Binding of Gamma-Glutamyl Transferase to TLR4 Signalling Allows Tissue Factor Activation in Monocytes

Gamma-glutamyl transferase (GGT) is involved in the progression of atherosclerosis, since its enzymatic activity promotes the generation of reactive oxygen species (ROS). Besides, GGT may act as a prothrombotic factor by inducing tissue factor (TF) expression, independently of its enzymatic activity. The aim of this study was to assess whether GGT-induced TF stimulation was a consequence of binding to toll-like receptor 4 (TLR4) expressed on monocytes, the precursors of macrophages and foam cells which colocalize with GGT activity within atherosclerotic plaques. Experiments were performed in human peripheral blood mononuclear cells (PBMCs), THP-1 cells (a monocytic cellular model), and HEK293 cells, which were genetically modified to study the activation of TLR4. TF procoagulant activity was assessed by a one-stage clotting time test, and TF protein expression was estimated by western blot. Human recombinant (hr) GGT protein increased TF procoagulant activity and protein expression in both PBMCs and THP-1 cells. The GGT-induced TF stimulation was prevented by cellular pretreatment with TLR4/NF-κB inhibitors (LPS-Rs, CLI-095, and BAY-11-7082), and HEK293 cells lacking TLR4 confirmed that TLR4 is essential for GGT-induced activation of NF-κB. In conclusion, hrGGT induced TF expression in monocytes through a cytokine-like mechanism that involved the activation of TLR4/NF-κB signaling.     

CD40 Pathway and IL-2 Expression Mediate the Differential Outcome of Colorectal Cancer Patients with Different CSF1R c.1085 Genotypes

Colony-stimulating factor 1 receptor (CSF-1R) acts as the receptor for colony stimulating factor 1, a cytokine that controls the production, differentiation, and function of macrophages. Prior studies showed cancer patients harboring germline CSF1R c.1085A>G genetic variant had better survival. Here, primary tumor samples from a stage III colorectal cancer (CRC) cohort were analyzed by a targeted gene expression assay containing 395 immune-related genes to study the immune mechanism underlying the different outcomes. CRC patients with CSF1R c.1085 genotype A_G had a better disease-free and overall survival than those with CSF1R genotype A_A. Compared to the group of patients without CSF1R variant, higher CD40LG expression, a surface marker of T cells, was found in the tumor tissues of patients with CSF1R c.1085 variant. In parallel with the higher CD40LG gene expression, immunofluorescent staining also showed more CD3⁺CD40L⁺ T cell infiltrates in tumors with CSF1R c.1085 genotype A_G. Moreover, higher IL-2 expression, known to be regulated by CD40 pathway, was also observed in tumors with CSF1R c.1085 genotype A_G than genotype A_A. Higher IL-2 expression generated by the interaction of CD40 ligand and CD40 between T cells and macrophages with CSF1R c.1085A>G variant is the potential mechanism explaining the different outcomes.     

Association between Serum Biomarkers and Peripheral Neuropathy in Microscopic Polyangiitis

This study aimed to elucidate the pathomechanism of peripheral neuropathy (PN) in microscopic polyangiitis (MPA) and to identify biomarkers useful for diagnosis and severity assessment. Patients with MPA (n = 37) and other non-inflammatory neurological diseases (ONDs; n = 12) were enrolled, and the peripheral nerves of all patients were evaluated using nerve conduction studies. We compared the clinical characteristics and 14 serum biomarker profiles among patients with MPA and PN, MPA without PN, and ONDs. Patients with MPA had a higher prevalence of motor neuropathy than patients with ONDs. Among the patients with MPA, those with motor neuropathy had significantly higher total Birmingham Vasculitis Activity Scores and serum levels of C-reactive protein (CRP), tissue inhibitor of metalloproteinase-1 (TIMP-1), and interleukin-6 than patients without motor neuropathy. Multivariable analyses adjusted for age, serum CRP level, and diabetes mellitus showed that high serum levels of TIMP-1 were independently related to a diagnosis of motor neuropathy in MPA. Additionally, there were significant negative correlations between the serum levels of TIMP-1 and compound muscle action potential amplitudes. Serum levels of TIMP-1 may be associated with the pathomechanism of motor neuropathy in MPA and could be a useful biomarker for diagnosing and evaluating the severity of motor neuropathy in MPA.     

Adenovirus-Mediated Inducible Expression of a PD-L1 Blocking Antibody in Combination with Macrophage Depletion Improves Survival in a Mouse Model of Peritoneal Carcinomatosis

Immune checkpoint inhibitors (ICIs) have demonstrated remarkable efficacy in a growing number of malignancies. However, overcoming primary or secondary resistances is difficult due to pharmacokinetics issues and side effects associated with high systemic exposure. Local or regional expression of monoclonal antibodies (mAbs) using gene therapy vectors can alleviate this problem. In this work, we describe a high-capacity adenoviral vector (HCA-EFZP-aPDL1) equipped with a mifepristone-inducible system for the controlled expression of an anti-programmed death ligand 1 (PD-L1) blocking antibody. The vector was tested in an immune-competent mouse model of colorectal cancer based on implantation of MC38 cells. A single local administration of HCA-EFZP-aPDL1 in subcutaneous lesions led to a significant reduction in tumor growth with minimal release of the antibody in the circulation. When the vector was tested in a more stringent setting (rapidly progressing peritoneal carcinomatosis), the antitumor effect was marginal even in combination with other immune-stimulatory agents such as polyinosinic-polycytidylic acid (pI:C), blocking mAbs for T cell immunoglobulin, mucin-domain containing-3 (TIM-3) or agonistic mAbs for 4-1BB (CD137). In contrast, macrophage depletion by clodronate liposomes enhanced the efficacy of HCA-EFZP-aPDL1. These results highlight the importance of addressing macrophage-associated immunoregulatory mechanisms to overcome resistance to ICIs in the context of colorectal cancer.     

Asprosin Exerts Pro-Inflammatory Effects in THP-1 Macrophages Mediated via the Toll-like Receptor 4 (TLR4) Pathway

Adipose tissue is a dynamic endocrine organ, secreting a plethora of adipokines which play a key role in regulating metabolic homeostasis and other physiological processes. An altered adipokine secretion profile from adipose tissue depots has been associated with obesity and related cardio-metabolic diseases. Asprosin is a recently described adipokine that is released in response to fasting and can elicit orexigenic and glucogenic effects. Circulating asprosin levels are elevated in a number of cardio-metabolic diseases, including obesity and type 2 diabetes. In vitro studies have reported pro-inflammatory effects of asprosin in a variety of tissues. The present study aimed to further elucidate the role of asprosin in inflammation by exploring its potential effect(s) in THP-1 macrophages. THP-1 monocytes were differentiated to macrophages by 48 h treatment with dihydroxyvitamin D3. Macrophages were treated with 100 nM recombinant human asprosin, 100 ng/mL lipopolysaccharide (LPS), and 10 μM caffeic acid phenethyl ester (CAPE; an inhibitor of NFκB activation) or 1 µM TAK-242 (a Toll-like receptor 4, TLR4, inhibitor). The expression and secretion of pertinent pro-inflammatory mediators were measured by qPCR, Western blot, ELISA and Bioplex. Asprosin stimulation significantly upregulated the expression and secretion of the pro-inflammatory cytokines: tumour necrosis factor α (TNFα), interleukin-1β (IL-1β), IL-8 and IL-12 in vitro. This pro-inflammatory response in THP-1 macrophages was partly attenuated by the treatments with CAPE and was significantly inhibited by TAK-242 treatment. Asprosin-induced inflammation is significantly counteracted by TLR4 inhibition in THP-1 macrophages, suggesting that asprosin exerts its pro-inflammatory effects, at least in part, via the TLR4 signalling pathway.     

Pharmacological Evidence on Augmented Antiallodynia Following Systemic Co-Treatment with GlyT-1 and GlyT-2 Inhibitors in Rat Neuropathic Pain Model

The limited effect of current medications on neuropathic pain (NP) has initiated large efforts to develop effective treatments. Animal studies showed that glycine transporter (GlyT) inhibitors are promising analgesics in NP, though concerns regarding adverse effects were raised. We aimed to study NFPS and Org-25543, GlyT-1 and GlyT-2 inhibitors, respectively and their combination in rat mononeuropathic pain evoked by partial sciatic nerve ligation. Cerebrospinal fluid (CSF) glycine content was also determined by capillary electrophoresis. Subcutaneous (s.c.) 4 mg/kg NFPS or Org-25543 showed analgesia following acute administration (30–60 min). Small doses of each compound failed to produce antiallodynia up to 180 min after the acute administration. However, NFPS (1 mg/kg) produced antiallodynia after four days of treatment. Co-treatment with subanalgesic doses of NFPS (1 mg/kg) and Org-25543 (2 mg/kg) produced analgesia at 60 min and thereafter meanwhile increased significantly the CSF glycine content. This combination alleviated NP without affecting motor function. Test compounds failed to activate G-proteins in spinal cord. To the best of our knowledge for the first time we demonstrated augmented analgesia by combining GlyT-1 and 2 inhibitors. Increased CSF glycine content supports involvement of glycinergic system. Combining selective GlyT inhibitors or developing non-selective GlyT inhibitors might have therapeutic value in NP.     

Glucagon-like Peptide-1 Receptor Agonists in the Management of Type 2 Diabetes Mellitus and Obesity: The Impact of Pharmacological Properties and Genetic Factors

Glucagon-like peptide-1 (GLP-1) receptor agonists are a new class of antihyperglycemic drugs that enhance appropriate pancreatic β-cell secretion, pancreatic α-cell (glucagon) suppression, decrease liver glucose production, increase satiety through their action on the central nervous system, slow gastric emptying time, and increase insulin action on peripheral tissue. They are effective in the management of type 2 diabetes mellitus and have a favorable effect on weight loss. Their cardiovascular and renal safety has been extensively investigated and confirmed in many clinical trials. Recently, evidence has shown that in addition to the existing approaches for the treatment of obesity, semaglutide in higher doses promotes weight loss and can be used as a drug to treat obesity. However, some T2DM and obese patients do not achieve a desired therapeutic effect of GLP-1 receptor agonists. This could be due to the multifactorial etiologies of T2DM and obesity, but genetic variability in the GLP-1 receptor or signaling pathways also needs to be considered in non-responders to GLP-1 receptor agonists. This review focuses on the pharmacological, clinical, and genetic factors that may influence the response to GLP-1 receptor agonists in the treatment of type 2 diabetes mellitus and obesity.     

Simultaneous Use of ROCK Inhibitors and EP2 Agonists Induces Unexpected Effects on Adipogenesis and the Physical Properties of 3T3-L1 Preadipocytes

To elucidate the additive effects of an EP2 agonist, omidenepag (OMD) or butaprost (Buta) on the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) on adipose tissue, two- or three-dimension (2D or 3D) cultures of 3T3-L1 cells were analyzed by lipid staining, the mRNA expression of adipogenesis-related genes, extracellular matrix (ECM) molecules including collagen (Col) -1, -4 and -6, and fibronectin (Fn), and the sizes and physical properties of 3D organoids, as measured by a micro-squeezer. The results indicate that adipogenesis induced (1) an enlargement of the 3D organoids; (2) a substantial enhancement in lipid staining as well as the expression of the Pparγ, Ap2 and Leptin genes; (3) a significant softening of the 3D organoids, the effects of which were all enhanced by Rip except for Pparγ expression; and (4) a significant downregulation in Col1 and Fn, and a significant upregulation in Col4, Col6, the effects of which were unchanged by Rip. When adding the EP2 agonist to Rip, (1) the sizes of the 3D organoids were reduced substantially; (2) lipid staining was increased (OMD), or decreased (Buta); (3) the stiffness of the 3D organoids was substantially increased in Buta; (4-1) the expression of Pparγ was suppressed (2D, OMD) or increased (2D, Buta), and the expressions of Ap2 were downregulated (2D, 3D) and Leptin was increased (2D) or decreased (3D), (4-2) all the expressions of four ECM molecules were upregulated in 2D (2D), and in 3D, the expression of Col1, Col4 was upregulated. The collective findings reported herein indicate that the addition of an EP2 agonist, OMD or Buta significantly but differently modulate the Rip-induced effects on adipogenesis and the physical properties of 2D and 3D cultured 3T3-L1 cells.     

Roles of Fatty Acids in Microglial Polarization: Evidence from In Vitro and In Vivo Studies on Neurodegenerative Diseases

Microglial polarization to the M1 phenotype (classically activated) or the M2 phenotype (alternatively activated) is critical in determining the fate of immune responses in neurodegenerative diseases (NDs). M1 macrophages contribute to neurotoxicity, neuronal and synaptic damage, and oxidative stress and are the first line of defense, and M2 macrophages elicit an anti-inflammatory response to regulate neuroinflammation, clear cell debris, and promote neuroregeneration. Various studies have focused on the ability of natural compounds to promote microglial polarization from the M1 phenotype to the M2 phenotype in several diseases, including NDs. However, studies on the roles of fatty acids in microglial polarization and their implications in NDs are a rare find. Most of the studies support the role of polyunsaturated fatty acids (PUFAs) in microglial polarization using cell and animal models. Thus, we aimed to collect data and provide a narrative account of microglial types, markers, and studies pertaining to fatty acids, particularly PUFAs, on microglial polarization and their neuroprotective effects. The involvement of only PUFAs in the chosen topic necessitates more in-depth research into the role of unexplored fatty acids in microglial polarization and their mechanistic implications. The review also highlights limitations and future challenges.     

Peripheral and Central Effects of Melatonin on Blood Pressure Regulation

The pineal hormone, melatonin (N-acetyl-5-methoxytryptamine), shows potent receptor-dependent and -independent actions, which participate in blood pressure regulation. The antihypertensive effect of melatonin was demonstrated in experimental and clinical hypertension. Receptor-dependent effects are mediated predominantly through MT1 and MT2 G-protein coupled receptors. The pleiotropic receptor-independent effects of melatonin with a possible impact on blood pressure involve the reactive oxygen species (ROS) scavenging nature, activation and over-expression of several antioxidant enzymes or their protection from oxidative damage and the ability to increase the efficiency of the mitochondrial electron transport chain. Besides the interaction with the vascular system, this indolamine may exert part of its antihypertensive action through its interaction with the central nervous system (CNS). The imbalance between the sympathetic and parasympathetic vegetative system is an important pathophysiological disorder and therapeutic target in hypertension. Melatonin is protective in CNS on several different levels: It reduces free radical burden, improves endothelial dysfunction, reduces inflammation and shifts the balance between the sympathetic and parasympathetic system in favor of the parasympathetic system. The increased level of serum melatonin observed in some types of hypertension may be a counter-regulatory adaptive mechanism against the sympathetic overstimulation. Since melatonin acts favorably on different levels of hypertension, including organ protection and with minimal side effects, it could become regularly involved in the struggle against this widespread cardiovascular pathology.     

Neuroprotective and Symptomatic Effects of Cannabidiol in an Animal Model of Parkinson’s Disease

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the Substantia Nigra pars compacta, leading to classical PD motor symptoms. Current therapies are purely symptomatic and do not modify disease progression. Cannabidiol (CBD), one of the main phytocannabinoids identified in Cannabis Sativa, which exhibits a large spectrum of therapeutic properties, including anti-inflammatory and antioxidant effects, suggesting its potential as disease-modifying agent for PD. The aim of this study was to evaluate the effects of chronic treatment with CBD (10 mg/kg, i.p.) on PD-associated neurodegenerative and neuroinflammatory processes, and motor deficits in the 6-hydroxydopamine model. Moreover, we investigated the potential mechanisms by which CBD exerted its effects in this model. CBD-treated animals showed a reduction of nigrostriatal degeneration accompanied by a damping of the neuroinflammatory response and an improvement of motor performance. In particular, CBD exhibits a preferential action on astrocytes and activates the astrocytic transient receptor potential vanilloid 1 (TRPV1), thus, enhancing the endogenous neuroprotective response of ciliary neurotrophic factor (CNTF). These results overall support the potential therapeutic utility of CBD in PD, as both neuroprotective and symptomatic agent.     

Synthesis of Novel Spiro-Tetrahydroquinoline Derivatives and Evaluation of Their Pharmacological Effects on Wound Healing

A highly diastereoselective method for the synthesis of novel spiro-tetrahydroquinoline derivatives is reported here, using a one-pot reaction method. All compounds were characterized by ¹H-nuclear magnetic resonance (NMR) and mass spectroscopy, and their stereo configurations were confirmed by X-ray analysis. These activities of these derivatives were then tested in human keratocyte cells. The responses of cells to treatment with selected compounds were studied using scratch analysis, and the compounds were tested in a mouse excision wound model. Three of the derivatives demonstrated significant wound-healing activities.     

Toll-like Receptor 7 (TLR7) Is Expressed in Adipocytes and the Pharmacological TLR7 Agonist Imiquimod and Adipocyte-Derived Cell-Free Nucleic Acids (cfDNA) Regulate Adipocyte Function

Endosome-localized Toll-like receptors (TLRs) 3 and 9 are expressed and functionally active in adipocytes. The functionality and role of TLR7 in adipocyte biology and innate immunity of adipose tissue (AT) is poorly characterized. We analyzed TLR7 mRNA and protein expression in murine 3T3-L1 and primary adipocytes, in co-cultures of 3T3-L1 adipocytes with murine J774A.1 monocytes and in human AT. The effects of TLR7 agonists imiquimod (IMQ) and cell-free nucleic acids (cfDNA) on adipokine concentration in cell-culture supernatants and gene expression profile were investigated. We found that TLR7 expression is strongly induced during adipocyte differentiation. TLR7 gene expression in adipocytes and AT stroma-vascular cells (SVC) seems to be independent of TLR9. IMQ downregulates resistin concentration in adipocyte cell-culture supernatants and modulates gene expression of glucose transporter Glut4. Adipocyte-derived cfDNA reduces adiponectin and resistin in cell-culture supernatants and potentially inhibits Glut4 gene expression. The responsiveness of 3T3-L1 adipocytes to imiquimod is preserved in co-culture with J774A.1 monocytes. Obesity-related, adipocyte-derived cfDNA engages adipocytic pattern recognition receptors (PRRs), modulating AT immune and metabolic homeostasis during adipose inflammation.     

NEIL1 and NEIL2 Are Recruited as Potential Backup for OGG1 upon OGG1 Depletion or Inhibition by TH5487

DNA damage caused by reactive oxygen species may result in genetic mutations or cell death. Base excision repair (BER) is the major pathway that repairs DNA oxidative damage in order to maintain genomic integrity. In mammals, eleven DNA glycosylases have been reported to initiate BER, where each recognizes a few related DNA substrate lesions with some degree of overlapping specificity. 7,8-dihydro-8-oxoguanine (8-oxoG), one of the most abundant DNA oxidative lesions, is recognized and excised mainly by 8-oxoguanine DNA glycosylase 1 (OGG1). Further oxidation of 8-oxoG generates hydantoin lesions, which are recognized by NEIL glycosylases. Here, we demonstrate that NEIL1, and to a lesser extent NEIL2, can potentially function as backup BER enzymes for OGG1 upon pharmacological inhibition or depletion of OGG1. NEIL1 recruitment kinetics and chromatin binding after DNA damage induction increase in cells treated with OGG1 inhibitor TH5487 in a dose-dependent manner, whereas NEIL2 accumulation at DNA damage sites is prolonged following OGG1 inhibition. Furthermore, depletion of OGG1 results in increased retention of NEIL1 and NEIL2 at damaged chromatin. Importantly, oxidatively stressed NEIL1- or NEIL2-depleted cells show excessive genomic 8-oxoG lesions accumulation upon OGG1 inhibition, suggesting a prospective compensatory role for NEIL1 and NEIL2. Our study thus exemplifies possible backup mechanisms within the base excision repair pathway.     

Overexpression of Adiponectin Receptor 1 Inhibits Brown and Beige Adipose Tissue Activity in Mice

Adult humans and mice possess significant classical brown adipose tissues (BAT) and, upon cold-induction, acquire brown-like adipocytes in certain depots of white adipose tissues (WAT), known as beige adipose tissues or WAT browning/beiging. Activating thermogenic classical BAT or WAT beiging to generate heat limits diet-induced obesity or type-2 diabetes in mice. Adiponectin is a beneficial adipokine resisting diabetes, and causing “healthy obese” by increasing WAT expansion to limit lipotoxicity in other metabolic tissues during high-fat feeding. However, the role of its receptors, especially adiponectin receptor 1 (AdipoR1), on cold-induced thermogenesis in vivo in BAT and in WAT beiging is still elusive. Here, we established a cold-induction procedure in transgenic mice over-expressing AdipoR1 and applied a live 3-D [¹⁸F] fluorodeoxyglucose-PET/CT (¹⁸F-FDG PET/CT) scanning to measure BAT activity by determining glucose uptake in cold-acclimated transgenic mice. Results showed that cold-acclimated mice over-expressing AdipoR1 had diminished cold-induced glucose uptake, enlarged adipocyte size in BAT and in browned WAT, and reduced surface BAT/body temperature in vivo. Furthermore, decreased gene expression, related to thermogenic Ucp1, BAT-specific markers, BAT-enriched mitochondrial markers, lipolysis and fatty acid oxidation, and increased expression of whitening genes in BAT or in browned subcutaneous inguinal WAT of AdipoR1 mice are congruent with results of PET/CT scanning and surface body temperature in vivo. Moreover, differentiated brown-like beige adipocytes isolated from pre-adipocytes in subcutaneous WAT of transgenic AdipoR1 mice also had similar effects of lowered expression of thermogenic Ucp1, BAT selective markers, and BAT mitochondrial markers. Therefore, this study combines in vitro and in vivo results with live 3-D scanning and reveals one of the many facets of the adiponectin receptors in regulating energy homeostasis, especially in the involvement of cold-induced thermogenesis.     

Genetic Deletion of Rheb1 in the Brain Reduces Food Intake and Causes Hypoglycemia with Altered Peripheral Metabolism

Excessive food/energy intake is linked to obesity and metabolic disorders, such as diabetes. The hypothalamus in the brain plays a critical role in the control of food intake and peripheral metabolism. The signaling pathways in hypothalamic neurons that regulate food intake and peripheral metabolism need to be better understood for developing pharmacological interventions to manage eating behavior and obesity. Mammalian target of rapamycin (mTOR), a serine/threonine kinase, is a master regulator of cellular metabolism in different cell types. Pharmacological manipulations of mTOR complex 1 (mTORC1) activity in hypothalamic neurons alter food intake and body weight. Our previous study identified Rheb1 (Ras homolog enriched in brain 1) as an essential activator of mTORC1 activity in the brain. Here we examine whether central Rheb1 regulates food intake and peripheral metabolism through mTORC1 signaling. We find that genetic deletion of Rheb1 in the brain causes a reduction in mTORC1 activity and impairs normal food intake. As a result, Rheb1 knockout mice exhibit hypoglycemia and increased lipid mobilization in adipose tissue and ketogenesis in the liver. Our work highlights the importance of central Rheb1 signaling in euglycemia and energy homeostasis in animals.     

Adipose-Derived Extract Suppresses IL-1β-Induced Inflammatory Signaling Pathways in Human Chondrocytes and Ameliorates the Cartilage Destruction of Experimental Osteoarthritis in Rats

We investigated the effects of adipose-derived extract (AE) on cultured chondrocytes and in vivo cartilage destruction. AE was prepared from human adipose tissues using a nonenzymatic approach. Cultured human chondrocytes were stimulated with interleukin-1 beta (IL-1β) with or without different concentrations of AE. The effects of co-treatment with AE on intracellular signaling pathways and their downstream gene and protein expressions were examined using real-time PCR, Western blotting, and immunofluorescence staining. Rat AE prepared from inguinal adipose tissues was intra-articularly delivered to the knee joints of rats with experimental osteoarthritis (OA), and the effect of AE on cartilage destruction was evaluated histologically. In vitro, co-treatment with IL-1β combined with AE reduced activation of the p38 and ERK mitogen-activated protein kinase (MAPK) pathway and nuclear translocation of the p65 subunit of nuclear factor-kappa B (NF-κB), and subsequently downregulated the expressions of matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, IL-6, and IL-8, whereas it markedly upregulated the expression of IL-1 receptor type 2 (IL-1R2) in chondrocytes. Intra-articular injection of homologous AE significantly ameliorated cartilage destruction six weeks postoperatively in the rat OA model. These results suggested that AE may exert a chondroprotective effect, at least in part, through modulation of the IL-1β-induced inflammatory signaling pathway by upregulation of IL-1R2 expression.     

The Enhanced Cytotoxic Effects in B-Cell Leukemia and Lymphoma Following Activation of Prostaglandin EP4 Receptor and Targeting of CD20 Antigen by Monoclonal Antibodies

Anti-CD20 monoclonal antibodies (MAbs) have revolutionized the treatment of B-cell leukemia and lymphoma. However, many patients do not respond to such treatment due to either deficiency of the complementary immune response or resistance to apoptosis. Other currently available treatments are often inadequate or induce major side effects. Therefore, there is a constant need for improved therapies. The prostaglandin E2 receptor 4 (EP4) receptor has been identified as a promising therapeutic target for hematologic B-cell malignancies. Herein, we report that EP4 receptor agonists PgE1-OH and L-902688 have exhibited enhanced cytotoxicity when applied together with anti-CD20 MAbs rituximab, ofatumumab and obinutuzumab in vitro in Burkitt lymphoma cells Ramos, as well as in p53-deficient chronic lymphocytic leukemia (CLL) cells MEC-1. Moreover, the enhanced cytotoxic effects of EP4 receptor agonists and MAbs targeting CD20 have been identified ex vivo on primary lymphocytes B obtained from patients diagnosed with CLL. Incubation of cells with PgE1-OH and L-902688 preserved the expression of CD20 molecules, further confirming the anti-leukemic potential of EP4 receptor agonists in combination with anti-CD20 MAbs. Additionally, we demonstrated that the EP4 receptor agonist PgE-1-OH induced apoptosis and inhibited proliferation via the EP4 receptor triggering in CLL. This work has revealed very important findings leading towards the elucidation of the anticancer potential of PgE1-OH and L-902688, either alone or in combination with MAbs. This may contribute to the development of potential therapeutic alternatives for patients with B-cell malignancies.     

Interleukin-34-CSF1R Signaling Axis Promotes Epithelial Cell Transformation and Breast Tumorigenesis

IL-34 has been recently identified as a ligand for CSF1R that regulates various cellular processes including cell proliferation, survival, and differentiation. Although the binding of IL-34 to CSF1R modulates several cancer-driving signaling pathways, little is known about the role of IL-34/CSF1R signaling in breast cancer. Herein, we report that IL-34 induces epithelial cell transformation and breast tumorigenesis through activation of MEK/ERK and JNK/c-Jun pathways. IL-34 increased the phosphorylation of MEK1/2, ERK1/2, JNK1/2, and c-Jun through CSF1R in mouse skin epidermal JB6 C141 cells and human breast cancer MCF7 cells. IL-34 enhanced c-Fos and c-Jun promoter activity, resulting in increased AP-1 transactivation activity in JB6 Cl41 and MCF7 cells. Moreover, PIN1 enhanced IL-34-induced phosphorylation of MEK1/2, ERK1/2, JNK1/2, and c-Jun in JB6 Cl41 and MCF7 cells. Inhibition of PIN1 using juglone prevented the IL-34-induced transformation of JB6 C141 cells. Similarly, silencing of PIN1 reduced the IL-34-induced tumorigenicity of MCF7 cells. Consistent with these results, the synergistic model showed that treatment with juglone suppressed the IL-34-induced growth of tumors formed by 4T1 cells in BALB/c mice. Our study demonstrates the role of IL-34-induced MEK/ERK and JNK/c-Jun cascades in breast cancer and highlights the regulatory role of PIN1 in IL-34-induced breast tumorigenesis.     

Combined Therapy with a CCR2/CCR5 Antagonist and FGF21 Analogue Synergizes in Ameliorating Steatohepatitis and Fibrosis

(1) Background: With new potential drug targets emerging, combination therapies appear attractive to treat non-alcoholic steatohepatitis (NASH) and fibrosis. Chemokine receptor CCR2/5 antagonists can improve fibrosis by reducing monocyte infiltration and altering hepatic macrophage subsets. Fibroblast growth factor 21 (FGF21) may improve NASH by modulating lipid and glucose metabolism. We compared effects of single drug to combination treatment as therapeutic strategies against NASH. (2) Methods: We analyzed serum samples and liver biopsies from 85 nonalcoholic fatty liver disease (NAFLD) patients. A CCR2/5 inhibitor (BMS-687681-02-020) and a pegylated FGF21 agonist (BMS-986171) were tested in male C57BL/6J mice subjected to dietary models of NASH and fibrosis (choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) up to 12 weeks; short- (2w) or long-term (6w) treatment). (3) Results: In NAFLD patients, chemokine CCL2 and FGF21 serum levels correlated with inflammatory serum markers, only CCL2 was significantly associated with advanced liver fibrosis. In rodent NASH, CCR2/5 inhibition significantly reduced circulating Ly6C+ monocytes and hepatic monocyte-derived macrophages, alongside reduced hepatic inflammation and fibrosis. FGF21 agonism decreased body weight, liver triglycerides and histological NASH activity. Combination treatment reflected aspects of both compounds upon short- and long-term application, thereby amplifying beneficial effects on all aspects of steatohepatitis and fibrosis. (4) Conclusions: CCR2/5 inhibition blocks hepatic infiltration of inflammatory monocytes, FGF21 agonism improves obesity-related metabolic disorders. Combined therapy ameliorates steatohepatitis and fibrosis more potently than single drug treatment in rodent NASH, corroborating the therapeutic potential of combining these two approaches in NASH patients.