Inflammasomes and the Maintenance of Hematopoietic Homeostasis: New Perspectives and Opportunities

Hematopoietic stem cells (HSCs) regularly produce various blood cells throughout life via their self-renewal, proliferation, and differentiation abilities. Most HSCs remain quiescent in the bone marrow (BM) and respond in a timely manner to either physiological or pathological cues, but the underlying mechanisms remain to be further elucidated. In the past few years, accumulating evidence has highlighted an intermediate role of inflammasome activation in hematopoietic maintenance, post-hematopoietic transplantation complications, and senescence. As a cytosolic protein complex, the inflammasome participates in immune responses by generating a caspase cascade and inducing cytokine secretion. This process is generally triggered by signals from purinergic receptors that integrate extracellular stimuli such as the metabolic factor ATP via P2 receptors. Furthermore, targeted modulation/inhibition of specific inflammasomes may help to maintain/restore adequate hematopoietic homeostasis. In this review, we will first summarize the possible relationships between inflammasome activation and homeostasis based on certain interesting phenomena. The cellular and molecular mechanism by which purinergic receptors integrate extracellular cues to activate inflammasomes inside HSCs will then be described. We will also discuss the therapeutic potential of targeting inflammasomes and their components in some diseases through pharmacological or genetic strategies.     

Identification and functional study of cytokines and chemokines involved in tumorigenesis

Tumorigenesis may be affected by various cellular components in the tumor cells and/or by tumor microenvironmental factors. Cytokines, including chemokines (chemotactic cytokines) are polypeptides or small soluble proteins generated by leukocytes and non-leukocytes, including cancer cells and stromal cells, for example, fibroblasts, mesenchymal stem cells (MSCs) and epithelial cells. Cytokines exert their functions on the cells that secrete them, on nearby cells, or on distant cells. Chemokines have expanded beyond their initial roles in impinging on every aspect of the immune system and leukocyte biology. They display multifunctional effects for regulating angiogenesis, tumor cell proliferation and apoptosis, mediating tumor cell metastasis in an organ-specific manner. This review will focus on the structural and functional aspects of chemokines as well as the roles that cytokines and their receptors play in angiogenesis, tumor invasion and metastasis, and discuss their potential value as important therapeutic targets for intervention in cancer.     

Targeting Human C‐Type Lectin‐like Molecule‐1 (CLL1) with a Bispecific Antibody for Immunotherapy of Acute Myeloid Leukemia

Acute myeloid leukemia (AML), which is the most common acute adult leukemia and the second most common pediatric leukemia, still has a poor prognosis. Human C‐type lectin‐like molecule‐1 (CLL1) is a recently identified myeloid lineage restricted cell surface marker, which is overexpressed in over 90 % of AML patient myeloid blasts and in leukemic stem cells. Here, we describe the synthesis of a novel bispecific antibody, αCLL1‐αCD3, using the genetically encoded unnatural amino acid, p‐acetylphenylalanine. The resulting αCLL1‐αCD3 recruits cytotoxic T cells to CLL1 positive cells, and demonstrates potent and selective cytotoxicity against several human AML cell lines and primary AML patient derived cells in vitro. Moreover, αCLL1‐αCD3 treatment completely eliminates established tumors in an U937 AML cell line xenograft model. These results validate the clinical potential of CLL1 as an AML‐specific antigen for the generation of a novel immunotherapeutic for AML.     

C-type lectins on dendritic cells and their interaction with pathogen-derived and endogenous glycoconjugates

Human C-type lectin receptors (CLRs) characteristically bind glycosylated ligands in a Ca(2+)-dependent way via their carbohydrate recognition domain (CRD). Their carbohydrate preference is dependent on the amino acid sequence in the CRD domain and on the ability and flexibility of the CRD domain to accommodate sugar moieties that are located at different distances from each other in the glycoconjugate. Although microbial and vertebrate cells are able to produce similar polysaccharide chains, the density of carbohydrates on microbes is much higher compared to vertebrate cells. Despite this difference, carbohydrates present on both cell types can be recognized by the CLRs. These receptors are predominantly expressed by antigen presenting cells such as dendritic cells. In addition to the Toll-like receptor family, CLRs function as pattern recognition receptors by recognizing glycosylated patterns on pathogens. This usually results in internalization of the pathogen, lysosomal degradation and subsequent loading of pathogen-derived peptides into major histocompatibility complex molecules for antigen presentation. However, several pathogens have developed ways to exploit the CLRs to evade immune eradication by for example escaping from the lysosomal degradation pathway or by inducing anti-inflammatory cytokines. When CLRs bind endogenous glycosylated ligands they mediate several processes like cell-cell adhesion and clearance of aberrant cells like tumor cells or apoptotic cells.     

Single-cell sphingosine kinase activity measurements in primary leukemia

Sphingosine kinase (SK) is a promising therapeutic target in a number of cancers, including leukemia. Traditionally, SK has been measured in bulk cell lysates, but this technique obscures the cellular heterogeneity present in this pathway. For this reason, SK activity was measured in single cells loaded with a fluorescent sphingosine reporter. An automated capillary electrophoresis (CE) system enabled rapid separation and quantification of the phosphorylated and nonphosphorylated sphingosine reporter in single cells. SK activity was measured in tissue-cultured cells derived from chronic myelogenous leukemia (K562), primary peripheral blood mononuclear cells (PBMCs) from three patients with different forms of leukemia, and enriched leukemic blasts from a patient with acute myeloid leukemia (AML). Significant intercellular heterogeneity existed in terms of the degree of reporter phosphorylation (as much as an order of magnitude difference), the amount of reporter uptake, and the metabolites formed. In K562 cells, the average amount of reporter converted to the phosphorylated form was 39?±?26 % per cell. Of the primary PBMCs analyzed, the average amount of phosphorylated reporter was 16?±?25 %, 11?±?26 %, and 13?±?23 % in a chronic myelogenous leukemia (CML) patient, an AML patient, and a B-cell acute lymphocytic leukemia (B-ALL) patient, respectively. These experiments demonstrated the challenge of studying samples comprised of multiple cell types, with tumor blasts present at 5 to 87 % of the cell population. When the leukemic blasts from a fourth patient with AML were enriched to 99 % of the cell population, 19?±?36 % of the loaded sphingosine was phosphorylated. Thus, the diversity in SK activity remained even in a nearly pure tumor sample. These enriched AML blasts loaded significantly less reporter (0.12?±?0.2 amol) relative to that loaded into the PBMCs in the other samples (≥1 amol). The variability in SK signaling may have important implications for SK inhibitors as therapeutics for leukemia and demonstrates the value of single-cell analysis in characterizing the nature of oncogenic signaling in cancer. Figure

Phosphorylation of a fluorescent sphingosine kinase reporter was used to measure single-cell SK activity in primary cells from leukemic patients. Peripheral blood mononuclear cells as well as enriched leukemic blasts were analyzed.     

Monocyte-specific esterase isoenzyme demonstrated by isoelectric focusing

Using horizontal thin-layer isoelectric focusing in polyacrylamide gels, we separated the isoenzymes of carboxylic esterase (EC 3.1.1.1) of cell extracts prepared from human hematopoietic cells. Isoenzyme bands were visualized by staining with naphthol ester as substrate and coupling to an azo dye. Staining intensities of isoenzymes were quantified by densitometric scanning. On isoelectric focusing in a pH 2-11 gradient, distinct esterase isoenzyme profiles could be discerned and correlated to various types of normal hematopoietic cells and their leukemic counterparts. One unique isoenzyme, termed monoband, could be clearly identified on the basis of its isoelectric point (pI 6.0), its strong expression by normal and malignant monocytes and its complete and selective inhibition by sodium fluoride. This band was only found in monocytes of either normal or leukemic origin, but not in lymphoid or myeloid cells. The monocyte esterase could be inhibited by sodium fluoride whereas other isoenzyme bands were resistant to this inhibition. However, the specificity of this inhibitory reaction was relative, depending on the concentration of sodium fluoride. Compared with normal monocytes, leukemic monocytes often showed an overexpression of the mono-bands. Dilution experiments established the distinct prominence of the mono-band which could be detected among the other isoenzymes even when only 1% of the total cell population consisted of monocytes. Immature myeloid, but mono-band negative leukemic cells whose arrest of differentiation can be overcome by in vitro 12-O-tetradecanoylphorbol 13-acetate-promoted differentiation to more mature cells, could be induced to express the mono-band which paralleled their maturation to monocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

ArtinM Binding Effinities and Kinetic Interaction with Leukemia Cells: A Quartz Crystal Microbalance Bioelectroanalysis on the Cytotoxic Effect

ArtinM, a D‐mannose‐binding lectin from Artocarpus heterophyllus (jackfruit), interacts with N‐glycosylated receptors on the surface of several cells of hematopoietic origin, triggering cell migration, degranulation, and cytokine release. Because malignant transformation is often associated with altered expression of cell surface glycans, we evaluated the interaction of ArtinM with human myelocytic leukemia cells by Quartz Cristal Microbalance (QCM) and cross compared this investigation with cellular responses (cytotoxicity) to lectin binding by the colorimetric assay MTT, which is a standard method in biology. QCM analysis was able to provide additional information (not possible to be obtained by MTT) that impact on the knowledge of medical biology related to leukemic cells. For instance, it was shown that association rate constant of ArtinM and cellular membranes (obtained from QCM) of leukemia cell varies across the myeloid leukemia cell lines, decreasing as cytotoxic effect increases. Meanwhile no differences were observed for dissociation rate constants so that the equilibrium binding affinity constant was observed to be a direct function of the association rate event. It was supposed that ArtinM cytotoxicity is affected by the association time with glycans on the cellular membranes of leukemia cells in a way that the higher it is the association time (the lower was the association rate constant) the higher is the ArtinM cytotoxicity over the leukemia cell lines.     

The potential use of mesenchymal stem cells in hematopoietic stem cell transplantation

In the last 10 years, mesenchymal stem cells (MSCs) have emerged as a therapeutic approach to regenerative medicine, cancer, autoimmune diseases, and many more due to their potential to differentiate into various tissues, to repair damaged tissues and organs, and also for their immunomodulatory properties. Findings in vitro and in vivo have demonstrated immune regulatory function of MSCs and have facilitated their application in clinical trials, such as those of autoimmune diseases and chronic inflammatory diseases. There has been an increasing interest in the role of MSCs in allogeneic hematopoietic stem cell transplantation (HSCT), including hematopoietic stem cell engraftment and the prevention and treatment of graft-versus-host disease (GVHD), and their therapeutic potential has been reported in numerous clinical trials. Although the safety of clinical application of MSCs is established, further modifications to improve their efficacy are required. In this review, we summarize advances in the potential use of MSCs in HSCT. In addition, we discuss their use in clinical trials of the treatment of GVHD following HSCT, the immunomodulatory capacity of MSCs, and their regenerative and therapeutic potential in the field of HSCT.     

The Anti-Leukemic Activity of Natural Compounds

The use of biologically active compounds has become a realistic option for the treatment of malignant tumors due to their cost-effectiveness and safety. In this review, we aimed to highlight the main natural biocompounds that target leukemic cells, assessed by in vitro and in vivo experiments or clinical studies, in order to explore their therapeutic potential in the treatment of leukemia: acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL). It provides a basis for researchers and hematologists in improving basic and clinical research on the development of new alternative therapies in the fight against leukemia, a harmful hematological cancer and the leading cause of death among patients.     

Intervention with costimulatory pathways as a therapeutic approach for graft-versus-host disease

Graft-versus-host disease (GVHD) is mediated by mature donor T cells contained in the hematopoietic stem cell graft. During the development of GVHD, signaling through a variety of costimulatory receptors plays an important role in allogeneic T cell responses. Even though delivery of costimulatory signals is a prerequisite for full activation of donor T cells in the phase of their interactions with host APCs, their involvement with GVHD might occur over multiple stages. Like many other aspects of GVHD, promise of therapeutic interventions with costimulatory pathways has been gleaned from preclinical models. In this review, I summarize some of the advances in roles of costimulatory molecules in GVHD pathophysiology and discuss preclinical approaches that warrant further exploration in the clinic, focusing on novel strategies to delete pathogenic T cells.     

Monitoring the Cascade of Tumor-specific Immune Response in vivo via Chemoenzymatic Proximity Labeling

Monitoring the highly dynamic and complex immune response remains a great challenge owing to the lack of reliable and specific approaches. Here, we develop a strategy to monitor the cascade of tumor immune response through the cooperation of pore-forming alginate gel with chemoenzymatic proximity-labeling. A macroporous gel containing tumor-associated antigens, adjuvants, and pro-inflammatory cytokines is utilized to recruit endogenous DCs and enhance their maturation in vivo. The mature DCs are then modified with GDP-fucose-fucosyltransferase (GDP-Fuc-Fuct) via the self-catalysis of fucosyltransferase (Fuct). Following the migration of the obtained Fuct-DCs to the draining lymph nodes (dLNs), the molecular recognition mediated interaction of DCs and T cells leads to the successful decoration of T cells with GDP-Fuc-azide through the Fuct catalyzed proximity-labeling. Therefore, the activated tumor-specific T cells in dLNs and tumors can be identified through bioorthogonal labeling, opening up a new avenue for studying the immune mechanism of tumors in situ.     

Ryk modulates the niche activity of mesenchymal stromal cells by fine-tuning canonical Wnt signaling

The importance of modulating the intensity of Wnt signaling has been highlighted in various biological models, but their mechanisms remain unclear. In this study, we found that Ryk—an atypical Wnt receptor with a pseudokinase domain—has a Wnt-modulating effect in bone marrow stromal cells to control hematopoiesis-supporting activities. We first found that Ryk is predominantly expressed in the mesenchymal stromal cells (MSCs) of the bone marrow (BM) compared with hematopoietic cells. Downregulation of Ryk in MSCs decreased their clonogenic activity and ability to support self-renewing expansion of primitive hematopoietic progenitors (HPCs) in response to canonical Wnt ligands. In contrast, under high concentrations of Wnt, Ryk exerted suppressive effects on the transactivation of target genes and HPC-supporting effects in MSCs, thus fine-tuning the signaling intensity of Wnt in BM stromal cells. This ability of Ryk to modulate the HPC-supporting niche activity of MSCs was abrogated by induction of deletion mutants of Ryk lacking the intracellular domain or extracellular domain, indicating that the pseudokinase-containing intracellular domain mediates the Wnt-modulating effects in response to extracellular Wnt ligands. These findings indicate that the ability of the BM microenvironment to respond to extracellular signals and support hematopoiesis may be fine-tuned by Ryk via modulation of Wnt signaling intensity to coordinate hematopoietic activity.Subject terms: Mesenchymal stem cells, Mesenchymal stem cells, Haematopoietic stem cells     

Micropatterned supported membranes as tools for quantitative studies of the immunological synapse

In living cells, membrane receptors transduce ligand binding into signals that initiate proliferation, specialization, and secretion of signaling molecules. Spatial organization of such receptors regulates signaling in several key immune cell interactions. In the most extensively studied of these, a T cell recognizes membrane-bound antigen presented by another cell, and forms a complex junction called the "immunological synapse" (IS). The importance of spatial organization at the IS and the quantification of its effect on signaling remain controversial topics. Researchers have successfully investigated the IS using lipid bilayers supported on solid substrates as model antigen-presenting membranes. Recent technical developments have enabled micron- and nanometre-scale patterning of supported lipid bilayers (SLBs) and their application to immune cell studies with provocative results, including spatial mutation of the IS. In this tutorial review, we introduce the IS; discuss SLB techniques, including micropatterning; and discuss various methods used to perturb and quantify the IS.     

The Intestinal Fatty Acid-Enteroendocrine Interplay,Emerging Roles for Olfactory Signaling and Serotonin Conjugates

Intestinal enteroendocrine cells (EECs) respond to fatty acids from dietary and microbial origin by releasing neurotransmitters and hormones with various paracrine and endocrine functions. Much has become known about the underlying signaling mechanisms, including the involvement of G-protein coupled receptors (GPCRs), like free fatty acids receptors (FFARs). This review focusses on two more recently emerging research lines: the roles of odorant receptors (ORs), and those of fatty acid conjugates in gut. Odorant receptors belong to a large family of GPCRs with functional roles that only lately have shown to reach beyond the nasal-oral cavity. In the intestinal tract, ORs are expressed on serotonin (5-HT) and glucagon-like-peptide-1 (GLP-1) producing enterochromaffin and enteroendocrine L cells, respectively. There, they appear to function as chemosensors of microbiologically produced short-, and branched-chain fatty acids. Another mechanism of fatty acid signaling in the intestine occurs via their conjugates. Among them, conjugates of unsaturated long chain fatty acids and acetate with 5-HT, N-acyl serotonins have recently emerged as mediators with immune-modulatory effects. In this review, novel findings in mechanisms and molecular players involved in intestinal fatty acid biology are highlighted and their potential relevance for EEC-mediated signaling to the pancreas, immune system, and brain is discussed.     

Interactions between tumor-derived proteins and Toll-like receptors

Damage-associated molecular patterns (DAMPs) are danger signals (or alarmins) alerting immune cells through pattern recognition receptors (PRRs) to begin defense activity. Moreover, DAMPs are host biomolecules that can initiate a noninflammatory response to infection, and pathogen-associated molecular pattern (PAMPs) perpetuate the inflammatory response to infection. Many DAMPs are proteins that have defined intracellular functions and are released from dying cells after tissue injury or chemo-/radiotherapy. In the tumor microenvironment, DAMPs can be ligands for Toll-like receptors (TLRs) expressed on immune cells and induce cytokine production and T-cell activation. Moreover, DAMPs released from tumor cells can directly activate tumor-expressed TLRs that induce chemoresistance, migration, invasion, and metastasis. Furthermore, DAMP-induced chronic inflammation in the tumor microenvironment causes an increase in immunosuppressive populations, such as M2 macrophages, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs). Therefore, regulation of DAMP proteins can reduce excessive inflammation to create an immunogenic tumor microenvironment. Here, we review tumor-derived DAMP proteins as ligands of TLRs and discuss their association with immune cells, tumors, and the composition of the tumor microenvironment.Subject terms: Immune cell death, Cancer microenvironment     

The role of adenosine receptor agonists in regulation of hematopoiesis

The review summarizes data evaluating the role of adenosine receptor signaling in murine hematopoietic functions. The studies carried out utilized either non-selective activation of adenosine receptors induced by elevation of extracellular adenosine or by administration of synthetic adenosine analogs having various proportions of selectivity for a particular receptor. Numerous studies have described stimulatory effects of non-selective activation of adenosine receptors, manifested as enhancement of proliferation of cells at various levels of the hematopoietic hierarchy. Subsequent experimental approaches, considering the hematopoiesis-modulating action of adenosine receptor agonists with a high level of selectivity to individual adenosine receptor subtypes, have revealed differential effects of various adenosine analogs. Whereas selective activation of A? receptors has resulted in suppression of proliferation of hematopoietic progenitor and precursor cells, that of A? receptors has led to stimulated cell proliferation in these cell compartments. Thus, A? and A? receptors have been found to play a homeostatic role in suppressed and regenerating hematopoiesis. Selective activation of adenosine A? receptors has been found to act curatively under conditions of drug- and radiation-induced myelosuppression. The findings in these and further research areas will be summarized and mechanisms of hematopoiesis-modulating action of adenosine receptor agonists will be discussed.     

Recent advances with TLR2-targeting lipopeptide-based vaccines

The next generation of vaccines are being rationally designed according to rules that govern the way in which antigen is recognised by and stimulates the immune system. Amongst the first cells that encounter potentially dangerous agents such as viruses and bacteria are cells of the innate immune system, such as dendritic cells, that are widely distributed throughout the body including the skin. These cells patrol most tissues and have on their surface an array of receptors that have evolved to recognise many of the surface features of pathogens including the lipids and carbohydrates of structural lipoproteins, glycolipids and glycoproteins. Once encountered, recognised and engaged by a particular receptor on the dendritic cell, pathogenic material may then be transported inside the cell and processed for presentation to cells of the adaptive immune system. The result of this concert of events is a specific cellular or antibody response to particular epitopes of the invading pathogen. If then ways can be found to specifically target dendritic cells, through their specific receptors, then the efficacy and potency of vaccines could well be greatly improved. This review covers some of the approaches that we and others are pursuing in order to achieve this result.     

Chrysophanol Attenuates Manifestations of Immune Bowel Diseases by Regulation of Colorectal Cells and T Cells Activation In Vivo

Inflammatory bowel disease (IBD) is an immune disorder that develops due to chronic inflammation in several cells. It is known that colorectal and T cells are mainly involved in the pathogenesis of IBD. Chrysophanol is an anthraquinone family member that possesses several bioactivities, including anti-diabetic, anti-tumor, and inhibitory effects on T cell activation. However, it is unknown whether chrysophanol suppresses the activity of colorectal cells. In this study, we found that chrysophanol did not induce cytotoxicity in HT-29 colorectal cells. Pre-treatment with chrysophanol inhibited the mRNA levels of pro-inflammatory cytokines in tumor necrosis factor-α (TNF-α)-stimulated HT-29 cells. Western blot analysis revealed that pre-treatment with chrysophanol mitigates p65 translocation and the mitogen-activated protein kinase (MAPK) pathway in activated HT-29 cells. Results from the in vivo experiment confirmed that oral administration of chrysophanol protects mice from dextran sulfate sodium (DSS)-induced IBD. Chrysophanol administration attenuates the expression of pro-inflammatory cytokines in colon tissues of the DSS-induced IBD model. In addition, we found that oral administration of chrysophanol systemically decreased the expression of effector cytokines from mesenteric lymph nodes. Therefore, these data suggest that chrysophanol has a potent modulatory effect on colorectal cells as well as exhibiting a beneficial potential for curing IBD in vivo.     

Assessment of Growth Factors,Cytokines, and Cellular Markers in Saliva of Patients with Trigeminal Neuralgia

We proposed to perform a comparative analysis of growth factors, cytokines, and chemokine receptors on the salivary cells in the saliva obtained from trigeminal neuralgia (TN) and normal subjects. Saliva was collected from TN and healthy subjects. Salivary cells were isolated by centrifugation. The expression of the cell surface marker was analyzed by flow cytometry. A cytometric bead array was done to measure the levels of cytokines and growth factors on the flow cytometer. Saliva from TN subjects showed lower growth factor levels of Angiopoietin-2, bFGF, HGF, SCF, TGF-α, and VEGF and higher cytokine levels of IL-1β, TNF-α, CCL2, IL-17A, IL-6, and CXCL8, as well as higher expression levels of chemokine receptors CCR1 (CD191), CR3 (CD11b), CCR2 (CD192), CXCR5 (CD185), and CCR5 (CD196) in the cells from TN saliva. A certain set of cytokines and growth factors in the saliva, as well as chemokine receptors on salivary cells, could be a useful tool in the diagnostics and prognostics of trigeminal neuralgia. Trigeminal neuralgia is one of the significant pathological conditions in the class of chronic diseases around the world. Many targeted approaches are being tried by various research groups to utilize the information of the inflammatory microenvironment to resolve the pathology of chronic TN.