preparations for therapeutics. Isolating glycoproteins from agricultural by- products with the intention to use them against Visceral Leishmaniasis (VL) was the focus in this doctoral study. The two nut shells, Arachis hypogea L. (Groundnut) and Cocos nucifera L. (Coconut), were used for the isolation of glycoprotein used in the study. Crude glycoproteins from the A. hypogea L. (AHP-C) were isolated and fractioned. AHP-F2 was identified as 28.37 kDa Mannose/Glucose binding lectin after peptide finger printing and MS/MS analysis. Its glycosylation pattern was identified using bioinformatic servers.

AHP-F2 induced nitrite generation, IL-12, and IFN-γ release from macrophages, which were the indicators of its immunomodulatory activity. CNP-F1 (43.61 kDa), and CNP-F2 (46.21) were also successfully purified from nut shell of C. nucifera L. and were identified as gibberellin-20-oxidase, and Serine- threonine kinase 11 (STK11), respectively, after peptide finger printing and MS/MS analysis. The identity of those ii molecules are reported for the for the first time in this dissertation. Presence of several xx glycosylation sites were confirmed in Bioinformatic servers indicating their nature as glycoprotein. CNP-F2 stimulated significant (p< 0.001) NO generation, IL-12, and IFN-γ release as compared with untreated cells. Comparison of CNP-F1 and CNP-F2 showed that CNP-F2 was more effective than the CNP-F1.

In silico interaction and mRNA expression study indicated that AHP-F2 and CNP-F2 might utilize TLR-4 for the activation of macrophage. All the crude and fractionated proteins from the two sources showed negligible toxicity to macrophages, as well as to promastigote forms of L. donovani parasites, within the studied dose ranges. But AHP-F2 (0.5 μg/mL) and CNP-F2 (0.5 μg/mL) significantly (p< 0.01) decreased the parasite burden in infected macrophages by 72.23% and 86.68%, respectively. In contrast the standard drugs, miltefosine (10 μg/mL) and amphotericin- B (1 μg/mL) decreased the parasite load in macrophages by 91.3% and 96%, respectively. Hence, the standard drugs were still more effective compared with isolated proteins.

However, CNP-F2 increased NO generation significantly (p< 0.001), which is essential for the intracellular killing of Leishmania parasites, compared with AHP-F2, and the standard drugs, miltefosine and amphotericin B. In this context, in vitro efficacy of CNP-F2 was also comparable with the standard drugs. Hence, we selected the CNP-F2 for the further study. CNP-F2 significantly (p< 0.01) increased the iNOS2 expression and suppressed the arginase-1 expression both in mRNA and protein level. Moreover, the pro- inflammatory cytokines i.e., IL-12 and IFN-γ increased; in contrast, anti-inflammatory cytokines, IL10 and TGF-β mRNA transcript levels decreased in CNP-F2 treated L. donovani infected macrophages.

These are well proven signatures of anti- leishmanial immune responses. We also checked the TLRs expression in L. donovani- infected macrophages after CNP-F2 exposure, as CNP-F2 interacted and modulated the innate immune iii receptors, TLRs. The results indicated the enhanced TLR-2, TLR-4, and TLR-9 mRNA expression in infected macrophages by 2.93-fold, 6.55-fold, and 2.09-fold, respectively, after treatment with CNP-F2 (0.5 μg/mL). We further confirmed these TLRs at the protein level by Western blotting. Hence, TLR-4 might be considered as the target of CNP-F2 for its anti-leishmanial immune response.

Phosphorylation of p38MAPK, NF- κBp65 and dephosphorylation of ERK-1/2 might enhance the NOS-2 expression during CNP-F2 treatment in infected macrophages. The pharmacological inhibitors of iNOS- 2, p38MAPK, and NF-κBp65 also confirmed their involvement in CNP-F2 mediated NO generation dependent parasite killing in macrophages. In addition, CNP-F2 (1 mg/kg B.W.) restricted the LDU significantly (p< 0.01) by 73.61% in the liver and 75.96% in the spleen tissue, which were also comparable with miltefosine. CNP-F2 also increased mature granuloma and prompt DTH responses in infected mice.

Moreover, CNP-F2 amended T-cell proliferation with increased IL-2 generation, as well as, Th1 responses in splenocytes, indicating the activation of the cell-mediated anti-VL immune response. Hence, CNP-F2 could able to induce anti-VL immune response that restrict the in vivo parasite load in infected BALB/c mice. Taken together, these findings suggested that CNP-F2 could be considered as a potential candidature for the modulation of VL immunity and could be. It deserves to be investigated further, as an immune-protective and therapeutic treatment against Visceral Leishmaniasis.

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