Current Status of the Zebrafish Euthanasia and Humane Endpoint in the Republic of Korea and Guideline Suggestion from Nationwide Expert Elicitation: A Model for Other Countries.

Since its introduction as a model organism in the 1980s, the use of zebrafish (Danio rerio) in research has expanded worldwide. Despite its now widespread use in research, guidelines to safeguard the ethical treatment of zebrafish, particularly with regard to euthanasia and humane endpoint practices, remain inadequate. One well-recognized example is the use of excess tricaine methanesulfonate (MS-222) as a means to euthanize zebrafish, regardless of life stage. In this study, through nationwide expert elicitation, we provide a detailed account of zebrafish research practices within the Republic of Korea and the challenges of implementing appropriate methods for euthanasia as a humane endpoint, with many opting for hypothermic shock. We report a local expert consensus for establishing national guidelines to improve zebrafish welfare and good research practice. Suggestions and recommendations for national guidelines were offered. Taken together, our findings raise awareness broadly among zebrafish research practitioners in the field, offer an accurate account of the welfare and treatment of zebrafish in research within the Republic of Korea, and advocate for the development and implementation of national guidelines. As such, our study is useful as a model to adopt the expert elicitation approach to investigate, quantify, and address welfare concerns in zebrafish research, and to establish best practice guidelines.

Comprehensive characterization of early-programmed tumor microenvironment by tumor-associated macrophages reveals galectin-1 as an immune modulatory target in breast cancer.

Background: In recent years, there has been considerable interest in the therapeutic targeting of tumor-associated macrophages (TAMs) to modulate the tumor microenvironment (TME), resulting in antitumoral phenotypes. However, key mediators suitable for TAM-mediated remodeling of the TME remain poorly understood. Methods: In this study, we used single-cell RNA sequencing analyses to analyze the landscape of the TME modulated by TAMs in terms of a protumor microenvironment during early tumor development. Results: Our data revealed that the depletion of TAMs leads to a decreased epithelial-to-mesenchymal transition (EMT) signature in cancer cells and a distinct transcriptional state characterized by CD8+ T cell activation. Moreover, notable alterations in gene expression were observed upon the depletion of TAMs, identifying Galectin-1 (Gal-1) as a crucial molecular factor responsible for the observed effect. Gal-1 inhibition reversed immune suppression via the reinvigoration of CD8+ T cells, impairing tumor growth and potentiating immune checkpoint inhibitors in breast tumor models. Conclusion: These results provide comprehensive insights into TAM-mediated early tumor microenvironments and reveal immune evasion mechanisms that can be targeted by Gal-1 to induce antitumor immune responses.

Effect of Lactobacillus Rhamnosus GG for Regulation of Inflammatory Response in Radiation-Induced Enteritis.

The purpose of this study was to investigate the role of Lactobacillus rhamnosus GG (LGG) probiotics in radiation enteritis using in vivo mice. A total of 40 mice were randomly assigned to four groups: control, probiotics, radiotherapy (RT), and RT + probiotics. For the group of probiotics, 0.2 mL of solution that contained 1.0 × 108 colony-forming units (CFU) of LGG was used and orally administered daily until sacrifice. For RT, a single dose of 14 Gy was administered using a 6 mega-voltage photon beam to the abdominopelvic area. Mice were sacrifice at day 4 (S1) and day 7 (S2) after RT. Their jejunum, colon, and stool were collected. A multiplex cytokine assay and 16 s ribosomal RNA amplicon sequencing were then performed. Regarding cytokine concentrations in tissues, pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin-6 and monocyte chemotactic protein-1, showed significantly decreased protein levels in colon tissues of the RT + probiotics group than in the RT alone group (all p < 0.05). As for comparing microbial abundance through alpha-diversity and beta-diversity, no significant differences were observed between the RT + probiotics and RT alone groups, except for an increase in alpha-diversity in the stool of the RT + probiotics group. Upon analysis of differential microbes based on treatment, the dominance of anti-inflammatory-related microbes, such as Porphyromonadaceae, Bacteroides acidifaciens, and Ruminococcus, was observed in the jejunum, colon, and stool of the RT + probiotics group. With regard to predicted metabolic pathway abundances, the pathways associated with anti-inflammatory processes, such as biosynthesis of pyrimidine nucleotides, peptidoglycans, tryptophan, adenosylcobalamin, and propionate, were differentially identified in the RT + probiotics group compared to the RT alone group. Protective effects of probiotics on radiation enteritis were potentially derived from dominant anti-inflammation-related microbes and metabolites.

Multiple Injections of Adipose-Derived Stem Cells Improve Graft Survival in Human-to-Rat Skin Xenotransplantation through Immune Modulation.

BACKGROUND: Adipose-derived stem cells (ADSCs) exert immunomodulatory effects in the treatment of transplant rejection. This study aimed to evaluate the effects of ADSCs on the skin graft survival in a human-to-rat xenograft transplantation model and to compare single and multiple injections of ADSCs. METHODS: Full-thickness human skin xenografts were transplanted into the backs of Sprague-Dawley rats. The rats were injected subcutaneously on postoperative days 0, 3, and 5. The injections were as follows: triple injections of phosphate-buffered saline (PBS group), a single injection of ADSCs and double injections of PBS (ADSC × 1 group), and triple injections of ADSCs (ADSC × 3 group). The immunomodulatory effects of ADSCs on human skin xenografts were assessed. RESULTS: Triple injections of ADSCs considerably delayed cell-mediated xenograft rejection compared with the PBS and ADSC × 1 groups. The vascularization and collagen type 1-3 ratios in the ADSC × 3 group were significantly higher than those in the other groups. In addition, intragraft infiltration of CD3-, CD4-, CD8-, and CD68-positive cells was reduced in the ADSC × 3 group. Furthermore, in the ADSC × 3 group, the expression levels of proinflammatory cytokine interferon-gamma (IFN-γ) were decreased and immunosuppressive prostaglandin E synthase (PGES) was increased in the xenograft and lymph node samples. CONCLUSION: This study presented that triple injections of ADSCs appeared to be superior to a single injection in suppressing cell-mediated xenograft rejection. The immunomodulatory effects of ADSCs are associated with the downregulation of IFN-γ and upregulation of PGES in skin xenografts and lymph nodes.

A new era of macrophage-based cell therapy.

Macrophages are essential innate immune cells found throughout the body that have protective and pathogenic functions in many diseases. When activated, macrophages can mediate the phagocytosis of dangerous cells or materials and participate in effective tissue regeneration by providing growth factors and anti-inflammatory molecules. Ex vivo-generated macrophages have thus been used in clinical trials as cell-based therapies, and based on their intrinsic characteristics, they outperformed stem cells within specific target diseases. In addition to the old methods of generating naïve or M2 primed macrophages, the recently developed chimeric antigen receptor-macrophages revealed the potential of genetically engineered macrophages for cell therapy. Here, we review the current developmental status of macrophage-based cell therapy. The findings of important clinical and preclinical trials are updated, and patent status is investigated. Additionally, we discuss the limitations and future directions of macrophage-based cell therapy, which will help broaden the potential utility and clinical applications of macrophages.

Pyruvate dehydrogenase kinase inhibitor dichloroacetate augments autophagy mediated constraining the replication of Mycobacteroides massiliense in macrophages.

Increasing evidence indicates a strong interaction between cellular metabolism and innate macrophage immunity. Here, we show that the intracellular replication of Mycobacteroides massiliense in macrophages depends on host pyruvate dehydrogenase kinase (PDK) activity. Infection with M. massiliense induced a metabolic switch in macrophages by increasing glycolysis and decreasing oxidative phosphorylation. Treatment with dichloroacetate (DCA), a PDK inhibitor, converts this switch in M. massiliense-infected macrophages and restricts intracellular bacterial replication. Mechanistically, DCA resulted in AMPKα1 activation via increased AMP/ATP ratio, consequently inducing autophagy to constrain bacterial proliferation in the phagolysosome. This study suggests that the pharmacological inhibition of PDK could be a strategy for host-directed therapy to control virulent M. massiliense infections.

Development of a transcriptome-based determination of innate immune suppressor (TDIS) assay as an in vitro test for immunotoxicity.

Immunotoxicity has been an important topic in toxicology since inadvertent exposures to xenobiotics were found to alter immune functions in humans. While rodent toxicity tests can reveal some levels of immunotoxicity, alternative methods must be developed to identify the detailed mechanisms. In this study, a method of in vitro prediction of innate immune suppression by substances was developed using a genomics approach. The primary selection of immune suppressors was based on their ability to downregulate MCP-1, CCL3, TNF, IL-8, and IL-12p40 expression levels in lipopolysaccharide (LPS)-stimulated THP-1 cells. Among 11 substances classified as potent immune suppressors, six including dexamethasone, tacrolimus, tofacitinib, prednisolone, sodium lauryl sulfate, and benzoic acid were used to create a dataset by transcriptomics of chemical-treated THP-1 cells using bulk RNA sequencing. We selected genes that were significantly upregulated by suppressor treatment while filtering out genes also upregulated in LPS-treated THP-1 cells. We identified a 226-gene immunosuppressive gene set (ISG). Innate immune suppressor signature scores were calculated as the median expression of the ISG. In a validation dataset, the signature score predicted acyclovir, cyclosporine, and mercuric chloride as immune suppressors, while selecting genistein as a non-immune suppressor. Although more dataset integration is needed in the future, our results demonstrated the possibility and utility of a novel genomics-based approach, the transcriptome-based determination of innate immune suppressor (TDIS) assay, to evaluate innate immune suppression by different substances. This provides insight into the development of future alternative testing methods because it reflects a comprehensive genetic signature derived from multiple substances rather than one cytokine.

Circulation Time-Optimized Albumin Nanoplatform for Quantitative Visualization of Lung Metastasis via Targeting of Macrophages.

The development of molecular imaging probes to identify key cellular changes within lung metastases may lead to noninvasive detection of metastatic lesions in the lung. In this study, we constructed a macrophage-targeted clickable albumin nanoplatform (CAN) decorated with mannose as the targeting ligand using a click reaction to maintain the intrinsic properties of albumin in vivo. We also modified the number of mannose molecules on the CAN and found that mannosylated serum albumin (MSA) harboring six molecules of mannose displayed favorable pharmacokinetics that allowed high-contrast imaging of the lung, rendering it suitable for in vivo visualization of lung metastases. Due to the optimized control of functionalization and surface modification, MSA enhanced blood circulation time and active/passive targeting abilities and was specifically incorporated by mannose receptor (CD206)-expressing macrophages in the metastatic lung. Moreover, extensive in vivo imaging studies using single-photon emission computed tomography (SPECT)/CT and positron emission tomography (PET) revealed that blood circulation of time-optimized MSA can be used to discern metastatic lesions, with a strong correlation between its signal and metastatic burden in the lung.

Liposomal Dexamethasone Reduces A/H1N1 Influenza-Associated Morbidity in Mice.

Re-emerging viral threats have continued to challenge the medical and public health systems. It has become clear that a significant number of severe viral infection cases are due to an overreaction of the immune system, which leads to hyperinflammation. In this study, we aimed to demonstrate the therapeutic efficacy of the dexamethasone nanomedicine in controlling the symptoms of influenza virus infection. We found that the A/Wisconsin/WSLH34939/2009 (H1N1) infection induced severe pneumonia in mice with a death rate of 80%, accompanied by significant epithelial cell damage, infiltration of immune cells, and accumulation of pro-inflammatory cytokines in the airway space. Moreover, the intranasal delivery of liposomal dexamethasone during disease progression reduced the death rate by 20%. It also significantly reduced the protein level of tumor necrosis factor-alpha (TNFα), interleukin-1ß (IL-1ß), IL-6, and the C-X-C motif chemokine ligand 2 (CXCL2) as well as the number of infiltrated immune cells in the bronchoalveolar lavage fluids as compared to the control and free dexamethasone. The liposomal dexamethasone was mainly distributed into the monocyte/macrophages as a major cell population for inducing the cytokine storm in the lungs. Taken together, the intranasal delivery of liposomal dexamethasone may serve as a novel promising therapeutic strategy for the treatment of influenza A-induced pneumonia.

Prostaglandin E2 receptor PTGER4-expressing macrophages promote intestinal epithelial barrier regeneration upon inflammation.

OBJECTIVE: Dysfunctional resolution of intestinal inflammation and altered mucosal healing are essential features in the pathogenesis of inflammatory bowel disease (IBD). Intestinal macrophages are vital in the process of inflammation resolution, but the mechanisms underlying their mucosal healing capacity remain elusive. DESIGN: We investigated the role of the prostaglandin E2 (PGE2) receptor PTGER4 on the differentiation of intestinal macrophages in patients with IBD and mouse models of intestinal inflammation. We studied mucosal healing and intestinal epithelial barrier regeneration in Csf1r-iCre Ptger4fl/fl mice during dextran sulfate sodium (DSS)-induced colitis. The effect of PTGER4+ macrophage secreted molecules was investigated on epithelial organoid differentiation. RESULTS: Here, we describe a subset of PTGER4-expressing intestinal macrophages with mucosal healing properties both in humans and mice. Csf1r-iCre Ptger4fl/fl mice showed defective mucosal healing and epithelial barrier regeneration in a model of DSS colitis. Mechanistically, an increased mucosal level of PGE2 triggers chemokine (C-X-C motif) ligand 1 (CXCL1) secretion in monocyte-derived PTGER4+ macrophages via mitogen-activated protein kinases (MAPKs). CXCL1 drives epithelial cell differentiation and proliferation from regenerating crypts during colitis. Specific therapeutic targeting of macrophages with liposomes loaded with an MAPK agonist augmented the production of CXCL1 in vivo in conditional macrophage PTGER4-deficient mice, restoring their defective epithelial regeneration and favouring mucosal healing. CONCLUSION: PTGER4+ intestinal macrophages are essential for supporting the intestinal stem cell niche and regeneration of the injured epithelium. Our results pave the way for the development of a new class of therapeutic targets to promote macrophage healing functions and favour remission in patients with IBD.

Human Cytomegalovirus-Induced Interleukin-10 Production Promotes the Proliferation of Mycobacterium massiliense in Macrophages.

Human cytomegalovirus (HCMV) exploits the interleukin-10 (IL-10) pathway as a part of its infection cycle through the manipulation of the host IL-10 signaling cascade. Based on its immunomodulatory nature, HCMV attenuates the host immune response and facilitates the progression of co-infection with other pathogens in an immune-competent host. To investigate the impact of HCMV infection on the burden of non-tuberculous mycobacteria (NTM), whose prevalence is growing rapidly worldwide, macrophages were infected with HCMV and further challenged with Mycobacterium massiliense in vitro. The results showed that HCMV infection significantly increased host IL-10 synthesis and promoted the proliferation of M. massiliense in an IL-10-dependent manner. Transcriptomic analysis revealed that HCMV infection dampened the regulatory pathways of interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-1 (IL-1), consequently abrogating the immune responses to M. massiliense coinfection in macrophages. These findings provide a mechanistic basis of how HCMV infection may facilitate the development of pathogenic NTM co-infection by upregulating IL-10 expression.

Protein Kinase A Catalytic Subunit Is a Molecular Switch that Promotes the Pro-tumoral Function of Macrophages.

As current therapies benefit only a minority of cancer patients, additional therapeutic targets are needed. Tumor-associated macrophages (TAMs) have attracted attention for improving therapeutic responses, yet regulatory strategies remain elusive. Here, we show that the protein kinase A catalytic subunit (PKA-C) acts as a molecular switch, inducing a pro-tumoral immunosuppressive macrophage phenotype within tumors. In human and murine breast cancer, overactivated PKA in TAMs creates a detrimental microenvironment for cancer progression by inducing vascular endothelial growth factor A (VEGFA), interleukin-10 (IL-10), and macrophage-derived arginase 1 (ARG1) expression. Macrophages with genetic deletion of PKA-C are prone to be pro-inflammatory, suggesting a possible immunotherapeutic target. Delivery of liposomal PKA inhibitor facilitates tumor regression and abrogates pro-tumoral TAM functions in mice. The therapeutic effect of targeting PKA is pronounced when combined with αCTLA-4 antibody, increasing cluster of differentiation 8 (CD8)+GranzymeB+ T cells by about 60-fold. Our findings demonstrate critical roles of TAM PKA-C in tumor progression and suggest that targeting PKA-C efficiently augments cancer treatment responses.

Pyruvate dehydrogenase kinase is a negative regulator of interleukin-10 production in macrophages.

Interleukin-10 (IL-10) is the most potent anti-inflammatory cytokine in the body and plays an essential role in determining outcomes of many inflammatory diseases. Cellular metabolism is a critical determinant of immune cell function; however, it is currently unclear whether metabolic processes are specifically involved in IL-10 production. In this study, we aimed to find the central metabolic molecule regulating IL-10 production of macrophages, which are the main producers of IL-10. Transcriptomic analysis identified that metabolic changes were predominantly enriched in Kupffer cells at the early inflammatory phase of a mouse endotoxemia model. Among them, pyruvate dehydrogenase kinase (PDK)-dependent acute glycolysis was negatively involved in IL-10 production. Inhibition or knockdown of PDK selectively increased macrophage IL-10 expression. Mechanistically, PDK inhibition increased IL-10 production via profound phosphorylation of adenosine monophosphate (AMP)-activated protein kinase alpha 1 (AMPKα1) by restricting glucose uptake in lipopolysaccharide-stimulated macrophages. AMPKα1 consequently activated p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and cyclic AMP-responsive element-binding protein to regulate IL-10 production. Our study uncovers a previously unknown regulatory mechanism of IL-10 in activated macrophages involving an immunometabolic function of PDK.

Identification of FES as a Novel Radiosensitizing Target in Human Cancers.

PURPOSE: The identification of novel targets for developing synergistic drug-radiation combinations would pave the way to overcome tumor radioresistance. We conducted cell-based screening of a human kinome siRNA library to identify a radiation-specific kinase that has a synergistic toxic effect with radiation upon inhibition and is not essential for cell survival in the absence of radiation. EXPERIMENTAL DESIGN: Unbiased RNAi screening was performed by transfecting A549 cells with a human kinome siRNA library followed by irradiation. Radiosensitizing effects of a target gene and involved mechanisms were examined. RESULTS: We identified the nonreceptor protein tyrosine kinase FES (FEline Sarcoma oncogene) as a radiosensitizing target. The expression of FES was increased in response to irradiation. Cell viability and clonogenic survival after irradiation were significantly decreased by FES knockdown in lung and pancreatic cancer cell lines. In contrast, FES depletion alone did not significantly affect cell proliferation without irradiation. An inducible RNAi mouse xenograft model verified in vivo radiosensitizing effects. FES-depleted cells showed increased apoptosis, DNA damage, G2-M phase arrest, and mitotic catastrophe after irradiation. FES depletion promoted radiation-induced reactive oxygen species formation, which resulted in phosphorylation of S6K and MDM2. The radiosensitizing effect of FES knockdown was partially reversed by inhibition of S6K activity. Consistent with the increase in phosphorylated MDM2, an increase in nuclear p53 levels was observed, which appears to contribute increased radiosensitivity of FES-depleted cells. CONCLUSIONS: We uncovered that inhibition of FES could be a potential strategy for inducing radiosensitization in cancer. Our results provide the basis for developing novel radiosensitizers.

Macrophages in intestinal inflammation and resolution: a potential therapeutic target in IBD.

Macrophages are the gatekeepers of intestinal immune homeostasis as they discriminate between innocuous antigens and potential pathogens to maintain oral tolerance. However, in individuals with a genetic and environmental predisposition, regulation of intestinal immunity is impaired, leading to chronic relapsing immune activation and pathologies of the gastrointestinal tract, such as IBD. As evidence suggests a causal link between defects in the resolution of intestinal inflammation and altered monocyte-macrophage differentiation in patients with IBD, macrophages have been considered as a novel potential target to develop new treatment approaches. This Review discusses the molecular and cellular mechanisms involved in the differentiation and function of intestinal macrophages in homeostasis and inflammation, and their role in resolving the inflammatory process. Understanding the molecular pathways involved in the specification of intestinal macrophages might lead to a new class of targets that promote remission in patients with IBD.

Lactic Acid Upregulates VEGF Expression in Macrophages and Facilitates Choroidal Neovascularization.

Purpose: Lactic acid, the end product of glycolysis, has emerged as an immune-modulating metabolite in various diseases. In this study, we aimed to examine whether lactic acid contributes to the disease pathogenesis of choroidal neovascularization (CNV) and to investigate the role of macrophages in CNV pathogenesis. Methods: CNV was induced by laser photocoagulation in C57BL/6J mice. Lactic acid concentration was measured in the RPE-choroid region. Macrophage infiltration and VEGF were quantified by flow cytometry. VEGF-positive areas and CNV lesions were measured by flat-mount immunofluorescence staining. To inhibit lactic acid uptake in vivo, alpha-cyano-4-hydroxycinnamic acid (α-CHC), a monocarboxylate transporter (MCT) blocker, was injected intravitreally 1 day after laser. VEGF productions were measured in ARPE-19, THP-1 cells, and human umbilical vein endothelial cells (HUVECs) by quantitative PCR and ELISA. Angiogenic activity of lactic acid-treated macrophages was assessed by HUVEC tube formation assay. Results: Lactic acid was significantly increased in the RPE-choroid region of CNV-induced mice. Lactic acid upregulated VEGFA mRNA and VEGF protein expressions in THP-1 macrophages, but did not in ARPE-19 or HUVECs. THP-1 macrophages treated with lactic acid increased the angiogenesis of endothelial cells independent of MCT activity. Intravitreal injection of α-CHC substantially reduced the VEGF-positive area that colocalized with F4/80-positive macrophages. CNV lesions were also significantly reduced following α-CHC injection compared with vehicle-injected controls. Conclusions: To our knowledge, these results show for the first time the role of lactic acid in facilitating neovascularization through macrophage-induced angiogenesis. We suggest that targeting macrophage metabolism can be a promising strategy for CNV treatment.

Fasiglifam (TAK-875), a G Protein-Coupled Receptor 40 (GPR40) Agonist, May Induce Hepatotoxicity through Reactive Oxygen Species Generation in a GPR40-Dependent Manner.

Fasiglifam (TAK-875) a G-protein coupled receptor 40 (GPR40) agonist, significantly improves hyperglycemia without hypoglycemia and weight gain, the major side effects of conventional anti-diabetics. Unfortunately, during multi-center Phase 3 clinical trials, unexpected liver toxicity resulted in premature termination of its development. Here, we investigated whether TAK-875 directly inflicts toxicity on hepatocytes and explored its underlying mechanism of toxicity. TAK-875 decreased viability of 2D and 3D cultures of HepG2, a human hepatocarcinoma cell line, in concentration- (＞50 µM) and time-dependent manners, both of which corresponded with ROS generation. An antioxidant, N-acetylcysteine, attenuated TAK-875-mediated hepatotoxicity, which confirmed the role of ROS generation. Of note, knockdown of GPR40 using siRNA abolished the hepatotoxicity of TAK-875 and attenuated ROS generation. In contrast, TAK-875 induced no cytotoxicity in fibroblasts up to 500 µM. Supporting the hepatotoxic potential of TAK-875, exposure to TAK-875 resulted in increased mortality of zebrafish larvae at 25 µM. Histopathological examination of zebrafish exposed to TAK-875 revealed severe hepatotoxicity as manifested by degenerated hypertrophic hepatocytes with cytoplasmic vacuolation and acentric nuclei, confirming that TAK-875 may induce direct hepatotoxicity and that ROS generation may be involved in a GPR40-dependent manner.

Metabolic features of macrophages in inflammatory diseases and cancer.

Macrophages are now considered to be important players in various inflammatory diseases as well as tumor progression. Emerging evidence reveals that macrophage metabolic features are deeply associated with their immune functions. Understanding the interaction between cellular metabolism and immune signaling pathways in macrophages can help us to develop appropriate therapeutic approaches for inflammatory diseases. In this review, we briefly summarize key metabolic features of M1 and M2 macrophages as well as signaling interactions between major metabolic molecules with TLRs and NLRs. Current knowledges of cellular metabolism are focused on macrophages in various disease situations including sepsis, atherosclerosis, obesity, tuberculosis and cancer. Novel insights and present targets for regulating macrophage metabolism are also discussed.

GM-CSF Grown Bone Marrow Derived Cells Are Composed of Phenotypically Different Dendritic Cells and Macrophages.

Granulocyte-macrophage colony stimulating factor (GM-CSF) has a role in inducing emergency hematopoiesis upon exposure to inflammatory stimuli. Although GM-CSF generated murine bone marrow derived cells have been widely used as macrophages or dendritic cells in research, the exact characteristics of each cell population have not yet been defined. Here we discriminated GM-CSF grown bone marrow derived macrophages (GM-BMMs) from dendritic cells (GM-BMDCs) in several criteria. After C57BL/6J mice bone marrow cell culture for 7 days with GM-CSF supplementation, two main populations were observed in the attached cells based on MHCII and F4/80 marker expressions. GM-BMMs had MHCIIlowF4/80high as well as CD11c+CD11bhighCD80-CD64+MerTK+ phenotypes. In contrast, GM-BMDCs had MHCIIhighF4/80low and CD11chighCD8α- CD11b+CD80+CD64-MerTKlow phenotypes. Interestingly, the GM-BMM population increased but GM-BMDCs decreased in a GM-CSF dose-dependent manner. Functionally, GM-BMMs showed extremely high phagocytic abilities and produced higher IL-10 upon LPS stimulation. GM-BMDCs, however, could not phagocytose as well, but were efficient at producing TNFα, IL-1ß, IL-12p70 and IL-6 as well as inducing T cell proliferation. Finally, whole transcriptome analysis revealed that GM-BMMs and GM-BMDCs are overlap with in vivo resident macrophages and dendritic cells, respectively. Taken together, our study shows the heterogeneicity of GM-CSF derived cell populations, and specifically characterizes GM-CSF derived macrophages compared to dendritic cells.