A novel self-assembled nucleobase-nanofiber platform of CDN to activate the STING pathway for synergistic cancer immunotherapy

2', 3'-cGAMP (CDN) as cGAS-STING pathway agonist is extensively used in tumor treatment. However, due to its negatively charged nature (containing two phosphate groups) and high hydrophilicity, CDN faces challenges in crossing cell membranes, resulting in reduced efficiency of its use. Additionally, CDN is susceptible to inactivation through phosphodiesterase hydrolysis. Therefore, the development of a new drug delivery system for CDN is necessary to prevent hydrolysis and enhance targeted... http://dlvr.it/Sxlccq

Sociable along with Conduct Well being Components Linked to Crazy along with Adult Game playing in Early Teenage life

Atherosclerosis is seen as a fancy resistant response within the vessel walls, concerning both infection and also autoinumme techniques. Epstein-Barr virus-induced gene Three or more (Ebi3) is owned by your interleukin (Illinois)-12 heterodimeric cytokine household, containing crucial immunomodulatory features. To date, little is well known regarding the role regarding Ebi3 throughout vascular ailment. We all reviewed the particular expression regarding Ebi3 in human being atheromatous lesions on the skin and assessed its transcriptional regulation in vascular tissue. The inside situ appearance associated with Ebi3 within individual endarterectomy specimens has been reviewed by immunohistochemistry. During these lesions, clean muscle cells portrayed Ebi3 as well as the IL-27 alpha/p28 and IL-12 alpha/p35 subunits. Primary aortic easy muscle tissues up-regulated Ebi3 as a result of proinflammatory stimulus like cancer necrosis factor-a and also interferon-gamma. Strangely enough, pretreatment of such cells using the peroxisome proliferator-activated receptor-gamma agonist rosiglitazone strongly diminished Ebi3 induction. Chromatin immunoprecipitation experiments says this specific inhibition is caused by disturbance together with p65/RelA hiring to the Ebi3 supporter. The information assist a prospective position involving Ebi3 within atherogenesis possibly as homodimer as well as because IL-27/IL-35 heterodimer, along with suggest that Ebi3 could be an fascinating target regarding restorative manipulation within vascular disease. (Are M Pathol 09,A hundred seventy five:440-447; DOI: Ten.2353/ajpath.Last year.080752)This study looked at the impact of diazepam for the joining characteristics associated with adrenoceptor, muscarinic along with benzodiazepine receptors within rat parotid human gland tissue layer utilizing a radioligand presenting assay. With a power of >15(-6) Mirielle, diazepam taken part along with [H-3]dihydroalprenolol pertaining to beta-adrenoceptor, although not [H-3] prazosin for et-adrenoceptor or [H-3]quinuclidinyl benzilate pertaining to muscarinic receptor. Ongoing government regarding diazepam from amounts of 3',3'-cGAMP clinical trial 2.Several mg/kg/day, my partner and i.s. for Seven days within rat drastically lowered pilocarpine (Four.0 mg/kg, i.p.)-induced parotid salivary flow. Diazepam additionally made a tremendous rise in the particular dissociation regular (Kd) benefit for [H-3]dihydroalprenolol presenting, yet absolutely no alternation in the particular maximal binding capacity (Bmax) value, plus a decline in the Kd benefit with regard to [H-3]diazepam joining to benzodiazepine receptors, but simply no alternation in the Kd or even Bmax values pertaining to [H-3]prazosin or even [H-3]quinuclidinyl benzilate joining. These types of outcomes suggest that constant government regarding diazepam modifies interest in [beta-adrenoceptor as well as benzodiazepine receptor presenting websites inside parotid glandular tissue layer understanding that alterations in these joining sites could be carefully associated with diazepam-induced elimination regarding salivary secretion. (C) This year Elsevier B.V. Almost all legal rights set-aside.All of us searched for to recognize key specifics within cellular buildings along with composition that may explain observed variants the particular indirect transport attributes of modest particle medications around distinct airway epithelial mobile sorts. Propranolol (PR) had been selected as being a weakly simple, product compound to compare your transfer components associated with major (NHBE) as opposed to. tumor-derived (Calu-3) tissues.

Structural basis of innate immune sensors

Cyclic GMP-AMP synthase (cGAS) is an innate immune protein which detects the presence of DNA in the cytosol as a potential indicator of infection. cGAS-like receptors (cGLRs) recognize divergent molecular patterns and synthesize distinct nucleotide second messenger signals to activate an immune response. While animal genomes encode multiple proteins with sequence homology to cGAS, the function of these important enzymes remained unknown.

SBGrid member Philip Kranzusch and other researchers have been working to understand this group of proteins. They determined that the cGAS-like receptors in humans and insects have a nucleotidyltransferase signaling core shared with cGAS. Further, these proteins have varied primary ligand-binding surfaces characterized by insertions and deletions which enables altered ligand specificity. As a demonstration of this, the authors used a forward biochemical screen to identify cGLR1 as a double-stranded RNA sensor in Drosophila, a model organism, which induces an enhanced antiviral state.

Above: Drosophila STING-3′2′-cGAMP complex. CC BY SBGrid.

This sets the basis for cGLRs as a diverse family of metazoan pattern recognition receptors, and it points to cGLRs as receptors in animals capable of recognizing diverse pathogen-associated molecular patterns.

Read more about this exciting work in Nature.

THP-1 Knockout Cell Lines: A powerful tool for immunity and inflammation study

A human leukemic cell line (THP-1) cultured from the blood of a boy with acute monocytic leukemia. Since its establishment in 1980, THP-1 cells have been widely used in the research of monocyte and macrophage related mechanisms, signaling pathways, nutrient and drug transportation. The morphological and functional characteristics of THP-1 are very similar to human primary monocytes (including cell differentiation markers). Compared with human peripheral blood monocytes (PBMC), THP-1 is easier to be cultured and has a more consistent background. Therefore, THP-1 is a commonly used acute monocytic leukemia cell line in various laboratories, and it is an ideal tool for studying immunity and inflammation.

Application of THP-1: macrophage and inflammation model

THP-1can be differentiated into M1/M2 macrophages and release corresponding cytokines.

M1 macrophage polarization:

THP-1 can be induced to differentiate into macrophages by Phorbol 12-myristate 13-acetate (PMA), and then M1 polarization can be induced by lipopolysaccharide (LPS) and IFN -γ, releasing TNF -α, IL-6 and other cytokines. This is a typical inflammatory model.

M2 macrophage polarization:

M2 polarization can beinduced by IL-4, IL-13 and macrophage colony-stimulating factor (M-CSF). TGF - β, IL-10 and other inhibitory cytokines can be released. This is similar to the process of tissue repair and reconstruction in the late stage of inflammation.

Atherosclerotic inflammation model: Under the action of oxidized low-density lipoprotein (ox-LDL), macrophages can further become foam cells. This is a pathological cell in atherosclerotic plaques and is a chronic inflammation model.The combination of THP-1 and CRISPR/Cas9 technology is helpful for the study of immune and inflammatory diseases. 

THP-1 is a near-tetraploid suspension cell, and the success rate of THP-1 is very low by conventional gene-editing methods. CRISPR/Cas9 is widely used to construct gene-editing THP-1 model because of its simple, high efficiency and low toxicity. Key genes for macrophagesclearing pathogens were found by CRISPR/Cas9 mediated gene knock-out THP-1 model Phagosome acidification of macrophages is an essential step to eliminate pathogens. Phagosome acidification is closely related to the metabolism of macrophages and the transportation of nutrients. And the transportation of metabolites is closely related to solute carrier (SLC)protein. The researchers found that the bicarbonate transporter SLC4A7 in the SLC family is an essential gene for phagosome acidification of macrophages. In CRISPR/Cas9 mediated SLC4A7

knockout THP-1 cell line

, the ability of phagosome acidification and killing bacteria was reduced. The acidity of the phagocyte was increased after the supplementation of SLC4A7. This indicates that SLC4A7 mediated bicarbonate driven in macrophages is essential for the maintenance of cytoplasmic pH and phagosome acidification. CRISPR-UTM can efficiently transfer gRNA and Cas9 into THP-1 cells by nucleofection. After drug screening, single clones would be generated. Positive clones would be validated by sequencing.

CRISPR/Cas9 mediated Chronic granulomatous disease (CGD) THP-1 cell line model is helpful to develop better disease treatmentsChronic granulomatous disease (CGD) is a rare X-linked genetic disease. Due to the mutation or deficient of CYBB gene, macrophages lack nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and cannot produce hydrogen peroxide to effectively kill the invading microorganisms. This usually leads to serious repeated infections caused by bacteria, fungi and other microorganisms. Some researchers used CRISPR/Cas9 technology to knockout CYBB gene and generate point mutation c.90c>G (found in a CGD patient) in THP-1 cells, and successfully constructed the CGD model. Compared with wild-type THP-1 cells, two KO clones (#3 and #27) and a point mutation clone (#2, c.90c > G) showed decrease in H2O2 level after PMA and LPS induction, and a significant increase in IL-1β, TNF-α and IL-6 release, which was consistent with the behavior of macrophages in CGD. This CGD model provides a powerful tool for disease study and will help to develop better treatments.

By using CRISPR-UTM,  THP-1 cell line would be efficiently co-transfected with gRNA, Cas9  and ssODN. After drug screening, single clones would be generated. Positive clones would be validated by sequencing.

The signal pathway of intracellular antiviral response was confirmed by the THP-1 Cell Models of gene knock-out and knock-in.The abnormal location of DNA in the cytoplasm is usually related to virus infection or tumor. The cGAS-cGAMP-STING pathway can detect the existence of cytosolic dsDNA, and induce a strong immune response, producing interferon and activating other immune response genes. RIG1-MAVS can detect pppRNA (dsRNA, the genome of some viruses) in cytoplasm and induce immune response. Sometimes there is a complex of RNA and DNA in the cytoplasm, which usually occurs in the case of some virus infection. In order to study which pathway that the RNA-DNA complex activates the immune response, the researchers generatedMAVS, cGAS, STING knockout THP-1 cell lines, and introduced dsDNA, ppRNA and RNA-DNA complex into the cells respectively. It was found that the RNA-DNA complex is activated by the cGAS-cGAMP-STING pathway.

Then, the researchers used CRISPR/Cas9 technology to insert2A-GLuc into the IFIT1 gene. IFIT1 is a typical interferon activated gene. Subsequent experiments showed that after the introduction of RNA-DNA complex, the expression of Gluc was driven by the activation of IFIT1 promoter due to the expression of interferon. These results further proved that the RNA-DNA complex in the cytoplasm activated the immune response through the cGAS-cGAMP-STING pathway. By using CRISPR-UTM, THP-1 cell line would be co-transfected with gRNA, Cas9 and donor vector. After drug screening, single clones would be generated. Positive clones would be validated by sequencing.

References:[1]Tsuchiya S, Yamabe M, Yamaguchi Y, et al. Establishment and characterization of a human acute monocytic leukemia cell line (THP‐1)[J]. International journal of cancer, 1980, 26(2): 171-176.[2] Chanput W, Mes J J, Wichers H J. THP-1 cell line: an in vitro cell model for immune modulation approach[J]. International immunopharmacology, 2014, 23(1): 37-45.[3] Sedlyarov V, Eichner R, Girardi E, et al. The bicarbonate transporter SLC4A7 plays a key role in macrophage phagosome acidification[J]. Cell host & microbe, 2018, 23(6): 766-774. e5.[4] Benyoucef A, Marchitto L, Touzot F. CRISPR gene-engineered CYBBko THP-1 cell lines highlight the crucial role of NADPH-induced reactive oxygen species for regulating inflammasome activation[J]. Journal of Allergy and Clinical Immunology, 2020.[5] Mankan A K, Schmidt T, Chauhan D, et al. Cytosolic RNA: DNA hybrids activate the cGAS–STING axis[J]. The EMBO journal, 2014, 33(24): 2937-2946.

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Development of Enpp1 Inhibitors as a Strategy to Activate Stimulator of Interferon Genes (STING) in Cancers and Other Diseases- Juniper Publishers

Abstract

Ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1/NPP1) is a membrane-bound nucleotide metabolizing enzyme that is implicated in a variety of physiological and pathological conditions. Recently, ENPP1 was discovered as the dominant 2’3’-cGAMP hydrolyzing enzyme. 2’3’-cGAMP is the endogenous STING agonist, generated from breakdown of cytosolic DNA by cGAS. Hydrolysis resistant 2’3’-cGAMP’s have been demonstrated to be potent activators of STING-dependent innate immunity and these are currently undergoing clinical trials in cancer. Here we discuss ENPP1 as a potential therapeutic target for activation of STING-dependent innate immune response.

Keywords: Innate immunity; STING; ENPP1/NPP1; Cytokines; Immunotherapies; Interferon; T-cell priming

Introduction

Innate immunity is the first response in the human body against pathogenic, or disease-causing stimuli. These stimuli can vary, and include viruses, perturbed normal tissue, and dying cancer cells. It is an important response, as it prevents continued proliferation of these pathogens and maintains a state of homeostasis within the body. It can also accommodate the development of a specific induced immune response during the first, or primary infection and, can therefore, establish inflammatory conditions. This induced response is specific because of the many different expressions that the cell surface gives off in the form of pattern recognition receptors, which can identify many of the molecules of life, such as, polysaccharides, glycoproteins, glycolipids, and nucleic acids [1].

The definition of innate immunity has altered over time. In earlier years, it was believed that innate immune response was premeditated. However, recent studies have shown that innate immunity is actually a specific response that results from damage or pathogen-associated molecular patterns (DAMP/PAMPs) [2]. In the initial phase, the innate immune system is able to coordinate inflammatory responses through cells of the hematopoietic compartment (neutrophils, macrophages and monocytes) and create conditions suitable for microbial clearance. In the second phase, other cells like dendritic cells are able to process antigens and present them on the surface in concert with major histocompatibility complex (MHC) to prime T-cells. This also allows the body to more effectively fight against infections of the same or similar type in the future. This “memory” is dependent on two specific types of cells: natural killer (NK) cells and macrophages. These cells provide crucial protection against reinfection in the immune system [3]. This “memory” found in innate immune systems is present in both vertebrate and invertebrate organisms.

Cytokines in Innate Immune Response

Cytokines are possibly the most indispensable component of the innate immune response. Cytokines are secreted by cells of the immune system and facilitate interaction between different types of cells. There are many different types of cytokines, and they are classified mainly by their biological functions. The main types of cytokines are: interferons (INFs), interleukins (ILs), transforming growth factors (TGFs), and tumor necrosis factors (TNFs) [4]. Interferons are the most commonly found type of cytokine in vertebrates and mammals and are crucial to mediate antiviral defense. To date, there have been three types of interferons discovered in vertebrates, and specifically mammals: Types 1, 2 and 3. Type 1 IFNs typically facilitate the antiviral response against microbial infection-causing pathogens. Type 2 IFNs also facilitate antiviral response, but at the same time, vitalize the process of phagocytosis and inhibit cell growth. Type 3 IFNs have been demonstrated to be strikingly similar in function to Type 1 IFNs [5,6]. Interleukins are a type of cytokines that also facilitate inflammatory responses in the immune system and help to stimulate cell growth [7]. Transforming growth factors (TGFs) regulate cell growth, help stimulate the growth of oocyte cells (which are found in the ovum), repair wounds inflicted upon the body, participate in immunosuppression, or reduce the activity of the immune system when naturally required [8]. Finally, tumor necrosis factors (TNFs) help to stimulate macrophages as they participate in the biological process of phagocytosis [9].

STING (Stimulator of interferon genes) as a DNA sensor

STING has been identified as a major signaling molecule that plays a pivotal role in innate immune response by inducing the production of interferons. STING is a cytoplasmic pattern recognition receptor activated by nucleic acid ligands known as cyclic dinucleotides (CDNs). These CDNs are generated by the DNA sensor cyclic GMP-AMP synthase (cGAS) using cytosolic DNA from extrinsic pathogens or endogenous aberrant self-DNA [10-12]. In case of tumors, it is probable that dying tumor cells are sources of dsDNA in the cytoplasm. In addition to CDN’s, STING can directly sense DNA and this dual sensing has been uncoupled with specific mutations in STING [10]. Activation of STING induces its binding with a kinase TBK1 (TANK-binding kinase 1) and further phosphorylation and dimerization of IRF3 (Interferon regulatory factor 3). IRF3 and another transcription factor that is activated by STING (STAT6) translocate to nucleus and bind to interferon promoters leading to production of type I interferons.

It is suggested that STING pathway is the main innate immune sensing pathway within tumor microenvironment and the main cell types in the tumor microenvironment that produce type I interferons are the dendritic cells [13,14]. In addition to the activation of STING pathway in response to tumor-derived DNA, dendritic cells prime T-cells by presenting tumor- associated antigens. These effects then create a signaling pathway, which allows T-cells, a main feature of the active immune response, to neutralize tumor cells [15,16]. Some tumor cells are able to “disguise” themselves to the innate immune response by upregulating immune checkpoints, or by having a lack of innate immune response within the tumor. A recent study reported that STING is epigenetically silenced in some cancers [17]. Additionally, oncoproteins from viruses such as human papillomavirus can bind and block activation of STING [18]. Thus, a cytosolic DNA sensing pathway is important for activation of innate immune response. In recent years, there has been considerable interest in the field of immune-oncology as well as an increase in the number of immunotherapies available [19,20].

ENPP1(Ectonucleotide Pyrophosphatase/Phosphodiesterase- 1) And Its Role in Innate Immunity

ENPP1 is a membrane bound enzyme that is an important regulator of extracellular inorganic pyrophosphate in osteoblasts and chondrocytes [21]. It is essential for prevention of soft tissue mineralization and ENPP1 deficient mice can have abnormal gait and progressive calcification in ectopic sites [22]. ENPP1 is responsible for hydrolysis of extracellular nucleotide triphosphates to produce inorganic pyrophosphates (PPi) [23]. Recent investigations have shown that ENPP1 plays a much larger role in limiting the innate immune response of the human body. It has been discovered that STING pathway is regulated by ENPP1[24]. ENPP1 was identified as the major hydrolase for the most potent endogenous CDN ligand for STING: 2’3’-cGAMP [25]. Importantly, it was demonstrated that denaturation of 2’3’-cGAMP can control the activation of the STING pathway [26]. Phosphothioate analogs of 2’3’-cGAMP resistant to ENPP1- mediated hydrolysis potently activate STING [25] and mediate anti-tumor responses. These analogs have now entered clinical trials as intra-tumoral injections in various advance cancers (Figure 1).

In another study, it was shown that Mycobacterium tuberculosis evades host immune response through a bacterial phosphodiesterase (CdnP) which inactivates host 2’3’-cGAMP. Loss of ENPP1 attenuated Mycobacterium tuberculosis infection, as did the inhibition of CdnP, the phosphodiesterase of Mycobacterium tuberculosis [27] More recently, inactivation of porcine ENPP1 was shown to attenuate pseudorabies infection through an interferon-β dependent response [28]. Many viruses generate antagonist proteins that can inactivate cGAS-STING pathway [29]. ENPP1 is differentially expressed in immune cells with low levels in NK cells, DC and macrophages and high levels in neutrophils [30]. ENPP1 is also expressed in a small subset of B-cells and studies suggest that these cells may be involved in modulation of T-cell activity [31]. Interestingly, ENPP1 expression was reported to be elevated in the M2 subtype of macrophages that are known to play a role in tumor promotion [28,32,33]. Other studies have indicated that expression of ENPP1 is increased in astrocytic tumors, breast cancers, and head and neck cancers [34-36]. Thus, inhibition of ENPP1 in humans may provide opportunities for treatment of cancers and pathogenic infections.

Challenges in Development of Inhibitors of ENPP1 for Human Use

Given the various functions for ENPP1 in regulating host immune responses, there is interest in development of ENPP1 inhibitors for human use. These inhibitors may have promising activity in human cancers and infectious pathologies. There are various practical challenges in development of these inhibitors. ENPP1 is a type II transmembrane glycoprotein that belongs to a family of ectonucleotide pyrophosphatase/phosphodiesterase (Enpp) family and consist of seven distinct proteins with distinct functions [37]. Thus, any inhibitor strategy will have to consider development challenges for specificity. In the published crystal structure of mouse ENPP1, there are important structural differences between ENPP2 and ENPP1. The N-terminal somatomedin-like (SMB) domains of ENPP1 do not interact with catalytic domains unlike those in ENPP2 [38,39]. ENPP1 appears to lack a hydrophobic pocket in contrast to ENPP2 although interdomain interactions are preserved [37-40]. Despite these challenges, our group and others have described novel selective and orally bioavailable inhibitors of ENPP1 [41-45].

Fundamental effects of ENPP1 inhibition on host immune response are still being determined. It is not known, for instance, if ENPP1 deficiency in mouse models impairs anti-tumor growth. Thus, optimal duration and intensity of ENPP1 inhibition is still being developed. This is important since systemic administration of these inhibitors can cause unwanted side effects due to excessive release of interferons. Interestingly, ENPP1 knockout mice are viable, thus pointing to possible avenues for development of such inhibitors. Prolonged administration of ENPP1 inhibitors may lead to unwanted effects on bony tissues and ectopic calcifications although this has been disputed in various studies in literature [46]. This is because bone and cartilage effects may not be entirely mediated by ENPP1. In other studies, oral administration of pyrophosphate can attenuate the connective tissue calcifications mediated by ENPP1 mutations in mouse models [47].

Conclusion

As hyper-activation of STING pathway may lead to production of abnormally high levels of proinflammatory cytokines, it is necessary to develop therapeutics that target STING pathway indirectly. Inhibition of ENPP1 activity is one approach that may result in optimal activation of STING pathway, enough to have anti-tumor effects, and minimize unintended consequences. Given the role of ENPP1 in immune modulation and tumor promotion, there is an increased interest to develop novel therapies based on inhibition of the ENPP1 activity and this will emerge as an interesting area in the coming years.

Acknowledgments

We thank Dr. Hariprasad Vankayalapati for advice in developing this review.

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Viral and metazoan poxins are cGAMP-specific nucleases that restrict cGAS–STING signalling

Viral and metazoan poxins are cGAMP-specific nucleases that restrict cGAS–STING signalling, Published online: 06 February 2019; doi:10.1038/s41586-019-0928-6

Poxvirus immune nucleases (poxins) degrade 2′,3′-cyclic GMP–AMP that is produced by cyclic GMP–AMP synthase (cGAS) to evade the innate immune system of the host. from Nature - Issue - nature.com science feeds https://go.nature.com/2WLpC6n

Intratumoral administration of cGAMP transiently accumulates potent macrophages for anti-tumor immunity at a mouse tumor site

Abstract

Stimulator of IFN genes (STING) spontaneously contributes to anti-tumor immunity by inducing type I interferons (IFNs) following sensing of tumor-derived genomic DNAs in the tumor-bearing host. Although direct injection of STING ligands such as cyclic diguanylate monophosphate (c-di-GMP) and cyclic [G(2′,5′)pA(3′,5′)p] (cGAMP) into the tumor microenvironment exerts anti-tumor effects through strong induction of type I IFNs and activation of innate and adaptive immunity, the precise events caused by STING in the tumor microenvironment remain to be elucidated. We describe here our finding that a CD45+ CD11bmid Ly6C+ cell subset transiently accumulated in mouse tumor microenvironment of 4T1 breast cancer, squamous cell carcinomas, CT26 colon cancer, or B16F10 melanoma tissue after intratumoral injection of cGAMP. The accumulated cells displayed a macrophage (M ) phenotype since the cells were positive for F4/80 and MHC class II and negative for Ly6G. Intratumoral cGAMP treatment did not induce Mφ accumulation in STING-deficient mice. Depletion of CD8+ T cell using anti-CD8 mAb impaired the anti-tumor effects of cGAMP treatment. Depletion of the Mφ using clodronate liposomes impaired the anti-tumor effects of cGAMP treatment. Functional analysis indicated that the STING-triggered tumor-migrating Mφ exhibited phagocytic activity, production of tumor necrosis factor alpha TNFα), and high expression levels of T cell-recruiting chemokines, Cxcl10 and Cxcl11, IFN-induced molecules, MX dynamin-like GTPase 1 (Mx1) and 2′-5′ oligoadenylate synthetase-like 1 (Oasl1), nitric oxide synthase 2 (Nos2), and interferon beta 1 (Ifnb1). These results indicate that the STING-triggered tumor-migrating Mφ participate in the anti-tumor effects of STING-activating compounds.

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SLC19A1 transports immunoreactive cyclic dinucleotides.

Related Articles SLC19A1 transports immunoreactive cyclic dinucleotides. Nature. 2019 Sep 11;: Authors: Luteijn RD, Zaver SA, Gowen BG, Wyman SK, Garelis NE, Onia L, McWhirter SM, Katibah GE, Corn JE, Woodward JJ, Raulet DH Abstract The accumulation of DNA in the cytosol serves as a key immunostimulatory signal associated with infections, cancer and genomic damage1,2. Cytosolic DNA triggers immune responses by activating the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway3. The binding of DNA to cGAS activates its enzymatic activity, leading to the synthesis of a second messenger, cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP)4-7. This cyclic dinucleotide (CDN) activates STING8, which in turn activates the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), promoting the transcription of genes encoding type I interferons and other cytokines and mediators that stimulate a broader immune response. Exogenous 2'3'-cGAMP produced by malignant cells9 and other CDNs, including those produced by bacteria10-12 and synthetic CDNs used in cancer immunotherapy13,14, must traverse the cell membrane to activate STING in target cells. How these charged CDNs pass through the lipid bilayer is unknown. Here we used a genome-wide CRISPR-interference screen to identify the reduced folate carrier SLC19A1, a folate-organic phosphate antiporter, as the major transporter of CDNs. Depleting SLC19A1 in human cells inhibits CDN uptake and functional responses, and overexpressing SLC19A1 increases both uptake and functional responses. In human cell lines and primary cells ex vivo, CDN uptake is inhibited by folates as well as two medications approved for treatment of inflammatory diseases, sulfasalazine and the antifolate methotrexate. The identification of SLC19A1 as the major transporter of CDNs into cells has implications for the immunotherapeutic treatment of cancer13, host responsiveness to CDN-producing pathogenic microorganisms11 and-potentially-for some inflammatory diseases. PMID: 31511694 [PubMed - as supplied by publisher] http://dlvr.it/RD1xmB

THP-1 Cell Line Gene-editing

A human leukemic cell line (THP-1) cultured from the blood of a boy with acute monocytic leukemia. Since its establishment in 1980, THP-1 cells have been widely used in the research of monocyte and macrophage related mechanisms, signaling pathways, nutrient and drug transportation. The morphological and functional characteristics of THP-1 are very similar to human primary monocytes (including cell differentiation markers). Compared with human peripheral blood monocytes (PBMC), THP-1 is easier to be cultured and has a more consistent background. Therefore, THP-1 is a commonly used acute monocytic leukemia cell line in various laboratories, and it is an ideal tool for studying immunity and inflammation.

Application of THP-1: macrophage and inflammation model

THP-1 can be differentiated into M1/M2 macrophages and release corresponding cytokines.

l M1 macrophage polarization：THP-1 can be induced to differentiate into macrophages by Phorbol 12-myristate 13-acetate (PMA), and then M1 polarization can be induced by lipopolysaccharide (LPS) and IFN -γ, releasing TNF -α, IL-6 and other cytokines. This is a typical inflammatory model.

l M2 macrophage polarization：M2 polarization can be induced by IL-4, IL-13 and macrophage colony-stimulating factor (M-CSF). TGF - β, IL-10 and other inhibitory cytokines can be released. This is similar to the process of tissue repair and reconstruction in the late stage of inflammation.

l Atherosclerotic inflammation model：Under the action of oxidized low-density lipoprotein (ox-LDL), macrophages can further become foam cells. This is a pathological cell in atherosclerotic plaques and is a chronic inflammation model.

 CRISPR-U™可在THP-1细胞中进行高效的基因编辑

THP-1 is a near-tetraploid suspension cell, and the success rate of THP-1 is very low by conventional gene-editing methods. CRISPR/Cas9 is widely used to construct gene-editing THP-1 model because of its simple, high efficiency and low toxicity.

CRISPR-U™, developed by Ubigene, is more efficient than general CRISPR/Cas9 in double-strand breaking, and CRISPR-U™ can greatly improve the efficiency of homologous recombination, easily achieve knockout (KO), point mutation (PM) and knockin (KI).

Ubigene can customize the gene-editing THP-1 cell line and other monocytes that you are interested in, as well as generate various genes overexpression in THP-1 cell line.

 THP-1 Cell Line Gene-editing Services：

 Knockout

CRISPR-UTM gene knockout THP-1 cell line: gRNA and Cas9 are transferred into THP-1 cells by nucleofection. After drug screening, single clones would be generated. Positive clones would be validated by sequencing.

Knockout Strategies：

Strategy

Application

Short fragment removal

Guide RNAs target introns at both sides of exon 2 and the number of bases in exon 2 is not a multiple of 3, which can cause frame-shift mutation.

Study of gene function through gene defect

Frame-shift mutation

Guide RNA targets the exon, and the base number of deletion is not a multiple of 3. After knockout, frame-shift mutation would cause gene knockout.

Large fragment removal.

Complete removal of the coding sequence to achieve gene knockout.

Case Study：

Key genes for macrophages clearing pathogens was found by CRISPR/Cas9 mediated gene knock-out THP-1 model

Phagosome acidification of macrophages is an essential step to eliminate pathogens. Phagosome acidification is closely related to the metabolism of macrophages and the transportation of nutrients. And the transportation of metabolites is closely related to solute carrier (SLC) protein. The researchers found that the bicarbonate transporter SLC4A7 in the SLC family is an essential gene for phagosome acidification of macrophages. In CRISPR/Cas9 mediated SLC4A7 knockout THP-1 cell line, the ability of phagosome acidification and killing bacteria was reduced. The acidity of the phagocyte was increased after the supplementation of SLC4A7. This indicates that SLC4A7 mediated bicarbonate driven in macrophages is essential for the maintenance of cytoplasmic pH and phagosome acidification.

Reference：

Sedlyarov V, Eichner R, Girardi E, et al. The bicarbonate transporter SLC4A7 plays a key role in macrophage phagosome acidification[J]. Cell host & microbe, 2018, 23(6): 766-774. e5.

Point mutation

CRISPR-UTM Point Mutation THP-1 Cell Line：THP-1 cell line would be efficiently co-transfected with gRNA, Cas9 and ssODN. After drug screening, single clones would be generated. Positive clones would be validated by sequencing.

细胞疾病模型建模

通过HDR，携带点突变序列的donor oligo (ssODN)替换WT对应的碱基。

细胞疾病模型修复

通过HDR，携带WT序列的donor oligo (ssODN)替换对应的突变碱基。

 Case Study:

CRISPR/Cas9 mediated Chronic granulomatous disease (CGD) THP-1 cell line model is helpful to develop better disease treatments

Chronic granulomatous disease (CGD) is a rare X-linked genetic disease. Due to the mutation or deficient of CYBB gene, macrophages lack nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and cannot produce hydrogen peroxide to effectively kill the invading microorganisms. This usually leads to serious repeated infections caused by bacteria, fungi and other microorganisms. Some researchers used CRISPR/Cas9 technology to knockout CYBB gene and generate point mutation c.90c>G (found in a CGD patient) in THP-1 cells, and successfully constructed the CGD model. Compared with wild-type THP-1 cells, two KO clones (#3 and #27) and a point mutation clone (#2, c.90c > G) showed decrease in H2O2 level after PMA and LPS induction, and a significant increase in IL-1β, TNF-α and IL-6 release, which was consistent with the behavior of macrophages in CGD. This CGD model provides a powerful tool for disease study and will help to develop better

Reference：

Benyoucef A, Marchitto L, Touzot F. CRISPR gene-engineered CYBBko THP-1 cell lines highlight the crucial role of NADPH-induced reactive oxygen species for regulating inflammasome activation[J]. Journal of Allergy and Clinical Immunology, 2020.

Knockin

CRISPR-UTM Gene Knockin THP-1 Cell Line： THP-1 cell line would be co-transfected with gRNA, Cas9 and donor vector. After drug screening, single clones would be generated. Positive clones would be validated by sequencing.

 Knockin Strategies :

Protein fusion：

Safe harbor knockin:

Gene knockin at Safe harbors such as hROSA26 and AAVS1 not only avoids random insertion in genome, but also achieves overexpression of target gene.

Case Study：

The signal pathway of intracellular antiviral response was confirmed by the THP-1 Cell Models of gene knock-out and knock-in.

The abnormal location of DNA in the cytoplasm is usually related to virus infection or tumor. The cGAS-cGAMP-STING pathway can detect the existence of cytosolic dsDNA, and induce a strong immune response, producing interferon and activating other immune response genes. RIG1-MAVS can detect pppRNA (dsRNA, the genome of some viruses) in cytoplasm and induce immune response. Sometimes there is a complex of RNA and DNA in the cytoplasm, which usually occurs in the case of some virus infection. In order to study which pathway that the RNA-DNA complex activates the immune response, the researchers generated MAVS, cGAS, STING knockout THP-1 cell lines, and introduced dsDNA, ppRNA and RNA-DNA complex into the cells respectively. It was found that the RNA-DNA complex is activated by the cGAS-cGAMP-STING pathway.

Then, the researchers used CRISPR/Cas9 technology to insert 2A-GLuc into the IFIT1 gene. IFIT1 is a typical interferon activated gene. Subsequent experiments showed that after the introduction of RNA-DNA complex, the expression of Gluc was driven by the activation of IFIT1 promoter due to the expression of interferon. These results further proved that the RNA-DNA complex in the cytoplasm activated the immune response through the cGAS-cGAMP-STING pathway.

Reference：

Mankan A K, Schmidt T, Chauhan D, et al. Cytosolic RNA: DNA hybrids activate the cGAS–STING axis[J]. The EMBO journal, 2014, 33(24): 2937-2946.

Structure and mechanism of a Hypr GGDEF enzyme that activates cGAMP signaling to control extracellular metal respiration.

Structure and mechanism of a Hypr GGDEF enzyme that activates cGAMP signaling to control extracellular metal respiration. Elife. 2019 Apr 09;8: Authors: Hallberg ZF, Chan CH, Wright TA, Kranzusch PJ, Doxzen KW, Park JJ, Bond DR, Hammond MC Abstract A newfound signaling pathway employs a GGDEF enzyme with unique activity compared to the majority of homologs associated with bacterial cyclic di-GMP signaling. This system provides a rare opportunity to study how signaling proteins natively gain distinct function. Using genetic knockouts, riboswitch reporters, and RNA-Seq, we show that GacA, the Hypr GGDEF in Geobacter sulfurreducens, specifically regulates cyclic GMP-AMP (3',3'-cGAMP) levels in vivo to stimulate gene expression associated with metal reduction separate from electricity production. To reconcile these in vivo findings with prior in vitro results that showed GacA was promiscuous, we developed a full kinetic model combining experimental data and mathematical modeling to reveal mechanisms that contribute to in vivo specificity. A 1.4 Å-resolution crystal structure of the Geobacter Hypr GGDEF domain was determined to understand the molecular basis for those mechanisms, including key cross-dimer interactions. Together these results demonstrate that specific signaling can result from a promiscuous enzyme. PMID: 30964001 [PubMed - in process] http://dlvr.it/R2XCBC

Intratumoral administration of cGAMP transiently accumulates potent macrophages for anti-tumor immunity at a mouse tumor site.

Related Articles Intratumoral administration of cGAMP transiently accumulates potent macrophages for anti-tumor immunity at a mouse tumor site. Cancer Immunol Immunother. 2017 Feb 27;: Authors: Ohkuri T, Kosaka A, Ishibashi K, Kumai T, Hirata Y, Ohara K, Nagato T, Oikawa K, Aoki N, Harabuchi Y, Celis E, Kobayashi H Abstract Stimulator of IFN genes (STING) spontaneously contributes to anti-tumor immunity by inducing type I interferons (IFNs) following sensing of tumor-derived genomic DNAs in the tumor-bearing host. Although direct injection of STING ligands such as cyclic diguanylate monophosphate (c-di-GMP) and cyclic [G(2',5')pA(3',5')p] (cGAMP) into the tumor microenvironment exerts anti-tumor effects through strong induction of type I IFNs and activation of innate and adaptive immunity, the precise events caused by STING in the tumor microenvironment remain to be elucidated. We describe here our finding that a CD45(+) CD11b(mid) Ly6C(+) cell subset transiently accumulated in mouse tumor microenvironment of 4T1 breast cancer, squamous cell carcinomas, CT26 colon cancer, or B16F10 melanoma tissue after intratumoral injection of cGAMP. The accumulated cells displayed a macrophage (M ) phenotype since the cells were positive for F4/80 and MHC class II and negative for Ly6G. Intratumoral cGAMP treatment did not induce Mφ accumulation in STING-deficient mice. Depletion of CD8(+) T cell using anti-CD8 mAb impaired the anti-tumor effects of cGAMP treatment. Depletion of the Mφ using clodronate liposomes impaired the anti-tumor effects of cGAMP treatment. Functional analysis indicated that the STING-triggered tumor-migrating Mφ exhibited phagocytic activity, production of tumor necrosis factor alpha TNFα), and high expression levels of T cell-recruiting chemokines, Cxcl10 and Cxcl11, IFN-induced molecules, MX dynamin-like GTPase 1 (Mx1) and 2'-5' oligoadenylate synthetase-like 1 (Oasl1), nitric oxide synthase 2 (Nos2), and interferon beta 1 (Ifnb1). These results indicate that the STING-triggered tumor-migrating Mφ participate in the anti-tumor effects of STING-activating compounds. PMID: 28243692 [PubMed - as supplied by publisher] http://dlvr.it/NW48qV

Intratumoral administration of cGAMP transiently accumulates potent macrophages for anti-tumor immunity at a mouse tumor site

Abstract

Stimulator of IFN genes (STING) spontaneously contributes to anti-tumor immunity by inducing type I interferons (IFNs) following sensing of tumor-derived genomic DNAs in the tumor-bearing host. Although direct injection of STING ligands such as cyclic diguanylate monophosphate (c-di-GMP) and cyclic [G(2′,5′)pA(3′,5′)p] (cGAMP) into the tumor microenvironment exerts anti-tumor effects through strong induction of type I IFNs and activation of innate and adaptive immunity, the precise events caused by STING in the tumor microenvironment remain to be elucidated. We describe here our finding that a CD45+ CD11bmid Ly6C+ cell subset transiently accumulated in mouse tumor microenvironment of 4T1 breast cancer, squamous cell carcinomas, CT26 colon cancer, or B16F10 melanoma tissue after intratumoral injection of cGAMP. The accumulated cells displayed a macrophage (M ) phenotype since the cells were positive for F4/80 and MHC class II and negative for Ly6G. Intratumoral cGAMP treatment did not induce Mφ accumulation in STING-deficient mice. Depletion of CD8+ T cell using anti-CD8 mAb impaired the anti-tumor effects of cGAMP treatment. Depletion of the Mφ using clodronate liposomes impaired the anti-tumor effects of cGAMP treatment. Functional analysis indicated that the STING-triggered tumor-migrating Mφ exhibited phagocytic activity, production of tumor necrosis factor alpha TNFα), and high expression levels of T cell-recruiting chemokines, Cxcl10 and Cxcl11, IFN-induced molecules, MX dynamin-like GTPase 1 (Mx1) and 2′-5′ oligoadenylate synthetase-like 1 (Oasl1), nitric oxide synthase 2 (Nos2), and interferon beta 1 (Ifnb1). These results indicate that the STING-triggered tumor-migrating Mφ participate in the anti-tumor effects of STING-activating compounds.

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