Afsar Raza Naqvi

miR-24, miR-30b, and miR-142-3p Regulate Phagocytosis in Myeloid Inflammatory Cells

Micro-RNAs (miRNAs) are small noncoding RNAs that regulate various biological pathways. As their role in phagocytosis remains poorly understood, we investigated their impact on phagocytosis in myeloid inflammatory cells. Seven miRNAs (miR-24, -30b, -101, 142-3p, -652-3p, -652-5p, and -1275) that were differentially expressed during monocyte to macrophage (Mφ) and monocyte to dendritic cell (DC) differentiation were screened for their potential role in phagocytosis. Among these, overexpression of miR-24, miR-30b, and miR-142-3p in human monocyte-derived Mφ, DC, monocytes, and PBMCs significantly attenuate phagocytosis of Escherichia coli and Staphylococcus aureus, as well as the secretion of inflammatory mediators, including TNF-α, IL-6, and IL-12p40. miRNA-mediated changes in cytokine profiles were observed at transcriptional and/or posttranscriptional levels and importantly exhibit miRNA-specific impact. To examine the underlying mechanism, we monitored the expression of phagocytosis pathway-associated genes and identified several genes that were altered in Mφ and DC transfected with miR-24, miR-30b, and miR-142-3p mimics. Some of these genes with altered expression also harbor putative miRNA binding sites. We show that miR-142-3p directly regulates protein kinase Cα (PKCα), a key gene involved in phagocytosis. Interestingly, miR-142-3p and PKCα exhibit antagonistic expression during Mφ and DC differentiation. Short interfering RNA-mediated knockdown of PKCα in Mφ leads to reduced bacterial uptake, further highlighting the role of the gene in phagocytosis. Overall, these results demonstrate that miR-24, miR-30b, and miR-142-3p regulate phagocytosis and associated cytokine production in myeloid inflammatory cells through modulation of various genes involved in the pathway.

Regulation of miR-24, miR-30b, and miR-142-3p during macrophage and dendritic cell differentiation potentiates innate immunity

miRNAs are ubiquitous regulators of human biology. Parallel profiling of in vitro monocyte‐to‐Mφ and monocyte‐to‐DC differentiation revealed static, convergent, and divergent expression of miRNA. Bioinformatic and network analysis of differentially expressed miRNAs implicated miR‐24, miR‐30b, and miR‐142‐3p as negative regulators of intracellular signaling pathways, triggered not only by differentiation factors (M‐CSF/GM‐CSF/IL‐4) but also from PRRs. Manipulation of miR‐24, miR‐30b, and miR‐142‐3p expression during the differentiation of mD‐Mφ and mD‐DC differentiation had minimal impact on the acquisition of phenotype but significantly abrogated the ability of these cells to mount inflammatory responses to pathogen‐associated stimuli. Forced expression of these miRNAs, which are down‐regulated during differentiation, inhibited release of inflammatory cytokines [TNF‐α, IL‐12(p40), IL‐6] upon stimulation with LPS. Functional analysis revealed overlapping mechanisms of inhibition, including surface expression of TLR4/CD14/MD‐1 and intracellular PKCα/NF‐κB activation. Potential intermediary targets of the TLR4‐NF‐κB axis included members of the PI3K and MAPK families and PKC isoforms. These results demonstrate the requirement of miR‐24, miR‐30b, and miR‐142‐3p down‐regulation for the generation of fully functional Mφs and DCs.

The HIV Nef protein modulates cellular and exosomal miRNA profiles in human monocytic cells

Introduction The HIV Nef protein is a multifunctional virulence factor that perturbs intracellular membranes and signalling and is secreted into exosomes. While Nef-containing exosomes have a distinct proteomic profile, no comprehensive analysis of their miRNA cargo has been carried out. Since Nef functions as a viral suppressor of RNA interference and disturbs the distribution of RNA-induced silencing complex proteins between cells and exosomes, we hypothesized that it might also affect the export of miRNAs into exosomes. Method Exosomes were purified from human monocytic U937 cells that stably expressed HIV-1 Nef. The RNA from cells and exosomes was profiled for 667 miRNAs using a Taqman Low Density Array. Selected miRNAs and their mRNA targets were validated by quantitative RT-PCR. Bioinformatics analyses were used to identify targets and predict pathways. Results Nef expression affected a significant fraction of miRNAs in U937 cells. Our analysis showed 47 miRNAs to be selectively secreted into Nef exosomes and 2 miRNAs to be selectively retained in Nef-expressing cells. The exosomal miRNAs were predicted to target several cellular genes in inflammatory cytokine and other pathways important for HIV pathogenesis, and an overwhelming majority had targets within the HIV genome. Conclusions This is the first study to report miRnome analysis of HIV Nef expressing monocytes and exosomes. Our results demonstrate that Nef causes large-scale dysregulation of cellular miRNAs, including their secretion through exosomes. We suggest this to be a novel viral strategy to affect pathogenesis and to limit the effects of RNA interference on viral replication and persistence.

The HIV-1 Nef protein binds argonaute-2 and functions as a viral suppressor of RNA interference.

The HIV-1 accessory protein Nef is an important virulence factor. It associates with cellular membranes and modulates the endocytic machinery and signaling pathways. Nef also increases the proliferation of multivesicular bodies (MVBs), which are sites for virus assembly and budding in macrophages. The RNA interference (RNAi) pathway proteins Ago2 and GW182 localize to MVBs, suggesting these to be sites for assembly and turnover of the miRNA-induced silencing complex (miRISC). While RNAi affects HIV replication, it is not clear if the virus encodes a suppressor activity to overcome this innate host response. Here we show that Nef colocalizes with MVBs and binds Ago2 through two highly conserved Glycine-Tryptophan (GW) motifs, mutations in which abolish Nef binding to Ago2 and reduce virus yield and infectivity. Nef also inhibits the slicing activity of Ago2 and disturbs the sorting of GW182 into exosomes resulting in the suppression of miRNA-induced silencing. Thus, besides its other activities, the HIV-1 Nef protein is also proposed to function as a viral suppressor of RNAi (VSR).

Leukocyte Production of Inflammatory Mediators Is Inhibited by the Antioxidants Phloretin, Silymarin, Hesperetin, and Resveratrol

Antioxidants possess significant therapeutic potential for the treatment of inflammatory disorders. One such disorder is periodontitis characterised by an antimicrobial immune response, inflammation, and irreversible changes to the supporting structures of the teeth. Recognition of conserved pathogen-associated molecular patterns is a crucial component of innate immunity to Gram-negative bacteria such as Escherichia coli, as well as the periodontal pathogen Aggregatibacter actinomycetemcomitans. In this study, we investigated the antioxidants Phloretin, Silymarin, Hesperetin, and Resveratrol to ascertain whether they altered the production of inflammatory mediators by innately-activated leukocytes. Peripheral blood mononuclear cells were stimulated with lipopolysaccharide purified from Aggregatibacter actinomycetemcomitans, and the production of cytokines, chemokines, and differentiation factors was assayed by enzyme-linked immunosorbent assay, cytometric bead array, and RT-PCR. Significant inhibition of these factors was achieved upon treatment with Phloretin, Silymarin, Hesperetin, and Resveratrol. These data further characterise the potent anti-inflammatory properties of antioxidants. Their ability to inhibit the production of inflammatory cytokines, chemokines, and differentiation factors by a heterogeneous population of leukocytes has clear implications for their therapeutic potential in vivo.

MiRNA-181a regulates Toll-like receptor agonist-induced inflammatory response in human fibroblasts.

MicroRNAs (miRNAs) regulate the synthesis of cytokines in response to Toll-like receptor (TLR) activation. Our recent microarray study comparing normal and inflamed human dental pulps showed that miRNA-181 (miR-181) family is differentially expressed in the presence of inflammation. Prior studies have reported that the dental pulp, which is composed primarily of TLR4/2+ fibroblasts, expresses elevated levels of cytokines including interleukin-8 (IL-8) when inflamed. In this study, we employed an in-vitro model to determine the role of the miRNA-181 family in the TLR agonist-induced response in human fibroblasts. TLR4/2+ primary human dental pulp fibroblasts were stimulated with lipopolysaccharide from Porphyromonas gingivalis (Pg LPS), a known oral pathogen, and IL-8 and miR-181 expression measured. An inversely proportional relationship between IL-8 and miR-181a was observed. In-silico analysis identified a miR-181a-binding site on the 3′ untranslated region (UTR) of IL-8, which was confirmed by dual-luciferase assays. MiR-181a directly binds to the 3′UTR of IL-8, an important inflammatory component of the immune response, and modulates its levels. This is the very first report demonstrating miR-181a regulation of IL-8.

MicroRNA: Dynamic Regulators of Macrophage Polarization and Plasticity

The ability of a healthy immune system to clear the plethora of antigens it encounters incessantly relies on the enormous plasticity displayed by the comprising cell types. Macrophages (MΦs) are crucial member of the mononuclear phagocyte system (MPS) that constantly patrol the peripheral tissues and are actively recruited to the sites of injury and infection. In tissues, infiltrating monocytes replenish MΦ. Under the guidance of the local micro-milieu, MΦ can be activated to acquire specialized functional phenotypes. Similar to T cells, functional polarization of macrophage phenotype viz., inflammatory (M1) and reparative (M2) is proposed. Equipped with diverse toll-like receptors (TLRs), these cells of the innate arm of immunity recognize and phagocytize antigens and secrete cytokines that activate the adaptive arm of the immune system and perform key roles in wound repair. Dysregulation of MΦ plasticity has been associated with various diseases and infection. MicroRNAs (miRNAs) have emerged as critical regulators of transcriptome output. Their importance in maintaining health, and their contribution toward disease, encompasses virtually all aspects of human biology. Our understanding of miRNA-mediated regulation of MΦ plasticity and polarization can be utilized to modulate functional phenotypes to counter their role in the pathogenesis of numerous disease, including cancer, autoimmunity, periodontitis, etc. Here, we provide an overview of current knowledge regarding the role of miRNA in shaping MΦ polarization and plasticity through targeting of various pathways and genes. Identification of miRNA biomarkers of diagnostic/prognostic value and their therapeutic potential by delivery of miRNA mimics or inhibitors to dynamically alter gene expression profiles in vivo is highlighted.

miR-24 Regulates Macrophage Polarization and Plasticity

Objective MicroRNAs (miRNA) are ubiquitous regulators of human biology and immunity. Previously, we have demonstrated an inhibitory role for miR-24 in the phagocytosis of Escherichia coli and Staphylococcus aureus bioparticles and the induction of cytokine secretion in response to lipopolysaccharide (LPS) of the same origin; also, we have identified divergent and convergent miRNA responses to LPS from the periodontopathic pathogens Aggregatibacter actinomycetemcomitams (Aa) and Porphyromonas gingivalis (Pg), and revealed cigarette smoke extract as an environmental modifier of Pg LPS structure (Pg CSE) impacting macrophage miRNA responses. This study was designed to investigate the role of miR-24 on macrophage polarization and plasticity. Methods Primary human macrophages were differentiated from CD14+ monocytes isolated from peripheral blood mononuclear cells (PBMCs) by MACS positive selection and transfected with miR-24 miRNA mimics, inhibitors, or negative control mimic; followed by stimulation with cytokines and/or LPS under various conditions representing key stages of macrophage activation. Macrophage activation and polarization was assessed using assays for cytokine production (ELISA) and protein expression (flow cytometry, immunoblot). MiR-24 expression was assessed by RT-PCR. Results Stimulation of macrophages with LPSs of Aa, Pg, and Pg CSE origin resulted in dissimilar levels of cytokine expression and differential expression of miR-24. Overexpression of miR-24 inhibited cytokine secretion in response to LPS. Priming of macrophages with interferon gamma (IFN-γ) did not overcome this inhibitory effect, but classical activation of macrophages with IFN-γ plus TNF-α, TNF-β, or IL-17, modulated the pattern of miR-24 mediated suppression in a cytokine-specific fashion. Overexpression of miR-24 enhanced CD206 upregulation during alternative macrophage activation and inhibited its downregulation in macrophage transitioning from alternative to classical activation states. Overexpression of miR-24 resulted in reduced expression of the Class 1A PI 3-kinase subunit p110 delta (p110δ). Conclusion Pathogen- and environment-specific modifications in LPS alter the expression of cytokines and miR-24 in human macrophages. MiR-24 is a negative regulator of macrophage classical activation by LPS and promotes alternative activation under conditions of polarization and plasticity. MiR-24 mediated inhibition of LPS-induced cytokine secretion is dependent upon macrophage activation state at the point of stimulation, and this may be due to the degree to which p110δ is involved in the intracellular signaling pathway/s that transduce receptor ligation into cytokine induction. While important differences were observed in the effect of miR-24 on macrophages, these data indicate that overexpression of miR-24 would be predominantly anti-inflammatory

Prediction and characterization of Tomato leaf curl New Delhi virus (ToLCNDV) responsive novel microRNAs in Solanum lycopersicum.

Tomato leaf curl New Delhi virus (ToLCNDV) infects tomato (Solanum lycopersicum) plants and causes severe crop losses. As the microRNAs (miRNAs) are deregulated during stressful events, such as biotic stress, we wanted to study the effect of ToLCNDV infection on tomato miRNAs. We constructed two libraries, isolating small RNAs (sRNAs) from healthy (HT) and ToLCNDV infected (IT) tomato leaves, and sequenced the library-specific sRNAs using the next generation sequencing (NGS) approach. These data helped predict 112 mature miRNA sequences employing the miRDeep-P program. A substantial number (58) of the sequences were 24-mer in size, which was a bit surprising. Based on the calculation of precision values, 53 novel miRNAs were screened from the predicted sequences. Nineteen of these were chosen for expression analysis; a northern blot analysis showed 15 to be positive. Many of the predicted miRNAs were up-regulated following viral infection. The target genes of the miRNAs were also predicted and the expression analysis of selected transcripts showed a typical inverse relation between the accumulation of target transcripts and the abundance of corresponding miRNAs. Furthermore, the cleavage sites of the target transcripts for three novel miRNAs were mapped, confirming the correct annotation of the miRNA-targets. The sRNA deep sequencing clearly revealed that the virus modulated global miRNA expression in the host. The validated miRNAs (Tom_4; Tom_14; Tom_17; Tom_21; Tom_29; Tom_43) could be valuable tools for understanding the ToLCNDV-tomato interaction, ultimately leading to the development of a virus-resistant tomato plant.

MiR-24, miR-30b and miR-142-3p interfere with antigen processing and presentation by primary macrophages and dendritic cells

Antigen uptake, processing and presentation by antigen presenting cells (APCs) are tightly coupled processes which consequently lead to the activation of innate and adaptive immune responses. However, the regulatory role of microRNA (miRNAs) in these critical pathways is poorly understood. In this study, we show that overexpression of miR-24, miR-30b and miR-142-3p attenuates uptake and processing of soluble antigen ovalbumin (Ova) in primary human macrophages and dendritic cells. MiRNA mimic transfected APCs exhibit defects in antigen presentation (Ova and CMV antigen) to CD4+ T-cells leading to reduced cell proliferation. Using transgenic OT-II mice we demonstrated that this impairment in T-cell proliferation is specific to antigen provided i.e., Ova. Further, human T-cells co-cultured with miRNA transfected dendritic cells secrete low levels of T helper (Th)-1 polarization associated cytokines. Analysis of molecules regulating APC and T-cell receptor interaction shows miRNA-mediated induced expression of Programmed Death-Ligand 1 (PD-L1) which inhibits T-cell proliferation. Blocking PD-L1 with antibodies rescues miRNA-mediated inhibition of T cell priming by DCs. These results uncover regulatory functions of miR-24, miR-30b and miR-142-3p in pairing innate and adaptive components of immunity.