Bone Marrow-derived Precursors Research Articles

40 Preclinical evaluation of a novel dendritic cell vaccine for kidney cancer

Abstract Background Dendritic cell (DC)-based vaccines have been previously shown to promote therapeutic T cell responses in both preclinical tumor models and in cancer patients, where extended patient overall survival has been noted in many cases. The goal of the present study is to develop a novel DC-based vaccine as an effective immuno-oncology (IO) agent for the prevention/treatment of kidney cancer. We previously demonstrated that actin-binding protein profilin1 (Pfn1) is overexpressed in tumor-associated vascular endothelial cells (ECs), where it may serve as a prognostic factor in human clear cell renal cancer (ccRCC). We further established a direct causal relationship between EC Pfn1 dysregulation, immune microenvironment alterations, and tumor progression in mouse models of RCC. The current work specifically explores whether Pfn1-targeted DC-based vaccines are effective in preventing orthotopic RCC in mice. Methods To generate Pfn1-targeted DC vaccines, we harvested and matured DCs from Balb/C mouse bone marrow-derived precursor cells in cultures containing rmIL4 and rmGM-CSF. DCs loaded with individual Pfn1 synthetic peptides (these sequences were identified using three MHC class I/II peptide-binding algorithms) were injected into syngeneic mice on days 0, 7 and 14. Spleens were harvested from immunized mice on day 21 to isolate CD4+ and CD8+ T cells, with T cells then stimulated with unmanipulated DCs or DCs pulsed with individual immunizing peptides to detect specific T cell activation, as measured by IFN-gamma release quantitated by ELISA. Tissue histology was performed to assess immune-related adverse events (iRAE) in vaccinated mice as a safety index. For cancer studies, Balb/c mice were immunized with DCs loaded with pooled Pfn1 peptides a few days prior to establishing either subcutaneous or orthotopic RCC tumors. Tumor-bearing mice were subjected to an identical booster vaccine prior one week later in advance of euthanasia and study end-point analyses. Results Our studies show that vaccination of Balb/c mice with DCs pulsed with Pfn1 peptides elicit specific CD4+ and/or CD8+ T cell responses without promoting irAEs. When applied in the prophylactic setting, DC-Pfn1 peptide vaccines substantially slow the growth of subcutaneous RCC tumors. In support of these findings, we have also developed preliminary data supporting the therapeutic effects of DC/Pfn1-peptide vaccines in a murine orthotopic model of RCC. Conclusions In summary, our studies provide first evidence for Pfn1-targeted IO agent in the cancer setting. Given that the survival benefit for dual immune-check point inhibitors (ICI)/anti-angiogenic therapy remains modest and is limited to a small minority of ccRCC patients, Pfn1-targeted vaccines could represent a novel therapeutic agent for improved patient outcomes when applied alone or in combination with in-clinic ICI agents. DOD CDMRP Funding: yes

Cortactin controls bone homeostasis through regulating the differentiation of osteoblasts and osteoclasts.

Cortactin (CTTN), a cytoskeletal protein and substrate of Src kinase, is implicated in tumor aggressiveness. However, its role in bone cell differentiation remains unknown. The current study revealed that CTTN was upregulated during osteoblast and adipocyte differentiation. Functional experiments demonstrated that CTTN promoted the in vitro differentiation of mesenchymal stem/progenitor cells into osteogenic and adipogenic lineages. Mechanistically, CTTN was able to stabilize the protein level of mechanistic target of rapamycin kinase (mTOR), leading to the activation of mTOR signaling. In-depth investigation revealed that CTTN could bind with casitas B lineage lymphoma-c (c-CBL) and counteract the function of c-CBL, a known E3 ubiquitin ligase responsible for the proteasomal degradation of mTOR. Silencing c-Cbl alleviated the impaired differentiation of osteoblasts and adipocytes caused by CTTN siRNA, while silencing mTOR mitigated the stimulation of osteoblast and adipocyte differentiation induced by CTTN overexpression. Notably, transplantation of CTTN-silenced bone marrow stromal cells (BMSCs) into the marrow of mice led to a reduction in trabecular bone mass, accompanied by a decrease in osteoblasts and an increase in osteoclasts. Furthermore, CTTN-silenced BMSCs expressed higher levels of receptor activator of nuclear factor κB ligand (RANKL) than control BMSCs did and promoted osteoclast differentiation when cocultured with bone marrow-derived osteoclast precursor cells. This study provides evidence that CTTN favors osteoblast differentiation by counteracting the c-CBL-induced degradation of mTOR and inhibits osteoclast differentiation by downregulating the expression of RANKL. It also suggests that maintaining an appropriate level of CTTN expression may be advantageous for maintaining bone homeostasis.

Tissue-Resident Macrophages in Solid Organ Transplantation: Harmful or Protective?

Transplanted organs carry donor immune cells into the recipient, the majority of which are tissue-resident macrophages (TRMs). The role they play in guiding the fate of the transplanted organ toward acceptance or rejection remains elusive. TRMs originate from both embryonic and bone marrow-derived precursors. Embryo-derived TRMs retain the embryonic capability to proliferate, so they are able to self-renew and, theoretically, persist for extended periods of time after transplantation. Bone marrow-derived TRMs do not proliferate and must constantly be replenished by adult circulating monocytes. Recent studies have aimed to clarify the different roles and interactions between donor TRMs, recipient monocytes, and monocyte-derived macrophages (MFs) after organ transplantation. This review aims to shed light on how MFs affect the fate of a transplanted organ by differentiating between the role of donor TRMs and that of MFs derived from graft infiltrating monocytes.

In situ co-deposition synthesis for collagen-Astragalus polysaccharide composite with intrafibrillar mineralization as potential biomimetic-bone repair materials.

A hybrid material possessing both componential and structural imitation of bone tissue is the preferable composites for bone defect repair. Inspired by the microarchitecture of native bone, this work synthesized in vitro a functional mineralized collagen fibril (MCF) material by utilizing the method of in situ co-precipitation, which was designed to proceed in the presence of Astragalus polysaccharide (APS), thus achieving APS load within the biomineralized collagen-Astragalus polysaccharide (MCAPS) fibrils. Transmission electron microscope (TEM), selected area electron diffraction (SAED) and scanning electronic microscopy (SEM) identified the details of the intrafibrillar mineralization of the MCAPS fibrils, almost mimicking the secondary level of bone tissue microstructure. A relatively uniform and continuous mineral layer formed on and within all collagen fibrils and the mineral phase was identified as typical weak-crystalline hydroxyapatite (HA) with a Ca/P ratio of about 1.53. The proliferation of bone marrow-derived mesenchymal stem cells (BMSC) and mouse embryo osteoblast precursor cells (MC3T3-E1) obtained a significant promotion by MCAPS. As for the osteogenic properties of MCAPS, a distinct increase in the alkaline phosphatase (ALP) activity and the number of calcium nodules (CN) in BMSC and MC3T3-E1 was detected. The up-regulation of three osteogenic-related genes of RUNX-2, BMP-2 and OCN were confirmed via reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to further verify the osteogenic performance promotion of MCAPS. A period of 14 days of culture demonstrated that MCAPS-L exhibited a preferable efficacy in enhancing ALP activity and CN quantity, as well as in promoting the expression of osteogenic-related genes over MCAPS-M and MCAPS-H, indicating that a lower dose of APS within the material of MCAPS is more appropriate for its osteogenesis promotion properties.

Flow cytometry-assisted analysis of phenotypic maturation markers on an immortalized dendritic cell line.

Dendritic cells (DCs), and especially so conventional type I DCs (cDC1s), are fundamental regulators of anticancer immunity, largely reflecting their superior ability to engulf tumor-derived material and process it for cross-presentation on MHC Class I molecules to CD8+ cytotoxic T lymphocytes (CTLs). Thus, investigating key DC functions including (but not limited to) phagocytic capacity, expression of CTL-activating ligands on the cell surface, and cross-presentation efficacy is an important component of multiple immuno-oncology studies. Unfortunately, DCs are terminally differentiated cells, implying that they cannot be propagated indefinitely in vitro and hence must be generated ad hoc from circulating or bone marrow-derived precursors, which presents several limitations. Here, we propose a simple, cytofluorometric method to quantify phenotypic activation markers including CD80, CD86 and MHC class II molecules on the surface of a conditionally immortalized immature DC line that can be indefinitely propagated in vitro but also driven into maturation at will with a simple change in culture conditions. Upon appropriate scaling and automatization, this approach is compatible with high-throughput screening programs for the discovery of novel DC activators that do not suffer from batch variability and other limitations associated with the generation of fresh DCs.

Differentiation-inducing effect of osteoclast microgrooves for the purpose of three-dimensional design of regenerated bone.

In vivo bone remodeling is promoted by the balance between osteoclast and osteoblast activity. Conventional research on bone regeneration has mainly focused on increasing osteoblast activity, with limited studies on the effects of scaffold topography on cell differentiation. Here, we examined the effect of microgroove-patterned substrate with spacings ranging from 1 to 10 μm on the differentiation of rat bone marrow-derived osteoclast precursors. Tartrate-resistant acid phosphatase (TRAP) staining and relative gene expression quantification showed that osteoclast differentiation was enhanced in substrate with 1 µm microgroove spacing compared with that in the other groups. Additionally, the ratio of podosome maturation stages in substrate with 1 μm microgroove spacing exhibited a distinct pattern, which was characterized by an increase in the ratio of belts and rings and a decrease in that of clusters. However, myosin II abolished the effects of topography on osteoclast differentiation. Overall, these showed that the reduction of myosin II tension in the podosome core by an integrin vertical vector increased podosome stability and promoted osteoclast differentiation in substrates with 1 μm microgroove spacing, including that microgroove design plays an important role in scaffolds for bone regeneration. STATEMENT OF SIGNIFICANCE: Reduction of myosin II tension in the podosome core, facilitated by an integrin vertical vector, resulted in an enhanced osteoclast differentiation, concomitant with an increase in podosome stability within 1-μm-spaced microgrooves. These findings are anticipated to serve as valuable indicators for the regulation of osteoclast differentiation through the manipulation of biomaterial surface topography in tissue engineering. Furthermore, this study contributes to the lucidation of the underlying mechanisms governing cellular differentiation by providing insights into the impact of the microtopographical environment.

Platelet-Derived Growth Factor-BB Priming Enhances Vasculogenic Capacity of Bone Marrow-Derived Endothelial Precursor Like Cells.

Human endothelial progenitor cells (EPCs) were first identified in the peripheral blood and later in the cord blood and bone marrow (BM) with different vascularization capacity and different surface marker profiles. However, their identity and functional roles in neovascularization have not been clearly demonstrated in vivo and in vitro. Characterization of BM-EPC like cells were performed by fluorescence-activated cell sorting, immunofluorescence staining, enzyme-linked immunosorbent assay, Matrigel tube formation assay,andwestern blot analysis. BM-EPC like cells were identified by selective adhesion to fibronectin and collagen from BM mononuclear cells, which generate fast-growing colonies with spindle morphology, express surface markers of CD105, vWF, UEA-I lectin binding, secrete HGF, VEGF, TGF-beta1 but can be distinguished from circulating EPC and endothelial cells by no expression of surface markers such as CD31, CD309, CD45, and CD34. These BM-EPC like cells shared many cell surface markers of BM-mesenchymal stem cells (MSC) but also can be distinguished by their vasculogenic property and other unique surface markers. Furthermore, the vasculogenic capacity of BM-EPC like cells were enhanced by co-culture of BM-MSC or PDGF-BB priming. PDGF-BB stimulated cell migration, proliferation, and secretion of laminin β-1, which was proposed as one of the mechanisms involved in the better vascularization of BM-EPC like cells. PDGF-BB priming may be applied to improve the potency and function of BM-EPC like cells as vasculogenic cell therapy for the ischemic vascular repair.

Tingible body macrophages arise from lymph node-resident precursors and uptake B cells by dendrites.

Antibody affinity maturation depends on the formation of germinal centers (GCs) in lymph nodes. This process generates a massive number of apoptotic B cells, which are removed by a specialized subset of phagocytes, known as tingible body macrophages (TBMs). Although defects in these cells are associated with pathological conditions, the identity of their precursors and the dynamics of dying GC B cell disposal remained unknown. Here, we demonstrate that TBMs originate from pre-existing lymph node-resident precursors that enter the lymph node follicles in a GC-dependent manner. Intravital imaging shows that TBMs are stationary cells that selectively phagocytose GC B cells via highly dynamic protrusions and accommodate the final stages of B cell apoptosis. Cell-specific depletion and chimeric mouse models revealed that GC B cells drive TBM formation from bone marrow-derived precursors stationed within lymphoid organs prior to the immune challenge. Understanding TBM dynamics and function may explain the emergence of various antibody-mediated autoimmune conditions.

A Beginner's Guide to T Cell Development.

T lymphocytes (T cells) are essential components of the adaptive immune system; they serve multiple functions in responses to pathogens and to ensure immune homeostasis. Written for readers first entering this field of study, this chapter is a brief overview of the development of T cells in the thymus, from the entry of thymus-settling bone marrow-derived precursors to the egress of mature T cells. Surveyed topics include the differentiation and expansion of early precursors, the generation of the T cell antigen receptor repertoire, the selection of αβ T cell precursors, and their acquisition of functional competency.

Short-Term Administration of HIV Protease Inhibitor Saquinavir Improves Skull Bone Healing with Enhanced Osteoclastogenesis.

Using immunomodulatory methods to address the challenging issue of craniofacial bone repair may be a potentially effective approach. The protease inhibitor saquinavir has been shown to inhibit the inflammatory response by targeting the toll-like receptor 4/myeloid differentiation primary response complex. Independently, inhibition of toll-like receptor 4 or myeloid differentiation primary response led to enhanced skull bone repair. Therefore, the authors aimed to investigate the effects of saquinavir on skull bone healing. The effects of saquinavir on skull bone healing were assessed by means of gene expression, histology, immunohistochemistry, and tomography in a mouse calvarial defect model. Subsequently, the role of saquinavir in cell viability, migration, and osteogenic and osteoclastogenic differentiation was also evaluated in vitro. One-week saquinavir administration improved skull bone healing based on micro-computed tomographic and histomorphometric analyses. Compared to the vehicle control, 1-week saquinavir treatment (1) enhanced osteoclast infiltration (tartrate-resistant acid phosphatase staining) at day 7, but not at days 14 and 28; (2) induced more CD206 + M2 macrophage infiltration, but not F4/80 + M0 macrophages at days 7, 14, and 28; and (3) elevated osteoclastogenic gene RANKL (quantitative polymerase chain reaction) expression and other osteogenic and cytokine expression. Furthermore, in vitro data showed that saquinavir administration did not influence MC3T3-E1 cell migration or mineralization, whereas higher concentrations of saquinavir inhibited cell viability. Saquinavir treatment also enhanced the osteoclastic differentiation of bone marrow-derived precursors, and partially reversed high-mobility group box 1-driven osteoclastogenesis inhibition and elevated proinflammatory cytokine expression. The improved skull bone repair following short-term saquinavir treatment may involve enhanced osteoclastogenesis and modulated inflammatory response following skull injury. The authors' work demonstrates improved skull bone healing by short-term application of saquinavir, a drug traditionally used in the treatment of acquired immunodeficiency syndrome. As such, saquinavir may be repurposed for skeletal repair.

Fate mapping analysis reveals a novel murine dermal migratory Langerhans-like cell population.

Dendritic cells residing in the skin represent a large family of antigen-presenting cells, ranging from long-lived Langerhans cells (LC) in the epidermis to various distinct classical dendritic cell subsets in the dermis. Through genetic fate mapping analysis and single-cell RNA-sequencing, we have identified a novel separate population of LC-independent CD207+CD326+ LClike cells in the dermis that homed at a slow rate to the lymph nodes (LNs). These LClike cells are long-lived and radio-resistant but, unlike LCs, they are gradually replenished by bone marrow-derived precursors under steady state. LClike cells together with cDC1s are the main migratory CD207+CD326+ cell fractions present in the LN and not, as currently assumed, LCs, which are barely detectable, if at all. Cutaneous tolerance to haptens depends on LClike cells, whereas LCs suppress effector CD8+ T-cell functions and inflammation locally in the skin during contact hypersensitivity. These findings bring new insights into the dynamism of cutaneous dendritic cells and their function opening novel avenues in the development of treatments to cure inflammatory skin disorders.

Rab11A Functions as a Negative Regulator of Osteoclastogenesis through Dictating Lysosome-Induced Proteolysis of c-fms and RANK Surface Receptors.

Osteoclast differentiation and activity are controlled by two essential cytokines, macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear factor-κB ligand (RANKL). Rab11A GTPase, belonging to Rab11 subfamily representing the largest branch of Ras superfamily of small GTPases, has been identified as one of the crucial regulators of cell surface receptor recycling. Nevertheless, the regulatory role of Rab11A in osteoclast differentiation has been completely unknown. In this study, we found that Rab11A was strongly upregulated at a late stage of osteoclast differentiation derived from bone marrow-derived macrophages (BMMs) or RAW-D murine osteoclast precursor cells. Rab11A silencing promoted osteoclast formation and significantly increased the surface levels of c-fms and receptor activator of nuclear factor-κB (RANK) while its overexpression attenuated osteoclast formation and the surface levels of c-fms and RANK. Using immunocytochemical staining for tracking Rab11A vesicular localization, we observed that Rab11A was localized in early and late endosomes, but not lysosomes. Intriguingly, Rab11A overexpression caused the enhancement of fluorescent intensity and size-based enlargement of early endosomes. Besides, Rab11A overexpression promoted lysosomal activity via elevating the endogenous levels of a specific lysosomal protein, LAMP1, and two key lysosomal enzymes, cathepsins B and D in osteoclasts. More importantly, inhibition of the lysosomal activity by chloroquine, we found that the endogenous levels of c-fms and RANK proteins were enhanced in osteoclasts. From these observations, we suggest a novel function of Rab11A as a negative regulator of osteoclastogenesis mainly through (i) abolishing the surface abundance of c-fms and RANK receptors, and (ii) upregulating lysosomal activity, subsequently augmenting the degradation of c-fms and RANK receptors, probably via the axis of early endosomes–late endosomes–lysosomes in osteoclasts.

Novel method for gain-of-function analyses in primary osteoclasts using a non-viral gene delivery system.

Overexpression studies have been commonly used to yield significant advances in cell biology. In vitro osteoclast culturing involves the differentiation of bone marrow-derived monocyte macrophage precursors (BMMs) in medium supplemented with macrophage colony-stimulating factor and receptor activator of nuclear factor-kB ligand (RANKL) into mature osteoclasts. Retroviral vectors are the gold standards for efficient gene delivery into BMMs. While this strategy is effective in BMMs that are in the early stages of differentiation, it is ineffective in RANKL-treated BMMs such as mono- and multinucleated osteoclasts. This study attempted to enhance gene delivery into differentiated BMMs using liposome-mediated RNA transfection. BMMs were transfected with an EYFP overexpression plasmid or EYFP RNA by lipofection, or transduced with a retroviral vector expressing EYFP. EYFP expression was assessed by flow cytometry. We performed overexpression analyses using enhanced yellow fluorescent protein (EYFP). Although EYFP expression was observed 24 h after infection of BMMs with a recombinant retrovirus containing EYFP, expression of EYFP was observed within 3 h of transfection with EYFP RNA. Moreover, the efficiency of EYFP RNA for gene delivery into BMMs was comparable to that of retroviral transduction of EYFP. In contrast, while very few BMMs stimulated by RANKL for two days expressed EYFP after retroviral infection, more than half of the cells expressed EYFP after transfection with EYFP RNA. RNA-mediated gene delivery is quick and easy method for performing gain-of-function analyses in primary osteoclast precursors and mature osteoclasts.

Incomplete response to Anti-VEGF therapy in neovascular AMD: Exploring disease mechanisms and therapeutic opportunities.

Intravitreal anti-vascular endothelial growth factor (VEGF) drugs have revolutionized the treatment of neovascular age-related macular degeneration (NVAMD). However, many patients suffer from incomplete response to anti-VEGF therapy (IRT), which is defined as (1) persistent (plasma) fluid exudation; (2) unresolved or new hemorrhage; (3) progressive lesion fibrosis; and/or (4) suboptimal vision recovery. The first three of these collectively comprise the problem of persistent disease activity (PDA) in spite of anti-VEGF therapy. Meanwhile, the problem of suboptimal vision recovery (SVR) is defined as a failure to achieve excellent functional visual acuity of 20/40 or better in spite of sufficient anti-VEGF treatment. Thus, incomplete response to anti-VEGF therapy, and specifically PDA and SVR, represent significant clinical unmet needs. In this review, we will explore PDA and SVR in NVAMD, characterizing the clinical manifestations and exploring the pathobiology of each. We will demonstrate that PDA occurs most frequently in NVAMD patients who develop high-flow CNV lesions with arteriolarization, in contrast to patients with capillary CNV who are highly responsive to anti-VEGF therapy. We will review investigations of experimental CNV and demonstrate that both types of CNV can be modeled in mice. We will present and consider a provocative hypothesis: formation of arteriolar CNV occurs via a distinct pathobiology, termed neovascular remodeling (NVR), wherein blood-derived macrophages infiltrate the incipient CNV lesion, recruit bone marrow-derived mesenchymal precursor cells (MPCs) from the circulation, and activate MPCs to become vascular smooth muscle cells (VSMCs) and myofibroblasts, driving the development of high-flow CNV with arteriolarization and perivascular fibrosis. In considering SVR, we will discuss the concept that limited or poor vision in spite of anti-VEGF may not be caused simply by photoreceptor degeneration but instead may be associated with photoreceptor synaptic dysfunction in the neurosensory retina overlying CNV, triggered by infiltrating blood-derived macrophages and mediated by Müller cell activation Finally, for each of PDA and SVR, we will discuss current approaches to disease management and treatment and consider novel avenues for potential future therapies.

Bovine Milk Extracellular Vesicles Are Osteoprotective by Increasing Osteocyte Numbers and Targeting RANKL/OPG System in Experimental Models of Bone Loss

Studying effects of milk components on bone may have a clinical impact as milk is highly associated with bone maintenance, and clinical studies provided controversial associations with dairy consumption. We aimed to evaluate the impact of milk extracellular vesicles (mEVs) on the dynamics of bone loss in mice. MEVs are nanoparticles containing proteins, mRNA and microRNA, and were supplemented into the drinking water of mice, either receiving diet-induced obesity or ovariectomy (OVX). Mice receiving mEVs were protected from the bone loss caused by diet-induced obesity. In a more severe model of bone loss, OVX, higher osteoclast numbers in the femur were found, which were lowered by mEV treatment. Additionally, the osteoclastogenic potential of bone marrow-derived precursor cells was lowered in mEV-treated mice. The reduced stiffness in the femur of OVX mice was consequently reversed by mEV treatment, accompanied by improvement in the bone microarchitecture. In general, the RANKL/OPG ratio increased systemically and locally in both models and was rescued by mEV treatment. The number of osteocytes, as primary regulators of the RANKL/OPG system, raised in the femur of the OVX mEVs-treated group compared to OVX non-treated mice. Also, the osteocyte cell line treated with mEVs demonstrated a lowered RANKL/OPG ratio. Thus, mEVs showed systemic and local osteoprotective properties in two mouse models of bone loss reflected in reduced osteoclast presence. Data reveal mEV potential in bone modulation, acting via osteocyte enhancement and RANKL/OPG regulation. We suggest that mEVs could be a therapeutic candidate for the treatment of bone loss.

The glycosyltransferase EXTL2 promotes proteoglycan deposition and injurious neuroinflammation following demyelination

BackgroundChondroitin sulfate proteoglycans (CSPGs) are potent inhibitors of axonal regrowth and remyelination. More recently, they have also been highlighted as a modulator of macrophage infiltration into the central nervous system in experimental autoimmune encephalomyelitis, an inflammatory model of multiple sclerosis.MethodsWe interrogated results from single nucleotide polymorphisms (SNPs) lying in or close to genes regulating CSPG metabolism in the summary results from two publicly available systematic studies of multiple sclerosis (MS) genetics. A demyelinating injury model in the spinal cord of exostosin-like 2 deficient (EXTL2-/-) mice was used to investigate the effects of dysregulation of EXTL2 on remyelination. Cell cultures of bone marrow-derived macrophages and primary oligodendrocyte precursor cells and neurons were supplemented with purified CSPGs or conditioned media to assess potential mechanisms of action.ResultsThe strongest evidence for genetic association was seen for SNPs mapping to the region containing the glycosyltransferase exostosin-like 2 (EXTL2), an enzyme that normally suppresses CSPG biosynthesis. Six of these SNPs showed genome-wide significant evidence for association in one of the studies with concordant and nominally significant effects in the second study. We then went on to show that a demyelinating injury to the spinal cord of EXTL2−/− mice resulted in excessive deposition of CSPGs in the lesion area. EXTL2−/− mice had exacerbated axonal damage and myelin disruption relative to wild-type mice, and increased representation of microglia/macrophages within lesions. In tissue culture, activated bone marrow-derived macrophages from EXTL2−/− mice overproduce tumor necrosis factor α (TNFα) and matrix metalloproteinases (MMPs).ConclusionsThese results emphasize CSPGs as a prominent modulator of neuroinflammation and they highlight CSPGs accumulating in lesions in promoting axonal injury.

The Anti-Angiogenic Effects of Anti-Human Immunodeficiency Virus Drugs.

The growth and metastasis of malignant tumors benefit from the formation of blood vessels within the tumor area. There, new vessels originate from angiogenesis (the sprouting of pre-existing neighboring vessels) and/or vasculogenesis (the mobilization of bone marrow-derived endothelial cell precursors which incorporate in tumor vasculature and then differentiate into mature endothelial cells). These events are induced by soluble molecules (the angiogenic factors) and modulated by endothelial cell interactions with the perivascular matrix. Given angiogenesis/vasculogenesis relevance to tumor progression, anti-angiogenic drugs are often employed to buttress surgery, chemotherapy or radiation therapy in the treatment of a wide variety of cancers. Most of the anti-angiogenic drugs have been developed to functionally impair the angiogenic vascular endothelial growth factor: however, this leaves other angiogenic factors unaffected, hence leading to drug resistance and escape. Other anti-angiogenic strategies have exploited classical inhibitors of enzymes remodeling the perivascular matrix. Disappointingly, these inhibitors have been found toxic and/or ineffective in clinical trials, even though they block angiogenesis in pre-clinical models. These findings are stimulating the identification of other anti-angiogenic compounds. In this regard, it is noteworthy that drugs utilized for a long time to counteract human immune deficiency virus (HIV) can directly and effectively hamper molecular pathways leading to blood vessel formation. In this review the mechanisms leading to angiogenesis and vasculogenesis, and their susceptibility to anti-HIV drugs will be discussed.

CRISPR Cas9 ribonucleoprotein-mediated genomic editing in primary innate immune cells

Abstract CRISPR (clustered, regularly interspaced, short palindromic repeats) genome engineering has become a powerful tool to functionally investigate complex mechanisms of immune system regulation. While decades of work have aimed to genetically reprogram innate immunity for therapeutic purposes, current approaches are often inefficient or nonspecific, limiting their use in primary innate immune cells. Here, we describe an optimized strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of primary innate lymphocytes (ILCs) and myeloid lineage cells that results in an almost complete loss of single or double target gene expression from a single electroporation. Furthermore, we describe in vivo adoptive transfer mouse models that can be utilized to screen for gene function during viral infection using cRNP-edited naïve NK cells and bone marrow-derived dendritic cell precursors. Using these methods, we demonstrate that XCR1+ conventional dendritic cells (cDC1) are sufficient and necessary for host protection to mouse cytomegalovirus (MCMV) in a MyD88-intrinsic manner. This scalable method will enhance target gene discovery and offer a specific and simplified approach to gene editing in the innate immune system.

Inhibitory effect of Chaenomelis Fructus ethanol extract on receptor activator of nuclear factor-kappa B ligand-mediated osteoclastogenesis

The fruit of Chaenomeles sinensis (Thouin) Koehne (Chaenomelis Fructus) known as “Mo-Gua” in Korea has been commonly used in traditional medicine to treat inflammatory diseases, such as sore throat. However, its effect on bone metabolism has not been elucidated yet. Here, we examined the effect of Chaenomelis Fructus ethanol extract (CFE) on receptor activator of nuclear factor (NF)-κB ligand (RANKL)-mediated osteoclast differentiation and formation. CF-E considerably inhibited osteoclast differentiation and tartrate-resistant acid phosphatase-positive multinuclear cell formation from bone marrow-derived macrophages and osteoclast precursor cells in a dose-dependent manner. In addition, the formation of actin rings and resorption pits were significantly suppressed in CF-E-treated osteoclasts as compared with the findings in non-treated control cells. Consistent with these phenotypic inhibitory results, the expressions of osteoclast differentiation marker genes (Acp5, Atp6v0d2 , Oscar, CtsK, and Tm7sf4) and Nfatc1 , a pivotal transcription factor for osteoclastogenesis, were markedly decreased by CF-E treatment. The inhibitory effect of CF-E on RANKL-induced osteoclastogenesis was associated with the suppression of NFATc1 expression, not by regulation of mitogen-activated protein kinases and NF-κB activation but by the inactivation of phospholipase C gamma 1 and 2. These results indicate that CF-E has an inhibitory effect on osteoclast differentiation and formation, and they suggest the possibility of CF-E as a traditional therapeutic agent against bone-resorptive diseases, such as osteoporosis, rheumatoid arthritis, and periodontitis.

Adhesion, motility and matrix-degrading gene expression changes in CSF-1-induced mouse macrophage differentiation.

Migratory macrophages play critical roles in tissue development, homeostasis and disease, so it is important to understand how their migration machinery is regulated. Whole-transcriptome sequencing revealed that CSF-1-stimulated differentiation of bone marrow-derived precursors into mature macrophages is accompanied by widespread, profound changes in the expression of genes regulating adhesion, actin cytoskeletal remodeling and extracellular matrix degradation. Significantly altered expression of almost 40% of adhesion genes, 60-86% of Rho family GTPases, their regulators and effectors and over 70% of extracellular proteases occurred. The gene expression changes were mirrored by changes in macrophage adhesion associated with increases in motility and matrix-degrading capacity. IL-4 further increased motility and matrix-degrading capacity in mature macrophages, with additional changes in migration machinery gene expression. Finally, siRNA-induced reductions in the expression of the core adhesion proteins paxillin and leupaxin decreased macrophage spreading and the number of adhesions, with distinct effects on adhesion and their distribution, and on matrix degradation. Together, the datasets provide an important resource to increase our understanding of the regulation of migration in macrophages and to develop therapies targeting disease-enhancing macrophages.