Biology subjects

Naqvi, R.

Publications and source records attributed to Naqvi, R..

5 recordsLinked to original sources

bioRxiv2023

Global Profiling of Differentiating Macrophages Identifies Novel Functional Long Non-coding RNAs Regulating Polarization and Innate Immune Responses

Macrophages (M{varphi}) are functionally dynamic immune cells that bridge innate and adaptive immune responses. However, the underlying epigenetic mechanisms that control the macrophage plasticity and innate immune functions are not well-elucidated. Here we performed transcriptome profiling of differentiating M1M{varphi} and M2M{varphi} and identified thousands of previously known and novel lncRNAs. We characterized three M{varphi}-enriched lncRNAs (LRRC75A-As1, GAPLINC and AL139099.5) with novel functions in M{varphi} differentiation, polarization and innate immunity. Knockdown of LRRC75A-As1, and GAPLINC downregulated M{varphi} differentiation markers CDw93 and CD68, and skewed macrophage polarization by decreasing M1 markers but had no significant impact on M2 markers. LRRC75A-As1, and GAPLINC RNAi in M{varphi} attenuated bacterial phagocytosis, antigen processing and inflammatory cytokine secretion supporting their functional role in potentiating innate immune functions. Mechanistically, lncRNA knockdown perturbed the expression of multiple cytoskeleton signaling thereby impairing M{varphi} migration suggesting their critical role in regulating macrophage polarity and motility. Together, our results show that M{varphi} acquire a unique repertoire of lncRNAs to shape differentiation, polarization and innate immune functions.

immunology↗

bioRxiv2023

Long noncoding RNA AL109754.1 Regulates Myeloid Dendritic Cell Differentiation and Potentiates TLR signaling

Dendritic cells (DCs) are key antigen presentation cells (APC) that bridge innate and adaptive immune functions to contain the pathogenic threats. Long noncoding RNAs (lncRNAs) are implicated in functional regulation of various biological processes including inflammation and immunity. However, the knowledge on myeloid DC expressed lncRNA repertoire and their regulatory functions is limited. In this study, we have reconnoitered the time-kinetics of lncRNA expression profiles during monocyte-to-DC differentiation and their roles in shaping DC functions. Our RNA-seq data identified thousands of differentially expressed lncRNAs associated with primary human monocyte-to-DC differentiation in vitro. We selected two lncRNAs viz., AL109754.1 and AC093278.2 that were enriched during DC differentiation. Knockdown of AL109754.1 but not AC093278 affects DCs differentiation as observed by marked reduction of surface markers CD1a, CD93 and CD209. These DCs also exhibit significant reduction in the expression of TLR 2, 4, 5, 7 and 9, suggesting that AL109754.1 expression is critical in maintaining TLR expression in DCs. Furthermore, reduced phosphorylation of NF-{kappa}B, IRF3 and IRF7 in AL109754.1 knockdown DCs treated with TLR agonists further substantiate their role in potentiating TLR signaling. Mechanistically, AL109754.1 knockdown DC showed significant downregulation of multiple NF-{kappa}B-induced genes and time-dependent inhibition of pro-inflammatory cytokine (IL-1{beta}, IL-6, IL-8 and TNF) secretion upon challenge with TLR 4, 5, or 7 agonists. Overall, this study characterized novel functions of AL109754.1 that regulates DC differentiation, and TLR-dependent innate immune activation.

immunology↗

bioRxiv2023

Dynamic Changes in Macrophage Polarization during the Resolution Phase of Periodontal Disease

Periodontal inflammation is largely governed by infiltration of myeloid cells, in particular macrophages. Polarization of M{varphi} within the gingival tissues is a well-controlled axis and has considerable consequences for how M{varphi} participate in inflammatory and resolution (tissue repair) phases. We hypothesize that periodontal therapy may instigate a pro-resolution environment favoring M2 M{varphi} polarization and contribute towards resolution of inflammation post-therapy. We aimed to evaluate the markers of macrophage polarization before and after periodontal therapy. Gingival biopsies were excised from human subjects with generalized severe periodontitis, undergoing routine non-surgical therapy. A second set of biopsies were excised after 4-6 weeks to assess the impact of therapeutic resolution at the molecular level. As controls, gingival biopsies were excised from periodontally healthy subjects, undergoing crown lengthening. Total RNA was isolated from gingival biopsies to evaluate pro- and anti-inflammatory markers associated with macrophage polarization by RT-qPCR. Mean periodontal probing depths, CAL and BOP reduced significantly after therapy and corroborated with the reduced levels of periopathic bacterial transcripts after therapy. Compared to heathy and treated biopsies, higher load of Aa and Pg transcripts were observed in disease. Lower expression of M1M{varphi} markers (TNF-, STAT1) were observed after therapy as compared to diseased samples. Conversely, M2M{varphi} markers (STAT6, IL-10) were highly expressed in post-therapy as opposed to pre-therapy, which correlated with clinical improvement. These findings corroborated with murine ligature-induced periodontitis and resolution model, comparing the respective murine M{varphi} polarization markers (M1 M{varphi}: cox2, iNOS2 and M2 M{varphi}: tgm2 and arg1). Our findings suggest that imbalance in M1 and M2 polarized macrophages by assessment of their markers can provide relevant clinical information on the successful response of periodontal therapy and can be used to target non-responders with exaggerated immune responses.

immunology↗

bioRxiv2023

LncRNA MALAT1/microRNA-30b axis regulate macrophage polarization and function

IntroductionMacrophages (M{varphi}) can polarize towards the proinflammatory M1 or proresolving M2 phenotype to control diverse biological processes such as inflammation, and tissue regeneration. Noncoding RNAs play critical roles in numerous biological pathways; however, their functional interaction in the regulation of M{varphi} polarization and immune responses remain unclear. ObjectivesTo examine relationship between lncRNA (MALAT1) and microRNA (miR-30b) in shaping macrophage polarization and immune functions. MethodsExpression of MALAT1 and miR-30b was examined in differentiating M1/M2 M{varphi}, human and murine inflamed gingival biopsies by RT-qPCR. MALAT1 and miR-30b direct interaction was examined by dual luciferase assays. Impact of MALAT1 knockdown and miR-30b overexpression was examined on macrophage polarization markers, bacterial phagocytosis, antigen uptake/processing and cytokine profiles. ResultsMALAT1 expression displays a time-dependent induction during M{varphi} differentiation and, upon challenge with TLR4 agonist (E. coli LPS). Knockdown of MALAT1 enhanced the expression of M2M{varphi} markers without affecting the M1M{varphi} markers, suggesting that MALAT1 favors the M1 phenotype by suppressing M2 polarization. MALAT1 knockdown M{varphi} exhibit reduced antigen uptake and processing, bacterial phagocytosis, and bactericidal activity, strongly supporting its critical role in regulating innate immune functions. Consistent with this, MALAT1 knockdown showed impaired cytokine secretion upon challenge with LPS. Importantly, MALAT1 exhibit an antagonistic expression pattern with all five members of the miR-30 family during M2M{varphi} differentiation. Dual-luciferase assays validated a novel sequence on MALAT1 that interacts with miR-30b, a microRNA that promotes the M2 phenotype. Phagocytosis and antigen processing assays unequivocally demonstrated that MALAT1 and miR-30b are functionally antagonistic. In human subjects with periodontal disease and murine model of ligature-induced periodontitis, we observed higher levels of MALAT1, and downregulation of miR-30b that correlates with higher M1M{varphi} markers expression in gingival tissues suggesting a pro-inflammatory function of MALAT1. ConclusionMALAT1/miR-30b antagonistic interaction shapes M{varphi} polarization in vitro and in inflamed gingival biopsies.

immunology↗

bioRxiv2022

Direct conversion of somatic cells into insulin-producing-cells by user-defined multiplex-epigenetic-engineering vector (MEEV-b)

We demonstrate here a single-step and user-friendly approach to generate insulin producing cells by gRNA driven specific-activation of PDX1, NKX6.1, MAFA, Insulin and Glut2 genes in somatic cells via multiplex-epigenetic-engineering-vector (MEEV-{beta}) containing dCas9.P300core developed by us. Sorted Glut2+ cells could secrete insulin in response to glucose challenge and showed expression of {beta}-cell specific transcription factors: NKX2.2, and aforementioned genes. Expression of Cav1.3, GSK3{beta},, KJNC11, and SLC30A8 genes substantiated the functional insulin secreting machinery genes in these Glut2+ cells. Also, absence of ARX and GCG expression in these cells highlighted the specificity of the conversion.

synthetic biology↗