M2-like Phenotype Research Articles

Resilience of dermis resident macrophages to inflammatory challenges.

The skin serves as a complex barrier organ populated by tissue-resident macrophages (TRMs), which play critical roles in defense, homeostasis, and tissue repair. This review examines the functions of dermis resident TRMs in different inflammatory settings, their embryonic origins, and their long-term self-renewal capabilities. We highlight the M2-like phenotype of dermal TRMs and their specialized functions in perivascular and perineuronal niches. Their interactions with type 2 immune cells, autocrine cytokines such as IL-10, and their phagocytic clearance of apoptotic cells have been explored as mechanisms for M2-like dermal TRM self-maintenance and function. In conclusion, we address the need to bridge murine models with human studies, with the possibility of targeting TRMs to promote skin immunity or restrain cutaneous pathology.

All-Natural Gelatin-Based Nanoemulsion Loaded with TLR 7/8 Agonist for Efficient Modulation of Macrophage Polarization for Immunotherapy.

Macrophages are multifunctional immune cells essential for both innate and adaptive immune responses. Tumor-associated macrophages (TAMs) often adopt a tumor-promoting M2-like phenotype, aiding tumor progression and immune evasion. Reprogramming TAMs to a tumoricidal M1-like phenotype is an emerging target for cancer immunotherapy. Resiquimod, a TLR7/8 agonist, can repolarize macrophages from the M2- to M1-like phenotype but is limited by poor solubility. We developed a gelatin nanoemulsion for the loading and delivery of resiquimod, utilizing eugenol oil as the liquid phase and riboflavin-crosslinked gelatin as a scaffold. These nanoemulsions showed high stability, low toxicity, and effective macrophage repolarization, significantly enhancing pro-inflammatory markers and anticancer activity in co-culture models.

The Systemic Effect of Ischemia Training and Its Impact on Bone Marrow-Derived Monocytes.

Monocytes are innate immune cells that play a central role in inflammation, an essential component during neovascularization. Our recent publication demonstrated that ischemia training by 24 h unilateral occlusion of the femoral artery (FA) can modify bone marrow-derived monocytes (BM-Mono), allowing them to improve collateral remodeling in a mouse model of hindlimb ischemia. Here, we expand on our previous findings, investigating a potential systemic effect of ischemia training and how this training can impact BM-Mono. BM-Mono from mice exposed to ischemia training (24 h) or Sham (same surgical procedure without femoral artery occlusion-ischemia training) procedures were used as donors in adoptive transfer experiments where recipients were subjected to hindlimb ischemia. Donor cells were divided corresponding to the limb from which they were isolated (left-limb previously subjected to 24 h ischemia and right-contralateral limb). Recipients who received 24 h ischemic-trained monocytes isolated from either limb had remarkable blood flow recovery compared to recipients with Sham monocytes (monocytes isolated from Sham group-no ischemia training). Since these data suggested a systemic effect of ischemic training, circulating extracellular vesicles (EVs) were investigated as potential players. EVs were isolated from both groups, 24 h-trained and Sham, and the former showed increased expression of histone deacetylase 1 (HDAC1), which is known to downregulate 24-dehydrocholesterol reductase (Dhcr24) gene expression. Since we previously revealed that ischemia training downregulates Dhcr24 in BM-Mono, we incubated EVs from 24 h-trained and Sham groups with wild-type (WT) BM-Mono and demonstrated that WT BM-Mono incubated with 24 h-trained EVs had lower gene expression of Dhcr24 and an HDAC1 inhibitor blunted this effect. Next, we repeated the adoptive transfer experiment using Dhcr24 KO mice as donors of BM-Mono for WT mice subjected to hindlimb ischemia. Recipients who received Dhcr24 KO BM-Mono had greater limb perfusion than those who received WT BM-Mono. Further, we focused on the 24 h-trained monocytes (which previously showed downregulation of Dhcr24 gene expression and higher desmosterol) to test the expression of a few genes downstream of the desmosterol pathway, confirm the Dhcr24 protein level and assess its differentiation in M2-like macrophage phenotype. We found that 24 h-trained BM-Mono had greater expression of key genes in the desmosterol pathway, such as liver X receptors (LXRs) and ATP-binding cassette transporter (ABCA1), and we confirmed low protein expression of Dhcr24. Further, we demonstrated that ischemic-trained BM-Mono polarized towards an anti-inflammatory M2 macrophage phenotype. Finally, we demonstrated that 24 h-trained monocytes adhere less to endothelial cells, and the same pattern was shown by WT BM-Mono treated with Dhcr24 inhibitor. Ischemia training leads to a systemic effect that, at least in part, involves circulating EVs and potential epigenetic modification in BM-Mono. These ischemic-trained BM-Mono demonstrated an anti-inflammatory phenotype towards M2 macrophage differentiation and less ability to adhere to endothelial cells, which is associated with the downregulation of Dhcr24 in those cells. These data together suggest that Dhcr24 might be an important target within monocytes to improve the outcomes of hindlimb ischemia.

ARL11 knockdown alleviates spinal cord injury by inhibiting neuroinflammation and M1 activation of microglia in mice

Spinal cord injury (SCI) is a severe central nervous system injury and microglia are major participants in neuroinflammation after injury. ADP-ribosylation factor-like GTPase 11 (ARL11) is a GTP-binding protein. Whether ARL11 is involved in the SCI progression is unknown. In the impactor-induced moderate SCI mouse model, ARL11 protein and mRNA expression were significantly increased in the injury site. LPS (100 ng/mL) and IFN-γ (20 ng/mL) were incubated with BV2 cells (immortalized mouse microglial cell line) to drive them into an M1-like phenotype. ARL11 up-regulation was also observed in activated microglia in SCI mice and LPS and IFN-γ treated BV2 cells. Basso Mouse Scale scores and inclined plate test revealed that ARL11 deletion promoted motor function recovery in SCI mice. Pathological examination showed ARL11 knockdown reduced spinal cord tissue damage, increased neuron numbers, and inhibited neuronal apoptosis in SCI mice. ARL11 knockdown notably inhibited IL-1β and IL-6 production in vivo and in vitro. Furthermore, ARL11 deletion significantly inhibited iNOS protein and mRNA expression in vivo and in vitro, and COX-2 expression in vivo. Mechanism studies revealed that ARL11 silencing decreased phosphorylated ERK1/2 protein expression. Additionally, ELF1 knockdown significantly inhibited ARL11 protein and mRNA expression in vitro. ELF1 acted as a transcription activator in regulating ARL11 expression by binding to the promoter. In conclusion, ARL11 knockdown protects neurons by inhibiting M1 microglia-induced neuroinflammation, thereby promoting motor functional recovery in SCI mice. This may occur in part under the regulation of ELF1. Our study provides a new molecular target for SCI treatment.

M2 Macrophage Exosomes Reverse Cardiac Functional Decline in Mice with Diet-Induced Myocardial Infarction by Suppressing Type 1 Interferon Signaling in Myeloid Cells.

Effective treatment strategies to alleviate heart failure that develops as a consequence of myocardial infarction (MI) remain an unmet need in cardiovascular medicine. In this study, we uncovered that exosomes produced by human THP-1 macrophages cultured with the cytokine IL-4 (THP1-IL4-exo), reverse cardiac functional decline in mice that develop MI as a consequence of diet-induced occlusive coronary atherosclerosis. Therapeutic benefits of THP1-IL4-exo stem from their ability to reprogram circulating Ly-6Chi monocytes into an M2-like phenotype and suppress Type 1 Interferon signaling in myeloid cells within the bone marrow, the circulation, and cardiac tissue. Collectively, these benefits suppress myelopoiesis, myeloid cell recruitment to cardiac tissue, and preserve populations of resident cardiac macrophages that together mitigate cardiac inflammation, adverse ventricular remodeling, and heart failure. Our findings introduce THP1-IL4-exo, one form of M2-macrophage exosomes, as novel therapeutics to preserve cardiac function subsequent to MI.

Proteomic profiling of oleamide-mediated polarization in a primary human monocyte-derived tumor-associated macrophages (TAMs) model: a functional analysis.

Tumor-associated macrophages (TAMs) play a critical function in the development of tumors and are associated with protumor M2 phenotypes. Shifting TAMs towards antitumor M1 phenotypes holds promise for tumor immunotherapy. Oleamide, a primary fatty acid amide, has emerged as a potent anticancer and immunomodulatory compound. However, the regulatory effects of oleamide on TAM phenotypes remain unclear. We used real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) techniques to study the influence of oleamide on primary human monocyte-derived TAM phenotypes, and we investigated the protein expression profiles based on mass spectrometry to analyze the effect of oleamide on macrophage polarization. Moreover, the advantageous binding scores between oleamide and these target candidate proteins are examined using molecular docking. Our study revealed that oleamide effectively suppressed the M2-like TAM phenotype by reducing interleukin (IL)-10 production and downregulating M2-like markers, including vascular endothelial growth factor A (VEGFA), MYC proto-oncogene, bHLH transcription factor (c-Myc), and mannose receptor C-type 1 (CD206). Moreover, the conditioned medium derived from oleamide-treated TAMs induces apoptosis of MDA-MB-231 breast cancer cells. Proteomic analysis identified 20 candidate up- and down-regulation proteins targeted by oleamide, showing modulation activity associated with the promotion of the M1-like phenotype. Furthermore, molecular docking demonstrated favorable binding scores between oleamide and these candidate proteins. Collectively, our findings suggest that oleamide exerts a potent antitumor effect by promoting the antitumor M1-like TAM phenotype. These novel insights provide valuable resources for further investigations into oleamide and macrophage polarization which inhibit the progression of breast cancer, which may provide insight into immunotherapeutic approaches for cancer.

Long-term expanded hepatic progenitor cells ameliorate D-GalN/LPS-induced acute liver failure through repolarizing M1 macrophage to M2-Like phenotype via activation of the IL-10/JAK2/STAT3 signaling pathway

Acute liver failure (ALF) is a devastating liver disease characterized by the rapid deterioration of hepatocytes, which causes a series of clinical complications, including hepatic dysfunction, coagulopathy, encephalopathy, and multiorgan failure. Cell-based therapy is a promising alternative as it can bridge patients until their livers regenerate, releasing immunomodulatory molecules to suppress inflammation. This study reports an iPSCs-derived long-term expanded hepatic progenitor cell (LTHepPCs), which can differentiate into hepatocyte-like cells (HLCs) in vivo. When introduced into drug-induced ALF models, LTHepPCs mitigate liver damage by modulating the local immune microenvironment. This is achieved by shifting macrophages/Kupffer cells towards an anti-inflammatory state, resulting in a decrease in the expression of inflammatory cytokines such as TNF-a, IL-1β, and IL-8, and an increase in the expression of anti-inflammatory cytokines such as IL-10 and ARG-1. In vitro co-culturing of THP-1 or mBMDMs with LTHepPCs suggested that LTHepPCs could activate the anti-inflammatory state of macrophages/Kupffer cells via the IL-10/JAK2/STAT3 signaling pathway. Therefore, LTHepPC transplantation is a promising therapy for ALF patients.

Single-cell RNA sequencing integrated with bulk RNA sequencing analysis reveals the protective effects of lactate-mediated lactylation of microglia-related proteins on spinal cord injury.

Spinal cord injury (SCI) results in significant neurological deficits, and microglia play the critical role in regulating the immune microenvironment and neurological recovery. Protein lactylation has been found to modulate the function of immune cells. Therefore, this study aimed to elucidate the effects of glycolysis-derived lactate on microglial function and its potential neuroprotective mechanisms via lactylation after SCI. Single-cell RNA sequencing (scRNA-seq) data were obtained from figshare to analyze cellular and molecular alterations within the spinal cord post-SCI, further focusing on the expression of microglia-related genes for cell sub-clustering, trajectory analysis, and glycolysis function analysis. We also evaluated the expression of lactylation-related genes in microglia between day 7 after SCI and sham group. Additionally, we established the mice SCI model and performed the bulk RNA sequencing in a time-dependent manner. The expression of glycolysis- and lactylation-related genes was evaluated, as well as the immune infiltration analysis based on the lactylation-related genes. Then, we investigated the bio-effects of lactate on the inflammation and polarization phenotype of microglia. Finally, adult male C57BL/6 mice were subjected to exercise first to increase lactate level, before SCI surgery, aiming to evaluate the protective effects of lactate-mediated lactylation of microglia-related proteins on SCI. scRNA-seq identified a subcluster of microglia, recombinant chemokine C-X3-C-motif receptor 1+ (CX3CR1+) microglia, which is featured by M1-like phenotype and increased after SCI. KEGG analysis revealed the dysfunctional glycolysis in microglia after SCI surgery, and AUCell analysis suggested that the decreased glycolysis an increased oxidative phosphorylation in CX3CR1+ microglia. Differential gene analysis suggested that several lactylation-related genes (Fabp5, Lgals1, Vim, and Nefl) were downregulated in CX3CR1+ microglia at day 7 after SCI, further validated by the results from bulk RNA sequencing. Immunofluorescence staining indicated the expression of lactate dehydrogenase A (LDHA) in CX3CR1+ microglia also decreased at day 7 after SCI. Cellular experiments demonstrated that the administration of lactate could increase the lactylation level and inhibit the pro-inflammatory phenotype in microglia. Functionally, exercise-mediated lactate production resulted in improved locomotor recovery and decreased inflammatory markers in SCI mice compared to SCI alone. In the subacute phase of SCI, metabolic remodeling in microglia may be key therapeutic targets to promote nerve regeneration, and lactate contributed to neuroprotection after SCI by influencing microglial lactylation and inflammatory phenotype, which offered a novel approach for therapeutic intervention.

Nano-Ayurvedic Medicine Approaches Using Ginkgo biloba-Phytochemicals Functionalized Gold Nanoparticles Against Breast Cancer.

Breast cancer is a significant global health issue, contributing to 15% of cancer-related deaths. Our laboratory has pioneered a novel approach, combining Ayurvedic principles with green nanotechnology, to develop a scientifically rigorous medical modality referred to as Nano-Ayurvedic Medicine, recently approved by the US Patents and Trademarks Office. Here in we report a new Nano-Ayurvedic medicine agent derived from gold nanoparticles encapsulated with phytochemicals from Ginkgo biloba plant (GB-AuNPs). We have developed biocompatible gold nanoparticles using electron-rich phytochemicals from Ginkgo biloba as reducing agent cocktail. Ginkgo biloba phytochemical-encapsulated gold nanoparticles (GB-AuNPs) were fully characterized, and their anticancer activity, including immunomodulatory profiles, were evaluated against breast (MDAMB-231) cancer cell lines. Characterization revealed spherical morphology for GB-AuNPs and possessed optimum in vitro stability through high zeta potential of -34 mV for optimum in vivo stability. The core size of GB-AuNPs of 19 nm allows for penetration into tumor cells through both EPR effects as well as through the receptor-mediated endocytosis. The Antitumor efficacy of this nano-ayurvedic medicine agent revealed strong antitumor effects of GB-AuNPs towards MDAMB-231. Our investigations reveal that GB-AuNPs enhance anti-tumor cytokines (IL-12, TNF-α, IFN-γ) and reduce pro-tumor cytokines (IL-10, IL-6), promoting the conversion of protumor M2 macrophages into M1-like macrophage antitumor phenotype. Cellular studies show that GB-AuNPs offer superior anti-tumor efficacy and a better safety profile against breast tumors compared to cisplatin. Our investigations have demonstrated that the nano-ayurvedic medicine agent, GB-AuNPs, treats cancers through an immunomodulatory mechanism facilitated by elevated levels of anti-tumor cytokines (TNF-α, IFN-γ and IL-12) with concomitant downregulation of pro-tumor cytokines expression (IL-6 and IL-10). The green nanotechnology approach for the development of nano-ayurvedic medicine agent (GB-AuNPs), as described in this paper, presents new and attractive opportunities for treating human cancers and other debilitating diseases and disorders.

Wharton's Jelly mesenchymal stem cell-derived extracellular vesicles induce liver fibrosis-resolving phenotype in alternatively activated macrophages.

The potential of extracellular vesicles (EVs) isolated from mesenchymal stromal cells in guiding macrophages toward anti-inflammatory immunophenotypes, has been reported in several studies. In our study, we provided experimental evidence of a distinctive effect played by Wharton Jelly mesenchymal stromal cell-derived EVs (WJ-EVs) on human macrophages. We particularly analyzed their anti-inflammatory effects on macrophages by evaluating their interactions with stellate cells, and their protective role in liver fibrosis. A three-step gradient method was used to isolate monocytes from umbilical cord blood (UCB). Two subpopulations of WJ-EVs were isolated by high-speed (20,000 g) and differential ultracentrifugation (110,000 g). Further to their characterization, they were designated as EV20K and EV110K and incubated at different concentrations with UCB-derived monocytes for 7 days. Their anti-fibrotic effect was assessed by studying the differentiation and functional levels of generated macrophages and their potential to modulate the survival and activity of LX2 stellate cells. The EV20K triggers the polarization of UCB-derived monocytes towards a peculiar M2-like functional phenotype more effectively than the M-CSF positive control. The EV20K treated macrophages were characterized by a higher expression of scavenger receptors, increased phagocytic capacity and production level of interleukin-10 and transforming growth factor-β. Conditioned medium from those polarized macrophages attenuated the proliferation, contractility and activation of LX2 stellate cells. Our data show that EV20K derived from WJ-MSCs induces activated macrophages to suppress immune responses and potentially play a protective role in the pathogenesis of liver fibrosis by directly inhibiting HSC's activation.

Morphine induces inflammatory responses via both TLR4 and cGAS-STING signaling pathways

BackgroundOpioid activation of the microglia or macrophage Toll-like receptor 4 (TLR4) and associated inflammatory cytokine release are implicated in opioid-induced hyperalgesia and tolerance. The cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS-STING) signaling pathway, activated by double-stranded DNA including mitochondrial DNA (mtDNA), has emerged as another key mediator of inflammatory responses. This study tested the hypothesis that morphine induces immune inflammatory responses in microglia and macrophages involving TLR4 and cGAS-STING pathway. MethodsBV2 microglia and Raw 264.7 (Raw) macrophage cells were exposed to morphine with and without a STING inhibitor (C176) for 6 h or TLR 4 inhibitor (TAK242) for 24 h. Western blotting and RT-qPCR analyses assessed TLR4, cGAS, STING, nuclear factor-kappa B (NF-κB), and pro-inflammatory cytokine expression. Morphine-induced mitochondria dysfunction was quantified by reactive oxygen species (ROS) release using MitoSOX, mtDNA release by immunofluorescence, and RT-qPCR. Polarization of BV2 and Raw cells was assessed by inducible nitric oxide (iNOS) and CD86 expression. The role of mtDNA on morphine-related inflammation was investigated by mtDNA depletion of the cells with ethidium bromide (EtBr) or cell transfection of mtDNA extracted from morphine-treated cells. ResultsMorphine significantly increased the expression of TLR4, cGAS, STING, p65 NF-κB, and cytokines (IL-6 and TNF-α) in BV2 and Raw cells. Morphine-induced mitochondrial dysfunction by increased ROS and mtDNA release; the increased iNOS and CD86 evidenced inflammatory M1-like phenotype polarization. TLR4 and STING inhibitors reduced morphine-induced cytokine release in both cell types. The transfection of mtDNA activated inflammatory signaling proteins, cytokine release, and polarization. Conversely, mtDNA depletion led to the reversal of these effects. ConclusionMorphine activates the cGAS-STING pathway in macrophage cell types. Inhibition of the STING pathway can be an additional method to overcome immune cell inflammation-related morphine tolerance and opioid-induced hyperalgesia.

Polyunsaturated fatty acids promote M2-like TAM deposition via dampening RhoA-YAP1 signaling in the ovarian cancer microenvironment

BackgroundPeritoneal metastases frequently occur in epithelial ovarian cancer (EOC), resulting in poor prognosis and survival rates. Tumor-associated-macrophages (TAMs) massively infiltrate into ascites spheroids and are multi-polarized as protumoral M2-like phenotype, orchestrating the immunosuppression and promoting tumor progression. However, the impact of omental conditioned medium/ascites (OCM/AS) on TAM polarization and its function in tumor progression remains elusive.MethodsThe distribution and polarization of TAMs in primary and omental metastatic EOC patients’ tumors and ascites were examined by m-IHC, FACS analysis, and immunofluorescence. QPCR, immunofluorescence, FACS analysis, lipid staining assay, ROS assay, and Seahorse real-time cell metabolic assay characterized TAMs as being polarized in the ascites microenvironment. The oncogenic role of TAMs in tumor cells was demonstrated by co-cultured migration/invasion, proliferation, and spheroid formation assays. Mechanistic studies of the regulations of TAM polarization were performed by using RNA-Seq, GTPase pull-down, G-LISA activation assays, and other biochemical assays. A Yap1 macrophages (MФs) conditional knockout (cKO) mouse model demonstrated the roles of YAP1 in TAM polarization status and its pro-metastatic function. Finally, the anti-metastatic potential of targeting TAMs through restoring YAP1 by pharmacological agonist XMU MP1 was demonstrated in vitro and in vivo.ResultsAbundant polyunsaturated fatty acids (PUFAs) in OCM/AS suppressed RhoA-GTPase activities, which, in turn, downregulated nuclear YAP1 in MФs, leading to increased protumoral TAM polarization accompanied by elevated OXPHOS metabolism. Abolishment of YAP1 in MФs further confirmed that a higher M2/M1 ratio of TAM polarization could alleviate CD8+ T cell infiltration and cytotoxicity in vivo. Consistently, the loss of YAP1 has been observed in EOC metastatic tissues, suggesting its clinical relevance. On the contrary, restoration of YAP1 expression by pharmaceutical inhibition of MST1/2 induced conversion of M2-to-M1-like polarized MФs, elevating the infiltration of CD8+ T cells and attenuating tumor growth.ConclusionThis study revealed that PUFAs-enriched OCM/AS of EOC promotes M2-like TAM polarization through RhoA-YAP1 inhibition, where YAP1 downregulation is required for accelerating protumoral M2-like TAM polarization, thereby causing immunosuppression and enhancing tumor progression. Conversion of M2-to-M1-like polarized MФs through Yap1 activation inhibits tumor progression and contributes to developing potential TAMs-targeted immunotherapies in combating EOC peritoneal metastases.

Injectable thermosensitive hydrogels loaded with irradiated tumor cell-derived microparticles and manganese activate anti-tumor immunity

Immunosuppressive tumor microenvironment and inadequate activation of the innate immune system are important reasons for the failure of systemic anti-tumor immunotherapy. Irradiated tumor cell-derived microparticles (RMPs) can induce immunogenic cell death (ICD) of tumor cells and inhibit the M2-like phenotype of tumor-associated macrophages (TAMs). The current study found that double-stranded DNA was enriched in RMPs, which combined with manganese ions (Mn2+), the natural activator of cGAS-STING signaling, and synergistically amplified cGAS-STING signaling cascade in antigen-presenting cells (APCs). On this basis, a polyamino acid thermosensitive hydrogel delivery system was designed to simultaneously load RMPs and Mn2+. The obtained RMPs@Mn2+ hydrogel exhibited thermosensitivity, biocompatibility and sustained release characteristics. The intratumoral injection of RMPs@Mn2+ slowly released RMPs and Mn2+, inducing ICD of tumor cells, continuously activating cGAS-STING signaling of APCs, reprograming TAMs to M1-like phenotype, and promoting the activation of dendritic cells in tumor draining lymph nodes. The full activation of innate immunity at the tumor site further promoted priming and tumor infiltration of T cells, leading to tumor regression. RMPs@Mn2+ combined with anti-PD-1 achieved strong anti-cancer efficacy in a refractory malignant ascites model, in which more than 50 % of mice with malignant ascites achieved complete regression and long-term immune memory.

Intermittent fasting attenuates cognitive dysfunction and systemic disease activity in mice with neuropsychiatric systemic lupus erythematosus

AimsCognitive dysfunction and systemic disease activity are common manifestations of neuropsychiatric systemic lupus erythematosus (NPSLE), a condition that affects a patient's health and quality of life. Clinical and preclinical studies have demonstrated that intermittent fasting (IF) improves health conditions and quality of life. Therefore, we aimed to test whether IF improves cognitive dysfunction and systemic disease activities in mice with NPSLE and to examine the underlying mechanisms. Main methodsNPSLE-prone MRL/lpr mice underwent 8 weeks of alternate-day fasting or ad libitum feeding, followed by behavioral tests to assess cognitive manifestations and biochemical tests to evaluate systemic disease activities. Key findingsIF significantly improved cognitive functionality, decreased blood-brain barrier permeability, and reduced the activation of astrocytes and microglia in the hippocampi of MRL/lpr mice. IF also improved systemic disease activities, including reduced kidney glomerular injury and interstitial inflammation, peripheral blood autoantibody titer, and splenic T lymphocyte contents. Mechanistic studies demonstrated that IF attenuates cognitive dysfunction by facilitating the microglial transition to the M2-like phenotype via the AMPK/PPARγ/NF-κB pathway. SignificanceTogether, observations from this study suggest a potential therapeutic benefit of IF in the treatment of cognitive dysfunction in patients with NPSLE.

Chondrosarcoma Co-Culture 3D Model─An Insight to Evaluate Drugs Acting on TAMs.

Chondrosarcoma (CHS), also known as malignant cartilage tumors, is the second most common bone cancer after osteosarcoma. This tumor is particularly chemo- and radioresistant, and the only therapeutic alternative is surgery with wide margins. The tumor immune microenvironment reveals an infiltration of tumor-associated macrophages (TAMs) sometimes approaching 50% of the tumor mass, mainly differentiated into M2-like phenotype and correlated with poor prognosis and metastasis. Thus, macrophage-targeting therapies could have an interest in the management of CHS. To evaluate these strategies, we propose here the development of a three-dimensional (3D) tumoroid co-culture model between two human CHS cell lines (JJ012 and CH2879) and a human leukemia monocytic cell line (THP-1) in a methylcellulose matrix. These two models were compared to the in vivo xenograft models in terms of macrophage phenotypes, proteoglycans, MMP-9, and COX-2 expression. Finally, mifamurtide, an immunomodulator acting on TAMs, was evaluated on the most in vitro relevant model: 3D co-culture CH2879 model. Our results showed that it is now possible to develop 3D models that very accurately mimic what is found in vivo with the possibility of evaluating treatments specific to a tumor cell component.

Ultrasound-Activated Probiotics Vesicles Coating for Titanium Implant Infections Through Bacterial Cuproptosis-Like Death and Immunoregulation.

Implant-associated infections (IAIs) are the main cause of prosthetic implant failure. Bacterial biofilms prevent antibiotic penetration, and the unique metabolic conditions in hypoxic biofilm microenvironment may limit the efficacy of conventional antibiotic treatment. Escaping survival bacteria may not be continually eradicated, resulting in the recurrence of IAIs. Herein, a sonosensitive metal-organic framework of Cu-TCPP (tetrakis(4-carboxyphenyl) porphyrin) nanosheets and tinidazole doped probiotic-derived membrane vesicles (OMVs) with high-penetration sonodynamic therapy (SDT), bacterial metabolic state interference, and bacterial cuproptosis-like death to eradicate IAIs is proposed. The Cu-TCPP can convert O2 to toxic 1O2 through SDT in the normoxic conditions, enhancing the hypoxic microenvironment and activating the antibacterial activity of tinidazole. The released Cu(II) under ultrasound can be converted to Cu(I) by exogenous poly(tannic acid) (pTA) and endogenous glutathione. The disruption of the bacterial membrane by SDT can enhance the Cu(I) transporter activity. Transcriptomics indicate that the SDT-enhanced Cu(I) overload and hypoxia-activated therapy hinder the tricarboxylic acid cycle (TCA), leading to bacterial cuproptosis-like death. Moreover, the OMVs-activated therapy can polarize macrophages to a M2-like phenotype and facilitate bone repair. The sonodynamic biofilm microenvironment modulation strategy, whereby the hypoxia-enhanced microenvironment is potentiated to synergize SDT with OMVs-activated therapy, provides an effective strategy for antibacterial and osteogenesis performance.

Hypoxia activates macrophage-NLRP3 inflammasome promoting atherosclerosis via PFKFB3-driven glycolysis.

The onset and progression of atherosclerosis are closely linked to the involvement of macrophages. While the contribution of NLRP3 inflammasome activation to the creation of a local highly inflammatory microenvironment is well recognized, the precise triggers remain unclear. In this study, we aimed to investigate the regulatory mechanism of NLRP3 inflammasome activation in response to hypoxia-induced glycolysis involving PFKFB3 in the development of atherosclerosis. To develop an atherosclerosis model, we selected ApoE knockout mice treated with a high-fat western diet. We then quantified the expression of HIF-1α, PFKFB3, and NLRP3. In addition, we administered the PFKFB3 inhibitor PFK158 during atherosclerosis modeling. The glycolytic activity was subsequently determined through 18F-FDG micro-PET/CT, exvivo glucose uptake, and ECAR analysis. Furthermore, we employed lipopolysaccharide (LPS) and TNF-α to induce the differentiation of bone marrow-derived macrophages (BMDMs) into M1-like phenotypes under both hypoxic and normoxic conditions. Our histological analyses revealed the accumulation of PFKFB3 in human atherosclerotic plaques, demonstrating colocalization with NLRP3 expression and macrophages. Treatment with PFK158 reduced glycolytic activity and NLRP3 inflammasome activation, thereby mitigating the occurrence of atherosclerosis. Mechanistically, hypoxia promoted glycolytic reprogramming and NLRP3 inflammasome activation in BMDMs. Subsequent blocking of either HIF-1α or PFKFB3 downregulated the NLRP3/Caspase-1/IL-1β pathway in hypoxic BMDMs. Our study demonstrated that the HIF-1α/PFKFB3/NLRP3 axis serves as a crucial mechanism for macrophage inflammation activation in the emergence of atherosclerosis. The therapeutic potential of PFKFB3 inhibition may represent a promising strategy for atheroprotection.

Abstract We069: Post-infarction Cardiac Function is Improved in Mice with TSC2 -/- Macrophages

Introduction: Macrophages (MF) play major roles in myocardial ischemia/reperfusion (I/R) injury, both beneficial and detrimental. The mechanistic target of rapamycin (mTOR) plays an important role in MF polarization and functionality. It is prominently regulated by tuberous sclerosis complex 2 (TSC2) which constitutively inhibits and regulates mTORC1. Little is known about how such mTORC1 regulation influences the effect of MFs in I/R injured hearts. Hypothesis: We tested if gene knockdown of TSC2 to constitutively enhance MF mTORC1 activity by TSC2 knockdown impairs or benefits the I/R heart. Methods/Results: MF-specific TSC2 knock-out mice (MF TSC2-/- ) were generated by crossing flox’d TSC2 mice with those expressing LysM-Cre. MTORC1 was constitutively active in bone marrow-derived MF TSC2-/- , reflected by increased S6K1 and 4E-BP1 phosphorylation. MF TSC2-/- exposed to liposaccharide had greater proinflammatory responses, e.g. increased TNF-a secretion, supporting an M1-like phenotype. MF TSC2-/- also exhibited less M2-like features (reduced Arg1, Mrc1, and Rentla expression). To test the impact of MF TSC2-/- on infiltrating myocardial MF subtypes post I/R, MF were isolated from the myocardium and sorted by flow-cytometry based on CD45 + , CD11b + , and CD64 + and then parsed as ±CCR2 and ±MHC-II. We found MF TSC2-/- resulted in an increase in infiltrating CCR2 - MHC-II - phenotypes, which are thought to be anti-inflammatory and to support healing. Consistent with this, we found MF TSC2-/- hearts exposed to I/R had higher ejection fractions (74.2±10.3%) vs controls (47.2±14.3%, P=0.002). MF TSC2-/- hearts also had smaller LV internal end-systolic diameter (P=0.009). As LysM is also expressed in neutrophils, we also performed this study in mice where neutrophils were first depleted using an anti-Ly6G antibody. Similar cardioprotective effects were observed with MF TSC2-/- after I/R: (EF: 56.9±12.3 vs 36.1±9.8, P=0.01). This supports the impact being conferred by MF. Conclusion: TSC2-deleted MF with constitutive mTORC1 activation confer cardio-protection to I/R injury. These findings help identify a novel signaling mechanism coupling mTORC1/TSC2 in MF to myocyte recovery, highlight novel avenues to improve post ischemic cardiac repair.

Patient-specific vascularized tumor model: Blocking monocyte recruitment with multispecific antibodies targeting CCR2 and CSF-1R

Tumor-associated inflammation drives cancer progression and therapy resistance, often linked to the infiltration of monocyte-derived tumor-associated macrophages (TAMs), which are associated with poor prognosis in various cancers. To advance immunotherapies, testing on immunocompetent pre-clinical models of human tissue is crucial. We have developed an in vitro model of microvascular networks with tumor spheroids or patient tissues to assess monocyte trafficking into tumors and evaluate immunotherapies targeting the human tumor microenvironment. Our findings demonstrate that macrophages in vascularized breast and lung tumor models can enhance monocyte recruitment via CCL7 and CCL2, mediated by CSF-1R. Additionally, a multispecific antibody targeting CSF-1R, CCR2, and neutralizing TGF-β (CSF1R/CCR2/TGF-β Ab) repolarizes TAMs towards an anti-tumoral M1-like phenotype, reduces monocyte chemoattractant protein secretion, and blocks monocyte migration. This antibody also inhibits monocyte recruitment in patient-specific vascularized tumor models. In summary, this vascularized tumor model recapitulates the monocyte recruitment cascade, enabling functional testing of innovative therapeutic antibodies targeting TAMs in the tumor microenvironment.

Tumoral Interferon Beta Induces an Immune-Stimulatory Phenotype in Tumor-Associated Macrophages in Melanoma Brain Metastases.

Our study shows that re-education of tumor-associated macrophages by tumoral IFNβ translates into improved clinical outcome in patients with melanoma brain metastases, providing pathomechanistic insights into synergistic type I interferon-inducing therapies with immunotherapies and warranting investigation of IFNβ as a predictive biomarker for combined radioimmunotherapy.