Mouse Peritoneal Research Articles

Berberine Induces Combined Cell Death in Gastrointestinal Cell Lines.

Berberine (BBR) is a plant alkaloid that has various biological activities. The effects of BBR on gastrointestinal cancer (GIC) have also been investigated and anti-tumor effects such as induction of cell death have been reported. However, the mechanism of BBR-induced cell death has not been fully elucidated. To this end, we investigated the effects of BBR using three GIC cell lines. Our analyses revealed that BBR inhibited cell proliferation, invasion, sphere formation, and anticancer drug resistance in all of the cell lines. BBR also induced an increase in mitochondrial superoxide, lipid peroxide and Fe2+ levels, decreased mitochondrial membrane potential and respiration, decreased glutathione peroxidase 4 expression and glutathione and induced Parkin/PINK1-associated mitophagy. BBR, as well as rotenone, inhibited mitochondrial complex I and enhanced complex II, which were associated with autophagy, reactive oxidative species production, and cell death. Inhibition of complex II by malonate abrogated these changes. BBR-induced cell death was partially rescued by ferrostatin-1, deferoxamine, Z-VAD-FMK, and ATG5 knockdown. Furthermore, oral administration of BBR significantly reduced tumor weight and ascites in a syngeneic mouse peritoneal metastasis model using CT26 GIC cells. These findings suggest that BBR induced a combined type of cell death via complex I inhibition and autophagy. The marked anti-tumor and anti-stemness effects are expected to be useful as a new cell death-inducing agent for the treatment of GIC.

Curcumin inhibits the pruritus in mice through mast cell MrgprB2 receptor.

Curcumin is a diketone compound extracted from the rhizomes of some plants in the Zingiberaceae and Araceae family. It possesses a variety of biological activities, including antioxidant, anti-inflammatory and anti-cancer properties. However, the cellular and molecular antipruritic mechanisms of curcumin remain to be explored. Our objective was to study the role of curcumin in pruritus and determine whether its antipruritic effect is related to MrgprB2 receptor. The effect of curcumin on pruritus in mice was examined by scratching behavior test. The antipruritic mechanism of curcumin was explored by using transgenic mice (MrgprB2-/- mice, MrgprB2CreTd/tomato mice), histological analysis, western blot and immunofluorescence. In addition, the relationship between curcumin and MrgprB2/X2 receptor was studied in vitro by using calcium imaging, plasmid transfection and molecular docking RESULTS: In the current study, we found that curcumin had obvious antipruritic effect. Its antipruritic effect was related to the regulation of MrgprB2 receptor activation and mast cells tryptase release. In vitro, mouse peritoneal mast cells activated by compound 48/80 could be inhibited by curcumin. In addition, curcumin was also found to suppress the calcium flux in MrgprX2 or MrgprB2-overexpression HEK cells induced by compound 48/80, substance P, and PAMP 9-20, displaying the specific relation with the MrgprB2/X2 receptor. Moreover, molecular docking results showed that curcumin had affinity to MrgprX2 protein. Overall, these results indicated that curcumin has the potential to treat pruritus induced by mast cell MrgprB2 receptor.

A flagellin-conjugate protein induces dual NLRC4- and NLRP3-inflammasome activation which modulates inflammatory cytokine secretion from macrophages.

A recombinant fusion protein combining the adjuvant and TLR5-ligand flagellin with the major birch pollen allergen Bet v 1 (rFlaA:Betv1) has been suggested to prevent the manifestation of birch allergy. Noteworthy, rFlaA:Betv1 induced both pro- and anti-inflammatory responses which were differentially regulated. However, the mechanism by which flagellin fusion proteins modulate allergen-specific immune responses, especially the mechanisms underlying IL-1β secretion and their contribution to the overall immune responses remains elusive. To investigate the mechanisms underlying the production of IL-1β from rFlaA:Betv1 stimulated macrophages. Macrophages were derived from mouse peritoneal-, human buffy-coat-, and PMA-differentiated THP-1 (wild type or lacking either ASC, NLRP3, or NLRC4) cells. Macrophages were stimulated with non-modified rFlaA:Betv1, mutant variants lacking either the flagellin DC0 domain or a sequence motif formerly described to mediate TLR5-activation, and respective controls in the presence or absence of inhibitors interfering with MAPK- and NFκB-signaling. Cytokine secretion was analyzed by ELISA and intracellular signaling by Western Blot. To study the contribution of IL-1β to the overall immune responses, IL1R-deficient mouse peritoneal macrophages were used. rFlaA:Betv1 consistently activated all types of investigated macrophages, inducing higher IL-1β secretion compared with the equimolar mixture of both proteins. rFlaA:Betv1-induced activation of THP-1 macrophages was shown to be independent of either the TLR5-activating sequence motif or the flagellin DC0 domain but depended on both NLRP3- and NLRC4-inflammasomes. In addition, NFκB and SAP/JNK MAP kinases regulated rFlaA:Betv1-induced inflammasome activation and cytokine secretion by modulating pro-Caspase-1- and pro-IL-1β-expression in THP-1 macrophages. Finally, lack of IL-1β positive feedback via the IL1R strongly diminished the rFlaA:Betv1-induced secretion of IL-1β, IL-6, and TNF-α from peritoneal macrophages. The mechanisms contributing to rFlaA:Betv1-induced IL-1β secretion from macrophages were shown to be complex, involving both NLRC4- and NLRP3-inflammsomes, as well as NFκB- and SAP/JNK MAP kinase-signaling. Better understanding the mechanisms regulating the activation of immune cells by novel therapeutic candidates like the rFlaA:Betv1 fusion protein will allow us to further improve and develop new treatment strategies when using flagellin as an adjuvant.

Trabectedin modulates macrophage polarization in the tumor-microenvironment. Role of KV1.3 and KV1.5 channels

Immune cells have an important role in the tumor-microenvironment. Macrophages may tune the immune response toward inflammatory or tolerance pathways. Tumor-associated macrophages (TAM) have a string of immunosuppressive functions and they are considered a therapeutic target in cancer. This study aimed to analyze the effects of trabectedin, an antitumor agent, on the tumor-microenvironment through the characterization of the electrophysiological and molecular phenotype of macrophages. Experiments were performed using the whole-cell configuration of the patch-clamp technique in resident peritoneal mouse macrophages. Trabectedin does not directly interact with KV1.5 and KV1.3 channels, but their treatment (16 h) with sub-cytotoxic concentrations of trabectedin increased their KV current due to an upregulation of KV1.3 channels. In vitro generated TAM (TAMiv) exhibited an M2-like phenotype. TAMiv generated a small KV current and express high levels of M2 markers. K+ current from TAMs isolated from tumors generated in mice is a mixture of KV and KCa, and in TAM isolated from tumors generated in trabectedin-treated mice, the current is mostly driven by KCa. We conclude that the antitumor capacity of trabectedin is not only due to its effects on tumor cells, but also to the modulation of the tumor microenvironment, due, at least in part, to the modulation of the expression of different macrophage ion channels.

In vitro Anti-Leishmanial Activities of Methanol Extract of Brucea antidysenterica J.F. Mill Seeds and Its Solvent Fractions.

Leishmaniasis is one of the neglected tropical diseases, threatening lives of about 350 million people globally. Brucea antidysenterica seeds are used for the treatment of cutaneous leishmaniasis in the traditional medicine in Ethiopia. This study aimed to evaluate Brucea antidysenterica seeds' anti-leishmanial activity in vitro. The crude (80% methanol) extract of Brucea antidysenterica seeds and its fractions were evaluated for their anti-leishmanial activities against promastigotes and intracellular amastigotes of Leishmania donovani and Leishmania aethiopica, and for their cytotoxic effects against mammalian cells. The quantitative estimations of total phenolic compounds (TPCs), flavonoids (TFCs) and alkaloids (TACs) were determined, spectrophotometrically. Median inhibitory concentration (IC50) and median cytotoxic concentration (CC50) of the extract and its solvent fractions were calculated using GraphPad Prism 9.1.0 computer software. Data was presented as mean ± standard error of the mean (SEM). The crude extract and its hexane, ethyl acetate and butanol fractions showed anti-leishmanial activities, with IC50 values of 4.14-60.12 µg/mL against promastigotes, and 6.16-40.12 µg/mL against amastigotes of both Leishmania species. They showed moderate cytotoxicity against Vero cell lines and peritoneal mice macrophages, with CC50 values of 100-500 µg/mL, but >1600 µg/mL against red blood cells. Selectivity indices ranged from 7.97 to 30.97. The crude extract, and its ethyl acetate and hexane fractions possessed 54.78-127.72 mg of gallic acid equivalent TPC, 18.30-79.21 mg of quercetin equivalent TFC, and 27.62-97.22 mg of atropine equivalent TAC per gram of extracts. The seeds of the plant possessed anti-leishmanial activities against L. aethiopica and L. donovani that might provide a scientific justification for its use in the treatment of leishmaniasis by traditional healers. Future works are recommended to isolate, purify and identify the possible secondary metabolites attributed to the anti-leishmanial activity.

Defining and targeting tumor-associated macrophages in malignant mesothelioma

Defining the ontogeny of tumor-associated macrophages (TAM) is important to develop therapeutic targets for mesothelioma. We identified two distinct macrophage populations in mouse peritoneal and pleural cavities, the monocyte-derived, small peritoneal/pleural macrophages (SPM), and the tissue-resident large peritoneal/pleural macrophages (LPM). SPM rapidly increased in tumor microenvironment after tumor challenge and contributed to the vast majority of M2-like TAM. The selective depletion of M2-like TAM by conditional deletion of the Dicer1 gene in myeloid cells (D-/-) promoted tumor rejection. Sorted SPM M2-like TAM initiated tumorigenesis invivo and invitro, confirming their capacity to support tumor development. The transcriptomic and single-cell RNA sequencinganalysis demonstrated that both SPM and LPM contributed to the tumor microenvironment by promoting the IL-2-STAT5 signaling pathway, inflammation, and epithelial-mesenchymal transition. However, while SPM preferentially activated the KRAS and TNF-α/NFkB signaling pathways, LPM activated the IFN-γ response. The importance of LPM in the immune response was confirmed by depleting LPM with intrapleural clodronate liposomes, which abrogated the antitumoral memory immunity. SPM gene signature could be identified in pleural effusion and tumor from patients with untreated mesothelioma. Five genes, TREM2, STAB1, LAIR1, GPNMB, and MARCO, could potentially be specific therapeutic targets. Accordingly, Trem2 gene deletion led to reduced SPM M2-like TAM with compensatory increase in LPM and slower tumor growth. Overall, these experiments demonstrate that SPM M2-like TAM play a key role in mesothelioma development, while LPM more specifically contribute to the immune response. Therefore, selective targeting of monocyte-derived TAM may enhance antitumor immunity through compensatory expansion of tissue-resident TAM.

Liquiritin inhibits MRGPRX2-mediated pseudo-allergy through the PI3K/AKT and PLCγ signaling pathways

Liquiritin is a natural flavone with a variety of pharmacological effects derived from the medicinal food homology plant Glycyrrhiza uralensis Fisch. As a kind of lethal allergic reactions, pseudo-allergic reactions (PARs) arise from the Mas-related G protein coupled receptor X2 (MRGPRX2)-triggered fast degranulation of mast cells (MCs). In the current work, the anti-pseudo-allergy action and potential mechanisms of liquiritin were explored in vivo and in vitro. Liquiritin suppressed the calcium influx and degranulation elicited by Compound 48/80 (C48/80) in mouse peritoneal mast cells (MPMCs). In mice, liquiritin also inhibited the C48/80-elicited hind paw extravasation, as well as the elevations in TNF-α and histamine levels. Molecular docking combined with detection of HEK293T cells expressing human MRGPRX2 showed that liquiritin was a potential MRGPRX2 antagonist and inhibited PARs through the PI3K/AKT and PLCγ signaling pathways downstream of MRGPRX2. The present work opens a new avenue for the PARs management.

Inactivation of the UL37 Deamidase Enhances Virus Replication and Spread of the HSV-1(VC2) Oncolytic Vaccine Strain and Secretion of GM-CSF.

The HSV-1 (VC2) live-attenuated vaccine strain was engineered with specific deletions in the amino termini of glycoprotein K (gK) and membrane protein UL20, rendering the virus unable to enter neurons and establish latency. VC2 replicates efficiently in epithelial cell culture but produces lower viral titers and smaller viral plaques than its parental HSV-1 (F) wild-type virus. VC2 is an effective live-attenuated vaccine against HSV-1 and HSV-2 infections in mice and guinea pigs and an anti-tumor immunotherapeutic and oncolytic virus against melanoma and breast cancer in mouse models. Previously, we reported that the gK/UL20 complex interacts with the UL37 tegument protein, and this interaction is essential for virion intracellular envelopment and egress. To investigate the potential role of the UL37 deamidase functions, the recombinant virus FC819S and VC2C819S were constructed with a C819S substitution to inactivate the UL37 predicted deamidase active site on an HSV-1(F) and HSV-1(VC2) genetic background, respectively. FC819S replicated to similar levels with HSV-1(F) and produced similar size viral plaques. In contrast, VC2C819S replication was enhanced, and viral plaques increased in size, approaching those of the wild-type HSV-1(F) virus. FC819S infection of cell cultures caused enhanced GM-CSF secretion in comparison to HSV-1(F) across several cell lines, including HEp2 cells and cancer cell lines, DU145 (prostate) and Panc 04.03 (pancreas), and primary mouse peritoneal cells. VC2 infection of these cell lines caused GM-CSF secretion at similar levels to FC819S infection. However, the VC2C819S virus did not exhibit any further enhancement of GM-CSF secretion compared to the VC2 virus. These results suggest that the UL37 deamidation functions in conjunction with the gK/UL20 complex to facilitate virus replication and GM-CSF secretion.

Chemosensor with Ultra-High Fluorescence Enhancement for Assisting in Diagnosis and Resection of Ovarian Cancer.

Fluorescence imaging-guided diagnostics is one of the most promising approaches for facile detection of tumors in situ owing to its simple operation and non-invasiveness. As a crucial biomarker for primary ovarian cancers, β-galactosidase (β-gal) has been demonstrated to be the significant molecular target for visualization of ovarian tumors. Herein, a membrane-permeable fluorescent chemosensor (namely, LAN-βgal) was synthesized for β-gal-specific detection using the d-galactose residue as a specific recognition unit and LAN-OH (ΦF = 0.47) as a fluorophore. After β-gal was digested, the fluorescence of the initially quenched LAN-βgal (ΦF < 0.001) was enhanced by up to more than 2000-fold, which exceeded the fluorescence enhancement of other previously reported probes. We also demonstrated that the chemosensor LAN-βgal could visualize endogenous β-gal and distinguish ovarian cancer cells from normal ovarian cells. Further, the chemosensor LAN-βgal was successfully applied to visualize the back tumor-bearing mouse model and peritoneal metastatic ovarian cancer model in vivo. More importantly, through in situ spraying, the proposed chemosensor was successfully employed to assist in the surgical resection of ovarian cancer tumors due to its high tumor-to-normal (T/N) tissue fluorescence ratio of 218. To the best of our knowledge, this is the highest T/N tissue fluorescence ratio ever reported. We believe that the LAN-βgal chemosensor can be utilized as a new tool for the clinical diagnosis and treatment of ovarian cancer.

Oleanolic Acid Acetate Inhibits Mast Cell Activation in Ovalbumin-Induced Allergic Airway Inflammation.

Asthma is a complex, heterogeneous chronic inflammatory airway disease with multiple phenotypes. There has been a great progress in managing asthma, but there are still unmet needs for developing uncontrolled asthma treatments. The present study aimed to determine the effectiveness of oleanolic acid acetate (OAA) from Vigna angularis against allergic airway inflammation and the underlying mechanism of action with a focus on mast cells. To investigate the effect of OAA in allergic airway inflammation, we used the ovalbumin (OVA)-sensitized and challenged mice. To examine allergic airway inflammation associated with immune responses of mast cell activation in vitro, various types of mast cells were used. Systemic and cutaneous anaphylaxis models were used for mast cell-mediated hyper-responsiveness in vivo. OAA reduced OVA-induced airway inflammatory responses such as bronchospasm, increase of immune cell infiltration and serum immunoglobulin E and G1 levels. Especially, OAA decreased the mast cell infiltration, and β-hexosaminidase release as a mast cell activation marker in the bronchoalveolar lavage fluid. OAA inhibited mast cell degranulation in mast cell line (RBL-2H3) and primary cells (rat peritoneal mast cell and mouse bone marrow-derived mast cell). Mechanistically, OAA suppressed intracellular signaling pathways including the phosphorylation of phospholipase Cγ and nuclear factor-κB, resulting from the suppression of intracellular calcium influx and pro-inflammatory cytokine expression. Further, oral administration of OAA attenuated mast cell-mediated systemic and cutaneous anaphylaxis. Our study showed that OAA can inhibit mast cell-mediated allergic reaction. Consequently, the application of OAA to mast cells for the allergic airway inflammation facilitate a new direction of treating allergic asthma.

Analysis of Human and Mouse Neutrophil Phagocytosis by Flow Cytometry.

Neutrophils are primary phagocytes that recognize their targets through surface chemistry, either through pattern recognition receptor (PPR) interaction with pathogen-associated molecular patterns (PAMPs) or through immunoglobulin (Ig) or complement mediated recognition. Opsonization can be important for target recognition and phagocytosis by neutrophils. Therefore, phagocytosis assays performed using neutrophils in whole blood, compared to isolated cells, will differ due to the presence of opsonizing blood serum components, as well as other blood components like platelets. Powerful and sensitive flow cytometry-based methods are presented to measure phagocytosis by human blood neutrophils and mouse peritoneal neutrophils.

Sonodynamic therapy suppresses matrix collagen degradation in vulnerable atherosclerotic plaque by modulating caspase 3 - PEDF/HIF-1α - MMP-2/MMP-9 signaling in macrophages.

The rupture of vulnerable atherosclerotic plaque is the main cause of acute ischemic vascular events, and is characterized by pathological degradation of matrix collagen in the fibrous cap. In a previous study, we reported that 5-aminolevulinic acid-mediated sonodynamic therapy suppressed collagen degradation in rabbit plaque. However, the underlying molecular mechanism has yet to be fully elucidated. We applied sinoporphyrin sodium-mediated sonodynamic therapy (DVDMS-SDT) to balloon-denuded rabbit and apolipoprotein E-deficient (ApoE-/-) mouse models to observe collagen content in plaque. Cultured human THP-1 and mouse peritoneal macrophage-derived foam cells were used for in vitro mechanistic studies. We observed that DVDMS-SDT decreased plaque area and increased the percentages of collagen and smooth muscle cells and reduced the percentage of macrophages in rabbit and ApoE-/- mouse advanced plaques. In vitro, DVDMS-SDT modulated the caspase 3-pigment epithelium-derived factor/hypoxia-inducible factor-1α (PEDF/HIF-1α)-matrix metalloprotease-2/9 (MMP-2/MMP-9) signaling in macrophage foam cells. Our findings show that DVDMS-SDT effectively inhibits matrix collagen degradation in advanced atherosclerotic plaque by modulating caspase 3-PEDF/HIF-1α-MMP-2/MMP-9 signaling in macrophage foam cells and therefore represents a suitable and promising clinical regimen to stabilize vulnerable plaques.

490. Brain-derived neurotrophic factor (BDNF) is a prognostic biomarker and therapeutic target through AMP-activated protein kinase (AMPK) signal pathway for sepsis

Abstract Background Brain-derived neurotrophic factor (BDNF) is a pleiotropic cytokine that not only acts on the central nervous system, but is also implicated in metabolic regulation, development of lung diseases, and support of hematopoiesis. Recently, a study report that the plasma levels of BDNF were independently associated with mortality in critically ill patients. We aimed to evaluate the role of BDNF in sepsis patients and animal sepsis model. Methods Prospectively enrolled adult sepsis patients (n=168) who visited the emergency room (ER) of Korea University Ansan Hospital from January 2017 to December 2018., and healthy volunteers (n=48) were included in the present study. Serum BDNF level was determined and analyzed in septic patients and healthy volunteers. The effects of BDNF on pro-inflammatory cytokines, AMP-activated protein kinase (AMPK) and NF-κB signaling in septic condition of RAW264.7 cells and mouse peritoneal macrophage, and mouse sepsis model by cecal ligation and puncture (CLP). Results Serum BDNF levels (mean) were significantly decreased in sepsis patients compared to healthy controls (11.78ng/ml vs 18.91ng/ml, p&amp;lt; 0.001) and significantly decreased in non-survivors compared to survivors at discharge (3.16ng/ml vs 13.86ng/ml, p&amp;lt; 0.05). BDNF treatment suppressed LPS-induced pro-inflammatory cytokine (TNF-α, iL-6, and iL-1β) in RAW264.7 cells. Although LPS stimulation diminished AMPK phosphorylation, BDNF was able to decrease the effects of LPS/toll-like receptor 4 (TLR4) engagement on down stream signaling events, particularly LPS-induced IκBα degradation in RAW cells and peritoneal macrophage. In CLP model, intra-muscular injection of BDNF after CLP significantly decreased serum levels of TNF-α which was increased compared to sham, and significantly improved survival of mouse (p&amp;lt; 0.01). Conclusion Sepsis results in a decreased production of BDNF, which might be a potential prognostic biomarker and therapeutic target through AMPK signal pathway for sepsis. Disclosures All Authors: No reported disclosures.

Rutin ameliorates inflammatory pain by inhibiting P2X7 receptor in mast cells.

Rutin is a natural anti-inflammatory ingredient widely found in medicinal plants. Studies have shown that rutin inhibits mast cell degranulation and the release of inflammatory mediators. Mast cell P2X7 receptor mediates mast cell degranulation and serves as a therapeutic target for inflammatory pain. Herein, the aim of this study was to investigate whether the anti-inflammatory mechanism of rutin is related to the mast cell P2X7 receptor. Our results showed that rutin could inhibit [Ca2+]i elevation induced by 5mM ATP or 30μM BZATP in a concentration-dependent manner in mouse peritoneal mast cells. Rutin also suppressed the inward current mediated by P2X7 receptor. In vivo, rutin could significantly inhibit the mechanical hypersensitivity induced by 100mM ATP that is associated with P2X7 receptor in mast cells. Moreover, molecular docking revealed the high affinity between rutin and the P2X7 receptor crystal structure. Collectively, this study demonstrated that rutin attenuated inflammatory pain by inhibiting the activity of P2X7 receptor in mast cells.

Production of reactive oxygen species by neutrophils and macrophages of F1 hybrid mice (C57Bl6xCBA) in response to stimulation with cucurbit(n)urils (n = 6, 7, 8)

Background. Due to their very small size, nanomaterials, in particular cucurbiturils, have unique physical and chemical properties that find their application in medicine. However, the toxicity of cucurbiturils is not fully understood; in particular, we are interested in the immunological safety of their use. One of the mechanisms of nanotoxicity is the formation of reactive oxygen species (ROS) by macrophages and neutrophils. Hyperproduction of ROS can lead to oxidative stress and further damage to cell DNA with loss of physiological function and development of pathology. The aim. Evaluation of the effect of cucurbit[n]urils (n = 6, 7, 8) on the production of reactive oxygen species by mice macrophages and neutrophils. Materials and methods. F1 hybrid mice (CBAxC57Bl/6) aged 2 months (n = 11) were used in the work. Evaluation of superoxide radical production by peritoneal mouse neutrophils and macrophages was carried out by spectrophotometric method for determining the reduction of p-nitroblue tetrazolium (NBT) to formazan. Results. It was shown that CB[6] and CB[7] at concentrations of 0.5 and 0.3 mM do not have an inhibitory effect on ROS synthesis, but, on the contrary, significantly increase ROS production by macrophages. In addition, CB[6] 0.3 mM increases the level of ROS in neutrophils. Conclusion. Cucurbiturils can lead to an increase in the production of ROS in immunocompetent cells, depending on the concentration used (0.3 mM and higher).

Pro-resolving and anti-arthritic properties of the MC1 selective agonist PL8177.

Melanocortins are peptides endowed with anti-inflammatory and pro-resolving activities. Many of these effects are mediated by the Melanocortin receptor 1 (MC1) as reported in several experimental settings. As such, MC1 can be a viable target for the development of new therapies that mimic endogenous pro-resolving mediators. The aim of this study was to assess the immunopharmacology of a selective MC1 agonist (PL8177) in vitro and in a mouse model of inflammatory arthritis. PL8177 and the natural agonist αMSH were tested for activation of mouse and human Melanocortin receptors (MC1,3,4,5), monitoring cAMP accumulation and ERK1/2 phosphorylation, using transiently transfected HEK293A cells. The anti-inflammatory and pro-resolving effects of PL8177 and αMSH were evaluated using mouse peritoneal Macrophages. Finally, a model of K/BxN serum transfer induced arthritis was used to determine the in vivo potential of PL8177. PL8177 activates mouse and human MC1 with apparent EC50 values of 0.01 and 1.49 nM, respectively, using the cAMP accumulation assay. Similar profiles were observed for the induction of ERK phosphorylation (EC50: 0.05 and 1.39 nM). PL8177 displays pro-resolving activity (enhanced Macrophage efferocytosis) and counteracts the inflammatory profile of zymosan-stimulated macrophages, reducing the release of IL-1β, IL-6, TNF-α and CCL-2. In the context of joint inflammation, PL8177 (3mg/kg i.p.) reduces clinical score, paw swelling and incidence of severe disease as well as the recruitment of immune cells into the arthritic joint. These results demonstrate that the MC1 agonism with PL8177 affords therapeutic effects in inflammatory conditions including arthritis. Drugs targeting the Melanocortin system have emerged as promising therapeutics for several conditions including inflammation or obesity. Multiple candidates are under clinical development, and some have already reached approval. Here we present the characterization of a novel drug candidate, PL8177, selective for the Melanocortin 1 receptor (MC1), demonstrating its selectivity profile on cAMP and ERK1/2 phosphorylation signaling pathways, of relevance as selective drugs will translate into lesser off-target effect. PL8177 also demonstrated, not only anti-inflammatory activity, but pro-resolving actions due to its ability to enhance efferocytosis (i.e. the phagocytosis of apoptotic cells), endowing this molecule with therapeutic advantages compared to classical anti-inflammatory drugs. Using a mouse model of inflammatory arthritis, the compound demonstrated in vivo efficacy by reducing clinical score, paw swelling and overall disease severity. Taken together, these results present Melanocortin-based therapies, and specifically targeting MC1 receptor, as a promising strategy to manage chronic inflammatory diseases.

P2X4 receptor stimulation enhances MrgprB2-mediated mast cell activation and pseudoallergic reactions in mice

Pseudoallergies caused by drugs make disease treatment difficult. Mas-relate G protein-coupled receptor X2 (MRGPRX2), which is specifically expressed in mast cells (MCs), has been implicated in pseudoallergies. High concentrations of therapeutic agents are typically required to stimulate MRGPRX2. Although regulatory mechanisms may enhance this response, the factors involved in this regulation are not well-understood. In this study, the effects of extracellular ATP on MC activation induced by MrgprB2, the mouse ortholog of human MRGPRX2, were examined in mouse peritoneal MCs (PMCs). ATP alone induced minimal PMC degranulation but markedly enhanced degranulation induced by the MrgprB2 agonist compound 48/80 (CP48/80), substance P, PAMP-12, and vancomycin. ATP promoted CP48/80-induced increase in intracellular Ca2+ in PMCs. This enhancement effect of ATP was absent in PMCs prepared from P2X4 receptor (P2X4R)-deficient mice and inhibited by the PI3K inhibitor wortmannin. In addition, P2X4R deficiency reduced the skin-specific and systemic anaphylactic responses to CP48/80 in vivo. In MC-deficient KitW-sh/W-sh mice, reconstitution with MCs obtained from wild-type mice led to a more severe anaphylactic response to CP48/80 compared to that from P2X4R-deficient mice. P2X4R-mediated effect may be involved in MrgprB2-mediated MC activation in vivo and is a potential target for alleviating pseudoallergic reactions.

Antileishmanial Activities of (Z)-2-(Nitroimidazolylmethylene)-3(2H)-Benzofuranones: Synthesis, In Vitro Assessment, and Bioactivation by NTR 1 and 2.

The antileishmanial activity of a series of (Z)-2-(heteroarylmethylene)-3(2H)-benzofuranone derivatives, possessing 5-nitroimidazole or 4-nitroimidazole moieties, was investigated against Leishmania major promastigotes and some analogues exhibited prominent activities. Compounds with IC50 values lower than 20 μM were further examined against L. donovani axenic amastigotes. Evaluated analogues in 5-nitroimidazole subgroup demonstrated significantly superior activity (~17-88-folds) against L. donovani in comparison to L. major. (Z)-7-Methoxy-2-(1-methyl-5-nitroimidazole-2-ylmethylene)-3(2H)-benzofuranone (5n) showed the highest L. donovani anti-axenic amastigote activity with IC50 of 0.016 μM. The cytotoxicity of these analogues was determined using PMM peritoneal mouse macrophage and THP-1 human leukemia monocytic cell lines and high selectivity indices of 26 to 431 were obtained for their anti-axenic amastigote effect over the cytotoxicity on PMM cells. Further studies on their mode of action showed that 5-nitroimidazole compounds were bioactivated predominantly by nitroreductase 1 (NTR1) and 4-nitroimidazole analogues by both NTR1 and 2. It is likely that this bioactivation results in the production of nitroso and hydroxylamine metabolites that are cytotoxic for the Leishmania parasite.

Mouse Ocilrp2/Clec2i negatively regulates LPS-mediated IL-6 production by blocking Dap12-Syk interaction in macrophage.

C-type lectin Ocilrp2/Clec2i is widely expressed in dendritic cells, lymphokine-activated killer cells and activated T cells. Previous studies have shown that Ocilrp2 is an important regulator in the activation of T cells and NK cells. However, the role of Ocilrp2 in the inflammatory responses by activated macrophages is currently unknown. This study investigated the expression of inflammatory cytokines in LPS-induced macrophages from primary peritoneal macrophages silenced by specific siRNA target Ocilrp2. Ocilrp2 was significantly downregulated in macrophages via NF-κB and pathways upon LPS stimuli or VSV infection. Silencing Ocilrp2 resulted in the increased expression of IL-6 in LPS-stimulated peritoneal macrophages and mice. Moreover, IL-6 expression was reduced in LPS-induced Ocilrp2 over-expressing iBMDM cells. Furthermore, we found that Ocilrp2-related Syk activation is responsible for expressing inflammatory cytokines in LPS-stimulated macrophages. Silencing Ocilrp2 significantly promotes the binding of Syk to Dap12. Altogether, we identified the Ocilrp2 as a critical role in the TLR4 signaling pathway and inflammatory macrophages’ immune regulation, and added mechanistic insights into the crosstalk between TLR and Syk signaling.

Inhibition of MRGPRX2 but not FcεRI or MrgprB2-mediated mast cell degranulation by a small molecule inverse receptor agonist.

Mas-related G protein-coupled receptor-X2 (MRGPRX2) expressed on mast cells (MCs) contributes to hypersensitivity reactions to cationic US-Food and Drug Administration (FDA) approved drugs such as the neuromuscular blocking agent, rocuronium. In addition, activation of MRGPRX2 by the neuropeptide substance P (SP) and the pro-adrenomedullin peptide (PAMP-12) is associated with a variety of cutaneous conditions such as neurogenic inflammation, pain, atopic dermatitis, urticaria, and itch. Thus, small molecules aimed at blocking MRGPRX2 constitute potential options for modulating IgE-independent MC-mediated disorders. Two inverse MRGPRX2 agonists, named C9 and C9-6, have recently been identified, which inhibit basal G protein activation and agonist-induced calcium mobilization in transfected HEK293 cells. Substance P serves as a balanced agonist for MRGPRX2 whereby it activates both G protein-mediated degranulation and β-arrestin-mediated receptor internalization. The purpose of this study was to determine if C9 blocks MRGPRX2’s G protein and β-arrestin-mediated signaling and to determine its specificity. We found that C9, but not its inactive analog C7, inhibited degranulation in RBL-2H3 cells stably expressing MRGPRX2 in response to SP, PAMP-12 and rocuronium with an IC50 value of ~300 nM. C9 also inhibited degranulation as measured by cell surface expression of CD63, CD107a and β-hexosaminidase release in LAD2 cells and human skin-derived MCs in response to SP but not the anaphylatoxin, C3a or FcϵRI-aggregation. Furthermore, C9 inhibited β-arrestin recruitment and MRGPRX2 internalization in response to SP and PAMP-12. We found that a G protein-coupling defective missense MRGPRX2 variant (V282M) displays constitutive activity for β-arrestin recruitment, and that this response was significantly inhibited by C9. Rocuronium, SP and PAMP-12 caused degranulation in mouse peritoneal MCs and these responses were abolished in the absence of MrgprB2 or cells treated with pertussis toxin but C9 had no effect. These findings suggest that C9 could provide an important framework for developing novel therapeutic approaches for the treatment of IgE-independent MC-mediated drug hypersensitivity and cutaneous disorders.