Inflammasomes, M2 cells, long non-coding RNAs and nanoparticles: The four wheels of multiple sclerosis?

Relapsing remitting multiple sclerosis (RRMS) hits around 85% MS patients. The main disease modifying therapies (DMTs) of this subtype are: Fingolimod (Gilenya), IFN-β A1(Rebif & Avonex), Teriflunomide(Aubagio), IFN-β 1B(β-feron) and Dimethyl Fumarate(Marovarex).The study aimed at: 1.Expression profiling of long non-coding RNA MALAT-1 in RRMS patients, MALAT1 knockdown and evaluating successful silencing of MALAT1 by specific siRNAs, 2.Expression profiling of Inflammasome components in RRMS patients and studying the impact of MALAT1-knock down on them, 3.Expression profiling of Calreticulin (CRT) and studying the impact of MALAT1-knock down on its protein expression, 4.Impact of CRT-knock down on the inflammasomes using Chitosan Nanoparticles (CS-NP) as transfection agents instead of Hiperfect.After collecting blood from 75 RRMS patients taking DMTs, the methods used were: PBMCs isolation by Ficoll separation, Mojosort Kit for monocytes negative selection and isolation, then differentiation to M2 macrophages and differentiation confirmation by microscope & flow cytometry. RNA was extracted followed by cDNA then PCR & ELISA to investigate the target genes and proteins. On the other hand, CS-NP were prepared by the ionotropic gelation technique followed by size verification with Zeta Sizer & Scanning electron Microscopy (SEM). CS-NP were delivered after measuring their encapsulation efficiency(EE).Results are shown in the following table:AimResult1.Expression profiling of long non-coding RNA MALAT-1 in RRMS patients, MALAT1 knockdown and evaluating successful silencing of MALAT1 by specific siRNAs MALAT-1 was overexpressed in RRMS patients.MALAT1-Knockdown was successful and efficient.2.Expression profiling of Inflammasome components in RRMS patients and studying the impact of MALAT1-knock down on them Caspase-1, IL-1β, IL-18 & NLRP3 were overexpressed in MS patients.The above mentioned four inflammasomes components have become downregulated on mRNA & protein level except for IL-1β which was upregulated on mRNA level.3.Expression profiling of Calreticulin (CRT) and studying the impact of MALAT1-knock down on its protein expression CRT was overexpressed in RRMS patients.CRT was downregulated after silencing MALAT-1 in healthy control.4.Impact of CRT-knock down on the inflammasomes using Chitosan Nanoparticles (CS-NP) as transfection agents instead of Hiperfect. Caspase-1, IL-1β, IL-18 & NLRP3 were downregulated as a result of silencing CRT using CS-NP and Hiperfect as transfection agents.The most impacted conditions were the ones where there was serum in the medium.To conclude, lncRNA MALAT-1 is pro-inflammatory in MS via regulating the CRT-Inflammasome pathway. Silencing this LncRNA has downregulated CRT, NLRP3, Caspase-1, IL-1β and IL-18. Moreover, successful transfection of PBMCs with siRNA loaded in CS-NP against CRT paves the road for efficient future targeted and personalized MS therapies.