scholarly journals Combination of Interleukin-27 and MicroRNA for Enhancing Expression of Anti-Inflammatory and Proosteogenic Genes

Arthritis ◽  
2017 ◽  
Vol 2017 ◽  
pp. 1-13 ◽  
Author(s):  
Manoel Figueiredo Neto ◽  
Marxa L. Figueiredo
Keyword(s):  
Immune Cell ◽  
Bone Erosion ◽  
Osteoclast Differentiation ◽  
Cell Types ◽  
Bone Cell ◽  
Inflammatory Gene Expression ◽  
Real Time Quantitative Pcr ◽  
Achievable Goal ◽  
Cell Balance

Remission of inflammation has become an achievable goal in inflammatory or rheumatoid arthritis (RA); however, bone erosion continues in many patients. Interleukin- (IL-) 27 regulates immune and bone cell balance and also suppresses activities of several inflammatory cell types in RA. Despite its promise, challenges to clinical translation of IL-27 have been its partial effects in vivo. Due to their ability to modulate plasticity of bone and immune cell differentiation, we examined the potential for several microRNA (miR) candidates in enhancing the effects of IL-27. Using differentiation, luciferase, and real time quantitative PCR assays, we show that IL-27 promotes osteoblast differentiation, reduces expression of osteoblast inhibitory genes, and reduces osteoclast differentiation, and results suggest a potential coordination with TGFβ/BMP/SMAD and JAK/STAT pathways. We selected miRNA regulators of these and related pathways to examine whether the effects of IL-27 could be augmented for therapeutic applications. miR-29b and miR-21 augmented IL-27 proosteogenic while downregulating osteoclastogenic signals and also worked to reduce inflammatory signaling in activated macrophages, while miR-21 and miR-20b worked with IL-27 to reduce inflammatory gene expression in fibroblasts and T cells. It appears that several miRNAs can be utilized to enhance IL-27’s impact on modulating osteogenesis and reducing proinflammatory signaling.

scholarly journals Systematic comparison of high-throughput single-cell RNA-seq methods for immune cell profiling

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Tracy M. Yamawaki ◽  
Daniel R. Lu ◽  
Daniel C. Ellwanger ◽  
Dev Bhatt ◽  
Paolo Manzanillo ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Immune Cell ◽  
Cell Types ◽  
Data Interpretation ◽  
Detection Sensitivity ◽  
Rna Seq ◽  
Cell Recovery

Abstract Background Elucidation of immune populations with single-cell RNA-seq has greatly benefited the field of immunology by deepening the characterization of immune heterogeneity and leading to the discovery of new subtypes. However, single-cell methods inherently suffer from limitations in the recovery of complete transcriptomes due to the prevalence of cellular and transcriptional dropout events. This issue is often compounded by limited sample availability and limited prior knowledge of heterogeneity, which can confound data interpretation. Results Here, we systematically benchmarked seven high-throughput single-cell RNA-seq methods. We prepared 21 libraries under identical conditions of a defined mixture of two human and two murine lymphocyte cell lines, simulating heterogeneity across immune-cell types and cell sizes. We evaluated methods by their cell recovery rate, library efficiency, sensitivity, and ability to recover expression signatures for each cell type. We observed higher mRNA detection sensitivity with the 10x Genomics 5′ v1 and 3′ v3 methods. We demonstrate that these methods have fewer dropout events, which facilitates the identification of differentially-expressed genes and improves the concordance of single-cell profiles to immune bulk RNA-seq signatures. Conclusion Overall, our characterization of immune cell mixtures provides useful metrics, which can guide selection of a high-throughput single-cell RNA-seq method for profiling more complex immune-cell heterogeneity usually found in vivo.

scholarly journals Revisiting the role of IRF3 in inflammation and immunity by conditional and specifically targeted gene ablation in mice

2018 ◽  
Vol 115 (20) ◽  
pp. 5253-5258 ◽  
Author(s):  
Hideyuki Yanai ◽  
Shiho Chiba ◽  
Sho Hangai ◽  
Kohei Kometani ◽  
Asuka Inoue ◽  
...  
Keyword(s):  
Immune Cell ◽  
Cell Types ◽  
Type I ◽  
Type I Ifn ◽  
Cell Type ◽  
Gene Ablation ◽  
Cell Type Specific

IFN regulatory factor 3 (IRF3) is a transcription regulator of cellular responses in many cell types that is known to be essential for innate immunity. To confirm IRF3’s broad role in immunity and to more fully discern its role in various cellular subsets, we engineered Irf3-floxed mice to allow for the cell type-specific ablation of Irf3. Analysis of these mice confirmed the general requirement of IRF3 for the evocation of type I IFN responses in vitro and in vivo. Furthermore, immune cell ontogeny and frequencies of immune cell types were unaffected when Irf3 was selectively inactivated in either T cells or B cells in the mice. Interestingly, in a model of lipopolysaccharide-induced septic shock, selective Irf3 deficiency in myeloid cells led to reduced levels of type I IFN in the sera and increased survival of these mice, indicating the myeloid-specific, pathogenic role of the Toll-like receptor 4–IRF3 type I IFN axis in this model of sepsis. Thus, Irf3-floxed mice can serve as useful tool for further exploring the cell type-specific functions of this transcription factor.

scholarly journals Bergamot Polyphenol Fraction Exerts Effects on Bone Biology by Activating ERK 1/2 and Wnt/β-Catenin Pathway and Regulating Bone Biomarkers in Bone Cell Cultures

Nutrients ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 1305 ◽  
Author(s):  
Arturo Pujia ◽  
Cristina Russo ◽  
Samantha Maurotti ◽  
Roberta Pujia ◽  
Vincenzo Mollace ◽  
...  
Keyword(s):  
Bone Cells ◽  
Citrus Fruit ◽  
Cell Types ◽  
Bone Cell ◽  
In Vivo Studies ◽  
Bone Biology ◽  
High Concentration ◽  
Mineral Density ◽  
Differentiation Markers

Epidemiological studies show that fruit consumption may modulate bone mineral density. However, data regarding the effect of the Citrus bergamia Risso (Bergamot orange), a citrus fruit containing a high concentration of flavonoids, on bone health are still lacking. In this study, we investigated the effects of Bergamot polyphenols on the Wnt/β-catenin pathway in two distinct bone cell types (Saos-2 and MG63). Findings showed that exposure to 0.01 and 0.1 mg/mL doses upregulate β-catenin expression (p = 0.001), osteoblast differentiation markers (e.g., RUNX2 and COL1A), and downregulate RANKL (p = 0.028), as compared to the control. Our results highlight, for the first time, that Bergamot polyphenols act on bone cells through the β-catenin pathway. In vivo studies are necessary to fully understand Bergamot’s role against bone resorption.

scholarly journals Administration of cardiac mesenchymal cells modulates innate immunity in the acute phase of myocardial infarction in mice

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yi Kang ◽  
Marjan Nasr ◽  
Yiru Guo ◽  
Shizuka Uchida ◽  
Tyler Weirick ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Innate Immunity ◽  
Mechanism Of Action ◽  
Cell Activation ◽  
Cell Function ◽  
Immune Cell ◽  
Mesenchymal Cell ◽  
Cell Types

Abstract Although cardiac mesenchymal cell (CMC) therapy mitigates post-infarct cardiac dysfunction, the underlying mechanisms remain unidentified. It is acknowledged that donor cells are neither appreciably retained nor meaningfully contribute to tissue regeneration—suggesting a paracrine-mediated mechanism of action. As the immune system is inextricably linked to wound healing/remodeling in the ischemically injured heart, the reparative actions of CMCs may be attributed to their immunoregulatory properties. The current study evaluated the consequences of CMC administration on post myocardial infarction (MI) immune responses in vivo and paracrine-mediated immune cell function in vitro. CMC administration preferentially elicited the recruitment of cell types associated with innate immunity (e.g., monocytes/macrophages and neutrophils). CMC paracrine signaling assays revealed enhancement in innate immune cell chemoattraction, survival, and phagocytosis, and diminished pro-inflammatory immune cell activation; data that identifies and catalogues fundamental immunomodulatory properties of CMCs, which have broad implications regarding the mechanism of action of CMCs in cardiac repair.

P151 DISRUPTION OF ENDOGENOUS ITACONATE PRODUCTION EXACERBATES EXPERIMENTAL COLITIS

2020 ◽  
Vol 26 (Supplement_1) ◽  
pp. S5-S6
Author(s):  
Ryan Frieler ◽  
Thomas Vigil ◽  
Richard Mortensen ◽  
Yatrik Shah
Keyword(s):  
Gene Expression ◽  
Immune Cell ◽  
Tricarboxylic Acid Cycle ◽  
Myeloid Cells ◽  
Experimental Colitis ◽  
Cell Types ◽  
Cell Phenotype ◽  
Inflammatory Gene Expression ◽  
Inflammatory Gene ◽  
Anti Inflammatory

Abstract Background Inflammation is a hallmark of inflammatory bowel disease and alterations in tricarboxylic acid cycle (TCA) metabolism have been identified as major regulators of immune cell phenotype during inflammation and hypoxia. The TCA cycle metabolite, itaconate, is produced by the enzyme aconitate decarboxylase 1 (Acod1) and is highly upregulated during classical macrophage activation and during experimental colitis. Itaconate and cell permeable derivatives have robust anti-inflammatory effects on macrophages, therefore we hypothesized that Acod1-produced itaconate has a protective, anti-inflammatory effect during experimental colitis. Methods and Results Wild type (WT) control and Acod1-/- mice were administered 3% Dextran Sulfate Sodium (DSS) in water for 7 days to induce experimental colitis. After DSS was discontinued, Acod1-/- mice had significantly reduced body weight recovery with increased macroscopic disease severity, and upon dissection had decreased colon length and more severe inflammation. To determine if myeloid cells are the critical Acod1/itaconate-producing cell types, we generated myeloid-specific Acod1 deficient mice, however no differences in weight loss, colon length or inflammatory gene expression were detected compared to WT controls. To test whether supplementation with exogenous itaconate could ameliorate colitis, WT mice were treated with the cell-permeable form of itaconate, dimethyl itaconate (DMI). Administration of DMI significantly improved recovery after 7 days of DSS treatment and significantly reduced inflammatory gene expression in the colon. Conclusion Our data suggest that Acod1-produced itaconate has an important role in the regulation of inflammation during experimental colitis. Although myeloid cells have been thought to be major producers of Acod1 and itaconate, our data indicate that other cell types are involved. These results highlight the importance of this immunometabolic pathway and suggest that preservation or enhancement of this pathway with natural metabolites or metabolite derivatives could have beneficial effects during colitis.

scholarly journals Fucoidan Prevents RANKL-Stimulated Osteoclastogenesis and LPS-Induced Inflammatory Bone Loss via Regulation of Akt/GSK3β/PTEN/NFATc1 Signaling Pathway and Calcineurin Activity

Marine Drugs ◽  
2019 ◽  
Vol 17 (6) ◽  
pp. 345 ◽  
Author(s):  
Sheng-Hua Lu ◽  
Yi-Jan Hsia ◽  
Kuang-Chung Shih ◽  
Tz-Chong Chou
Keyword(s):  
Bone Loss ◽  
Molecular Mechanisms ◽  
Bone Erosion ◽  
Osteoclast Differentiation ◽  
Bone Diseases ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Calcineurin Activity

Excessive osteoclast differentiation and/or function plays a pivotal role in the pathogenesis of bone diseases such as osteoporosis and rheumatoid arthritis. Here, we examined whether fucoidan, a sulfated polysaccharide present in brown algae, attenuates receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclastogenesis in vitro and lipopolysaccharide (LPS)-induced bone resorption in vivo, and investigated the molecular mechanisms involved. Our results indicated that fucoidan significantly inhibited osteoclast differentiation in RANKL-stimulated macrophages and the bone resorbing activity of osteoclasts. The effects of fucoidan may be mediated by regulation of Akt/GSK3β/PTEN signaling and suppression of the increase in intracellular Ca2+ level and calcineurin activity, thereby inhibiting the translocation of nuclear factor-activated T cells c1 (NFATc1) into the nucleus. However, fucoidan-mediated NFATc1 inactivation was greatly reversed by kenpaullone, a GSK3β inhibitor. In addition, using microcomputer tomography (micro-CT) scanning and bone histomorphometry, we found that fucoidan treatment markedly prevented LPS-induced bone erosion in mice. Collectively, we demonstrated that fucoidan was capable of inhibiting osteoclast differentiation and inflammatory bone loss, which may be modulated by regulation of Akt/GSK3β/PTEN/NFATc1 and Ca2+/calcineurin signaling cascades. These findings suggest that fucoidan may be a potential agent for the treatment of osteoclast-related bone diseases.

scholarly journals IMMU-08. MICROENVIRONMENT MODULATION BY TIM-3 BLOCKADE IMPROVES THE OUTCOME OF PRECLINICAL DIPG MODELS

2021 ◽  
Vol 23 (Supplement_1) ◽  
pp. i28-i28
Author(s):  
Iker Ausejo-Mauleon ◽  
Sara Labiano ◽  
Virginia Laspidea ◽  
Marc Garcia-Moure ◽  
Daniel de la Nava ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Microenvironment ◽  
Nk Cells ◽  
Immune Cell ◽  
Cell Types ◽  
Pediatric Brain Tumors ◽  
Immune Memory ◽  
Cd8 Cells ◽  
Functional Studies

Abstract Diffuse Midline Gliomas (DMGs), encompassing Diffuse Intrinsic Pontine Gliomas (DIPGs), are the most aggressive pediatric brain tumors. Their meagre survival has not changed despite the combination of radiotherapy with targeted therapies emphasizing the urgent need for effective treatments. Recent research suggested that the DIPG tumor microenvironment is neither highly immunosuppressive nor inflammatory. These analyses showed the lack of infiltrating lymphocytes and the abundance of CD11b+ cells. TIM-3 (HAVCR2) is a member of the T-cell immunoglobulin and mucin domain protein family which is expressed on multiple immune cell types including T cells, Tregs, NK cells, monocytes, dendritic cells and microglia, where it potently regulates not only adaptive immunity but also innate immunity. Therefore, the central hypothesis of this study is that TIM-3 inhibitors could stimulate a cytotoxic immune effect and challenge several components in the tumor microenvironment including microglia, thereby providing a potential effective treatment for DMGs. In silico assessment of TIM-3 expression in a DIPG datasets showed a robust expression of this gene. Moreover, single-cell sequencing analyses of DIPG biopsies uncover its expression on tumor cells, especially in the OPCs compartment. In vivo efficacy studies showed that treatment with anti-TIM-3 antibody significantly increase the overall survival in two DIPG immunocompetent orthotopic animal models (doubling the median), lead to long-term survivors (50%) and showed immune memory. Analyses of CD45+ populations in the tumor microenvironment showed a significant increase in B, NK and CD8+ cells corresponding with a T-cell activate phenotype in treated-mice. The potential therapeutic involvement of NK cells was certified using nude mice and functional studies. Involvement of microglia in currently being analysed. In summary, these data underscore TIM-3 as a potential target DIPGs and uncover the potential involvement of NKs and other immune mechanisms in the efficacy of anti-TIM-3 therapy.

scholarly journals Adipose-tissue derived signals control bone remodelling

2020 ◽  
Author(s):  
Maria-Bernadette Madel ◽  
He Fu ◽  
Dominique D. Pierroz ◽  
Mariano Schiffrin ◽  
Carine Winkler ◽  
...  
Keyword(s):  
Bone Erosion ◽  
Cell Formation ◽  
Cell Types ◽  
Whole Body ◽  
Long Bone ◽  
Bone Homeostasis ◽  
Multiple Cell ◽  
The One

SummaryLong bones from mammals host blood cell formation and contain multiple cell types, including adipocytes. Physiological functions of bone marrow adipocytes are poorly documented. Herein, we used adipocyte-deficient PPARγ-whole body null mice to investigate the consequence of total adipocyte deficiency on bone homeostasis in mice. We first highlight the dual bone phenotype of PPARγ null mice: on the one hand the increase bone formation and subsequent trabecularization extending in the long bone diaphysis, due to the well-known impact of PPARγ deficiency on osteoblasts formation and activity; on the other hand, an increased osteoclastogenesis in the cortical bone. We then further explore the cause of this unexpected increased osteoclastogenesis using two independent models of lipoatrophy, which recapitulated this phenotype. This demonstrates that hyperosteoclastogenesis is not intrinsically linked to PPARγ deficiency, but is a consequence of the total lipodystrophy. We further showed that adiponectin, a cytokine produced by adipocytes and mesenchymal stromal cells is a potent inhibitor of osteoclastogenesis in vitro and in vivo. Moreover, pharmacological activation of adiponectin receptors by the synthetic agonist AdipoRon inhibits mature osteoclast activity both in mouse and human cells by blocking podosome formation through AMPK activation. Finally, we demonstrated that AdipoRon treatment blocks bone erosion in vivo in a murine model of inflammatory bone loss, providing potential new approaches to treat osteoporosis.

scholarly journals Systematic Comparison of High-Throughput Single-Cell RNAseq Methods for Immune Cell Profiling

2020 ◽  
Author(s):  
Chi-Ming Kevin Li ◽  
Tracy M Yamawaki ◽  
Daniel R Lu ◽  
Daniel C Ellwanger ◽  
Dev Bhatt ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Immune Cell ◽  
Cell Types ◽  
Drop Out ◽  
Detection Sensitivity ◽  
Rna Seq ◽  
Cell Recovery

Abstract Background: Elucidation of immune populations with single-cell RNA-seq has greatly benefited the fieldof immunology by deepening the characterization of immune heterogeneity and leading to thediscovery of new subtypes. However, single-cell methods inherently suffer from limitations in therecovery of complete transcriptomes due to the prevalence of cellular and transcriptional dropoutevents. This issue is often compounded by limited sample availability and limited prior knowledge ofheterogeneity, which can confound data interpretation.Results: Here, we systematically benchmarked seven high-throughput single-cell RNA-seq methods. Weprepared 21 libraries under identical conditions of a defined mixture of two human and two murinelymphocyte cell lines, simulating heterogeneity across immune-cell types and cell sizes. We evaluatemethods by their cell recovery rate, library efficiency, sensitivity, and ability to recover expressionsignatures for each cell type. We observed higher mRNA detection sensitivity with the 10x Genomics 5’v1 and 3’ v3 methods. We demonstrate that these methods have fewer drop-out events whichfacilitates the identification of differentially-expressed genes and improves the concordance of singlecellprofiles to immune bulk RNA-seq signatures.Conclusion: Overall, our characterization of immune cell mixtures provides useful metrics, which canguide selection of a high-throughput single-cell RNA-seq method for profiling more complex immunecellheterogeneity usually found in vivo.

Cell-specific and targeted delivery of RNA moieties

2020 ◽  
Author(s):  
Aditi Bhargava ◽  
Peter Ohara ◽  
Luc Jasmin
Keyword(s):  
Nucleic Acids ◽  
Targeted Delivery ◽  
Immune Cell ◽  
Neuronal Cell ◽  
Cell Types ◽  
Specific Cell ◽  
Chemically Modified ◽  
Covalently Linked

AbstractDelivery of therapeutic moieties to specific cell types, such as neurons remains a challenge. Genes present in neurons are also expressed in non-neuronal cell types such as glia where they mediate non-targeted related functions. Thus, non-specific targeting of these proteins/channels has numerous unwanted side effects, as is the case with current small molecules or drug therapies. Current methodologies that use nanoparticles, lipid-mediated uptake, or mannitol in conjunction with lipids to deliver double-stranded RNA (dsRNA) have yielded mixed and unreliable results. We used a neuroanatomical tracer (B subunit of Cholera Toxin (CTB)) that binds to the ganglioside receptors (GM1) expressed on cells, including primary sensory neurons to deliver encapsulated dsRNA. This approach greatly improved delivery of dsRNA to the desired cells by enhancing uptake, reducing vehicle-mediated toxicity and protecting nucleotides from degradation by endonucleases. The delivery complex is internalized, and once inside the cell, the dsRNA naturally dissociates itself from the carrier complex and is very effective in knocking down cognate targets, both in vivo and in vitro. Past methods have used CTB-fusion proteins or chemically modified oligos or DNA moieties that have been covalently conjugated to CTB. Furthermore, CTB conjugated to an antigen, protein, or chemically modified nucleic acid is a potent activator of immune cell (T and B cells, macrophages) response, whereas CTB admixed with antigens or unmodified nucleic acids does not evoke this immune response. Importantly, in our method, the nucleic acids are not covalently linked to the carrier molecules. Thus, our method holds strong potential for targeted delivery of therapeutic moieties for cell types expressing GM1 receptors, including neuronal cell types.

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