MiR-19b-3p Attenuates Meningitic Escherichia Coli-induced Neuroinflammation Via Targeting TNFAIP3

Bacterial meningitis is a major cause of mortality and morbidity worldwide.Escherichia coli is an important gram-negative pathogen causing meningitis in the human population,mostly infants.Despite advance,anti-microbial therapy,meningitis E.coli endured as an important fatal disease around the globe.Limited knowledge regarding pathogenesis is the reason behind such morbidity and mortality.Meningitic E.coli infection usually characterized by three essential events i.e.bacteremia,blood-brainbarrier(BBB)invasion and initiation of prompt inflammatory response.For invasion into the brain,pathogen entered through human brain microvascular endothelial cells(h BMECs),multiple host as well as pathogen related factors,were involved in this complex mechanism.Numerous studies have been focusing on such factors involving bacteremia and BBB invasion in meningitic E.coli infection;however,the factors modulating inflammatory response upon E.coli infection have been studied infrequently in past.The rapid inflammatory response causes severe proliferation and tissue damage upon bacterial infection.Thus,there is need to identify novel therapies that not only promote survival and tissue homeostasis but also prevent from the excessive proliferative or inflammatory response.Micro RNAs(mi-RNAs)are small,noncoding RNAs of 18-25 nucleotides in length,can regulate gene expression by binding with the 3’untranslated-region(3UTR)of target m RNA.Mi-RNAs are now well-accepted fine tuners of regulators of the genome in different physiological and pathophysiological pathways.Mi-RNAs sculpted the expression on m RNA by binding them through sequence complementation and silent the expression of the target gene.Numerous studies have been demonstrated that mi-RNAs not only involved in cell proliferation,organogenesis differentiation,and apoptosis,but also act as key regulators of congenital immune response and pathogen infection.TNF-alpha-induced-protein-3(TNFAIP3)also known as A20,earlier acted as a primary response gene activated in TNF-alpha stimulation and protected cells from death.TNFAIP3 is now widely recognized as a key inhibitor of inflammation and immunity based activities in the nuclear-factor-κB(NF-κB)signaling pathway as well as a potent regulator of apoptosis.The host immune system encompasses multiple strategies against invading pathogens;an abrupt inflammatory response is one of them.Activation of the NF-κB transcription caused the spontaneous release of inflammatory cytokines and chemokines to eradicate the organism and promote the expression of cell-survival genes;however,some factors involved in negative feedback regulation of this ongoing lethal inflammatory response such as TNFAIP3.As our previous RNA-seq data,we have found that miR-19b-3p expression was significantly downregulated and TNFAIP3 expression was significantly upregulated in h BMECs after meningitic E.coli infection.In this study,we confirmed the inverse relationship between miR-19b-3p and TNFAIP3 in our in-vivo and in-vitro meningitic E.coli infection model by using technologies such as fluorescence quantitative PCR,western blot,dual-luciferase reporter assay,and CRISPR/Cas9 gene editing.Mechanistically,upon bacterial infection,miR-19b-3p down regulation and TNFAIP3 upregulation caused downward regulation of P65 phosphorylation,an essential subunit of NF-κB and corresponding inflammatory cytokines and chemokines like tumor necrosis factor-alpha(TNF-α),interleukin-6(IL-6),interleukin-1beta(IL-1β),and chemokine(CC motif)ligand 2(CCL-2).A substantial increase of these cytokines and chemokines were recorded either by overexpression of endogenous miR-19b-3p through mimics or by TNFAIP3 knock-out from h BMECs.Hence,we demonstrated that inflammatory response can be attenuated by signal cascade pathway of miR-19b-3p/TNFAIP3/NF-κB during meningitic E.coli infection in brain endothelial cells.