Bone Marrow-derived Macrophages Research Articles

Abstract C036: Pharmacological modulation of glutamine metabolism alters macrophage activity and tumor immune responses

Abstract As a critical component of innate and adaptive immunity, macrophages play an important role in shaping the tumor microenvironment (TME), which affects tumor initiation, progression, and response to treatment. Tumor-associated macrophages can display anti-tumor or pro-tumor activity, a cell fate attributed to macrophage polarization that depends on metabolomic regulation. Targeting macrophage metabolism could be a promising approach for cancer treatment by converting a pro-tumor TME into an anti-tumor TME. To examine in vivo mechanisms behind glutamine metabolism on macrophages, we treated immunocompetent murine ovarian cancer models with a clinical-grade glutamine prodrug, JHU083, and performed single-cell RNA sequencing (scRNAseq). In addition to the significant anti-tumor efficacy of JHU083, we found that JHU083 specifically suppressed the M2-like tumor macrophages while sparing M1-like macrophages. To better understand the molecular mechanism of how JHU083 causes macrophage polarization shift, we cultured an M1-like macrophage cell line with DON (6-Diazo-5-oxo-L-norleucine), the active component of JHU083, and performed bulk RNA sequencing. Using bulk RNAseq and scRNAseq transcriptome analyses on M1-like macrophages, glutamine inhibitor treatment significantly enhanced MHC class II genes, including CD74, H2-Aa, H2-Eb1, H2-Ea, H2-Ab1, and TNF family genes including Tnfsf12, and Tnfrsf14 which may promote T cell trafficking and cytotoxicity through reversing immunosuppressive tumor microenvironment. Moreover, DON-treated bone marrow-derived M1 macrophages promoted CD4+ T cell expansion. On the other hand, DON treatment significantly suppressed bone marrow-derived M2 macrophages. In conclusion, our findings suggest that glutamine metabolism reprogramming can promote anti-tumor functions in tumor macrophages. Therefore, targeting glutamine metabolism to regulate macrophage function and polarization can be a promising direction for future cancer therapy. Citation Format: Jiawei Fan, Sumeng Qi, Ie-Ming Shih, Tian-Li Wang. Pharmacological modulation of glutamine metabolism alters macrophage activity and tumor immune responses [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C036.

Abstract C024: MPLA+IFNg-activated tumor-associated macrophages alter fibroblast composition

Abstract In a previous study, we developed a strategy to reprogram tumor-associated macrophages (TAMs) and induce a collaborative innate-adaptive immune response by combining monophosphoryl lipid A (MPLA) with IFNγ. However, we also observed downregulation of extracellular matrix (ECM)-associated genes in MPLA+IFNγ-treated mammary tumors, indicating that the combination treatment impacts the tumor microenvironment beyond just the immune cells. In this study, we investigated how MPLA+IFNg treatment modulates the tumor ECM, with a particular focus on fibroblasts-the major ECM-producing cells. Using scRNA-seq to profile mammary carcinomas, we identified six macrophage populations (C1q+, Ccr2+, Spp1+, Lyz+, mt+ and ki67+ TAMs) and two fibroblast populations (Cd34+ and a-SMA+ fibroblasts) in MMTV-PyMT tumor. MPLA+IFNγ treatment increased the numbers and percentages of Lyz+ TAMs and Cd34+ fibroblasts, while the numbers and percentages of Spp1+ TAMs and α-SMA+ fibroblasts were decreased by the treatment. Thus, MPLA+IFNγ treatment induced significant changes in both macrophage and fibroblast populations. Further analysis revealed an unusual gene expression pattern in Lyz+ TAMs. Mgst1 and Gng12 expression was detected in Lyz+ TAMs at levels comparable to those in fibroblasts, despite these genes typically being expressed in fibroblasts but not in macrophages of normal human tissues. However, to our surprise, MPLA+IFNγ treatment did not significantly increase Mgst1 and Gng12 expression of mono-cultured TAMs or bone marrow-derived macrophages, suggesting that these genes were not directly upregulated by MPLA+IFNγ treatment. Instead, the Mgst1 and Gng12 transcripts may have originated from engulfed fibroblasts. In addition, following MPLA+IFNγ treatment, more iNOS+ macrophages (which we previously identified as tumoricidal macrophages) were found in very close proximity to α-SMA+ fibroblasts. To directly test whether MPLA+IFNg-treated TAMs kill and engulf fibroblasts, we performed ex vivo co-cultures of TAMs and fibroblasts. Following MPLA+IFNγ treatment, TAMs effectively killed and engulfed the fibroblasts, whereas minimal fibroblast engulfment was observed in the control co-cultures. Our data suggest that tumoricidal MPLA+IFNγ-treated TAMs can regulate the ECM of tumors by targeting and eliminating the major ECM-producing cells, the fibroblasts. Ongoing studies are determining the functional consequences of the TAM-mediated regulation of fibroblasts and ECM composition. Citation Format: Lijuan Sun, Bodu Liu, Yixin Zhao, Mikala Egeblad. MPLA+IFNg-activated tumor-associated macrophages alter fibroblast composition [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C024.

Abstract C051: Epigenetic reprogramming of brain-infiltrating myeloid cells deters brain metastasis outgrowth

Abstract The unique brain immune microenvironment (BrIME) co-evolves with brain metastasis (BrM) and is marked by immune suppression, posing a significant therapeutic challenge. BrIME is dominated by myeloid cells, with monocyte-derived macrophages (MDMs) comprising nearly half of the tumor-infiltrating leukocytes. Targeting MDMs remains challenging due to their inherent heterogeneity and high plasticity, which are regulated by epigenetic modifiers, including histone deacetylases (HDACs). Yet, the impact of HDAC inhibition in BrIME and its implications for BrM immunotherapy remains unclear. Here, we establish BrM syngenetic model by intracarotid artery (ICA) injection. Immune profiling by mass cytometry (CyTOF) analysis shows that selective class I HDAC inhibitor (HDACi) Entinostat (ENT) significantly enriches inflammatory MDMs in E0771 murine mammary tumor BrM model. Additionally, functional assay with bone marrow-derived macrophages (BMDMs) shows that ENT significantly enhances tumor cell phagocytosis and antigen presentation, increasing the proliferation of antigen-specific T cells in vitro. To test whether HDAC inhibition enhances antitumoral response in the BrIME, we designed a novel sequential treatment: with (1) ENT pre-conditioning the BrIME, (2) low- dose whole brain radiotherapy (LD-WBRT) to release tumor antigen followed by (3) anti-PD1 immune checkpoint therapy (ICT) to reinvigorate tumor-infiltrating. Our result shows that sequential triple treatment demonstrates significant BrM deterrence and induce a specific interferon-responsive MDM subset with robust type I interferon response in single-cell RNA sequencing, correlating with improved ICT clinical outcomes. Our data show that selective HDACi treatment reprograms the suppressive BrIME by steering the BrM-infiltrating MDMs toward immuno-stimulatory status. Sequential triple combinatorial therapy impedes BrM by activating innate and adaptive immunity simultaneously and is well tolerated in the animal model. Our findings highlight the potential of HDACi-mediated epigenetic reprogramming of the BrIME to enhance therapeutic efficacy in treating BrM. Citation Format: Shao-Ping Yang, Yimin Duan, Xiangliang Yuan, Lin Zhang, Wendao Liu, Jun Yao, Patrick Zhang, Jason T Huse, Zhongming Zhao, Dihua Yu. Epigenetic reprogramming of brain-infiltrating myeloid cells deters brain metastasis outgrowth [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C051.

MyD88 protein destabilization mitigates NF-κB-dependent protection against macrophage apoptosis.

Various signaling pathways are essential for both the innate immune response and the maintenance of cell homeostasis, requiring coordinated interactions among them. In this study, a mutation in the caspase-1 recognition site within MyD88 abolished inflammasome-dependent negative regulation, causing phenotypic changes in mice with some similarities to human NEMO-deficiencies. The MyD88D162E mutation reduced MyD88 protein levels and colon inflammation in DSS-induced colitis mice but did not affect cytokine expression in bone marrow-derived macrophages (BMDMs). However, compared to MyD88wt counterparts, MyD88D162E BMDMs had increased oxidative stress and dysfunctional mitochondria, along with reduced prosurvival Bcl-xL and BTK expression, rendering cells more prone to apoptosis, exacerbated by ibrutinib treatment. NF-κB activation by lipopolysaccharide mitigated this sensitive phenotype. These findings underscore the importance of MyD88wt signaling for NF-κB activation, protecting against macrophage premature apoptosis at resting state. Targeting MyD88 quantity rather than just its signaling could be a promising strategy for MyD88-driven lymphoma treatment.

Mycobacterium tuberculosis Antigen Rv1471 Induces Innate Immune Memory and Adaptive Immunity against Infection.

Protein subunit vaccines form a key pipeline for developing novel tuberculosis (TB) vaccines. Mycobacterium tuberculosis (Mtb) contains approximately 4000 individual proteins. However, only approximately 100 have been evaluated as antigens in the clinical and preclinical stages of vaccine development. Trained immunity-targeting vaccines induce innate immune memory against heterologous infections and enhance antigen-specific adaptive immune responses. However, no trained immunity-targeting subunit TB vaccine has been reported yet. This study tested Rv1471, a thioredoxin secreted by Mtb, as a candidate TB vaccine antigen due to its capacity to stimulate mouse bone marrow-derived macrophages (BMDMs). Rv1471 induced functional and phenotypic maturation of BMDMs in vitro, reflected by the increased production of inflammatory cytokines and surface expression of co-stimulatory molecules. Transcription analysis of Rv1471-trained BMDMs indicated that innate immune memory was activated through pathways of Akt-mTOR-HIF-1α and aerobic glycolysis. Rv1471 also enhanced innate immune memory responses and protection against intracellular infections of different mycobacteria. In a murine model of TB, immunization with Rv1471 formulated with liposomal DDA/MPLA adjuvant produced robust antigen-specific multi-functional CD4+ and CD8+ T-cell immune responses and had substantial protective efficacy against Mtb challenge. Analysis of recall immunity showed that the Rv1471 subunit vaccine triggered robust T-cell immunity post Mtb infection. These findings support the development of an innate immune memory-targeting subunit TB vaccine to increase TB vaccine efficacy.

Probiotic bacteria-released extracellular vesicles enhance macrophage phagocytosis in polymicrobial sepsis by activating the FPR1/2 pathway.

Sepsis-induced organ failure and high mortality are largely ascribed to the failure of bacterial clearance from the infected tissues. Recently, probiotic bacteria-released extracellular vesicles (BEVs) have been implicated as critical mediators of intercellular communication which are widely involved in the regulation of the inflammatory response. However, their functional role in macrophage phagocytosis during sepsis has never been explored. BEVs were collected from three different strains of probiotics including Lactiplantibacillus plantarum WCFS1 (LP WCFS1), Lactobacillus rhamnosus Gorbach-Goldin (LGG), and Escherichia coli Nissle 1917 (EcN), or from LGG cultured under three pH conditions (pH5-acid, pH6.5-standard, pH8-akaline) through differential centrifugation, filtration, and ultracentrifugation of their culture supernatants. In vitro phagocytosis was measured in Raw264.7 cells and bone marrow-derived macrophages using pHrodo red E. coli BioParticles. The in vivo therapeutic effects of BEVs were tested using a feces-injection-in-peritoneum (FIP) model of polymicrobial sepsis. LGG-derived EVs (BEVLGG) were the best among these three probiotics BEVs in stimulating macrophages to take up bacteria. Furthermore, BEVLGG collected from pH8 culture condition (BEVpH8) exhibited the strongest capacity of phagocytosis, compared with BEVpH5and BEVpH6.5. Treatment of septic mice with BEVpH8significantly prolonged animal survival; increased bacterial clearance from the blood, peritoneal lavage fluid, and multiple organs; and decreased serum levels of pro-inflammatory cytokines/chemokines, as well as reduced multiple organ injuries, in comparison with control-treated septic mice. Mechanistically, RNA-seq and bioinformatic analysis identified that the FPR1/2 signaling was remarkably activated, along with its downstream pathways (PI3K-Akt-MARCO and NADPH-ROS) in BEVpH8-treated macrophages, compared with control cells. Accordingly, pre-addition of Boc2, a specific antagonist of FPR1/FPR2, to macrophages significantly attenuated BEVpH8-mediated phagocytosis, compared to controls. This study demonstrates that LGG-derived BEVs may have therapeutic effects against sepsis-induced organ injury and mortality through enhancing FPR1/2-mediated macrophage phagocytosis.

Macrophage-Specific Lactate Dehydrogenase Expression Modulates Inflammatory Function In Vitro.

In acute kidney injury, macrophages play a major role in regulating inflammation. Classically activated macrophages (M1) undergo drastic metabolic reprogramming during their differentiation and upregulate the aerobic glycolysis pathway to fulfill their pro-inflammatory functions. NAD+ regeneration is crucial for the maintenance of glycolysis and the most direct pathway by which this occurs is via the fermentation of pyruvate to lactate, catalyzed by lactate dehydrogenase A (LDHA). Our previous study determined that LDHA is predominantly expressed in the proximal segments of the nephron in the mouse kidney and increases with hypoxia. This study investigates the potential of LDHA as a therapeutic target for inflammation by exploring its role in macrophage function in vitro. Bone-marrow-derived macrophages (BMDMs) were isolated from myeloid-specific LDHA knockout mice derived from crossbreeding LysM-Cre transgenic mice and LDHA floxed mice. RNA sequencing and LC-MS/MS metabolomics analyses were used in this study to determine the effect of LDHA deletion on BMDM following stimulation with IFN-γ. LDHA deletion in IFN-γ BMDMs resulted in a significant alteration of the macrophage activation and functional pathways, and change in glycolytic, cytokine, and chemokine gene expression. Metabolite concentrations associated with pro-inflammatory macrophage profiles were diminished while anti-inflammatory-associated ones were increased in LDHA KO BMDMs. Glutamate and amino sugars metabolic pathways were significantly affected by the LDHA deletion. A combined muti-omics analysis highlighted changes in Rap1 signaling, cytokine-cytokine receptor interaction, focal adhesion, and MAPK signaling metabolism pathways. Deletion of LDHA in macrophages results in a notable reduction in the pro-inflammatory profile and concurrent upregulation of anti-inflammatory pathways. These findings suggest that LDHA could serve as a promising therapeutic target for inflammation, a key contributor to the pathogenesis of acute kidney injury.

Kirenol Ameliorates Myocardial Ischemia-Reperfusion Injury by Promoting Mitochondrial Function and Inhibiting Inflammasome Activation.

Macrophage-mediated inflammation plays a crucial role in the pathophysiological process of myocardial ischemia/reperfusion (I/R) injury. Recent studies have highlighted the importance of mitochondrial function and inflammasome activation in the inflammatory process. Kirenol, a well-known natural compound, has been shown to regulate inflammation in various diseases. This study investigated whether Kirenol could exert anti-inflammatory effects on macrophages during myocardial I/R injury. Mouse myocardial I/R models were established by 45min of ischemia followed by 24h of reperfusion. Saline or Kirenol treatment was administered. In vivo assessments included the evaluation of cardiac function, infarcted area, and immune cell infiltration. Subsequently, bone marrow-derived macrophages (BMDMs) were isolated, and mitochondrial function and pyroptosis were assessed. Furthermore, the study compared the cardioprotective effects of Kirenol with a specific NOX1/NOX4 inhibitor, GKT137831. Kirenol gavage improved cardiac function, decreased infarct area, and alleviated inflammatory infiltration in mice subjected to myocardial I/R injury. Mechanistically, Kirenol inhibited NOX1 and NOX4 and enhanced mitochondrial function, ultimately attenuating the pyroptosis of macrophages. The therapeutic effects of Kirenol and GKT137831 were not significantly different. This study demonstrates that Kirenol mitigates myocardial I/R injury by inhibiting NOX1 and NOX4, restoring mitochondrial function, and ameliorating macrophage pyroptosis.

EXTH-43. ANTI-GLIOMA ACTIVITY OF BIMODAL CGAS-AGONISTIC AND STAT3-INHIBITORY SPHERICAL NUCLEIC ACIDS

Abstract Activation of the cGAS-STING (cyclic GMP-AMP synthase-stimulator of Interferon Genes) pathway enhances T cell activation and infiltration into tumors, reversing the immunosuppressive phenotype of myeloid cells. Targeting the STING receptor with synthetic cyclic dinucleotide (CDN) ligands offers a promising immunotherapeutic strategy for lymphocyte-depleted, myeloid cell-enriched tumors like Glioblastoma (GBM). However, this approach is constrained by the structural instability of CDNs and the presence of immune-evasive, STING-inhibitory mechanisms in the tumor microenvironment (TME), particularly those driven by the master transcriptional regulator STAT3 (Signal Transducer and Activator of Transcription 3). To activate cGAS anti-tumor responses and simultaneously inhibit STAT3-driven immunosuppression in the GBM TME, we developed Spherical Nucleic Acids (SNAs) consisting of a nanoparticle core densely functionalized with radially oriented stem-loop dsDNA oligonucleotides containing a guanine repeat element flanked by palindromic STAT3 decoy sequences. In vitro studies demonstrated that these SNAs effectively activate cGAS and inhibit STAT3 in reporter cell lines, exhibit high-affinity binding to both the cGAS enzyme and STAT3 proteins, and downregulate IL-6-induced STAT3 transcriptional targets. Furthermore, intratumoral injection of these bimodal SNA architectures induced type I interferon (IFN) and proinflammatory cytokines, activated T cells and B cells, and natural killer cells; and reprogrammed immunosuppressive tumor-associated macrophages (TAMs). Importantly, bimodal SNAs showed profound sex-based differential pharmacological action with therapeutic benefits seen in females but not in male mice in aggressive immune checkpoint-resistant murine glioma models. In the absence of cGAS transcriptional changes, western blot and flow cytometry analysis revealed that cGAS protein levels were approximately twice as high in female BMDMs (bone marrow-derived macrophages) and TAMs than males. In summary, our study establishes bimodal SNAs as a first-in-class, single-entity nucleic acid therapeutic capable of targeting multiple pathways for the immunological reprogramming of the GBM TME.

TMET-09. SEX-BIASED ROLE OF TUMOR-ASSOCIATED MACROPHAGES IN GLIOBLASTOMA IRON METABOLISM

Abstract Glioblastoma (GBM) is an aggressive brain cancer that is extremely difficult to treat. GBM is sexually dimorphic in nature, where females are less susceptible to developing GBM, respond to therapy better, and get a survival advantage. Sex-biased differences in tumor iron metabolism play a significant part in making this disease sexually dimorphic. Macrophages constitute a significant population in the GBM tumor microenvironment and are key players in tumor iron homeostasis. Here, we examine macrophage iron homeostasis as a potential driver of sex biases in glioblastoma using bone marrow-derived macrophages (BMDM) isolated from C57BL/6 mice. Immunoblot analysis revealed higher ferritin L-chain (FTL) expression in male BMDM than in females (Fold change: 3.18, P = 0.02). Flow cytometric analysis revealed that male BMDMs had a significantly higher labile iron pool (LIP) (Fold change: 1.62, P = 0.0081). We used a stable isotope of iron (Fe57) to determine macrophage iron uptake by ICP-MS, and observed that male BMDMs have a higher iron uptake (fold change: 1.623, P = 0.0018) than females. These results indicate that macrophage iron metabolism is sexually dimorphic in nature. Next, we used a transwell co-culture system to study how macrophages regulate the iron status of GL261 cells. We found that GL261 cells had significantly lower H-Ferritin (FTH1) expression when co-cultured with male BMDMs (Fold change: 0.789, P = 0.0045). However, this decrease in ferritin didn’t result in a decrease in cellular LIP. We discovered that there was an increase in secreted ferritin in the GL261 culture media. Further experiments with Fe57-loaded GBM cells showed that this increase in secreted ferritin correlated with increased iron release from the GL261 cells. These results indicate that tumor-associated macrophages may regulate tumor iron homeostasis by inducing ferritin-bound iron release from GBM cells in a sex-biased manner.

Monocyte/macrophage pyroptosis and C5b-9-induced cyst enlargement in Pkd1-/- mice.

The levels of C5b-9, terminal products of complement activation, were significantly elevated in autosomal dominant polycystic kidney disease (ADPKD). However, the precise mechanisms by which C5b-9 facilitates cyst growth remain incompletely elucidated. Three groups of chronic-onset Pkd1-/- mice were established: one group received intravenous injections of 0.5 mg/kg C5b-9, another was administered 1.0 mg/kg monoclonal anti-C9 antibodies, and a control group received 1 mg/kg IgG isotype control (IC). All treatments were administered biweekly for two months (postnatal day [PD] 180-240). Renal macrophages from distinct subsets were sorted using fluorescence-activated cell sorting (FACS). To deplete macrophages, liposome clodronate (LC) was injected intraperitoneally. Sublethal irradiation followed by bone marrow (BM) reconstruction was performed in Pkd1-/- mice to evaluate the role of BM-derived macrophages (BMDMs) in ADPKD progression. (1) In vitro, sublytic C5b-9 did not affect the viability of renal tubular epithelial cells (RTECs), but significantly induced M1-like polarization and pyroptosis of BMDMs. (2) In vivo, C5b-9 notably triggered pyroptosis of Ly6C+ monocytes and a reduction in circulating monocyte numbers as cysts enlarged. (3) Residual Ly6C+ monocytes infiltrated renal tissues and differentiated into Ly6C+ macrophages, which exhibited a greater susceptibility to pyroptosis compared to Ly6C- macrophages. (4) Although limited evidence has recently suggested that Ly6C- monocytes may also be affected by C5b-9, upregulation of CCR2 in Ly6C- macrophages was observed in C5b-9-treated Pkd1-/- mice, implying that Ly6C- monocytes could represent a significant source of M2 macrophages. C5b-9 infusion promoted RTEC proliferation by inducing pyroptosis of Ly6C+ monocytes/macrophages, contributing to progressive cyst enlargement in chronic-onset PKD mice.

Loss of O6-methylguanine DNA methyltransferase (MGMT) in macrophages alters responses to TLR3 stimulation and enhances DNA double-strand breaks and mitophagy.

O6-methylguanine-DNA methyltransferase (MGMT) is a DNA damage repair enzyme. The roles of this enzyme in immune cells remain unclear. In this study, we explored the roles of MGMT in bone marrow-derived murine macrophages (BMMs) via the use of MGMT knockout (KO) mice. Loss of MGMT altered the response to TLR3 agonists (poly (I:C)), such as dampening the production of TNFα and IL-6. Increased DNA double-strand breaks (DSBs) were observed in MGMT-KO macrophages but did not result in increased cell death. MGMT localized to both nuclei and mitochondria at increasing levels during poly (I:C) stimulation. MGMT deficiency increased the production of mitochondrial reactive oxygen species (mtROS), which was correlated with increased mitophagy. The underlying mechanism involves mediation through activation of the AMPKα pathway. Taken together, our findings reveal the roles of MGMT in macrophages in regulating the response to TLR3, which links DSBs to mtROS and mitophagy via the AMPKα pathway. These roles may have consequences for the inflammatory response and chronic inflammation.

Immunological Pre-Metastatic Niche in Dogs With Naturally Occurring Osteosarcoma.

Pre-metastatic niche (PMN) formation is essential for metastatic development and drives organotropism. Tumour-derived extracellular vesicles and soluble factors remodel the microenvironment of distant metastatic organs before subsequent metastasis. Dogs with osteosarcoma (OS) have proven to be excellent disease models for their human companions. Here, we show evidence of PMN formation in dogs with OS before metastasis. We necropsied and sampled lung tissues from dogs with naturally occurring treatment-naïve OS (n = 15) and control dogs without cancer (n = 10). We further divided dogs with OS into those having lung metastases (n = 5) and those without (n = 10). We stained formalin-fixed paraffin-embedded tissues using multiplex immunofluorescence to quantify the number of bone marrow-derived cells, monocytes and macrophages in the lung samples from each dog. The numbers of CD204+ macrophages, CD206+ macrophages and monocytes and CD11d+ bone marrow-derived cells (BMDCs) were significantly higher in the pre-metastatic lung of dogs with OS (n = 10) than in control dogs without cancer (n = 10). Furthermore, the total nucleated cell (DAPI+) density was higher before metastasis than in healthy lungs. In dogs with established metastases, the number of CD11d+ BMDCs was significantly lower than in the pre-metastatic lung, suggesting this recruitment is transient. Our study provides evidence of PMN existence in a naturally occurring cancer model similar to those observed in pre-clinical murine models. BMDCs are recruited to the lungs before metastases have developed. Dogs with OS may represent ideal candidates for assessing new PMN-targeting therapies.

ApoE Receptor-2 R952Q Variant in Macrophages Elevates Soluble LRP1 to Potentiate Hyperlipidemia and Accelerate Atherosclerosis in Mice.

apoER2 (apolipoprotein E receptor-2) is a transmembrane receptor in the low-density lipoprotein receptor (LDLR) family with unique tissue expression. A single-nucleotide polymorphism that encodes the R952Q sequence variant has been associated with elevated plasma cholesterol levels and increased myocardial infarction risk in humans. The objective of this study was to delineate the mechanism underlying the association between the apoER2 R952Q variant and increased atherosclerosis risk. An apoER2 R952Q mouse model was generated using a CRISPR/Cas9 strategy, intercrossed with LDLR knockout mice, followed by feeding a Western-type high-fat high-cholesterol diet for 16 weeks. Atherosclerosis was investigated by immunohistology. Plasma lipids and lipid distributions among the various lipoprotein classes were analyzed by colorimetric assay. Tissue-specific effects of the R952Q sequence variant on atherosclerosis were analyzed by bone marrow transplant studies. sLRP1 (soluble low-density lipoprotein receptor-related protein 1) was measured in plasma and conditioned media from bone marrow-derived macrophages by ELISA and GST-RAP (glutathione S-transferase-receptor-associated protein) pull-down, respectively. P38 MAPK (mitogen-activated protein kinase) phosphorylation in VLDL (very-low-density lipoprotein)-treated macrophages was determined by Western blot analysis. Consistent with observations in humans with this sequence variant, the apoER2 R952Q mutation exacerbated diet-induced hypercholesterolemia, via impediment of plasma triglyceride-rich lipoprotein clearance, to accelerate atherosclerosis in Western diet-fed LDLR knockout mice. Reciprocal bone marrow transplant experiments revealed that the apoER2 R952Q mutation in bone marrow-derived cells instead of non-bone marrow-derived cells was responsible for the increase in hypercholesterolemia and atherosclerosis. Additional data showed that the apoER2 R952Q mutation in macrophages promotes VLDL-induced LRP1 (low-density lipoprotein receptor-related protein 1) shedding in a p38 MAPK-dependent manner. The apoER2 R952Q mouse model recapitulates characteristics observed in human disease. The underlying mechanism is that the apoER2 R952Q mutation in macrophages exacerbates VLDL-stimulated sLRP1 production in a p38 MAPK-dependent manner, resulting in its competition with cell surface LRP1 to impede triglyceride-rich lipoprotein clearance, thereby resulting in increased hypercholesterolemia and accelerated atherosclerosis.

Mannose receptor (MRC1) mediates uptake of dextran by bone marrow-derived macrophages.

Macrophages survey their environment using receptor-mediated endocytosis and pinocytosis. Receptor-mediated endocytosis allows internalization of specific ligands, whereas pinocytosis non-selectively internalizes extracellular fluids and solutes. CRISPR/Cas9 whole-genome screens were used to identify genes regulating constitutive and growth factor-stimulated dextran uptake in murine bone-marrow derived macrophages (BMDM). The mannose receptor c-type 1 (MRC1/CD206) was a top hit in the screen. Targeted gene disruptions of Mrc1 reduced dextran uptake but had little effect on fluid-phase uptake of Lucifer yellow. Other screen hits also differentially affected the uptake of dextran and Lucifer yellow, indicating internalization by separate mechanisms. Visualization of dextran and Lucifer yellow uptake by microscopy showed enrichment of dextran in small puncta, which was inhibitable by mannan, a ligand of MRC1. In contrast, Lucifer yellow predominantly was internalized in larger macropinosomes. In addition, IL4-treated BMDMs internalized more dextran than untreated BMDM correlating with increased MRC1 expression. Therefore, dextran is not an effective marker for pinocytosis in BMDMs since it is internalized by receptor-mediated process. Numerous genes that regulate dextran internalization in primary murine macrophages were identified in the whole-genome screens, which can inform understanding of the regulation of MRC1 expression and MRC1-mediated uptake in macrophages.

Inhibition of GDNF-Driven Macrophage-to-Myofibroblast Transition Protects Against Colitis-Associated Intestinal Fibrosis.

Glial cell line-derived neurotrophic factor (GDNF) has been demonstrated to promote the development of liver fibrosis, but its role in intestinal fibrosis is unknown. Macrophage-to-myofibroblast transition (MMT) is an important pathway contributing to fibrosis diseases. However, whether MMT cells, characterized by co-expressing both macrophage (CD68 or F4/80) and myofibroblast (α-SMA) markers, occurs in intestinal fibrosis remain to be addressed. Here, we showed that GDNF expression and the infiltration of MMT cells in intestinal tissues from patients with fibrostenotic Crohn's disease (CD) and a mouse model of chronic dextran sodium salt-induced intestinal fibrosis were significantly increased. GDNF induced bone marrow-derived macrophages (BMDMs) differentiation into MMT cells in vitro. Mechanistically, the Src pathway was activated by GDNF stimulation and contributed to GDNF-induced MMT in BMDMs. Moreover, pharmacological inhibition of GDNF by using antibody markedly decreased the infiltration of MMT cells following the decrease of collagen deposition and α-SMA and Col1 expression in the mouse model of colitis-associated intestinal fibrosis. In conclusion, GDNF is able to induce MMT and contributes to intestinal fibrosis in the context of chronic intestinal inflammation. Pharmacological inhibition of GDNF-driven MMT might provide a novel approach for the treatment of fibrosis complication in CD.

Engineered self-regulating macrophages for targeted anti-inflammatory drug delivery

BackgroundRheumatoid arthritis (RA) is a systemic autoimmune disease characterized by increased levels of inflammation that primarily manifests in the joints. Macrophages act as key drivers for the progression of RA, contributing to the perpetuation of chronic inflammation and dysregulation of pro-inflammatory cytokines such as interleukin 1 (IL-1). The goal of this study was to develop a macrophage-based cell therapy for biologic drug delivery in an autoregulated manner.MethodsFor proof-of-concept, we developed “smart” macrophages to mitigate the effects of IL-1 by delivering its inhibitor, IL-1 receptor antagonist (IL-1Ra). Bone marrow-derived macrophages were lentivirally transduced with a synthetic gene circuit that uses an NF-κB inducible promoter upstream of either the Il1rn or firefly luciferase transgenes. Two types of joint like cells were utilized to examine therapeutic protection in vitro, miPSCs derived cartilage and isolated primary mouse synovial fibroblasts while the K/BxN mouse model of RA was utilized to examine in vivo therapeutic protection.ResultsThese engineered macrophages were able to repeatably produce therapeutic levels of IL-1Ra that could successfully mitigate inflammatory activation in co-culture with both tissue-engineered cartilage constructs and synovial fibroblasts. Following injection in vivo, macrophages homed to sites of inflammation and mitigated disease severity in the K/BxN mouse model of RA.ConclusionThese findings demonstrate the successful development of engineered macrophages that possess the ability for controlled, autoregulated production of IL-1 based on inflammatory signaling such as via the NF-κB pathway to mitigate the effects of this cytokine for applications in RA or other inflammatory diseases. This system provides proof of concept for applications in other immune cell types as self-regulating delivery systems for therapeutic applications in a range of diseases.

The VEGF-Mediated Cytoprotective Ability of MIF-Licensed Mesenchymal Stromal Cells in House Dust Mite-Induced Epithelial Damage.

Enhancing mesenchymal stromal cell (MSC) therapeutic efficacy through licensing with proinflammatory cytokines is now well established. We have previously shown that macrophage migration inhibitory factor (MIF)-licensed MSCs exerted significantly enhanced therapeutic efficacy in reducing inflammation in house dust mite (HDM)-driven allergic asthma. Soluble mediators released into the MSC secretome boast cytoprotective properties equal to those associated with the cell itself. In asthma, epithelial barrier damage caused by the inhalation of allergens like HDM drives goblet cell hyperplasia. Vascular endothelial growth factor (VEGF) plays a pivotal role in the repair and maintenance of airway epithelial integrity. Human bone marrow-derived MSCs expressed the MIF receptors CD74, CXCR2, and CXCR4. Endogenous MIF from high MIF expressing CATT7 bone marrow-derived macrophages increased MSC production of VEGF through the MIF CXCR4 chemokine receptor, where preincubation with CXCR4 inhibitor mitigated this effect. CATT7-MIF licensed MSC conditioned media containing increased levels of VEGF significantly enhanced bronchial epithelial wound healing via migration and proliferation in vitro. Blocking VEGFR2 or theuse of mitomycin C abrogated this effect. Furthermore, CATT7-MIF MSC CM significantly decreased goblet cell hyperplasia after the HDM challenge in vivo. This was confirmed to be VEGF-dependent, as the use of anti-human VEGF neutralising antibody abrogated this effect. Overall, this study highlights that MIF-licenced MSCs show enhanced production of VEGF, which has the capacity to repair the lung epithelium.

Alveolar and Bone Marrow-derived Macrophages Differ in Metabolism and Glutamine Utilization.

Changes in metabolic activity are key regulators of macrophage activity. Pro-inflammatory macrophages upregulate glycolysis, which promotes an inflammatory phenotype, whereas pro-repair macrophages rely upon oxidative metabolism and glutaminolysis to support their activity. Work to understand how metabolism regulates macrophage phenotype has been done primarily in macrophage cell lines and bone marrow-derived macrophages (BMDM). Our study sought to understand changes in metabolic activity of murine tissue-resident alveolar macrophages (AM) in response to LPS stimulation and to contrast them to BMDM. These studies also determined the contribution of glutamine metabolism using the glutamine inhibitor, DON. We found that compared to BMDM, AM have higher rates of oxygen consumption and contain a higher concentration of intracellular metabolites involved in fatty acid oxidation. In response to LPS, BMDM but not AM increased rates of glycolysis. Inhibition of glutamine metabolism using DON altered the metabolic activity of AM but not BMDM. Within AM, glutamine inhibition led to increases in intracellular metabolites involved in glycolysis, the TCA cycle, fatty acid oxidation, and amino acid metabolism. Glutamine inhibition also altered the metabolic response to LPS within AM but not BMDM. Our data reveal striking differences in the metabolic activity of AM and BMDM.

Pigment epithelium-derived factor exerts neuroprotection in oxygen-induced retinopathy by targeting endoplasmic reticulum stress and oxidative stress

Endoplasmic reticulum (ER) stress and oxidative stress have been involved in the occurrence of neuronal apoptosis in ischemic retinopathy. Pigment epitheliu-derived factor (PEDF) is well known for its multifunctional properties, including neuroprotection, anti-inflammation and antioxidant. However, the association between PEDF and ER stress or oxidative stress in ischemic retinopathy remain incompletely understood. In this study, the concentration of the key factor of ER stress C/EBP homologous protein (CHOP) in aqueous humor (AqH) and vitreous samples of proliferative diabetic retinopathy (PDR) patients were measured by ELISA assays. Oxygen-induced retinopathy (OIR) mice model was established and PEDF intravitreal injections were conducted. Primary bone marrow derived macrophages (BMDMs) were isolated and cultured under hypoxic conditions in vitro. Western blotting, real-time RT-PCR, immunofluorescence, transmission electron microscopy (TEM), TUNEL assays were performed to explore roles of PEDF on ER stress and oxidative stress, as well as subsequently neuronal apoptosis under hypoxic conditions in vivo and in vitro. The results revealed that ER stress and oxidative stress were notably activated under hypoxic conditions. We also observed that hypoxia evoked ultrastructural damage of ER and mitochondrion in the retina. However, PEDF significantly prevented ER stress and oxidative stress, as well as the damage of ultrastructure, resulting in diminution of photoreceptor apoptosis in OIR retinas. These results indicate that PEDF may play its neuroprotection role through inhibiting ER stress and oxidative stress in ischemic retinopathy, which is a novel molecular mechanism of PEDF protecting photoreceptors from ischemic damage, thereby suggesting that PEDF is an effective therapeutic agent for the treatment of neuron damage in ischemic retinal diseases.