Murine Peritoneal Macrophages Research Articles

Abstract 4284: Macrophage reprogramming and immune suppression through extracellular vesicles loaded eIF4A1in ovarian cancer

Abstract Background- In ovarian carcinoma (OvCa), macrophages are reprogrammed toward pro-tumorigenic phenotypes, including releasing anti-inflammatory cytokines and expressing immunosuppressive molecules. However, how these macrophages acquire their phenotype remains largely unexplored. To understand the mechanism driving pro-tumorigenic phenotype in macrophages, we studied the role of tumor cell-derived extracellular vesicles (EVs) in the cross-talk with macrophages in OvCa. We found that EVs from ovarian cancer cells contain eukaryotic translation initiation factor 4A1 (eIF4A1). eIF4A1 containing EVs from ovarian cells enhanced macrophage translational activity and PD-L1 expression. However, the detailed mechanism that explains how eIF4A1-packaged EVs induce immunosuppressive phenotype in macrophages is necessary to develop novel immunotherapy strategies for advanced OvCa treatment. Methods- We performed SUnSET assay (surface sensing of translation) to determine whether eIF4E-EVs affect global translation in macrophages. SILAC (Stable isotope labeling by amino acids) based mass spectrometry was used to identify the downstream targets of eIF4A1 in macrophages. We isolated murine peritoneal macrophages from naïve C57BL/6 female mice and co-cultured the macrophages with EVs derived from ovarian cancer cell lines. The expression of PD-L1, CD206, interleukin-6 (IL-6), and other inflammatory cytokines on EVs treated macrophages was evaluated using flow cytometry and a cytokine array kit. Further, we created a Rab27aKO ovarian cancer in vivo model to study the effect of eIF4A1 packaged EVs on the immune suppressive ability of macrophages and tumor progression. Results- Our study shows that tumor cells employ EVs to deliver eIF4A1 to the tumor microenvironment, facilitating the release of cytokines such as IL-6 and the expression of PD-L1 on macrophages and supporting tumor growth. In addition, our data shows that eIF4A1-EVs enhance protein synthesis in macrophages. Our data show that in the Rab27aKO ovarian cancer in vivo mouse model, intravenous injection of eIF4A1-packaged EVs increased the tumor burden, metastasis, and ascites accumulation as compared to PBS control. eIF4A1-EVs treatment increased the infiltration of tumor-associated macrophages (TAM) in ascites. We also found increased expression of PD-L1 and CD206 on TAM isolated from ascites Citation Format: Sonam Mittal, Ishaque Pulikkal Kadamberi, Sudhir Kumar, Jasmine George, Mona Singh, Anjali Geethadevi, Pradeep Chaluvally-Raghavan, Sunila Pradeep. Macrophage reprogramming and immune suppression through extracellular vesicles loaded eIF4A1in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4284.

Jaceosidin attenuates the progression of hepatic fibrosis by inhibiting the VGLL3/HMGB1/TLR4 signaling pathway

BackgroundJaceosidin (JA) is a natural flavone extracted from Artemisia that is used as a food and traditional medicinal herb. It has been reported to possess numerous biological activities. However, the regulatory mechanisms underlying amelioration of hepatic fibrosis remain unclear. Hypothesis/PurposeWe hypothesized that jaceosidin acid (JA) modulates hepatic fibrosis and inflammation. MethodsThioacetamide (TAA) was used to establish an HF mouse model. In vitro, mouse primary hepatocytes and HSC-T6 cells were induced by TGF-β, whereas mouse peritoneal macrophages received a treatment lipopolysaccharide (LPS)/ATP. ResultsJA decreased serum transaminase levels and improved hepatic histological pathology in TAA-treated mice stimulated by TAA. Moreover, the expression of pro-fibrogenic biomarkers associated with the activation of liver stellate cells was downregulated by JA. Likewise, JA down-regulated the expression of vestigial-like family member 3 (VGLL3), high mobility group protein B1 (HMGB1), toll-like receptors 4 (TLR4), and nucleotide-binding domain-(NOD-) like receptor protein 3 (NLRP3), thereby inhibiting the inflammatory response and inhibiting the release of mature-IL-1β in TAA-stimulated mice. Additionally, JA suppressed HMGB1 release and NLRP3/ASC inflammasome activation in LPS/ATP-stimulated murine peritoneal macrophages. JA decreases the expression of pro-fibrogenic biomarkers related to liver stellate cell activation and inhibits inflammasome activation in mouse primary hepatocytes. It also down-regulated α-SMA and VGLL3 expressions and also suppressed inflammasome activation in HSC-T6 cells. VGLL3 and α-SMA expression levels were decreased in TGF-β-stimulated HSC-T6 cells following Vgll3 knockdown. In addition, the expression levels of NLRP3 and cleaved-caspase-1 were decreased in Vgll3-silenced HSC-T6 cells. JA enhanced the inhibitory effects on Vgll3-silenced HSC-T6 cells. Finally, Vgll3 overexpression in HSC-T6 cells affected the expression levels of α-SMA, NLRP3, and cleaved-caspase-1. ConclusionJA effectively modulates hepatic fibrosis by suppressing fibrogenesis and inflammation via the VGLL3/HMGB1/TLR4 axis. Therefore, JA may be a candidate therapeutic agent for the management of hepatic fibrosis. Understanding the mechanism of action of JA is a novel approach to hepatic fibrosis therapy.

Dexamethasone nanomedicines with optimized drug release kinetics tailored for treatment of site-specific rheumatic musculoskeletal diseases

The application of polymer-based drug delivery systems is advantageous for improved pharmacokinetics, controlled drug release, and decreased side effects of therapeutics for inflammatory disease. Herein, we describe the synthesis and characterization of linear N-(2-hydroxypropyl)methacrylamide-based polymer conjugates designed for controlled release of the anti-inflammatory drug dexamethasone through pH-sensitive bonds. The tailored release rates were achieved by modifying DEX with four oxo-acids introducing reactive oxo groups to the DEX derivatives. Refinement of reaction conditions yielded four well-defined polymer conjugates with varied release profiles which were more pronounced at the lower pH in cell lysosomes. In vitro evaluations in murine peritoneal macrophages, human synovial fibroblasts, and human peripheral blood mononuclear cells demonstrated that neither drug derivatization nor polymer conjugation affected cytotoxicity or anti-inflammatory properties. Subsequent in vivo tests using a murine arthritis model validated the superior anti-inflammatory efficacy of the prepared DEX-bearing conjugates with lower release rates. These nanomedicines showed much higher therapeutic activity compared to the faster release systems or DEX itself.

Correlation between secondary metabolites of Iris confusa Sealy and Iris pseudacorus L. and their newly explored antiprotozoal potentials

BackgroundIn the last few decades, the use of plant extracts and their phytochemicals as candidates for the management of parasitic diseases has increased tremendously. Irises are aromatic and medicinal plants that have long been employed in the treatment of different infectious diseases by traditional healers in many cultures. This study aims to explore the potential of three common Iris species (I. confusa Sealy, I. pseudacorus L. and I. germanica L.) against infectious diseases. Their in vitro antiprotozoal potency against Plasmodium falciparum, Trypanosoma brucei brucei, T. b. rhodesiense, T. cruzi and Leishmania infantum beside their cytotoxicity on MRC-5 fibroblasts and primary peritoneal murine macrophages were examined.MethodsThe secondary metabolites of the tested extracts were characterized by UPLC-HRMS/MS and Pearsons correlation was used to correlate them with the antiprotozoal activity.ResultsOverall, the non-polar fractions (NPF) showed a significant antiprotozoal activity (score: sc 2 to 5) in contrast to the polar fractions (PF). I. confusa NPF was the most active extract against P. falciparum [IC50 of 1.08 μg/mL, selectivity index (S.I. 26.11) and sc 5] and L. infantum (IC50 of 12.7 μg/mL, S.I. 2.22 and sc 2). I. pseudacorus NPF was the most potent fraction against T. b. rhodesiense (IC50 of 8.17 μg/mL, S.I. 3.67 and sc 3). Monogalactosyldiacylglycerol glycolipid (18:3/18:3), triaceylglycerol (18:2/18:2/18:3), oleic acid, and triterpenoid irridals (spirioiridoconfal C and iso-iridobelamal A) were the top positively correlated metabolites with antiplasmodium and antileishmanial activities of I. confusa NPF. Tumulosic acid, ceramide sphingolipids, corosolic, maslinic, moreollic acids, pheophytin a, triaceylglycerols, mono- and digalactosyldiacylglycerols, phosphatidylglycerol (22:6/18:3), phosphatidylcholines (18:1/18:2), and triterpenoid irridal iso-iridobelamal A, were highly correlated to I. pseudacorus NPF anti- T. b. rhodesiense activity. The ADME study revealed proper drug likeness properties for certain highly corelated secondary metabolites.ConclusionThis study is the sole map correlating I. confusa and I. pseudacorus secondary metabolites to their newly explored antiprotozoal activity.

Danlou Recipe promotes cholesterol efflux in macrophages RAW264.7 and reverses cholesterol transport in mice with hyperlipidemia induced by P407

IntroductionLiver X Receptor (LXR) agonists could attenuate the development of atherosclerosis but bring excess lipid accumulation in the liver. Danlou Recipe was believed to be a benefit for improving the lipid profile. Thus, it is unclear whether Danlou Recipe could attenuate hyperlipidemia without excess lipid accumulated in the liver of mice. This study aimed to clarify if Danlou Recipe could alleviate the progression of hyperlipidemia in mice without extra lipids accumulated in the liver.MethodsMale murine macrophage RAW264.7 cells and murine peritoneal macrophages were used for the in vitro experiments. Cellular cholesterol efflux was determined using the fluorescent cholesterol labeling method. Those genes involved in lipid metabolism were evaluated by qRT‐PCR and western blotting respectively. In vivo, a mouse model of hyperlipidemia induced by P407 was used to figure out the effect of Danlou Recipe on reverse cholesterol transport (RCT) and hyperlipidemia. Ethanol extract of Danlou tablet (EEDL) was prepared by extracting the whole powder of Danlou Prescription from ethanol, and the chemical composition was analyzed by ultra-performance liquid chromatography (UPLC).ResultsEEDL inhibits the formation of RAW264.7 macrophage-derived foam cells, and promotes ABCA1/apoA1 conducted cholesterol efflux in RAW264.7 macrophages and mouse peritoneal macrophages. In the P407-induced hyperlipidemia mouse model, oral administration of EEDL can promote RCT in vivo and improve fatty liver induced by a high-fat diet. Consistent with the findings in vitro, EEDL promotes RCT by upregulating the LXR activities.ConclusionOur results demonstrate that EEDL has the potential for targeting RCT/LXR in the treatment of lipid metabolism disorders to be developed as a safe and effective therapy.

Vitexin along with verapamil downregulates efflux pump P-glycoprotein in macrophages and potentiate M1 to M2 switching via TLR4-NF-κB-TNFR2 pathway in lipopolysaccharide treated mice

The lipopolysaccharide, a microbial toxin, is one of the major causative agents of sepsis. P-gp expression and its functions are altered during inflammation. LPS has been known to impair the functions of P-gp, an efflux transporter. But the effect of LPS on P-gp expression in murine peritoneal macrophages is poorly understood. Molecular docking studies reveal that vitexin is a potent substrate and verapamil a potent inhibitor of P-gp. In the present experimental study, the curative potential of vitexin as a fruit component and verapamil treated as a control inhibitor of P-gp was examined in a murine LPS sepsis model. The effects of vitexin and verapamil on P-gp expression in macrophages correlating with changes in macrophage polarization and associated functional responses during LPS induced sepsis were studied. Peritoneal macrophages of LPS (10 mg/kg body weight) challenged mice exhibited elevated levels of H2O2, superoxide, and NO in parallel with lower antioxidant activity. LPS treatment increased P-gp expression through increased TLR4/expression. However, LPS challenged mice treated with vitexin (5 mg/kg body weight) + verapamil (5 mg/kg body weight) showed higher anti-oxidant enzyme activity (SOD, CAT and GRx) resulting in reduced oxidative stress. This combination treatment also elevated TNFR2, concomitant with down-regulation of TLR4, NF-κB and P-gp expression in murine peritoneal macrophages, resulting in a switch from M1 to M2 polarisation of macrophages and reduced inflammatory responses. In conclusion, combined vitexin and verapamil treatment could be used as a promising therapy to regulate P-gp expression and protection against LPS mediated sepsis and inflammatory damages.

Microgramma vacciniifolia Frond Lectin: In Vitro Anti-leishmanial Activity and Immunomodulatory Effects Against Internalized Amastigote Forms of Leishmania amazonensis.

The treatment of leishmaniasis, an anthropozoonosis caused by Leishmania protozoa, is limited by factors, such as adverse effects, toxicity, and excessive cost, which has highlighted the importance of novel drugs. In this context, natural products have been considered as sources of antileishmanial agents. This study investigated the leishmanicidal activity of Microgramma vacciniifolia frond lectin (MvFL) on promastigotes and amastigotes of Leishmania amazonensis. The effects of MvFL on promastigote proliferation and macrophage infection by amastigotes were evaluated and mean inhibitory concentrations (IC50) were calculated. As a safety assessment, the hemolytic capacity of MvFL (6.25-200µg/mL) against mouse and human erythrocytes was determined. Additionally, the ability of MvFL (6.25-100µg/mL) to modulate lysosomal and phagocytic activities and the nitric oxide (NO) production by murine peritoneal macrophages was also investigated. After 24h, MvFL inhibited the proliferation of L. amazonensis promastigotes, with anIC50 of 88µg/mL; however, hemolytic activity was not observed. MvFL also reduced macrophage infection by amastigotes with an IC50 of 52µg/mL. Furthermore, treatment with MvFL reduced the number of amastigotes internalized by infected murine peritoneal macrophages by up to 68.9% within 48h. At a concentration of 25µg/mL, MvFL stimulated lysosomal activity of macrophages within 72h, but did not alter phagocytic activity or induce NO production at any of the tested concentrations. MvFL exerts antileishmanial activity and further studies are needed to assess its therapeutic potential in in vivo experimental models of leishmaniasis.

Cyasterone ameliorates sepsis-related acute lung injury via AKT (Ser473)/GSK3β (Ser9)/Nrf2 pathway

BackgroundAcute lung injury (ALI) is a severe disease that can lead to acute respiratory distress syndrome (ARDS), characterized by intractable hypoxemia, poor lung compliance, and respiratory failure, severely affecting patients' quality of life. The pathogenesis of ALI has not been fully elucidated yet, and sepsis is an important cause of ALI. Among the organ injuries caused by sepsis, the lungs are the earliest damaged ones. Radix cyathulae is reported to have analgesic, anti-inflammatory, and anti-aging effects. Cyasterone is extracted from Radix cyathulae. However, it is not known whether cyasterone has protective effects for ALI. This study aims to investigate the effect of cyasterone on sepsis-related ALI and its mechanism.MethodsWe used the cecal ligation peferation (CLP) method to establish a mouse sepsis model, and cyasterone was given intraperitoneally on days 1–3 to observe its preventive effect on sepsis-related acute lung injury. Primary murine peritoneal macrophages were used to investigate the molecular mechanism of cyasterone in vitro.ResultsCyasterone pretreatment inhibits pro-inflammatory cytokine production, NLRP3 inflammasome activation, and oxidative stress in vivo and in vitro. In addition, cyasterone attenuates sepsis-induced ALI by activating nuclear factor erythroid2-related factor (Nrf2), which may be associated with AKT(Ser473)/GSK3β(Ser9) pathway activation.ConclusionsCyasterone defends against sepsis-induced ALI by inhibiting inflammatory responses and oxidative stress, which depends heavily on the upregulation of the Nrf2 pathway through phosphorylation of AKT(Ser473)/GSK3β(Ser9). These results suggest cyasterone may be a valuable drug candidate for preventing sepsis-related ALI.

Guggulsterone protects against lipopolysaccharide-induced inflammation and lethal endotoxemia via heme oxygenase-1

Guggulsterone (GS) is a phytosterol used to treat inflammatory diseases. Although many studies have examined the anti-inflammatory activities of GS, the detailed mechanisms of GS in lipopolysaccharide (LPS)-induced inflammation and endotoxemia have not yet been examined. Therefore, we investigated the anti-inflammatory effects of GS on LPS-induced inflammation. In murine peritoneal macrophages, the anti-inflammatory activity of GS was primarily mediated by heme oxygenase-1 (HO-1) induction. HO-1 induction by GS was mediated by GSH depletion and reactive oxygen species (ROS) production. The ROS generated by GS caused the phosphorylation of GSK3β (ser9/21) and p38, leading to the translocation of nuclear factor erythroid-related factor 2 (Nrf2), which ultimately induced HO-1. In addition, GS pretreatment significantly inhibited inducible nitric oxide synthase (iNOS), iNOS-derived NO, and COX-2 protein and mRNA expression, and production of COX-derived prostaglandin PGE2, interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α). In a mouse model of endotoxemia, GS treatment prolonged survival and inhibited the expression of inflammatory mediators, including IL-1β, IL-6, and TNF-α. GS treatment also inhibited LPS-induced liver injury. These results suggest that GS-induced HO-1 could exert anti-inflammatory effects via ROS-dependent GSK (ser21/9)-p38 phosphorylation and nuclear translocation of Nrf2.

SNASP Mutation Aggravates to the TLR4-Mediated Inflammation in SLE by TAK1 Pathway.

Genetic factors play an important role in the pathogenesis of systemic lupus erythematosus (SLE), and abnormal Toll-like receptor (TLR) signaling pathways are closely related to the onset of SLE. In previous studies, we found that the mutant somatic nuclear autoantigenic sperm protein (sNASP) gene in the mouse lupus susceptibility locus Sle2 can promote the development of lupus model mice, but the mechanism is still unclear. Here, we stimulated mouse peritoneal macrophages with different concentrations of lipopolysaccharide. The results showed that sNASP gene mutations can promote the response of the TLR4-TAK1 signaling pathway but have no significant effect on the TLR4-TBK1 signaling pathway. sNASP mutations enhanced TLR4-mediated nuclear factor-κ-gene binding and mitogen-activated protein kinase activation and IL-6, tumor necrosis factor secretion in murine peritoneal macrophages. Collectively, our study revealed the impact of sNASP gene mutation on the sensitivity of TLR4 receptors in mouse peritoneal macrophages and shed light on potential mechanisms underlying inflammation in autoimmune diseases.

Trichinella spiralis dipeptidyl peptidase 1 suppressed macrophage cytotoxicity by promoting M2 polarization via the STAT6/PPARγ pathway

Trichinella spiralis dipeptidyl peptidase 1 (TsDPP1), or cysteine cathepsin C, is a secretory protein that is highly expressed during the infective larvae and adult worm stages in the intestines. The aim of this study was to investigate the mechanism by which recombinant TsDPP1 (rTsDPP1) activates macrophages M2 polarization and decreases macrophage cytotoxicity to kill newborn larvae via ADCC. RAW264.7 macrophages and murine peritoneal macrophages were used in this study. The results of the immunofluorescence test (IFT) and confocal microscopy showed that rTsDPP1 specifically bound to macrophages, and the binding site was localized on the cell membrane. rTsDPP1 activated macrophage M2 polarization, as demonstrated by high expression levels of Arg1 (M2 marker) and M2-related genes (IL-10, TGF-β, CD206 and Arg1) and high numbers of CD206+ macrophages. Furthermore, the expression levels of p-STAT6, STAT6 and PPARγ were obviously increased in rTsDPP1-treated macrophages, which were evidently abrogated by using a STAT6 inhibitor (AS1517499) and PPARγ antagonist (GW9662). The results indicated that rTsDPP1 promoted macrophage M2 polarization through the STAT6/PPARγ pathway. Griess reaction results revealed that rTsDPP1 suppressed LPS-induced NO production in macrophages. qPCR and flow cytometry results showed that rTsDPP1 downregulated the expression of FcγR I (CD64) in macrophages. The ability of ADCC to kill newborn larvae was significantly decreased in rTsDPP1-treated macrophages, but AS1517499 and GW9662 restored its killing capacity. Our results demonstrated that rTsDPP1 induced macrophage M2 polarization, upregulated the expression of anti-inflammatory cytokines, and inhibited macrophage-mediated ADCC via activation of the STAT6/PPARγ pathway, which is beneficial to the parasitism and immune evasion of this nematode.

Pyrrolidine dithiocarbamate in combination with L-N-monomethyl arginine alleviates Staphylococcus aureus infection via regulation of CXCL8/CXCR1 axis in peritoneal macrophages in vitro

The CXCL8/CXCR1 axis in conjoint with the free radicals and anti-oxidants dictates the severity of inflammation caused by the bacteria, Staphylococcus aureus. S.aureus mediated inflammatory processes is regulated by NF-κB and its product, iNOS. The objective of this study was to examine the effects of inhibition of NF-κB and iNOS on CXCL8/CXCR1, alteration in M1/M2 polarization of macrophages and associated inflammatory responses during S.aureus infection in vitro. For this, the murine peritoneal macrophages were pretreated with NF-κB inhibitor, Pyrrolidine dithiocarbamate (PDTC) and iNOS inhibitor, L-N-monomethyl arginine (LNMMA), either alone or in combination, followed by time-dependent S.aureus infection. The chemotactic migrations of macrophages were determined by the agarose spot assay. The iNOS, NF-κB and CXCR1 protein expressions were evaluated. The ROS level (superoxide, H2O2, NO) and antioxidant activities (SOD, CAT, GSH, arginase) were measured. The intra-macrophage phagoctyic activity had been analyzed by confocal microscopy. S.aureus activated macrophages showed increased iNOS expression that symbolizes M1 characterization of macrophages. The results suggest that the combination treatment of LNMMA + PDTC was effective in diminution of CXCL8 production and CXCR1 expression through downregulation of NF-κB and iNOS signaling pathway. Consequently, there was decrement in macrophage migration, reduced ROS generation, elevated antioxidant enzyme activity as well as bacterial phagocytosis at 90 min post bacterial infection. The increased arginase activity further proves the switch from pro-inflammatory M1 to anti-inflammatory M2 polarization of macrophages. Concludingly, the combination of PDTC + LNMMA could resolve S.aureus mediated inflammation through mitigation of CXCL8/CXCR1 pathway switching from M1 to M2 polarization.

Tetracaine hydrochloride induces macrophage pyroptosis through caspase‑1/11‑GSDMD signaling pathways.

Tetracaine hydrochloride (TTC) is a long-lasting local anesthetic commonly used for topical anesthesia. Inappropriate dosage or allergic reactions to TTC can lead to local anesthetic toxicity. TTC exerts cytotoxic effects on certain cell types by inducing apoptosis and necrosis; however, the effects of TTC on macrophages are currently unclear. In the present study, the RAW 264.7 and BV2 cell lines, and murine peritoneal macrophages, were used to evaluate the cytotoxicity of TTC. The present study demonstrated that TTC caused a decrease in cell viability according to a Cell Counting Kit-8 assay, increased lactate dehydrogenase and IL-1β secretion according to ELISA, and induced morphological changes characteristic of pyroptosis according to western blotting. Moreover, TTC-induced macrophage pyroptosis was mediated by gasdermin (GSDM)D, and the cleavage of GSDMD was modulated by both caspase-1 and caspase-11. These results were experimentally validated using caspase-1 and caspase-11 inhibitors. Furthermore, it was observed that TTC and lipopolysaccharide (LPS) exerted similar effects on macrophages. However, the mechanism of induction of pyroptosis by TTC was different from that of LPS. The present study demonstrated that TTC alone could induce macrophage pyroptosis mediated by canonical and non-canonical inflammatory caspases. Therapies targeting pyroptosis may potentially provide a promising future strategy for the prevention and treatment of local anesthetic toxicity induced by TTC.

Effect of 3-Carene and the Micellar Formulation on Leishmania (Leishmania) amazonensis.

Leishmaniases are neglected tropical diseases caused by obligate intracellular protozoa of the genus Leishmania. The drugs used in treatment have a high financial cost, a long treatment time, high toxicity, and variable efficacy. 3-Carene (3CR) is a hydrocarbon monoterpene that has shown in vitro activity against some Leishmania species; however, it has low water solubility and high volatility. This study aimed to develop Poloxamer 407 micelles capable of delivering 3CR (P407-3CR) to improve antileishmanial activity. The micelles formulated presented nanometric size, medium or low polydispersity, and Newtonian fluid rheological behavior. 3CR and P407-3CR inhibited the growth of L. (L.) amazonensis promastigote with IC50/48h of 488.1 ± 3.7 and 419.9 ±1.5 mM, respectively. Transmission electron microscopy analysis showed that 3CR induces multiple nuclei and kinetoplast phenotypes and the formation of numerous cytosolic invaginations. Additionally, the micelles were not cytotoxic to L929 cells or murine peritoneal macrophages, presenting activity on intracellular amastigotes. P407-3CR micelles (IC50/72 h = 0.7 ± 0.1 mM) increased the monoterpene activity by at least twice (3CR: IC50/72 h >1.5 mM). These results showed that P407 micelles are an effective nanosystem for delivering 3CR and potentiating antileishmanial activity. More studies are needed to evaluate this system as a potential therapeutic option for leishmaniases.

CD40 Signaling in Mice Elicits a Broad Antiviral Response Early during Acute Infection with RNA Viruses.

Macrophages are critical in the pathogenesis of a diverse group of viral pathogens, both as targets of infection and for eliciting primary defense mechanisms. Our prior in vitro work identified that CD40 signaling in murine peritoneal macrophages protects against several RNA viruses by eliciting IL-12, which stimulates the production of interferon gamma (IFN-γ). Here, we examine the role of CD40 signaling in vivo. We show that CD40 signaling is a critical, but currently poorly appreciated, component of the innate immune response using two distinct infectious agents: mouse-adapted influenza A virus (IAV, PR8) and recombinant VSV encoding the Ebola virus glycoprotein (rVSV-EBOV GP). We find that stimulation of CD40 signaling decreases early IAV titers, whereas loss of CD40 elevated early titers and compromised lung function by day 3 of infection. Protection conferred by CD40 signaling against IAV is dependent on IFN-γ production, consistent with our in vitro studies. Using rVSV-EBOV GP that serves as a low-biocontainment model of filovirus infection, we demonstrate that macrophages are a CD40-expressing population critical for protection within the peritoneum and T-cells are the key source of CD40L (CD154). These experiments reveal the in vivo mechanisms by which CD40 signaling in macrophages regulates the early host responses to RNA virus infection and highlight how CD40 agonists currently under investigation for clinical use may function as a novel class of broad antiviral treatments.

CCR4 Blockade Diminishes Intratumoral Macrophage Recruitment and Augments Survival of Syngeneic Pancreatic Cancer-Bearing Mice.

Pancreatic cancer is known for its tumor microenvironment (TME), which is rich in stromal and immune cells supporting cancer growth and therapy resistance. In particular, tumor-associated macrophages (TAMs) are known for their angiogenesis- and metastasis-promoting properties, which lead to the failure of conventional therapies for pancreatic cancer. Hence, treatment options targeting TAMs are needed. The C-C chemokine receptor type 4 (CCR4) is critical for immune cell recruitment into the TME, and in this paper we explore the effects of its genetic or immunotherapeutic blockade in pancreatic-cancer-bearing mice. Murine PDA6606 pancreatic cancer cells and murine peritoneal macrophages were used for in vitro migration assays. In vivo, a syngeneic, orthotropic pancreatic cancer model was established. Tumor growth and survival were monitored under prophylactic and therapeutic application of a CCR4 antagonist (AF-399/420/18025) in wildtype (CCR4wt) and CCR4-knockout (CCR4-/-) mice. Immune infiltration was monitored in tumor tissue sections and via flow cytometry of lysed tumors. PDA6606 cells induced less migration in CCR4-/- than in CCR4wt macrophages in vitro. Pancreatic TAM infiltration was higher, and survival was reduced in CCR4wt mice compared to CCR4-/- mice. Antagonizing CCR4 in wildtype mice revealed similar results as in CCR4-/- mice without antagonization. Prophylactic CCR4 antagonist application in wildtype mice was more efficient than therapeutic antagonization. CCR4 seems to be critically involved in TAM generation and tumor progression in pancreatic cancer. CCR4 blockade may help prolong the relapse-free period after curative surgery in pancreatic cancer and improve prognosis.

Abstract 466: CD36 Mediates Mitochondrial Reactive Oxygen Species Production Through PKM2 In Macrophages

Introduction: Excessive mitochondria-derived reactive oxygen species (mtROS) are associated with foam cell formation and atherosclerosis progression in both animal models and human patients. We previously showed that oxLDL/CD36 signaling stimulated mtROS production and facilitated diet-induced atherosclerosis in mice. However, underlying mechanisms by which oxLDL/CD36 pathway mediates mtROS production during atherosclerosis remain elusive. Pyruvate kinase M2 (PKM2) is a glycolytic enzyme recently discovered to facilitate foam cell and atherosclerosis development. We hypothesize that oxLDL/CD36 signaling mediates mtROS production by stimulating PKM2 mitochondrial translocation. Methods and Results: We separated mitochondria and cytosol fractions in murine peritoneal macrophages. OxLDL treatment upregulated PKM2 protein level in mitochondria fractions in a time-dependent manner (peak at 3h, ~2.5 fold), but not in cytosol. Confocal imaging further confirmed a translocation of PKM2 to mitochondria induced by oxLDL. This phenomenon was not observed in CD36-deficient macrophages. To investigate the mechanism, we immunoprecipitated PKM2 followed by mass spectrometry analysis of proteins associated with PKM2. GRP75, a known chaperone protein that imports cytosol proteins to the mitochondria was identified. Co-IP assay confirmed the interaction between GRP75 and PKM2, which was augmented by oxLDL (~3 fold). Moreover, UQCRC1, a subunit of mitochondrial electron transport chain (ETC) complex III, a major site of mtROS production, was also identified from mass spectrometry. Increased interaction between PKM2 and UQCRC1 induced by oxLDL (~3 fold) was further validated by coIP and in situ proximity ligation assay, which was dependent on CD36.In addition, PKM2 inhibitor Shikonin and PKM2 siRNA both inhibited the oxLDL-induced mtROS generation (by 50% and 100% respectively) in murine macrophages and human monocytes-derived macrophages. Conclusions: OxLDL/CD36 axis promotes PKM2 translocation to mitochondrial which facilitates mtROS production in macrophages. It highlights the novel role of PKM2 in mtROS production during atherosclerosis and implicates GRP75-PKM2-UQCRC1 axis as a potential therapeutic target.

Metabololipidomic and proteomic profiling reveals aberrant macrophage activation and interrelated immunomodulatory mediator release during aging.

Macrophages adapt distinct pro-inflammatory (M1-like) and pro-resolving (M2-like) phenotypes with specific tasks in the immune response and tissue homeostasis. Altered macrophage responses with age are causative for unresolved inflammation, so-called inflammaging, and lead to higher infection susceptibility with unfavorable progression. Here, we reveal molecular determinants of age-related changes in phenotypic functions of murine peritoneal macrophages (PM) by employing comprehensive mass spectrometry-based proteomics (4746 protein groups) and metabololipidomics (>40 lipid mediators). Divergent expression of various macrophage-specific marker proteins and signaling pathways indicates aberrant PM phenotypes in old mice which detrimentally impact their capabilities to release immunomodulatory chemokines and cytokines. We show that aging strikingly compromises the polarization process of macrophages to adapt either pro-inflammatory or pro-resolving phenotypes, thereby yielding aberrant and afunctional macrophage subtypes that cannot be readily assigned to either a typical M1 or M2 phenotype. In particular, the phenotypic adaptation of the bacteria-challenged metabololipidome in macrophages related to inflammation is severely limited by age, which persists across ex vivo polarization towards M1 and M2a macrophages. Our results establish distinct age-associated PM phenotypes outside of the simplified M1 and M2 dichotomy and challenge the dogma of increased pro-inflammatory macrophage pre-activation due to aging by revealing maladaptive functions throughout all phases of inflammation, including resolution.

Apigenin is a promising molecule for treatment of visceral leishmaniasis.

Current treatment for visceral leishmaniasis is based on drugs such as pentavalent antimony and amphotericin B. However, this treatment remains mostly ineffective and expensive, resulting in several side effects and generating resistance. Apigenin, a flavonoid present in fruits and vegetables, has demonstrated several biological functions. In the present study, we observed a concentration-dependent inhibition of the L. infantum promastigote in the presence of apigenin, exhibiting an IC50 value of 29.9 µM. Its effect was also evaluated in L. infantum-infected murine peritoneal macrophages, presenting an C50 value against intracellular amastigotes of 2.3 µM and a selectivity index of 34.3. In a murine model of visceral leishmaniasis, the in vivo effect of apigenin was measured using short-term and long-term treatment schemes. Treatment with apigenin demonstrated 99.7% and 94% reductions in the liver parasite load in the short-term and long-term treatment schemes, respectively. Furthermore, no alterations in serological and hematological parameters were observed. Taken together, these results suggest that apigenin is a potential candidate for visceral leishmaniasis chemotherapy by oral administration.

Abstract 1350: Kupffer cells secrete CXCL5 to promote liver cancer

Abstract Liver carcinoma is the 6th most prevalent cancer worldwide in 2020. Moreover, it is the 3rd leading cause of cancer related deaths. In addition to the genomic and transcriptomic heterogeneity of liver tumor cells which is recognized as a major driver in liver cancer progression, the liver immune system is also fundamental to liver carcinogenesis and presents a promising target for therapy. The liver immune response is orchestrated by cytokines and chemokines. Recent studies suggest that chemokines not only recruit immune cells but also regulate various liver functions. In partial hepatectomy, CXCL2 has been shown to promote hepatocyte proliferation. CXCL1, 2, 5 and 8 can induce endothelial cells chemotaxis to promote angiogenesis through binding to CXCR2. These diverse functions suggest that chemokines could play multifaceted roles in liver cancer development. However, chemokines that are commonly associated with liver cancer is still unknown.We analyzed HCC patient data from the GEO database, and we categorized the datasets based on HCC etiologies including HBV, HCV, alcoholic and NASH. We identified CXCL5 as the only chemokine consistently upregulated in HCC with different etiologies compared to healthy or cirrhotic livers. Immunohistochemistry (IHC) analysis reveals that CXCL5 was produced by immune cells but not tumor cells in human HCC tissues. To further study HCC associated CXCL5 expression, the liver-specific Pten deletion mouse model (PM mice) that recapitulates NAFLD-NASH-HCC progression was used. A gradual increase of hepatic CXCL5 expression is observed during HCC development, reaching nearly 100-fold upregulation of CXCL5 mRNA expression in 12-month-old PM mice livers carrying tumors. Examination of liver immune cell populations showed that macrophages were significantly enriched in Pten deleted livers bearing tumors than wild type livers without tumors. Flow cytometry and IHC analysis further identified Kupffer cells (KCs), the liver resident macrophages as the source of CXCL5 in tumor bearing livers using these mice. Since increased LPS is a prominent feature in most chronic liver diseases, we isolated and treated mouse KCs with LPS and found that LPS treatment robustly increased CXCL5 expression by nearly 20-fold. Interestingly, neither murine macrophage cell lines nor primary peritoneal macrophages displayed induced CXCL5 expression in response to LPS. These data suggest that induction of CXCL5 in KCs is likely a unique function of the KCs but not of other macrophages. To explore the function of CXCL5 in HCC development, we treated mouse hepatocytes and HCC cells with CXCL5 and showed that CXCL5 induces the proliferation of these cells. This effect is further blocked by the inhibition of CXCR2, the receptor of CXCL5, demonstrating the specificity for CXCL5 mediated effects. Together we show here for the first time that CXCL5 expression is a unique property of Kupffer cells and the induction of CXCL5 promotes HCC progression. Citation Format: Taojian Tu, Handan Hong, Lina He, Mario Alba, Curtis T. Okamoto, Bangyan L. Stiles. Kupffer cells secrete CXCL5 to promote liver cancer [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 1350.