Macrophage Cell Cultures Research Articles

New insights into the role of the CHI3L2 protein in invasive ductal breast carcinoma

AbstractChitinase-like proteins have multiple biological functions that promote tumor growth, angiogenesis and metastasis. Expression of CHI3L2, which is similar in structure to CHI3L1, is detected in glioma cells and tumor-associated macrophages (TAMs) in glioma and breast cancer. However, its exact role remains unclear. We analyzed the expression of CHI3L2 in 74 invasive ductal breast carcinoma (IDC) tumors, breast cancer and macrophages cell cultures using immunohistochemistry, immunofluorescence, Western blot and PCR methods. Clinicopathologic data were included in the analysis. The results obtained show that CHI3L2 expression decreases with increasing degree of tumor grade and negative status of estrogen (ER) and progesterone receptors (PR). Furthermore, CHI3L2 is significantly and positively correlated with phosphorylation of STAT-3 and ERK1/2 signaling pathways, but negatively correlated with macrophage infiltration. CHI3L2 is expressed both in the cytoplasm of cancer cells and in macrophages and may regulate STAT-3 and ERK1/2 phosphorylation in breast cancer cell lines. Analysis of the clinicopathologic data revealed that CHI3L2 levels had no effect on patient survival. CHI3L2 expression may be specific for cancer cells in IDC and involved in cross-talk with the tumor microenvironment. Our study has shown that IDC cancer cells express the CHI3L2 protein, possibly indicating a novel function of this protein.

Leukocyte infiltration and cross-talk with cardiomyocytes exploit intracellular stress pathways in dilated cardiomyopathy of idiopathic origin.

Dilated cardiomyopathy (DCM) is a prevalent form of heart failure results in dilation and disruption of heart. Most strikingly a majority of the DCM cases do not have any identified etiology, hence known as idiopathic DCM (IDCM). Our study aimed to investigate the cross-talk between leukocytes and cardiomyocytes in terms of cardiac inflammation and stress response in IDCM. 60 IDCM patients and 60 age and sex matched healthy volunteers were recruited in this study based on the New York Heart Association (NYHA) guidelines. Their echocardiographic and biochemical markers were assessed and PBMCs were analyzed for leukocyte migration and inflammation. Also C2C12 myocyte cells were cultured with LPS-activated RAW264.7 monocytes to investigate the cross-talk between them. Left ventricular (LV) dysfunction was evident in the IDCM patients which were correlated with their physical discomfort level according to NYHA classification. Their serum levels of IL-1β and TNF-α (≈ 20 pg/ml) were found to be very high along with hs-CRP and IL-2. Elevated levels of ROCK, SMA and ICAM-1 proteins indicated activation and migration of the leukocytes. During monocyte-myocyte co-culture, robust diapedesis was observed in the cultured macrophage cells towards myocytes through the transwell pores (8 µM) in presence of IL-1β and TNF-α causing ER stress and cell death in the myocytes. Inhibition of this migration or by alleviating ER stress inhibits leukocyte recruitment and ensures protection to the myocytes. The present study showed that alleviating cellular stress and managing leukocyte migration promotes protection to the heart.

Natural Therapeutic Agents’ Efficacy in Preventive Strategies against the Periodontal Pathogen Aggregatibacter actinomycetemcomitans: An In Vitro Study

Adolescent carriers of the Aggregatibacter actinomycetemcomitans JP2 genotype have an increased risk of developing periodontitis, due to the bacterium’s high leukotoxin (LtxA) production. LtxA contributes to marginal bone loss by killing immunity cells, thus activating the proinflammatory interleukin-1β (IL-1β), which, in turn, activates the osteoclasts. A possible strategy to prevent periodontitis might be to neutralize LtxA in JP2-infected individuals. The aim of this study was to investigate whether extracts from Matcha or Guava leaves can prolong the viability of macrophages in cell cultures by neutralizing the highly leukotoxic JP2 genotype bacteria. The A. actinomycetemcomitans JP2 genotype was pretreated with extracts from either Matcha or Guava leaves. Later, the extracts were rinsed off, before JP2 bacteria were exposed to macrophage cell cultures. The experiment was repeated, where JP2 bacteria were persistently treated with the extracts instead, i.e., the extracts were not rinsed off. The macrophage viability after bacterial exposure was analyzed and compared with that of macrophages exposed to untreated JP2 bacteria. IL-1β secretion in the cell culture medium was quantified in all group samples. Pretreatment of the A. actinomycetemcomitans JP2 genotype with Matcha or Guava leaf extracts moderately neutralized LtxA activity, which resulted in prolonged macrophage viability and decreased IL-1β secretion. These effects of prolonged macrophage viability were enhanced when extracts were persistently present during the exposure period. The results indicate that Matcha and Guava leaf extracts have effects on the virulence of the A. actinomycetemcomitans JP2 genotype that may be useful in future treatment strategies to prevent periodontitis in JP2 bacterium carriers.

First detection of recombinant variant of African swine fever virus in the Russian Federation (brief communication)

As part of extensive molecular and genetic research into African swine fever virus isolates circulating in Russia, a recombinant variant with a mosaic genome structure has been identified. The one that caused an outbreak on a pig farm in the Primorsky Krai, in 2023. The characterized strain ASFV/Primorsky_2023/DP-4560.Recdemonstrates hemadsorption, active propagation in porcine primary macrophage cell culture, 99.9917% identity with the first recombinant isolates from the People’s Republic of China, recovered in 2021. Recombination sites included 79 open reading frames homologous to genotype II isolates; 49 ones homologous to genotype I and 12 mixed ones. Testing biomaterial from dead pigs in real-time polymerase chain reaction showed no changes in sensitivity or specificity, despite significant genetic distinctions between the recombinant and genotype II isolates that are enzootic to the Russian Federation. However, in 2023, D. Zhao et al. reported on high virulence of the virus related variants as revealed by the challenge tests in domestic pigs. Given the accelerating rates of AFSV molecular evolution in the East Asian countries (China, Vietnam and the Far Eastern regions of Russia), it is required to improve control measures, general and specific prevention, national and international surveillance over the economically significant animal disease.

In vitro studies on the effects of cryopreserved platelet-rich plasma on cells related to wound healing

Platelet-rich plasma (PRP) holds promise as a therapeutic modality for wound healing; however, immediate utilization encounters challenges related to volume, concentration, and consistency. Cryopreservation emerges as a viable solution, preserving PRP’s bioactive components and extending its shelf life. This study explores the practicality and efficacy of cryopreserved platelet-rich plasma (cPRP) in wound healing, scrutinizing both cellular mechanisms and clinical implications. Fresh PRP and cPRP post freeze-thaw underwent assessment in macrophage, fibroblast, and endothelial cell cultures. The impact of cPRP on active component release and cell behavior pertinent to wound healing was evaluated. Varied concentrations of cPRP (1%, 5%, 10%) were examined for their influence on cell polarization, migration, and proliferation. The results showed minimal changes in cPRP’s IL-1β levels, a slight decrease in PDGF-BB, and superior effects on macrophage M2 polarization and fibroblast migration, while no statistical significance was observed in endothelial cell angiogenesis and proliferation. Remarkably, 5% PRP exhibited the most significant stimulation among all cPRP concentrations, notably impacting cell proliferation, angiogenesis, and migration. The discussion underscores that cPRP maintains platelet phenotype and function over extended periods, with 5% cPRP offering the most favorable outcomes, providing a pragmatic approach for cold storage to extend post-thaw viability and amplify therapeutic effects.

Water-Soluble Trityl Radicals for Fluorescence Imaging.

Stable tris(trichlorophenyl)methyl radicals have gained interest as all-organic bioimaging agents combining fluorescent and paramagnetic properties. However, cellular uptake has so far only been reported for nanoparticles, because molecular hydrophobic trityl radicals are not soluble in aqueous media. Here, we report the synthesis and characterization of new water-soluble tris(trichlorophenyl)methyl radical derivatives exhibiting red doublet emission. Solubility in water is achieved through functionalization with oligoethylene glycol (OEG) chains. The emission behavior of OEG functionalized trityl radicals is studied in polar environments. Donor-functionalization with carbazole evokes a charge-transfer excited state that is efficiently quenched in polar solvents. In contrast, click-reaction mediated attachment of OEG-azide and trityl acetylene furnishes a triazole functionalized radical with locally excited states and emission in water. Confocal fluorescence microscopy proves successful uptake of the material by macrophages in cell culture, showing the potential of our water soluble trityl radical for fluorescence bioimaging.

Differential 5'-tRNA Fragment Expression in Circulating Preeclampsia Syncytiotrophoblast Vesicles Drives Macrophage Inflammation.

The relationship between placental pathology and the maternal syndrome of preeclampsia is incompletely characterized. Mismatch between placental nutrient supply and fetal demands induces stress in the syncytiotrophoblast, the layer of placenta in direct contact with maternal blood. Such stress alters the content and increases the release of syncytiotrophoblast extracellular vesicles (STB-EVs) into the maternal circulation. We have previously shown 5'-tRNA fragments (5'-tRFs) constitute the majority of small RNA in STB-EVs in healthy pregnancy. 5'-tRFs are produced in response to stress. We hypothesized STB-EV 5'-tRF release might change in preeclampsia. We perfused placentas from 8 women with early-onset preeclampsia and 6 controls, comparing small RNA expression in STB-EVs. We used membrane-affinity columns to isolate maternal plasma vesicles and investigate placental 5'-tRFs in vivo. We quantified 5'-tRFs from circulating STB-EVs using a placental alkaline phosphatase immunoassay. 5'-tRFs and scrambled RNA controls were added to monocyte, macrophage and endothelial cells in culture to investigate transcriptional responses. 5'-tRFs constitute the majority of small RNA in STB-EVs from both preeclampsia and normal pregnancies. More than 900 small RNA fragments are differentially expressed in preeclampsia STB-EVs. Preeclampsia-dysregulated 5'-tRFs are detectable in maternal plasma, where we identified a placentally derived load. 5'-tRF-Glu-CTC, the most abundant preeclampsia-upregulated 5'-tRF in perfusion STB-EVs, is also increased in preeclampsia STB-EVs from maternal plasma. 5'-tRF-Glu-CTC induced inflammation in macrophages but not monocytes. The conditioned media from 5'-tRF-Glu-CTC-activated macrophages reduced eNOS (endothelial NO synthase) expression in endothelial cells. Increased release of syncytiotrophoblast-derived vesicle-bound 5'-tRF-Glu-CTC contributes to preeclampsia pathophysiology.

Enhancing the correlation between in vitro and in vivo experiments in dental implant osseointegration: investigating the role of Ca ions.

This study delves into the osteogenic potential of a calcium-ion modified titanium implant surface, unicCa, employing state-of-the-art proteomics techniques both in vitro (utilizing osteoblasts and macrophage cell cultures) and in vivo (in a rabbit condyle model). When human osteoblasts (Hobs) were cultured on unicCa surfaces, they displayed a marked improvement in cell adhesion and differentiation compared to their unmodified counterparts. The proteomic analysis also revealed enrichment in functions associated with cell migration, adhesion, extracellular matrix organization, and proliferation. The analysis also underscored the involvement of key signalling pathways such as PI3K-Akt and mTOR. In the presence of macrophages, unicCa initially exhibited improvement in immune-related functions and calcium channel activities at the outset (1 day), gradually tapering off over time (3 days). Following a 5-day implantation in rabbits, unicCa demonstrated distinctive protein expression profiles compared to unmodified surfaces. The proteomic analysis highlighted shifts in adhesion, immune response, and bone healing-related proteins. unicCa appeared to influence the coagulation cascade and immune regulatory proteins within the implant site. In summary, this study provides a comprehensive proteomic analysis of the unicCa surface, drawing correlations between in vitro and in vivo results. It emphasizes the considerable potential of unicCa surfaces in enhancing osteogenic behavior and immunomodulation. These findings significantly contribute to our understanding of the intricate molecular mechanisms governing the interplay between biomaterials and bone cells, thereby facilitating the development of improved implant surfaces for applications in bone tissue engineering.

The interaction of human immunodeficiency virus-1 and human endogenous retroviruses in patients (primary cell cultures) and cell line models.

Human endogenous retroviruses (HERVs) integrated into the human genome millions of years ago, and their expression has been described both in healthy tissues and in pathology. In the present study, we study the differential expression of multiple HERV families in the presence of human immunodeficiency virus (HIV) infection in the peripheral blood mononuclear cells (PBMCs) of HIV-positive individuals, in HIV-infected cell lines, and in vitro HIV-infected macrophages/monocytes of healthy donors, utilizing publicly available data sets for secondary analysis using a pipeline appropriate for HERV transcription detection and comparison. We recognized significantly decreased expression of HERV-H in treatment-naïve HIV patients with viremia and increased expression of multiple HERV families in the PBMCs of HIV-infected individuals under combination antiretroviral therapy compared to healthy donors, in in vitro HIV-infected Stanford University pediatric T (SUP-T1) cells at 24 hours after HIV infection compared to non-infected SUP-T1 cells, and in in vitro HIV-infected MT4 T cells at 3 days after HIV infection compared to non-infected MT4 T cells. We recognized significantly increased expression of HERV-W, after HIV infection in monocyte-derived macrophage cell culture; no difference was found in HERV expression after HIV infection of monocyte cultures. We found a significantly decreased expression of HERV-K (HML-6) in donor-derived macrophage cultures at 36 hours after HIV infection, which does not persist at 6 days after infection. HIV infection modifies the expression of multiple HERV families in the PBMCs of HIV patients and in T cell-derived cultures, while the effect of HIV infection on HERV expression appears to be more restricted in cells of monocytic origin.IMPORTANCEIn this work, we demonstrated that human immunodeficiency virus (HIV) infection leads to the modification of the human endogenous retrovirus (HERV) expression. Differential expression of multiple HERVs was found in peripheral blood mononuclear cells derived from HIV-infected patients compared to healthy donors and HIV-infected T cell cultures compared to non-infected. The effect of HIV presence on HERV expression appears to be more restricted in cells of monocytic origin, as only deregulation of HERV-W and HERV-K (HML-6) was found in these cell cultures after their infection with HIV. Multiple factors contribute to this aberrant HERV expression, and its levels appear to be modified in a time-dependent manner. Further studies and the development of optimized in vitro protocols are warranted to elucidate the interactions between HIV and HERVs in detail.

In Vitro Anti-Inflammatory Activity of Methyl Derivatives of Flavanone

Inflammation plays an important role in the immune defense against injury and infection agents. However, the inflammatory chronic process may lead to neurodegenerative diseases, atherosclerosis, inflammatory bowel diseases, or cancer. Flavanones present in citrus fruits exhibit biological activities, including anti-oxidative and anti-inflammatory properties. The beneficial effects of flavanones have been found based on in vitro cell cultures and animal studies. A suitable in vitro model for studying the inflammatory process are macrophages (RAW264.7 cell line) because, after stimulation using lipopolysaccharide (LPS), they release inflammatory cytokines involved in the immune response. We determined the nitrite concentration in the macrophage cell culture and detected ROS using chemiluminescence. Additionally, we measured the production of selected cytokines using the Bio-Plex Magnetic Luminex Assay and the Bio-PlexTM 200 System. For the first time, we have shown that methyl derivatives of flavanone inhibit NO and chemiluminescence generated via LPS-stimulated macrophages. Moreover, the tested compounds at 1–20 µM dose-dependently modulate proinflammatory cytokine production (IL-1β, IL-6, IL-12p40, IL-12p70, and TNF-α) in stimulated RAW264.7 cells. The 2′-methylflavanone (5B) and the 3′-methylflavanone (6B) possess the strongest anti-inflammatory activity among all the tested flavanone derivatives. These compounds reduce the concentration of IL-6, IL-12p40, and IL12p70 compared to the core flavanone structure. Moreover, 2′-methylflavanone reduces TNF-α, and 3′-methylflavanone reduces IL-1β secreted by RAW264.7 cells.

Macrophage IL-1β-positive microvesicles exhibit thrombo-inflammatory properties and are detectable in patients with active juvenile idiopathic arthritis.

IL-1β is a leaderless cytokine with poorly known secretory mechanisms that is barely detectable in serum of patients, including those with an IL-1β-mediated disease such as systemic juvenile idiopathic arthritis (sJIA). Leukocyte microvesicles (MVs) may be a mechanism of IL-1β secretion. The first objective of our study was to characterize IL-1β-positive MVs obtained from macrophage cell culture supernatants and to investigate their biological functions in vitro and in vivo. The second objective was to detect circulating IL-1β-positive MVs in JIA patients. MVs were purified by serial centrifugations from PBMCs, or THP-1 differentiated into macrophages, then stimulated with LPS ± ATP. MV content was analyzed for the presence of IL-1β, NLRP3 inflammasome, caspase-1, P2X7 receptor, and tissue factor (TF) using ELISA, Western blot, or flow cytometry. MV biological properties were studied in vitro by measuring VCAM-1, ICAM-1, and E-selectin expression after HUVEC co-culture and factor-Xa generation test was realized. In vivo, MVs' ability to recruit leukocytes in a murine model of peritonitis was evaluated. Plasmatic IL-1β-positive MVs were studied ex vivo in 10 active JIA patients using flow cytometry. THP-1-derived macrophages stimulated with LPS and ATP released MVs, which contained NLRP3, caspase-1, and the 33-kDa precursor and 17-kDa mature forms of IL-1β and bioactive TF. IL-1β-positive MVs expressed P2X7 receptor and released soluble IL-1β in response to ATP stimulation in vitro. In mice, MVs induced a leukocyte peritoneal infiltrate, which was reduced by treatment with the IL-1 receptor antagonist. Finally, IL-1β-positive MVs were detectable in plasma from 10 active JIA patients. MVs shed from activated macrophages contain IL-1β, NLRP3 inflammasome components, and TF, and constitute thrombo-inflammatory vectors that can be detected in the plasma from active JIA patients.

Synaptamide modulates glial and neurotransmitter activity in the spinal cord during neuropathic pain

N-docosahexaenoylethanolamine, or synaptamide, is an endogenous metabolite of docosahexaenoic acid that is known for synaptogenic and neurogenic effects. In our previous studies we have shown that synaptamide attenuates neuropathic pain, facilitates remyelination, and reduces neuroinflammation after the chronic constriction injury (CCI) of the sciatic nerve in rats. In the current study, we show that daily synaptamide administration (4 mg/kg/day) within 14 days post-surgery: (1) decreases micro- and astroglia activity in the dorsal and ventral horns of the lumbar spinal cord; (2) modulates pro-inflammatory (IL1β, IL6) and anti-inflammatory (IL4, IL10) cytokine level in the serum and spinal cord; (3) leads to a rise in synaptamide and anandamide concentration in the spinal cord; (4) enhances IL10, CD206 and N-acylethanolamine-hydrolyzing acid amidase synthesis in macrophage cell culture following LPS-induced inflammation. Thus, the ability of synaptamide to modulate glial and cytokine activity indicates its potential for implementation in the treatment peripheral nerve injury.

Effect of Oleoylethanolamide-Based Dietary Supplement on Systemic Inflammation in the Development of Alimentary-Induced Obesity in Mice.

The complex effect of oleoylethanolamide-based dietary supplement (OEA-DS) was studied in a model of diet-induced obesity in mice. Physiological, biochemical, and immunohistochemical methods were used to reveal differences in the changes in the weight of experimental animals, morphological changes in the spleen tissues, and changes in the cytokine expression profile in the spleen, blood plasma, and macrophage cell culture. First, it is shown that a hypercaloric diet high in carbohydrates and cholesterol led to the development of systemic inflammation, accompanied by organ morphological changes and increased production of proinflammatory cytokines. In parallel, the use of OEA-DS reduced the intensity of cellular inflammatory reactions, accompanied by a decrease in markers of cellular inflammation and proliferation, such as CD68, Iba-1, and Ki67 in the spleen tissue, and stabilized the level of proinflammatory cytokines (IL-1β, IL-6, TNFα) both in animals and in cell culture. In addition, in the macrophage cell culture (RAW264.7), it was shown that OEA-DS also suppressed the production of reactive oxygen species and nitrites in LPS-induced inflammation. The results of this study indicate the complex action of OEA-DS in obesity, which includes a reduction of systemic inflammation.

In situ synthesis of gold nanoparticles on layered double hydroxide nanoparticles for multiplexing molecular imaging of single cells

Quasi-spherical gold nanoparticles (AuNPs) are the most widely used surface-enhanced Raman scattering (SERS) nanotags. However, their surface plasmon resonance properties are significantly restricted by their poor colloidal stability in biological media. Herein, we developed a new strategy for in situ synthesis of AuNPs on layered double hydroxide (LDH) nanoparticles to improve the colloidal stability of AuNPs for SERS imaging with multiplexing capability using single laser excitation. The hybridized LDH-AuNPs were constructed via in situ growth of AuNPs on lactic acid-LDH nanoparticles. The size of AuNPs was tuned by changing the concentration of LDH. The LDH-AuNPs were further functionalized with Raman reporter molecules (Ra) to obtain LDH-AuNPs-Ra as SERS nanotags, which were examined in terms of the colloidal stability and photostability in biological media as well as cellular uptake in J774A.1 macrophage cell culture by single cell mapping via confocal Raman microscopy. The cellular uptake of multiple LDH-AuNPs-Ra functionalized with different Raman reporter molecules were further investigated, where the multiplexing capability of SERS assay endows simultaneous detection of multiple LDH-AuNPs-Ra distribution after cellular uptake for cell imaging. In summary, the LDH-supported in situ synthesis of AuNPs have shown the merits of stabilizing AuNPs by using LDH for SERS imaging, as well as demonstrating the multiplexing molecular imaging of single cells. This work thus provides a promising strategy and a new bioimaging nanoplatform for various biomedical studies in the near future.

Cellular Antioxidant, Anti-Inflammatory, and Antiproliferative Activities from the Flowers, Leaves and Fruits of Gallesia integrifolia Spreng Harms.

Gallesia integrifolia, a notable species in the Atlantic Forest, has been traditionally employed in folk medicine for treating rheumatism, asthma, and worms. This study investigated the cellular antioxidant, antiproliferative, and anti-inflammatory activities of the essential oils (EOs) and crude extracts (CEs) from G. integrifolia flowers, fruits, and leaves. The chemical identification of EOs was performed by GC-MS and CEs by UHPLC-MS. Cellular antioxidant and anti-inflammatory activities were assessed through mouse macrophage cell culture. In addition, the antiproliferative potential was evaluated in gastric, colorectal, breast, and lung tumor cell lines and non-tumor VERO cells. EOs predominantly contained organosulfur compounds in flowers (96.29%), fruits (94.94%), and leaves (90.72%). We found the main compound is 2,2'-Disulfanediyldiethanethiol in the EOs of flowers (47.00%), leaves (41.82%), and fruits (44.39%). Phenolic compounds were identified in CEs. The EOs and CEs demonstrated potential against the tumor cell lines tested (GI50 between 51 and 230 µg/mL). The selectivity index values were greater than 1.0 (1.01 to 3.37), suggesting a relative safety profile. Moreover, the anti-inflammatory activity IC50 ranged from 36.00 to 268 µg/mL, and the cellular oxidation inhibition ranged from 69% to 82%. The results suggest that oils and extracts derived from G. integrifolia have potential for use in various industrial sectors.

Deletion of the CD2 Gene in the Virulent ASFV Congo Strain Affects Viremia in Domestic Swine, but Not the Virulence.

African swine fever (ASF) is an infectious disease that causes the most significant losses to the pig industry. One of the effective methods for combating this disease could be the development of vaccines. To date, experimental vaccines based on the use of live attenuated strains of the ASF virus (ASFV) obtained by the deletion of viral genes responsible for virulence are the most effective. Deletion of the EP402R gene encoding a CD2-like protein led to the attenuation of various strains of the ASFV, although the degree of attenuation varies among different isolates. Here we have shown that the deletion of the EP402R gene from the genome of a high-virulent Congo isolate did not change either the virulence of the virus or its ability to replicate in the swine macrophage cell cultures in vitro. However, in vivo, animals infected with ΔCongo-v_CD2v had a delay in the onset of the disease and viremia compared to animals infected with the parental strain. Thus, deletion of the CD2 gene in different isolates of the ASFV has a different effect on the virulence of the virus, depending on its genetic background.

Immunometabolism Modulation by Extracts from Pistachio Stalks Formulated in Phospholipid Vesicles.

Several studies have demonstrated the effectiveness of plant extracts against various diseases, especially skin disorders; namely, they exhibit overall protective effects. The Pistachio (Pistacia vera L.) is known for having bioactive compounds that can effectively contribute to a person's healthy status. However, these benefits may be limited by the toxicity and low bioavailability often inherent in bioactive compounds. To overcome these problems, delivery systems, such as phospholipid vesicles, can be employed. In this study, an essential oil and a hydrolate were produced from P. vera stalks, which are usually discarded as waste. The extracts were characterized by liquid and gas chromatography coupled with mass spectrometry and formulated in phospholipid vesicles intended for skin application. Liposomes and transfersomes showed small size (<100 nm), negative charge (approximately -15 mV), and a longer storage stability for the latter. The entrapment efficiency was determined via the quantification of the major compounds identified in the extracts and was >80%. The immune-modulating activity of the extracts was assayed in macrophage cell cultures. Most interestingly, the formulation in transfersomes abolished the cytotoxicity of the essential oil while increasing its ability to inhibit inflammatory mediators via the immunometabolic citrate pathway.

Abstract 663: Resistin mediates the immunoediting activity exerted by primary human macrophages towards mesothelioma cells

Abstract Tumor-associated macrophages (TAM) are particularly abundant (i.e. 27% ± 9%) in the microenvironment of malignant pleural mesothelioma (MPM). These macrophages do not correspond to classical (M1) and alternatively-activated (M2) phenotypes but, instead, are shaped by local inflammatory mediators released in the pleura. We previously showed that, besides classical functions exerted by macrophages (i.e. phagocytosis, cytokine expression and antigen presentation), murine RAW264.7 cells are directly cytotoxic to MPM tumors (Hamaidia et al, JCI Insight 4:e128474). Upon direct cell-to-cell contact, RAW264.7 macrophages kill mesothelioma cells by a mechanism of oxeiptosis involving the enhancer of zeste homolog 2 (EZH2) methyltransferase. We have now investigated this immune-editing activity in human mesothelioma. We show that M1-activated primary macrophages are more cytotoxic for M14K cells than M2. The killing activity of M1 macrophages is dependent on NADPH oxidase activity and peroxynitrite levels. Mesothelioma cells and M2 macrophages interact through an inhibitory synapse characterized by engagement of the PD-1 receptor. Consistently, the immune-editing activity of M2 macrophages is partially restored in presence of neutralizing anti-PD1 antibody. Primary human macrophages cultured in presence of pleural effusions of MPM patients are less cytotoxic than M1 and are unable to inhibit tumor growth in mice. Macrophages differentiated in pleural effusions display a broad spectrum of cytotoxic activities (i.e. from complete inactivity to rates similar to those of M2). Luminex profiling of pleural fluids has identified a series of key mediators associated with the cytotoxic phenotype. The best correlation is obtained with resistin (RETN, FIZZ3, ADSF). Recombinant human resistin improves the killing activity of pleural effusion macrophages in cell culture. Gene transduction of RETN in RAW264.7 impairs growth of AB12 tumors in BALB/c mice. In conclusion, we have shown that resistin is the main factor that mediates immunoediting activity exerted by primary human macrophages towards mesothelioma cells, opening new prospects for therapeutic intervention. Citation Format: Malik Hamaidia, Majeed Jamakhani, Jean-Rock Jacques, Alexis Fontaine, Arnaud Scherpereel, Eric Wasielewski, Louis Renaud, Vincent Heinen, Bernard Duysinx, Luc Willems. Resistin mediates the immunoediting activity exerted by primary human macrophages towards mesothelioma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 663.

Abstract 4635: Single cell definition of the immunogenic synapse between the tumorigenic schwann cells and tumor associated macrophages in Plexiform Neurofibroma

Abstract Background: Patients with Neurofibromatosis Type 1 (NF1) frequently develop benign tumors called Plexiform neurofibromas (PN). Macrophages compose 30-50% of the total cellular composition of PNs and have previously been shown to play a critical role in the PN development and growth. This study defines components of the immunogenic synapse between the neoplastic Schwann cell and the tumor associated myeloid compartment and investigates alterations of the synapse during treatment with MEK inhibition (MEKi). The IL34-CSF1R axis is nominated as a key node in the maintenance of the tumor associated macrophages in PN. Methods: In this work, we applied single cell RNA sequencing (scRNAseq) to human primary tumor PN samples to comprehensively evaluate tumor cellular populations and their interactions. Resulting cellular populations were compared to a single cell atlas of tumor associated immune cells taken from a panel of tumor types. Ligand and receptor interactions were informatically extracted from scRNAseq data and immunohistochemistry was used to validate scRNAseq observations. scRNAseq was also used on pre and post treatment samples taken from three patients receiving the MEKi Selumetinib. Human primary macrophage cell cultures and qPCR were used to interrogate the roles of the IL34-CSF1R signaling and MEKi in PN. Results: scRNAseq generated data on 45101 PN myeloid cells. Integration of these cells with a publicly available tumor atlas of 11 different tumors containing a total of 52195 cells discovered correlation of cell populations between the PN as compared to other tumor types. Tumor-promoting (M2) macrophages represented the largest portion (48%) of the PN myeloid population. Receptor-ligand analysis using cellphoneDB highlighted IL34-CSF1R (p&amp;lt;.0001), TNF-TNFRSF1A (p&amp;lt;.0001) and SIRPA-CD47 (p&amp;lt;.0001) as significant interactions between the neoplastic Schwann cell and macrophages. IL34 and CSF1R protein expression was confirmed in human PN tumors with immunohistochemistry. scRNAseq data from NF1 patients PN tumors taken pre and post MEK inhibitor treatment, demonstrated decreased M2 macrophage gene expression of CD163(p&amp;lt;.001), MRC1(p&amp;lt;.001), TREM2(p&amp;lt;.001) after treatment. Finally, IL34 stimulated primary human monocytes produced macrophages with an M2-like phenotype. MEKi treatment of these macrophages resulted in increased expression of inflammatory macrophage genes CXCL9 (p&amp;lt;.05), CXCL10(p&amp;lt;.001), IDO1(p&amp;lt;.001). Conclusions: PN myeloid populations contain large amounts of M2 macrophages with expression profiles consistent with TAM seen in a variety of malignant tumors. We postulate that IL34 expression by the neoplastic Schwann cell leads to expansion and maintenance of this TAM population. MEKi treatment on human PNs reduces number of macrophages in PNs and shifts macrophage gene expression towards inflammatory genes. Citation Format: Thomas J. On, Xiyuan Zhang, Shahroze Abbas, John Shern. Single cell definition of the immunogenic synapse between the tumorigenic schwann cells and tumor associated macrophages in Plexiform Neurofibroma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4635.

Composition and Bioactivity of a Placental Tissue Particulate (PTP-001) Indicate Greater Potential than Platelet-Rich Plasma for the Treatment of Osteoarthritis.

This study was conducted to compare therapeutically relevant properties of platelet-rich plasma (PRP), a commonly used autologous intra-articular treatment for osteoarthritis (OA), with those of a novel placental tissue particulate, PTP-001, which is in development as a regulated biologic treatment for knee OA. Quantitative immunoassays were performed to determine the content of key growth/regulatory biofactors in PTP-001, and in leukocyte-rich (LR)-PRP or leukocyte-poor (LP)-PRP. An anti-inflammatory bioassay was used to evaluate the effects of each treatment on pro-inflammatory cytokine (tumor necrosis factor (TNF)-α) production in a macrophage cell culture system. Gene expression experiments were conducted using a co-culture system of human synoviocytes (pre-stimulated with interleukin (IL)-1β) and articular chondrocytes, with quantitative polymerase chain reaction analyses of the separate cellular compartments. The concentrations of several biofactors (e.g., basic fibroblast growth factor, tissue inhibitor of metalloproteases-3, interleukin-1 receptor antagonist) representative of diverse disease-relevant mechanisms of action were significantly higher for PTP-001 relative to LR-PRP or LP-PRP. PTP-001 and PRP preparations were able to reduce TNF-α production in macrophage cell cultures; however, greater variability was observed for PRP in comparison with PTP-001. In the chondrocyte/synoviocyte co-culture experiments, PTP-001 and LR-PRP (but not LP-PRP) significantly reduced chondrocyte MMP13 expression in cultures containing IL-1-pretreated synoviocytes. In addition, ADAMTS5 expression was reduced in the chondrocyte compartment following treatment with PTP-001 relative to PRP. These findings support evidence of a potent, multifactorial mechanism of action for a consistently manufactured biologic (PTP-001), which may be of greater therapeutic benefit in comparison with more heterogeneous preparations of PRP which may be generated at the time of treatment.