Proinflammatory Responses In Macrophages Research Articles

Harnessing β-glucan conjugated quercetin nanocomplex to function as a promising anti-inflammatory agent via macrophage-targeted delivery

Quercetin, a promising anti-inflammatory agent, faces challenges related to poor bioavailability and limited practical applications. β-glucan, a natural polysaccharide, can be specifically recognized by macrophages, making it an ideal targeting carrier to enhance therapeutic efficacy for macrophage-related dysfunctions. In this study, β-glucan conjugated quercetin nano-complexes (CM-Cur@QT) were developed to target macrophage and alleviate pro-inflammatory response in M1-like macrophages. The results demonstrated that CM-Cur@QT exhibited a spheric shape with an average diameter around 200 nm. FT-IR, 1H NMR, XRD and XPS analyses confirmed the complexation of CM-Cur@QT. This complex showed excellent stability during stimulated digestion, protecting QT from degradation while maintaining favorable antioxidant activity. After complexation, CM-Cur@QT displayed sustained uptake kinetics and enhanced accumulation in macrophages, with a 61.88 % increase compared to individual quercetin after 5 h of incubation. Meanwhile, CM-Cur@QT administration induced evidently cell cycle phases transitions and altered phagocytotic activity in M1-like macrophages. Furthermore, CM-Cur@QT reduced intracellular ROS accumulation, achieving a ROS scavenging rate of up to 49.92 %, compared to 25.59 % in quercetin group. This complex also effectively modulated TNF-a, IL-6 and TGF-β secretion profiles in pro-inflammatory macrophages, outperforming individual QT treatment. Notably, CM-Cur@QT facilitated anti-inflammatory effects while minimizing impacts on inactivated M0 macrophages. These findings underscore the potential of CM-Cur@QT as a promising agent for mitigating inflammatory disorders.

Extracellular vesicles from human breast cancer-resistant cells promote acquired drug resistance and pro-inflammatory macrophage response.

Breast cancer is a significant public health problem around the world, ranking first in deaths due to cancer in females. The therapy to fight breast cancer involves different methods, including conventional chemotherapy. However, the acquired resistance that tumors develop during the treatment is still a central cause of cancer-associated deaths. One mechanism that induces drug resistance is cell communication via extracellular vesicles (EVs), which can carry efflux transporters and miRNA that increase sensitive cells' survivability to chemotherapy. Our study investigates the transcription changes modulated by EVs from tamoxifen- and doxorubicin-resistant breast cancer cells in sensitive cells and how these changes may induce acquired drug resistance, inhibit apoptosis, and increase survivability in the sensitive cells. Additionally, we exposed human macrophages to resistant EVs to understand the influence of EVs on immune responses. Our results suggest that the acquired drug resistance is associated with the ability of resistant EVs to upregulate several transporter classes, which are directly related to the increase of cell viability and survival of sensitive cells exposed to EVs before a low-dose drug treatment. In addition, we show evidence that resistant EVs may downregulate immune system factors to evade detection and block cell death by apoptosis in sensitive breast cancer cells. Our data also reveals that human macrophages in contact with resistant EVs trigger a pro-inflammatory cytokine secretion profile, an effect that may be helpful for future immunotherapy studies. These findings are the first transcriptome-wide analysis of cells exposed to resistant EVs, supporting that resistant EVs are associated with the acquired drug resistance process during chemotherapy by modulating different aspects of sensitive cancer cells that coffer the chemoresistance.

Drp1 Promotes Macrophage M1 Polarization and Inflammatory Response in Autoimmune Myocarditis by Driving Mitochondrial Fission.

Autoimmune myocarditis (AM) is characterized by an intricate inflammatory response within the myocardium. Dynamin-related protein 1 (Drp1), a pivotal modulator of mitochondrial fission, plays a role in the pathogenesis of various diseases. A myosin-induced experimental autoimmune myocarditis (EAM) mouse model was successfully established. Flow cytometry was employed to detect M1/M2-like macrophages. Mitochondrial fragmentation was assessed using Mito-Tracker Red CMXRos. Drp1 was upregulated and activated in EAM mice. Depletion of Drp1 was observed to mitigate inflammation, macrophage infiltration and M1 polarization within the cardiac tissue of EAM mice. In M1-like macrophages derived from the hearts of EAM mice, Drp1 was found to promote mitochondrial fission and diminish mitochondrial fusion. Furthermore, the depletion of Drp1 reduced the NF-κB-related pro-inflammatory response in EAM-associated M1-like macrophages. Drp1 drives mitochondrial fission in macrophages, driving their M1 polarization and the subsequent inflammatory response. Drp1 may represent an effective target for the prevention and treatment of AM.

MDM2 induces pro-inflammatory and glycolytic responses in M1 macrophages by integrating iNOS-nitric oxide and HIF-1α pathways in mice.

M1 macrophages induce protective immunity against infection, but also contribute to metabolic and inflammatory diseases. Here we show that the E3 ubiquitin ligase, MDM2, promotes the glycolytic and inflammatory activities of M1 macrophage by increasing the production of IL-1β, MCP-1 and nitric oxide (NO). Mechanistically, MDM2 triggers the ubiquitination and degradation of E3 ligase, SPSB2, to stabilize iNOS and increases production of NO, which s-nitrosylates and activates HIF-1α for triggering the glycolytic and pro-inflammatory programs in M1 macrophages. Myeloid-specific haplodeletion of MDM2 in mice not only blunts LPS-induced endotoxemia and NO production, but also alleviates obesity-induced adipose tissue-resident macrophage inflammation. By contrast, MDM2 haplodeletion induces higher mortality, tissue damage and bacterial burden, and also suppresses M1 macrophage response, in the cecal ligation and puncture-induced sepsis mouse model. Our findings thus identify MDM2 as an activator of glycolytic and inflammatory responses in M1 macrophages by connecting the iNOS-NO and HIF-1α pathways.

Keratinocyte derived extracellular vesicles mediated crosstalk between epidermis and dermis in UVB-induced skin inflammation.

Ultraviolet-B (UVB) light induces dermal inflammation, although it is mostly absorbed in the epidermis. Recent reports suggest extracellular vesicles (EVs) act as a mediator of photodamage signaling. Melatonin is reported to be a protective factor against UV-induced damage. We hypothesized that EVs derived from UVB-irradiated keratinocytes might trigger proinflammatory responses in dermal cells and tested whether melatonin can ameliorate UVB-induced inflammation. We used UVB-irradiated HaCaT cells, primary keratinocytes and STING knock-out mice to model production of EVs under photodamaging conditions and performed immunoblotting and ELISA to measure their effect on dermal macrophages. UVB-irradiated keratinocytes produce an increased number of EVs that contain higher concentrations of DNA and protein compared with controls. KC-derived EVs (KEVs) induced a STING- and inflammasome-mediated proinflammatory response in macrophages in vitro, and a pronounced inflammatory infiltrate in mouse dermis in vivo. Melatonin ameliorated KEVs inflammatory effect both in vitro and in vivo. This data suggests EVs are mediators in a crosstalk that takes place between keratinocytes and their neighboring cells as a result of photodamage. Further studies exploring EVs induced by damaging doses of UVB, and their impact on other cells will provide insight into photodamage and may help develop targeted therapeutic approaches.

Cabozantinib prevents the progression of metabolic dysfunction-associated steatohepatitis by inhibiting the activation of hepatic stellate cell and macrophage and attenuating angiogenic activity

Cabozantinib, a multiple tyrosine kinase inhibitor targeting AXL, vascular endothelial growth factor receptor (VEGFR), and MET, is used clinically to treat certain cancers, including hepatocellular carcinoma. This study aimed to assess the impact of cabozantinib on liver fibrosis and hepatocarcinogenesis in a rat model of metabolic dysfunction-associated steatohepatitis (MASH). MASH-based liver fibrosis and hepatocarcinogenesis were induced in rats by feeding them a choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) for eight and 16 weeks, respectively. Cabozantinib (1 or 2 mg/kg, daily) was administered concurrently with the diet in the fibrosis model and after eight weeks in the carcinogenesis model. Treatment with cabozantinib significantly attenuated hepatic inflammation and fibrosis without affecting hepatocyte steatosis and ballooning in CDAHFD-fed rats. Cabozantinib-treated rats exhibited a marked reduction in α-smooth muscle actin+ activated hepatic stellate cell (HSC) expansion, CD68+ macrophage infiltration, and CD34+ pathological angiogenesis, along with reduced hepatic AXL, VEGF, and VEGFR2 expression. Consistently, cabozantinib downregulated the hepatic expression of profibrogenic markers (Acta2, Col1a1, Tgfb1), inflammatory cytokines (Tnfa, Il1b, Il6), and proangiogenic markers (Vegfa, Vwf, Ang2). In a cell-based assay of human activated HSCs, cabozantinib inhibited Akt activation induced by GAS6, a ligand of AXL, leading to reduced cell proliferation and profibrogenic activity. Cabozantinib also suppressed lipopolysaccharide-induced proinflammatory responses in human macrophages, VEGFA-induced collagen expression and proliferation in activated HSCs, and VEGFA-stimulated proliferation in vascular endothelial cells. Meanwhile, administration of cabozantinib did not affect Ki67+ hepatocyte proliferation or serum albumin levels, indicating no negative impact on regenerative capacity. Treatment with cabozantinib also reduced the placental glutathione transferase+ preneoplastic lesions in CDAHFD-fed rats. In conclusion, cabozantinib shows promise as a novel option for preventing MASH progression.

Bioinspired micro-nano hierarchical delivery system for sequential targeted therapy of inflammatory bowel disease

Inflammatory bowel disease (IBD) constitutes an immense health and economic burden worldwide, highlighting the need for improved therapy. Up to now, various delivery systems have been proposed to enhance IBD treatment. Herein, inspired by cellular communication in nature, we present a novel micro-nano hierarchical microsphere system with the enhanced targeting capability for IBD treatment. The hierarchical microspheres are designed for oral delivery, with astaxanthin nanoparticles in the core and Zein protein shell. Similar to a stepwise process of cellular communication, microscale spheres can escape from the gastric acid due to Zein coating. Once reach into the intestine, mucoadhesive Zein coating and the core could be enzymatically digested, following the targeted release of astaxanthin nanoparticles. Particularly, these astaxanthin nanoparticles are endowed with the mitochondria targeting capability, thereby greatly attenuating pro-inflammatory response of macrophages. Based on these features, practical performances of the hierarchical delivery system have been investigated in both acute and chronic colitis model. Animal experiments have revealed the desired therapeutic effects of the hierarchical microspheres by inhibiting inflammatory responses and modulating gut microbiota. These results suggested the present hierarchical delivery system offers a stepwise strategy for nanodrug delivery to the intestine as well as the effective treatment of IBD.

Hemin-induced reactive oxygen species triggers autophagy-dependent macrophage differentiation and pro-inflammatory responses in THP-1 cells

The toxic effect of oxidized-heme, also known as hemin, is implicated in developing adverse clinical outcome in various hematolytic diseases. To simulate and reconstruct the molecular events associated with hemin exposure on circulating monocytes, we employed a THP-1 cell line based in vitro model. Flow cytometry and Western blot analyses were subsequently applied. Hemin-treated THP-1 produced ROS in a dose-dependent manner which resulted in 10–30 % of cell death primarily through apoptosis. Surviving cells induced autophagy which too was ROS-dependent, as revealed by application of N-acetyl-L-cysteine. Hemin-mediated autophagy promoted differentiation of CD14+ THP-1 cells into CD11b+ macrophages. Application of 3-methyladenine, reinforced that differentiation of THP-1 was an autophagy-dependent process. It was revealed that despite a higher polarization towards M2-macrophage, synthesis of pro-inflammatory cytokines namely TNF-α, IL-1A, IL-2, IL-8 and IL-17A predominated. IL-6, a pleiotropic cytokine, was also elevated. It may thus be surmised that hemin-induced pro-inflammatory response in THP-1 is downstream to ROS-dependent autophagy and monocyte differentiation. This finding is translationally meaningful as hemin is already approved by FDA for amelioration of acute porphyria and is actively considered as a therapeutic agent for other diseases. This study underscores the need of further research untangling the reciprocal regulation of inflammatory signaling and autophagy under oxidative stress.

LRG1 promotes atherosclerosis by inducing macrophage M1-like polarization

Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by the accumulation of cholesterol-rich lipoproteins in macrophages. How macrophages commit to proinflammatory polarization under atherosclerosis conditions is not clear. Report here that the level of a circulating protein, leucine-rich alpha-2 glycoprotein 1 (LRG1), is elevated in the atherosclerotic tissue and serum samples from patients with coronary artery disease (CAD). LRG1 stimulated macrophages to proinflammatory M1-like polarization through the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways. The LRG1 knockout mice showed significantly delayed atherogenesis progression and reduced levels of macrophage-related proinflammatory cytokines in a high-fat diet-induced Apoe-/- mouse atherosclerosis model. An anti-LRG1 neutralizing antibody also effectively blocked LRG1-induced macrophage M1-like polarization in vitro and conferred therapeutic benefits to animals with ApoE deficiency-induced atherosclerosis. LRG1 may therefore serve as an additional biomarker for CAD and targeting LRG1 could offer a potential therapeutic strategy for CAD patients by mitigating the proinflammatory response of macrophages.

Phosphatidylserine liposomes induce a phagosome acidification-dependent and ROS-mediated intracellular killing of Mycobacterium abscessus in human macrophages.

Mycobacterium abscessus (Mab) is an opportunistic nontuberculous mycobacterium responsible of difficult-to-treat pulmonary infections in vulnerable patients, such as those suffering from Cystic Fibrosis (CF), where it represents a major cause of morbidity and mortality. Additionally, due to the intrinsic extensive antimicrobial resistance spectrum displayed by this species and the side effects reported for some available antibiotics, the therapeutic management of such infections remains extremely difficult. In the present study, we show that phosphatidylserine liposomes (PS-L) enhance intracellular mycobacterial killing of Mab infected human macrophages with functional or pharmacologically inhibited cystic fibrosis conductance regulator (CFTR), by a mechanism involving phagosome acidification and reactive oxygen species (ROS) production. Additionally, PS-L significantly reduce proinflammatory response of Mab infected macrophages in terms of NF-kB activation and TNF-α production, irrespective of CFTR inhibition. Altogether, these results represent the proof of concept for a possible future development of PS-L as a therapeutic strategy against difficult-to-treat Mab infection.

ALDH2 deficiency augments atherosclerosis through the USP14-cGAS-dependent polarization of proinflammatory macrophages

The aldehyde dehydrogenase 2 (ALDH2) rs671 polymorphism commonly exists in the East Asian populations and is associated with high risks of cardiovascular disease (CVD). However, the cellular and molecular mechanisms that underlie the ALDH2 rs671 mutant-linked high CVD remain elusive. Here, we show that macrophages derived from human ALDH2 rs671 carriers and ALDH2 knockout mice exhibited an enhanced pro-inflammatory macrophage phenotype and an impaired anti-inflammatory macrophage phenotype. Transplanting bone marrow from ALDH2−/−ApoE−/− to ApoE−/− mice significantly increased atherosclerotic plaque growth and pro-inflammatory macrophage polarization in vivo. Mechanistically, ALDH2 inhibited activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway in macrophages. Pharmacological inhibition of cGAS by RU.521 completely neutralized ALDH2-deficiency-induced macrophage polarization. In-depth mechanistic investigation showed that ALDH2 accelerated cGAS K48-linked polyubiquitination degradation at lysine 282 in macrophages by reducing the interaction between ubiquitin-specific protease 14 (USP14) and cGAS, mainly through its enzymatic role in mitigating 4-hydroxy-2-nonenal (4-HNE) accumulation. Consistently, USP14 knockdown in bone marrow cells alleviated proinflammatory responses in macrophages and protected against atherosclerosis. Our findings provide new mechanistic insights of ALDH2 deficiency-associated proinflammation and atherosclerosis and new therapeutic and preventive paradigms for treatment of atherosclerosis-associated CVD.

Investigating the role of ER-stress in the regulation of macrophage lipid metabolism and pro-inflammatory response: Focus on atherogenesis

Investigating the role of ER-stress in the regulation of macrophage lipid metabolism and pro-inflammatory response: Focus on atherogenesis

TFEB-Mediated Pro-inflammatory Response in Murine Macrophages Induced by Acute Alpha7 Nicotinic Receptor Activation.

Transcription factors TFEB and TFE3 are crucial for regulating autophagy, lysosomal biogenesis, and lipid metabolism, and have significant roles in macrophage function and innate immunity. The alpha7 nicotinic acetylcholine receptor (α7nAChR), a ligand-gated Ca 2+ channel known for its therapeutic potential in neurological and inflammatory disorders, has been implicated in modulating immune responses by modulating macrophage function. Stimulation of α7nAChR with chemical agonists has been claimed to activate TFEB in pancreatic acinar cells and neurons. However, the impact of α7nAChR activation on TFEB and TFE3 in macrophages remained unknown, posing an important question due to the potential implications for inflammation regulation. This study investigates the effects of acute α7nAChR activation on TFEB-mediated responses in murine macrophages using the specific agonist PNU-282987. We demonstrate that α7nAChR stimulation triggers TFEB nuclear translocation and lysosomal expansion. Surprisingly, PNU-282987 induces a broad pro-inflammatory gene signature without concomitant cytokine secretion, suggesting an uncoupling of gene expression from cytokine release. Mechanistically, TFEB activation requires the lysosomal Ca 2+ exporter MCOLN1 and the Ca 2+ -dependent phosphatase PPP3/calcineurin. Additionally, PNU-282987 elevates reactive oxygen species (ROS) levels, and ROS are involved in TFEB activation by PNU-282987. Notably, even with α7nAChR deletion, compensatory ROS-mediated TFEB activation persists, suggesting the involvement of additional nicotinic receptors. Our findings reveal a novel α7nAChR-TFEB signaling axis in macrophages, offer new insights into the cholinergic regulation of immune responses, establish a baseline for comparison with disease states, and identify potential therapeutic targets for modulating inflammation.

Emodin inhibits M1 macrophage activation that related to acute and chronic kidney injury through EGFR/MAPK pathway.

Macrophages are the main inflammatory cells involved in kidney injury and play a significant role in the development of acute kidney injury (AKI) and progression of chronic kidney disease (CKD). Emodin is believed to stabilize macrophage homeostasis under pathological conditions. The objective of this study aimed to explore the underlying mechanisms and effects of Emodin on M1 macrophages. Network pharmacology methods were used to predict target proteins associated with renal injury and identify the pathways affected by emodin. RAW264.7 macrophages were induced into M1 polarization using LPS and then treated with emodin at 20, 40, and 80 µM. The effects of emodin on cell viability, cytokines (IL-1β, IL-6, TNF-α), M1 macrophage markers (F4/80 + CD86+), and the EGFR/MAPK pathway were evaluated. Additionally, we transfected RAW264.7 cells with an EGFR shRNA interference lentivirus to assess its effects on RAW264.7 cells function and MAPK pathway. After RAW264.7 cells were passaged to expanded culture and transfected with EGFR-interfering plasmid, macrophages were induced to polarize towards M1 with LPS and then treated with 80 µM emodin. CKD modeling was performed to test how emodin is regulated during CKD. There are 15 common targets between emodin and kidney injury, of which the EGFR/MAPK pathway is the pathway through which emodin affects macrophage function. Emodin significantly reduced the levels of IL-6, IL-1β and TNF-α (p < 0.05) and the ratio of M1 macrophage surface markers F4/80 + CD86+ (p < 0.01) in the supernatant of RAW264.7 cells in a dose-dependent manner. Furthermore, the inhibitory effect of emodin on RAW264.7 cells was achieved by interfering with the EGFR/MAPK pathway. Moreover, emodin also affected the mRNA and protein expression of EGFR and Ras, leading to a decrease in the rate of M1 macrophages, thus inhibiting the pro-inflammatory effect of M1 macrophages. The addition of emodin reduced the rate of M1 macrophages in CKD and inhibited the further polarization of M1 macrophages, thus maintaining the pro-inflammatory and anti-inflammatory homeostasis in CKD, and these effects were achieved by emodin through the control of the EGRF/ERK pathway. Emodin attenuates M1 macrophage polarization and pro-inflammatory responses via the EGFR/MAPK signalling pathway. And the addition of emodin maintains pro- and anti-inflammatory homeostasis, which is important for maintaining organ function and tissue repair.

The short peptide encoded by long non-coding RNA RNF217-AS1 inhibits stomach cancer tumorigenesis, macrophage recruitment, and pro-inflammatory responses

Certain long non-coding RNAs (lncRNAs) have potential peptide-coding abilities. Here, the role and molecular basis of the RNF217-AS1-encoded peptide in stomach cancer (SC) tumorigenesis were explored. Here, lncRNAs associated with SC pathogenesis and macrophage infiltration and lncRNAs with peptide-coding potential were searched by bioinformatics analysis. The gene mRNA and protein levels were examined by RT-qPCR and western blot assays, respectively. Cell viability, migratory, and invasive abilities were measured by CCK-8, Transwell migration, and Transwell invasion assays, respectively. The potential biological processes related to lncRNA RNF217-AS1 were identified by single-gene GSEA analysis. The effect of RNF217-AS1-encoded peptide on SC tumorigenesis was examined by mouse xenograft experiments. The results showed that lncRNA NR2F1-AS1 and RNF217-AS1 were differentially expressed and associated with macrophage infiltration in SC, and they had the ability to translate into short peptides. The RNF217-AS1 ORF-encoded peptide could reduce SC cell viability, inhibit cell migration and invasion, as well as hinder the development of SC xenograft tumors. The RNF217-AS1 ORF-encoded peptide in human SC AGS cells suppressed THP-1 cell migration, triggered the differential expression of CXCL1/CXCL2/CXCL8/CXCL12, and inactivated the TLR4/NF-κB/STAT1 signaling pathways. As a conclusion, the RNF217-AS1 ORF-encoded peptide hindered SC progression in vitro and in vivo and suppressed macrophage recruitment and pro-inflammatory responses in SC.

Adipocyte-specific FAK deletion promotes pancreatic β-cell apoptosis via adipose inflammatory response to exacerbate diabetes mellitus.

White adipose tissue (WAT) has a key role in maintaining energy balance throughout the body, and their dysfunction take part in the regulation of diabetes mellitus. However, the internal regulatory mechanisms underlying are still unknown. We generated adipocyte-specific FAK KO (FAK-AKO) mice and investigated their phenotype. The cascade of adipocyte, macrophage in adipocyte tissues, and pancreatic β-cells were proposed in FAK-AKO mice and validated by cell line studies using 3T3-L1, Raw264.7 and Min6. The FAK-AKO mice exhibited glucose intolerance, reduced adipose tissue mass and increased apoptosis, lipolysis and inflammatory response in adipose tissue. We further demonstrate that adipocyte FAK deletion increases β cell apoptosis and inflammatory infiltrates into islets, which is potentiated if mice were treated with STZ. In the STZ-induced diabetes model, FAK AKO mice exhibit less serum insulin content and pancreatic β cell area. Moreover, serum pro-inflammatory factors increased and insulin levels decreased after glucose stimulation in FAK AKO mice. In a parallel vitro experiment, knockdown or inhibition of FAK during differentiation also increased apoptosis, lipolysis and inflammatory in 3T3-L1 adipocytes, whereas the opposite was observed upon overexpression of FAK. Moreover, coculturing LPS-treated RAW264.7 macrophages with knockdown FAK of 3T3-L1 adipocytes increased macrophage pro-inflammatory response. Furthermore, conditioned medium from above stimulated Min6 cells apoptosis (with or without STZ), whereas the opposite was observed upon overexpression of FAK. Mechanistically, FAK protein interact with TRAF6 in adipocytes and knockdown or inhibition of FAK activated TRAF6/TAK1/NF-κB signaling, which exacerbates inflammation of adipocytes themselves. Adipocyte FAK deletion promotes both adipocyte apoptosis and adipose tissue inflammation. Pro-inflammatory factors released by the FAK-null adipose tissue further trigger apoptosis in pancreatic islets induced by the administration of STZ, thereby exacerbating the diabetes mellitus. This study reveals a link between FAK-mediated adipose inflammation and diabetes mellitus, a mechanism that has not been previously recognized.

NINJ1 Facilitates Abdominal Aortic Aneurysm Formation via Blocking TLR4-ANXA2 Interaction and Enhancing Macrophage Infiltration.

Abdominal aortic aneurysm (AAA) is a common and potentially life-threatening condition. Chronic aortic inflammation is closely associated with the pathogenesis of AAA. Nerve injury-induced protein 1 (NINJ1) is increasingly acknowledged as a significant regulator of the inflammatory process. However, the precise involvement of NINJ1 in AAA formation remains largely unexplored. The present study finds that the expression level of NINJ1 is elevated, along with the specific expression level in macrophages within human and angiotensin II (Ang II)-induced murine AAA lesions. Furthermore, Ninj1flox/flox and Ninj1flox/floxLyz2-Cre mice on an ApoE-/- background are generated, and macrophage NINJ1 deficiency inhibits AAA formation and reduces macrophage infiltration in mice infused with Ang II. Consistently, in vitro suppressing the expression level of NINJ1 in macrophages significantly restricts macrophage adhesion and migration, while attenuating macrophage pro-inflammatory responses. Bulk RNA-sequencing and pathway analysis uncover that NINJ1 can modulate macrophage infiltration through the TLR4/NF-κB/CCR2 signaling pathway. Protein-protein interaction analysis indicates that NINJ1 can activate TLR4 by competitively binding with ANXA2, an inhibitory interacting protein of TLR4. These findings reveal that NINJ1 can modulate AAA formation by promoting macrophage infiltration and pro-inflammatory responses, highlighting the potential of NINJ1 as a therapeutic target for AAA.

P53 lysine-lactylated modification contributes to lipopolysaccharide-induced proinflammatory activation in BV2 cell under hypoxic conditions

p53 has diversity functions in regulation of transcription, cell proliferation, cancer metastasis, etc. Recent studies have shown that p53 and nuclear factor-κB (NF-κB) co-regulate proinflammatory responses in macrophages. However, the role of p53 lysine lactylation (p53Kla) in mediating proinflammatory phenotypes in microglia under hypoxic conditions remains unclear. In the current study, we investigated the proinflammatory activation exacerbated by hypoxia and the levels of p53Kla in microglial cells. BV2 cells, an immortalized mouse microglia cell line, were divided into control, lipopolysaccharide (LPS)-induced, hypoxia (Hy), and LPS-Hy groups. The protein expression levels of p53 and p53Kla and the activation of microglia were compared among the four groups. Sodium oxamate and mutant p53 plasmids were transfected into BV2 cells to detect the effect of p53Kla on microglial proinflammatory activation. LPS-Hy stimulation significantly upregulated p53Kla levels in both the nucleus and the cytoplasm of BV2 cells. In contrast, the p53 protein levels were downregulated. LPS-Hy stimulation upregulated phosphorylated p65 protein levels in nuclear and activated the NF-κB pathway in BV2 cells, resulting in increased expression of pro-inflammatory cytokines (iNOS, IL6, IL1β, TNFα), enhanced cell viability, and concomitantly, increased cytotoxicity. In conclusion, p53 lysine-lactylated modification contributes to LPS-induced proinflammatory activation in BV2 cells under hypoxia through NF-κB pathway and inhibition of lactate production may alleviate neuroinflammatory injury.

DIPG-16. THE BIG AND SMALL OF PROTEIN POST-TRANSLATIONAL MODIFICATIONS: TARGETING SUMOYLATION AND CITRULLINATION IN DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Protein post-translational modifications (PTMs) alter the properties of protein through proteolytic cleavage, or the addition of modifying groups to one or more amino acids. Histones are major targets of PTMs, including through SUMOylation of lysine residues and citrullination of arginine residues, with both processes able to regulate gene transcription. We aimed to delineate the potential role of SUMOylation and citrullination in the epigenetic dysregulation that drives Diffuse Midline Glioma (DMG) tumorigenicity. METHODS AND RESULTS DMG tumours demonstrated an upregulation of SUMOylation machinery pathway components and increased expression of peptidylarginine deiminases (PADs), the enzymes responsible for citrullination. Nanomolar doses of the SUMOylation inhibitor TAK-981 blocks DMG cell cycle G2/M transition and results in potent decreases in cell viability and clonogenic potential, triggering ‘viral mimicry’ in DMG cells, resulting in increased expression of interferon stimulated genes and inflammatory mediators including IL-1β, IL-6 and TNFα, whilst also causing a pro-inflammatory M1 macrophage response. These data suggest that SUMOylation inhibition may lead to a double-punch effect against DMG by inhibiting tumorgenicity and stimulating an immune response. In further studies, we have shown that pharmacological inhibition of PAD activity in DMG decreases histone citrullination and restores histone methylation – considered the Achilles heel of DMG. This was accompanied by induction of reactive oxygen species and an unfolded protein response, resulting in DMG cell death in a dose dependent manner. In addition to fully characterising the epigenetic and global changes within DMG initiated through SUMOylation or PAD inhibition, we are currently performing preliminary drug efficacy studies in orthotopic DMG models, providing the necessary pre-clinical research pipeline towards an effective treatment for DMG. CONCLUSIONS Protein SUMOylation and citrullination are previously unrecognised therapeutic targets in DMG and potentially play key roles in tumour initiation and pathogenesis. Our data suggests a novel therapeutic approach for children with DMG.

Targeting macrophagic RasGRP1 with catechin hydrate ameliorates sepsis-induced multiorgan dysfunction

BackgroundThe proinflammatory response induced by macrophages plays a crucial role in the development of sepsis and the resulting multiorgan dysfunction. Identifying new regulatory targets for macrophage homeostasis and devising effective treatment strategies remains a significant challenge in contemporary research. PurposeThis study aims to identify new regulatory targets for macrophage homeostasis and develop effective strategies for treating sepsis. Study design and methodsMacrophage infiltration in septic patients and in lungs, kidneys, and brains of caecum ligation and puncture (CLP)-induced septic mice was observed using CIBERSORT and immunofluorescence (IF). Upon integrating the MSigDB database and GSE65682 dataset, differently expressed macrophage-associated genes (DEMAGs) were identified. Critical DEMAGs were confirmed through machine learning. The protein level of the critical DEMAG was detected in PBMCs of septic patients, RAW264.7 cells, and mice lungs, kidneys, and brains using ELISA, western blot, immunohistochemistry, and IF. siRNA was applied to investigate the effect of the critical DEMAG in RAW264.7 cells. A natural product library was screened to find a compound targeting the critical DEMAG protein. The binding of compounds and proteins was analyzed through molecular docking, molecular dynamics simulations, CETSA, and MST analysis. The therapeutic efficacy of the compounds against sepsis was then evaluated through in vitro and in vivo experiments. ResultsMacrophage infiltration was inversely correlated with survival in septic patients. The critical differentially expressed molecule RasGRP1 was frequently observed in the PBMCs of septic patients, LPS-induced RAW264.7 cells, and the lungs, kidneys, and brains of septic mice. Silencing RasGRP1 alleviated proinflammatory response and oxidative stress in LPS-treated RAW264.7 cells. Catechin Hydrate (CH) was identified as an inhibitor of RasGRP1, capable of maintaining macrophage homeostasis and mitigating lung, kidney, and brain damage during sepsis. ConclusionThis study demonstrates that RasGRP1, a novel activator of macrophage proinflammatory responses, plays a crucial role in the excessive inflammation and oxidative stress associated with sepsis. CH shows potential for treating sepsis by inhibiting RasGRP1.