Targeting IRE1α improves insulin sensitivity and thermogenesis and suppresses metabolically active adipose tissue macrophages in obesity.

Overnutrition engenders the expansion of adipose tissue and the accumulation of immune cells, in particular, macrophages, in the adipose tissue, leading to chronic low-grade inflammation and insulin resistance. In obesity, several proinflammatory subpopulations of adipose tissue macrophages (ATMs) identified hitherto include the conventional "M1-like" CD11C-expressing ATM and the newly discovered metabolically activated CD9-expressing ATM; however, the relationship among ATM subpopulations is unclear. The ER stress sensor inositol-requiring enzyme 1α (IRE1α) is activated in the adipocytes and immune cells under obesity. It is unknown whether targeting IRE1α is capable of reversing insulin resistance and obesity and modulating the metabolically activated ATMs. We report that pharmacological inhibition of IRE1α RNase significantly ameliorates insulin resistance and glucose intolerance in diet-induced obesity mice. IRE1α inhibition also increases thermogenesis and energy expenditure, and hence protects against high fat diet-induced obesity. Our study shows that the "M1-like" CD11c + ATMs are largely overlapping with but yet non-identical to CD9 + ATMs in obese white adipose tissue. Notably, IRE1α inhibition diminishes the accumulation of obesity-induced metabolically activated ATMs and "M1-like" ATMs, resulting in the curtailment of adipose inflammation and ensuing reactivation of thermogenesis, without augmentation of the alternatively activated M2 macrophage population. Our findings suggest the potential of targeting IRE1α for the therapeutic treatment of insulin resistance and obesity.

Blocking of doublecortin-like kinase 1-regulated SARS-CoV-2 replication cycle restores cell signaling network.

IMPORTANCE: Severe COVID-19 and post-acute sequelae often afflict patients with underlying co-morbidities. There is a pressing need for highly effective treatment, particularly in light of the emergence of SARS-CoV-2 variants. In a previous study, we demonstrated that DCLK1, a protein associated with cancer stem cells, is highly expressed in the lungs of COVID-19 patients and enhances viral production and hyperinflammatory responses. In this study, we report the pivotal role of DCLK1-regulated mechanisms in driving SARS-CoV-2 replication-transcription processes and pathogenic signaling. Notably, pharmacological inhibition of DCLK1 kinase during SARS-CoV-2 effectively impedes these processes and counteracts virus-induced alternations in global cell signaling. These findings hold significant potential for immediate application in treating COVID-19.

Methylation Array Signals are Predictive of Chronological Age Without Bisulfite Conversion.

DNA methylation data has been used to make "epigenetic clocks" which attempt to measure chronological and biological aging. These models rely on data derived from bisulfite-based measurements, which exploit a semi-selective deamination and a genomic reference to determine methylation states. Here, we demonstrate how another hallmark of aging, genomic instability, influences methylation measurements in both bisulfite sequencing and methylation arrays. We found that non-methylation factors lead to "pseudomethylation" signals that are both confounding of epigenetic clocks and uniquely age predictive. Quantifying these covariates in aging studies will be critical to building better clocks and designing appropriate studies of epigenetic aging.

Targeting Doublecortin-Like Kinase 1 (DCLK1)-Regulated SARS-CoV-2 Pathogenesis in COVID-19.

Host factors play critical roles in SARS-CoV-2 infection-associated pathology and the severity of COVID-19. In this study, we systematically analyzed the roles of SARS-CoV-2-induced host factors, doublecortin-like kinase 1 (DCLK1), and S100A9 in viral pathogenesis. In autopsied subjects with COVID-19 and pre-existing chronic liver disease, we observed high levels of DCLK1 and S100A9 expression and immunosuppressive (DCLK1+S100A9+CD206+) M2-like macrophages and N2-like neutrophils in lungs and livers. DCLK1 and S100A9 expression were rarely observed in normal controls, COVID-19-negative subjects with chronic lung disease, or COVID-19 subjects without chronic liver disease. In hospitalized patients with COVID-19, we detected 2 to 3-fold increased levels of circulating DCLK1+S100A9+ mononuclear cells that correlated with disease severity. We validated the SARS-CoV-2-dependent generation of these double-positive immune cells in coculture. SARS-CoV-2-induced DCLK1 expression correlated with the activation of ß-catenin, a known regulator of the DCLK1 promoter. Gain and loss of function studies showed that DCLK1 kinase amplified live virus production and promoted cytokine, chemokine, and growth factor secretion by peripheral blood mononuclear cells. Inhibition of DCLK1 kinase blocked pro-inflammatory caspase-1/interleukin-1ß signaling in infected cells. Treatment of SARS-CoV-2-infected cells with inhibitors of DCLK1 kinase and S100A9 normalized cytokine/chemokine profiles and attenuated DCLK1 expression and ß-catenin activation. In conclusion, we report previously unidentified roles of DCLK1 in augmenting SARS-CoV-2 viremia, inflammatory cytokine expression, and dysregulation of immune cells involved in innate immunity. DCLK1 could be a potential therapeutic target for COVID-19, especially in patients with underlying comorbid diseases associated with DCLK1 expression. IMPORTANCE High mortality in COVID-19 is associated with underlying comorbidities such as chronic liver diseases. Successful treatment of severe/critical COVID-19 remains challenging. Herein, we report a targetable host factor, DCLK1, that amplifies SARS-CoV-2 production, cytokine secretion, and inflammatory pathways via activation of ß-catenin(p65)/DCLK1/S100A9/NF-κB signaling. Furthermore, we observed in the lung, liver, and blood an increased prevalence of immune cells coexpressing DCLK1 and S100A9, a myeloid-derived proinflammatory protein. These cells were associated with increased disease severity in COVID-19 patients. Finally, we used a novel small-molecule inhibitor of DCLK1 kinase (DCLK1-IN-1) and S100A9 inhibitor (tasquinimod) to decrease virus production in vitro and normalize hyperinflammatory responses known to contribute to disease severity in COVID-19.

Cellular Carcinogenesis: Role of Polarized Macrophages in Cancer Initiation.

Inflammation is an essential hallmark of cancer. Macrophages are key innate immune effector cells in chronic inflammation, parainflammation, and inflammaging. Parainflammation is a form of subclinical inflammation associated with a persistent DNA damage response. Inflammaging represents low-grade inflammation due to the dysregulation of innate and adaptive immune responses that occur with aging. Whether induced by infection, injury, or aging, immune dysregulation and chronic macrophage polarization contributes to cancer initiation through the production of proinflammatory chemokines/cytokines and genotoxins and by modulating immune surveillance. This review presents pre-clinical and clinical evidence for polarized macrophages as endogenous cellular carcinogens in the context of chronic inflammation, parainflammation, and inflammaging. Emerging strategies for cancer prevention, including small molecule inhibitors and probiotic approaches, that target macrophage function and phenotype are also discussed.

Pharmacological Inhibition of Inositol-Requiring Enzyme 1α RNase Activity Protects Pancreatic Beta Cell and Improves Diabetic Condition in Insulin Mutation-Induced Diabetes.

ß-cell ER stress plays an important role in ß-cell dysfunction and death during the pathogenesis of diabetes. Proinsulin misfolding is regarded as one of the primary initiating factors of ER stress and unfolded protein response (UPR) activation in ß-cells. Here, we found that the ER stress sensor inositol-requiring enzyme 1α (IRE1α) was activated in the Akita mice, a mouse model of mutant insulin gene-induced diabetes of youth (MIDY), a monogenic diabetes. Normalization of IRE1α RNase hyperactivity by pharmacological inhibitors significantly ameliorated the hyperglycemic conditions and increased serum insulin levels in Akita mice. These benefits were accompanied by a concomitant protection of functional ß-cell mass, as shown by the suppression of ß-cell apoptosis, increase in mature insulin production and reduction of proinsulin level. At the molecular level, we observed that the expression of genes associated with ß-cell identity and function was significantly up-regulated and ER stress and its associated inflammation and oxidative stress were suppressed in islets from Akita mice treated with IRE1α RNase inhibitors. This study provides the evidence of the in vivo efficacy of IRE1α RNase inhibitors in Akita mice, pointing to the possibility of targeting IRE1α RNase as a therapeutic direction for the treatment of diabetes.

A novel peroxisome proliferator-activated receptor gamma ligand improves insulin sensitivity and promotes browning of white adipose tissue in obese mice.

OBJECTIVE: Nuclear receptor Peroxisome Proliferator-Activated Receptor γ (PPARγ) is a promising target for the treatment of type 2 diabetes. The antidiabetic drug thiazolidinediones (TZDs) are potent insulin sensitizers as full agonists of PPARγ, but cause unwanted side effects. Recent discoveries have shown that TZDs improve insulin sensitivity by blocking PPARγ phosphorylation at S273, which decouples the full agonism-associated side effects. PPARγ ligands that act through the blockage of PPARγ phosphorylation but lack the full agonist activity would be expected to improve insulin sensitivity without TZD-associated side effects, however, chemicals that carry such traits and bind to PPARγ with high-affinity are lacking. Moreover, TZDs are known to promote white-to-brown adipocyte conversion and energy expenditure and appear to require their full agonism on PPARγ for this activity. It is unknown whether a partial or non-TZD agonist of PPARγ is capable of promoting browning effect. In this study, we developed a novel non-TZD partial agonist of PPARγ and investigated its function on insulin sensitivity and white-to-brown conversion and energy expenditure in diet-induced obese mice. METHODS: A novel indole-based chemical WO95E was designed via medicinal chemistry and tested for PPARγ binding and activity and for the effect on PPARγ phosphorylation. Diet-induced obese mice were administered with WO95E for 4 weeks. Insulin sensitivity, glucose tolerance, body weight, fat tissue weight, adipocyte size, morphology, energy expenditure, and expression levels of genes involved in PPARγ activity, thermogenesis/browning, and TZD-related side effects were evaluated. RESULTS: WO95E binds to PPARγ with high affinity and acts as a PPARγ partial agonist. WO95E inhibits PPARγ phosphorylation and regulates PPARγ phosphorylation-dependent genes. WO95E ameliorates insulin resistance and glucose tolerance in mice of diet-induced obesity, with minimal TZD use-associated side effects. We found that WO95E promotes white-to-brown adipocyte conversion and energy expenditure and hence protects against diet-induced obesity. WO95E decreases the size of adipocytes and suppresses adipose tissue inflammation. WO95E also suppresses obesity-associated liver steatosis. CONCLUSIONS: WO95E improves insulin sensitivity and glucose homeostasis and promotes browning and energy expenditure by acting as a novel PPARγ phosphorylation inhibitor/partial agonist. Our findings suggest the potential of this compound or its derivative for the therapeutic treatment of insulin resistance and obesity.

It takes a village: microbiota, parainflammation, paligenosis and bystander effects in colorectal cancer initiation.

Sporadic colorectal cancer (CRC) is a leading cause of worldwide cancer mortality. It arises from a complex milieu of host and environmental factors, including genetic and epigenetic changes in colon epithelial cells that undergo mutation, selection, clonal expansion, and transformation. The gut microbiota has recently gained increasing recognition as an additional important factor contributing to CRC. Several gut bacteria are known to initiate CRC in animal models and have been associated with human CRC. In this Review, we discuss the factors that contribute to CRC and the role of the gut microbiota, focusing on a recently described mechanism for cancer initiation, the so-called microbiota-induced bystander effect (MIBE). In this cancer mechanism, microbiota-driven parainflammation is believed to act as a source of endogenous mutation, epigenetic change and induced pluripotency, leading to the cancerous transformation of colon epithelial cells. This theory links the gut microbiota to key risk factors and common histologic features of sporadic CRC. MIBE is analogous to the well-characterized radiation-induced bystander effect. Both phenomena drive DNA damage, chromosomal instability, stress response signaling, altered gene expression, epigenetic modification and cellular proliferation in bystander cells. Myeloid-derived cells are important effectors in both phenomena. A better understanding of the interactions between the gut microbiota and mucosal immune effector cells that generate bystander effects can potentially identify triggers for parainflammation, and gain new insights into CRC prevention.

MicroRNA function in megakaryocytes.

Megakaryocytes (MKs), the largest cells in the bone marrow, are generated from hematopoietic stem cells (HSCs) in a sequential process called megakaryocytopoiesis in which HSCs undergo MK-progenitor (MP) commitment and maturation to terminally differentiated MK. Megakaryocytopoiesis is controlled by a complex network of bone marrow niche factors. Traditionally, the studies on megakaryocytopoiesis were focused on different cytokines, growth factors and transcription factors as the regulators of megakaryocytopoiesis. Over the past two decades many research groups have uncovered the key role of microRNAs (miRNAs) in megakaryocytopoiesis. miRNAs are a class of small length non-coding RNAs which play key regulatory role in cellular processes such as proliferation, differentiation and development and are also known to be involved in disease development. This review summarizes the current state of knowledge of miRNAs which have changed expression during megakaryocytopoiesis, also focuses on miRNAs which are differentially regulated during developmental maturation of MKs. Further, we aimed to discuss potential mechanisms of miRNAs-mediated regulation underlying megakaryocytopoiesis and developmental maturation of MKs.

Epigenetic Mechanisms: Role in Hematopoietic Stem Cell Lineage Commitment and Differentiation.

Major breakthroughs in the last several decades have contributed to our knowledge of the genetic regulation in development. Although epigenetics is not a new concept, unfortunately, the role of epigenetics has not come to fruition in the past. But the field of epigenetics has exploded within the past decade. Now, growing evidences show a complex network of epigenetic regulation in development. The epigenetic makeup of a cell, tissue or individual is much more complex than their genetic complement. Epigenetic modifications are more important for normal development by maintaining the gene expression pattern in tissue- and context-specific manner. Deregulation of epigenetic mechanism can lead to altered gene expression and its function, which result in altered tissue specific function of cells and malignant transformation. Epigenetic modifications directly shape Hematopoietic Stem Cell (HSC) developmental cascades, including their maintenance of self-renewal and multilineage potential, lineage commitment, and aging. Hence, there is a growing admiration for epigenetic players and their regulatory function in haematopoiesis. Epigenetic mechanisms underlying these modifications in mammalian genome are still not completely understood. This review mainly explains 3 key epigenetics mechanisms including DNA methylation, histone modifications and non-coding RNAs inference in hematopoietic lineage commitment and differentiation.

LiCl regulates mitochondrial biogenesis during megakaryocyte development.

JAK-STAT, PI3K-AKT and MAPK signaling pathways are involved in platelet production process. Although wnt signaling has been reported in the biogenesis of platelets, but its role in megakaryocyte development is not well studied. We used an inducible canonical wnt signaling system that utilizes LiCl (GSK-3ß inhibitor). LiCl could activate wnt signaling pathway along with maturation of megakaryocytes. Mitochondrial staining showed an increase in mitochondrial mass upon induction with LiCl. Also, mitochondrial markers PGC-1α and TFAM were up regulated with increase in mitochondrial DNA content. LiCl leads to increase in the ROS production, suggesting significance of mitochondria in megakaryocyte development.

Erythropoietin and thrombopoietin mimetics: Natural alternatives to erythrocyte and platelet disorders.

Erythropoietin (EPO) and thrombopoietin (TPO) plays a major role in the regulation of hematopoietic development. Though, blood transfusion was the most widely used method to treat low blood count, over the years with advancements in recombinant technology and drug designing, the EPO and TPO mimetics are dominating the therapeutics industry. On the other hand, the recombinant human EPO and TPO are associated either with reduced half-life or immune reactions. The restoration of alternate medicine in recent years has the hope to overcome limitations associated with recombinant technology, to treat various disorder including blood diseases, with low to no side effects. The work in recent years on plant derived mimetics suggests a paradigm shift in the way diseases are treated. Here, we are providing a comprehensive review on the EPO and TPO recombinant counterparts and synthetic mimetics studied till date with a focus on the need for more natural alternatives.

Role of let-7b/Fzd4 axis in mitochondrial biogenesis through wnt signaling: In neonatal and adult megakaryocytes.

BACKGROUND: Megakaryocytes (MKs), a rare population of bone marrow cells, are responsible for the production of platelets. Sick neonates are predisposed to developing thrombocytopenia (platelet count <150×109/L) and neonates are affected by several megakaryocyte disorders as compared to adults. HYPOTHESIS: MicroRNAs (miRNAs) have been shown to crucially involve in the regulation of stem-cell differentiation in normal as well as malignant hematopoiesis, but their role in regulation of biological differences between adult and neonatal megakaryopoiesis is unknown. METHODS: To study this, we cultured human cord blood (CB) and peripheral blood (PB) derived CD34+ cells in the presence of thrombopoietin for 14days and collected cultures expressing>90% CD41+ by flow cytometry and studied 88 miRNAs involved in stem cell development and differentiation. miRNA validation studies were performed in Dami cell line. RESULTS: Out of 88 miRNAs involved in stem cell development, let-7b was the only miRNA down regulated (∼10-fold) in neonates compared to adult-MKs. Let-7b has not been previously described in MKs, however reduced expression of let-7b was found in several human cancers, suggesting that it functions as a tumor suppressor. Our results showed the inhibitory effect of let-7b on wnt signaling pathway by regulating Fzd4 (frizzled family receptor 4) and thereby regulating proliferation as well as differentiation. Let-7b down regulation induced mitochondrial biogenesis and its markers PGC-1α and NRF1 during megakaryocyte development. CONCLUSIONS: Our findings for the first time unveil the novel role of let-7b/Fzd4 axis through wnt signaling by regulating mitochondrial biogenesis during megakaryocyte development.

Toll-like receptor 2 signalling: Significance in megakaryocyte development through wnt signalling cross-talk and cytokine induction.

TLR2 is a toll-like receptor protein which is involved in innate immune responses. TLR2 recognize several virus, fungal and bacterial pathogens, upon their uptake cause internalization and cellular activation. During this process several cytokines participate including interleukins, IL6 and IL12. Interestingly, TLR2 is expressed on megakaryocytes (MKs) and platelets, which is crucial for immune mediated platelet activation. The role of TLR2 on MKs is not completely understood. We observed TLR2 induction leads to MK maturation and is involved in production of ROS which is essential for MK development. In Dami cells, TLR2 up-regulation causes increase in the cytokine production, particularly IL-6, which has been shown to stimulate CFU formation and CD41 expression. Additionally, TLR2 ligand induces wnt ß-catenin signalling pathway components suggesting a cross talk between wnt and TLR pathway leading to maturation of MKs. This study shows TLR2 signalling induce cytokine production and regulate wnt signalling thereby cause maturation of MKs.

Wnt Signaling: Role in Regulation of Haematopoiesis.

Hematopoietic stem cells (HSCs) are a unique population of bone marrow cells which are responsible for the generation of various blood cell lineages. One of the significant characteristics of these HSCs is to self-renew, while producing differentiating cells for normal hematopoiesis. Deregulation of self-renewal and differentiation leads to the hematological malignancies. Several pathways are known to be involved in the maintenance of HSC fate among which Wnt signaling is a crucial pathway which controls development and cell fate determination. Wnt signaling also plays a major role in differentiation, self-renewal and maintenance of HSCs. Wnt ligands activate three major pathways including planar cell polarity, Wnt/ß-catenin and Wnt/Ca(2+). It has been shown that Wnt/ß-catenin or canonical pathway regulates cell proliferation, survival and differentiation in HSCs, deregulation of this pathway leads to hematological malignancies. Wnt non-canonical pathway regulates calcium signaling and planar cell polarity. In this review, we discuss various signaling pathways induced by Wnt ligands and their potential role in hematopoiesis.

Apoptosis: role in myeloid cell development.

Hematopoiesis is the process that generates blood cells in an organism from the pluripotent stem cells. Hematopoietic stem cells are characterized by their ability to undergo self-renewal and differentiation. The self-renewing ability ensures that these pluripotent cells are not depleted from the bone marrow niche. A proper balance between cell death and cell survival is necessary to maintain a homeostatic condition, hence, apoptosis, or programmed cell death, is an essential step in hematopoiesis. Recent studies, however, have introduced a new aspect to this process, citing the significance of the apoptosis mediator, caspase, in cell development and differentiation. Extensive research has been carried out to study the possible role of caspases and other apoptosis related factors in the developmental processes. This review focuses on the various apoptotic factors involved in the development and differentiation of myeloid lineage cells: erythrocytes, megakaryocytes, and macrophages.

TLR-4 signalling pathway: MyD88 independent pathway up-regulation in chicken breeds upon LPS treatment.

Toll-like receptors (TLRs) that sense the microbial pathogens are important components of host immune system. TLRs play key roles in the innate defence mechanism against pathogens, in the development of adaptive immunity, and are possibly the major determinants of the susceptibility to infections. To study the resistance pattern in different breeds of chicken, a comprehensive understanding of TLR4 signalling pathways is required. We investigated the TLR-4 pathway regulated gene expressions in PBMCs of chicken breeds of Broiler (Cobb), Aseel, Dahlem Red and Ghagus upon LPS treatment using Quantitative RT-PCR approach. Several genes were found to be up regulated in both TLR-induced MyD88-dependent and MyD88-independent pathways. These genes include TLR4 (Toll-like receptor 4), MyD88 (Myeloid differentiation primary response gene 88), TRAF6 (TNF receptor associated factor 6), TRIF (TIR domain containing adapter inducing interferon beta), the transcription factors NFkB (Nuclear factor kappa B), IRF7 (Interferon regulatory factor 7) and IFN ß (Interferon beta). We have also studied inflammatory cytokines such as IL2, IL6, IL8, IL1 ß and TNF α to further understand the downstream signalling of TLR4 pathway. These results showed that higher expression of TLR signalling activation via both MyD88-dependent and TRIF-dependent pathways are more beneficial to chicken mononuclear cells mediated innate immunity. We observed TRIF dependent pathway in Aseel and Ghagus breeds. Our results are in concurrent with general observation that Aseel breed is comparatively more resistant, Ghagus and broilers are moderately resistant and Dahlem Red is comparatively more susceptible to bacterial infections.

MicroRNAs as Haematopoiesis Regulators.

The production of different types of blood cells including their formation, development, and differentiation is collectively known as haematopoiesis. Blood cells are divided into three lineages erythriod (erythrocytes), lymphoid (B and T cells), and myeloid (granulocytes, megakaryocytes, and macrophages). Haematopoiesis is a complex process regulated by several mechanisms including microRNAs (miRNAs). miRNAs are small RNAs which regulate the expression of a number of genes involved in commitment and differentiation of hematopoietic stem cells. Evidence shows that miRNAs play an important role in haematopoiesis; for example, myeloid and erythroid differentiation is blocked by the overexpression of miR-15a. miR-221, miR-222, and miR-24 inhibit the erythropoiesis, whereas miR-150 plays a role in B and T cell differentiation. miR-146 and miR-10a are downregulated in megakaryopoiesis. Aberrant expression of miRNAs was observed in hematological malignancies including chronic myelogenous leukemia, chronic lymphocytic leukemia, multiple myelomas, and B cell lymphomas. In this review we have focused on discussing the role of miRNA in haematopoiesis.