Granulocyte macrophage colony-stimulating factor-induced macrophages of individuals with autism spectrum disorder adversely affect neuronal dendrites through the secretion of pro-inflammatory cytokines.

BACKGROUND: A growing body of evidence suggests that immune dysfunction and inflammation in the peripheral tissues as well as the central nervous system are associated with the neurodevelopmental deficits observed in autism spectrum disorder (ASD). Elevated expression of pro-inflammatory cytokines in the plasma, serum, and peripheral blood mononuclear cells of ASD has been reported. These cytokine expression levels are associated with the severity of behavioral impairments and symptoms in ASD. In a prior study, our group reported that tumor necrosis factor-α (TNF-α) expression in granulocyte-macrophage colony-stimulating factor-induced macrophages (GM-CSF MΦ) and the TNF-α expression ratio in GM-CSF MΦ/M-CSF MΦ (macrophage colony-stimulating factor-induced macrophages) was markedly higher in individuals with ASD than in typically developed (TD) individuals. However, the mechanisms of how the macrophages and the highly expressed cytokines affect neurons remain to be addressed. METHODS: To elucidate the effect of macrophages on human neurons, we used a co-culture system of control human-induced pluripotent stem cell-derived neurons and differentiated macrophages obtained from the peripheral blood mononuclear cells of five TD individuals and five individuals with ASD. All participants were male and ethnically Japanese. RESULTS: Our results of co-culture experiments showed that GM-CSF MΦ affect the dendritic outgrowth of neurons through the secretion of pro-inflammatory cytokines, interleukin-1α and TNF-α. Macrophages derived from individuals with ASD exerted more severe effects than those derived from TD individuals. LIMITATIONS: The main limitations of our study were the small sample size with a gender bias toward males, the use of artificially polarized macrophages, and the inability to directly observe the interaction between neurons and macrophages from the same individuals. CONCLUSIONS: Our co-culture system revealed the non-cell autonomous adverse effects of GM-CSF MΦ in individuals with ASD on neurons, mediated by interleukin-1α and TNF-α. These results may support the immune dysfunction hypothesis of ASD, providing new insights into its pathology.

Single-Cell Analysis Reveals a Subset of High IL-12p40-Secreting Dendritic Cells within Mouse Bone Marrow-Derived Macrophages Differentiated with M-CSF.

Macrophages and dendritic cells (DCs), although ontogenetically distinct, have overlapping functions and exhibit substantial cell-to-cell heterogeneity that can complicate their identification and obscure innate immune function. In this study, we report that M-CSF-differentiated murine bone marrow-derived macrophages (BMDMs) exhibit extreme heterogeneity in the production of IL-12, a key proinflammatory cytokine linking innate and adaptive immunity. A microwell secretion assay revealed that a small fraction of BMDMs stimulated with LPS secrete most IL-12p40, and we confirmed that this is due to extremely high expression of Il12b, the gene encoding IL-12p40, in a subset of cells. Using an Il12b-YFP reporter mouse, we isolated cells with high LPS-induced Il12b expression and found that this subset was enriched for genes associated with the DC lineage. Single-cell RNA sequencing data confirmed a DC-like subset that differentiates within BMDM cultures that is transcriptionally distinct but could not be isolated by surface marker expression. Although not readily apparent in the resting state, upon LPS stimulation, this subset exhibited a typical DC-associated activation program that is distinct from LPS-induced stochastic BMDM cell-to-cell heterogeneity. Overall, our findings underscore the difficulty in distinguishing macrophages and DCs even in widely used in vitro murine BMDM cultures and could affect the interpretation of some studies that use BMDMs to explore acute inflammatory responses.

Radical-Generating Activity, Phagocytosis, and Mechanical Properties of Four Phenotypes of Human Macrophages.

Macrophages are the major players and orchestrators of inflammatory response. Expressed proteins and secreted cytokines have been well studied for two polar macrophage phenotypes-pro-inflammatory M1 and anti-inflammatory regenerative M2, but little is known about how the polarization modulates macrophage functions. In this study, we used biochemical and biophysical methods to compare the functional activity and mechanical properties of activated human macrophages differentiated from monocyte with GM-CSF (M0_GM) and M-CSF (M0_M) and polarized into M1 and M2 phenotypes, respectively. Unlike GM-CSF, which generates dormant cells with low activity, M-CSF confers functional activity on macrophages. M0_M and M2 macrophages had very similar functional characteristics-high reactive oxygen species (ROS) production level, and higher phagocytosis and survival compared to M1, while M1 macrophages showed the highest radical-generating activity but the lowest phagocytosis and survival among all phenotypes. All phenotypes decreased their height upon activation, but only M1 and M2 cells increased in stiffness, which can indicate a decrease in the migration ability of these cells and changes in their interactions with other cells. Our results demonstrated that while mechanical properties differ between M0 and polarized cells, all four phenotypes of monocyte-derived macrophages differ in their functional activities, namely in cytokine secretion, ROS production, and phagocytosis. Within the broad continuum of human macrophages obtained in experimental models and existing in vivo, there is a diversity of phenotypes with varying combinations of both markers and functional activities.

Keratinocyte integrin α3ß1 induces expression of the macrophage stimulating factor, CSF-1, through a YAP/TEAD-dependent mechanism.

The development of wound therapy targeting integrins is hampered by inadequate understanding of integrin function in cutaneous wound healing and the wound microenvironment. Following cutaneous injury, keratinocytes migrate to restore the skin barrier, and macrophages aid in debris clearance. Thus, both keratinocytes and macrophages are critical to the coordination of tissue repair. Keratinocyte integrins have been shown to participate in this coordinated effort by regulating secreted factors, some of which crosstalk to distinct cells in the wound microenvironment. Epidermal integrin α3ß1 is a receptor for laminin-332 in the cutaneous basement membrane. Here we show that wounds deficient in epidermal α3ß1 express less epidermal-derived macrophage colony-stimulating factor 1 (CSF-1), the primary macrophage-stimulating growth factor. α3ß1-deficient wounds also have fewer wound-proximal macrophages, suggesting that keratinocyte α3ß1 may stimulate wound macrophages through the regulation of CSF-1. Indeed, using a set of immortalized keratinocytes, we demonstrate that keratinocyte-derived CSF-1 supports macrophage growth, and that α3ß1 regulates Csf1 expression through Src-dependent stimulation of Yes-associated protein (YAP)-Transcriptional enhanced associate domain (TEAD)-mediated transcription. Consistently, α3ß1-deficient wounds in vivo display a substantially reduced number of keratinocytes with YAP-positive nuclei. Overall, our current findings identify a novel role for epidermal integrin α3ß1 in regulating the cutaneous wound microenvironment by mediating paracrine crosstalk from keratinocytes to wound macrophages, implicating α3ß1 as a potential target of wound therapy.

The Modulation of Phospho-Extracellular Signal-Regulated Kinase and Phospho-Protein Kinase B Signaling Pathways plus Activity of Macrophage-Stimulating Protein Contribute to the Protective Effect of Stachydrine on Acetaminophen-Induced Liver Injury.

Stachydrine, a prominent bioactive alkaloid derived from Leonurus heterophyllus, is a significant herb in traditional medicine. It has been noted for its anti-inflammatory and antioxidant characteristics. Consequently, we conducted a study of its hepatoprotective effect and the fundamental mechanisms involved in acetaminophen (APAP)-induced liver injury, utilizing a mouse model. Mice were intraperitoneally administered a hepatotoxic dose of APAP (300 mg/kg). Thirty minutes after APAP administration, mice were treated with different concentrations of stachydrine (0, 2.5, 5, and 10 mg/kg). Animals were sacrificed 16 h after APAP injection for serum and liver tissue assays. APAP overdose significantly elevated the serum alanine transferase levels, hepatic pro-inflammatory cytokines, malondialdehyde activity, phospho-extracellular signal-regulated kinase (ERK), phospho-protein kinase B (AKT), and macrophage-stimulating protein expression. Stachydrine treatment significantly decreased these parameters in mice with APAP-induced liver damage. Our results suggest that stachydrine may be a promising beneficial target in the prevention of APAP-induced liver damage through attenuation of the inflammatory response, inhibition of the ERK and AKT pathways, and expression of macrophage-stimulating proteins.

The ubiquitin ligases Cbl and Cbl-b regulate macrophage growth by controlling CSF-1R import into macropinosomes.

The ubiquitination of transmembrane receptors regulates endocytosis, intracellular traffic, and signal transduction. Bone marrow-derived macrophages from myeloid Cbl-/- and Cbl-b-/- double knockout (DKO) mice display sustained proliferation mirroring the myeloproliferative disease that these mice succumb to. Here, we found that the ubiquitin ligases Cbl and Cbl-b have overlapping functions for controlling the endocytosis and intracellular traffic of the CSF-1R. DKO macrophages displayed complete loss of ubiquitination of the CSF-1R whereas partial ubiquitination was observed for either single Cbl-/- or Cbl-b-/- macrophages. Unlike wild type, DKO macrophages were immortal and displayed slower CSF-1R internalization, elevated AKT signaling, and a failure to transport the CSF-1R into the lumen of nascent macropinosomes, leaving its cytoplasmic region available for signaling. CSF-1R degradation depended upon lysosomal vATPase activity in both WT and DKO macrophages, with this degradation confined to macropinosomes in WT but occurring in distributed/tubular lysosomes in DKO cells. RNA-sequencing comparison of Cbl-/-, Cbl-b-/- and DKO macrophages indicated that while the overall macrophage transcriptional program remained intact, DKO macrophages had alterations in gene expression associated with growth factor signaling, cell cycle, inflammation and senescence. Cbl-b-/- had minimal effect on the transcriptional program whereas Cbl-/- led to more alternations but only DKO macrophages demonstrated substantial changes in the transcriptome, suggesting overlapping but unique functions for the two Cbl-family members. Thus, Cbl/Cbl-b-mediated ubiquitination of CSF-1R regulates its endocytic fate, constrains inflammatory gene expression, and regulates signaling for macrophage proliferation.

Utilization of a Histoplasma capsulatum zinc reporter reveals the complexities of fungal sensing of metal deprivation.

Histoplasma capsulatum is a dimorphic fungal pathogen acquired via inhalation of soil-resident spores. Upon exposure to mammalian body temperatures, these fungal elements transform into yeasts that reside primarily within phagocytes. Macrophages (MΦ) provide a permissive environment for fungal replication until T cell-dependent immunity is engaged. MΦ activated by granulocyte macrophage colony stimulating factor (GM-CSF) induces metallothioneins (MTs) that bind zinc (Zn) and deprive yeast cells of labile Zn, thereby disabling fungal growth. Prior work demonstrated that the zinc transporter, ZRT2, was important for fungal survival in vivo. Hence, we constructed a yeast cell reporter strain that expresses green fluorescent protein (GFP) under control of the ZRT2 zinc-regulated promoter. This reporter accurately responds to a medium devoid of Zn. ZRT2 expression increased in GM-CSF, but not interferon-γ, stimulated MΦ. To examine the in vivo response, we infected mice with a reporter yeast strain and assessed ZRT2 expression at 0, 3, 7, and 14 days post-infection (dpi). ZRT2 expression minimally increased at 3 dpi and peaked at 7 dpi, corresponding with the onset of adaptive immunity. We discovered that the major MΦ populations that restrict Zn from the fungus are interstitial MΦ and exudate MΦ. Neutralizing GM-CSF blunted the control of infection but unexpectedly increased ZRT2 expression. This increase was dependent on another cytokine that activates MΦ to control H. capsulatum replication, M-CSF. These findings illustrate the reporter's ability to sense Zn in vitro and in vivo and correlate ZRT2 expression with GM-CSF and M-CSF activation of MΦ.IMPORTANCEPhagocytes use an arsenal of defenses to control the replication of Histoplasma yeasts, one of which is the limitation of trace metals. On the other hand, H. capsulatum combats metal restriction by upregulating metal importers such as the Zn importer ZRT2. This transporter contributes to H. capsulatum pathogenesis upon activation of adaptive immunity. We constructed a fluorescent ZRT2 transcriptional reporter to probe H. capsulatum Zn sensing during infection and exposed the role for M-CSF activation of macrophages when GM-CSF is absent. These data highlight the ways in which fungal pathogens sense metal deprivation in vivo and reveal the potential of metal-sensing reporters. The work adds a new dimension to study how intracellular pathogens sense and respond to the changing environments of the host.

The CSF1-CSF1R pathway in the trigeminal ganglion mediates trigeminal neuralgia via inflammatory responses in mice.

BACKGROUND: Trigeminal neuralgia (TN) is the most severe type of neuropathic pain. The trigeminal ganglion (TG) is a crucial target for the pathogenesis and treatment of TN. The colony-stimulating factor 1 (CSF1) - colony-stimulating factor 1 receptor (CSF1R) pathway regulates lower limb pain development. However, the effect and mechanism of the CSF1-CSF1R pathway in TG on TN are unclear. METHODS: Partial transection of the infraorbital nerve (pT-ION) model was used to generate a mouse TN model. Mechanical and cold allodynia were used to measure pain behaviors. Pro-inflammatory factors (IL-6, TNF-a) were used to measure inflammatory responses in TG. PLX3397, an inhibitor of CSF1R, was applied to inhibit the CSF1-CSF1R pathway in TG. This pathway was activated in naïve mice by stereotactic injection of CSF1 into the TG. RESULTS: The TN model activated the CSF1-CSF1R pathway in the TG, leading to exacerbated mechanical and cold allodynia. TN activated inflammatory responses in the TG manifested as a significant increase in IL-6 and TNF-a levels. After using PLX3397 to inhibit CSF1R, CSF1R expression in the TG declined significantly. Inhibiting the CSF1-CSF1R pathway in the TG downregulated the expression of IL-6 and TNF-α to reduce allodynia-related behaviors. Finally, mechanical allodynia behaviors were exacerbated in naïve mice after activating the CSF1-CSF1R pathway in the TG. CONCLUSIONS: The CSF1-CSF1R pathway in the TG modulates TN by regulating neuroimmune responses. Our findings provide a theoretical basis for the development of treatments for TN in the TG.

Validation of CSF-1 receptor (CD115) staining for analysis of murine monocytes by flow cytometry.

CD115, the receptor for colony stimulating factor 1, is essential for survival and differentiation of monocytes and macrophages and is therefore frequently used to define monocyte subsets and their progenitors in immunological assays. However, CD115 surface expression and detection by flow cytometry is greatly influenced by cell isolation and processing methods, organ source, and disease context. In a systematic analysis of murine monocytes, we define experimental conditions that preserve or limit CD115 surface expression and staining by flow cytometry. We also find that, independent of conditions, CD115 surface levels are consistently lower in Ly6Clo monocytes than in Ly6Chi monocytes, with the exception of Ly6Clo monocytes in the bone marrow. Furthermore, in contrast to IL-34, the presence of colony stimulating factor 1 impairs CD115 antibody staining in a dose-dependent manner, which, in a model of ischemic kidney injury with elevated levels of colony stimulating factor 1, influenced quantification of kidney monocytes. Thus, staining and experimental conditions affect quantitative and qualitative analysis of monocytes and may influence experimental conclusions.

Role of macrophage colony stimulating factor and interferon regulatory factor 7 in modulating the immune profile of mouse testicular macrophages.

Testicular macrophages (TM) are critical for the function of the testis by regulating homeostasis and inflammatory responses. However, the mechanisms by which TM fulfil these roles remain elusive. In this study, we explored the impact of two key testicular microenvironmental factors, namely 25-hydroxycholesterol (25HC), an oxysterol related to sex hormones and macrophage colony-stimulating factor (M-CSF), a factor crucial for macrophage survival and differentiation, on the regulation of the TM phenotype. Specifically, we examined their role in controlling the expression of the transcription factor interferon regulatory factor 7 (Irf7), a factor critical for maintaining the alternative macrophage phenotype. To achieve this, we used an in vitro bone marrow-derived macrophage (BMDM) model as a surrogate for TM to investigate the roles of 25HC and M-CSF in regulating the expression of Irf7 during the polarization of murine TM. M-CSF was identified as the main regulator of Irf7 expression, while 25HC production is a consequence of Irf7 activation in BMDM. In turn, 25HC plays a role in a negative feedback loop on the expression levels of Irf7 in BMDM. Using flow cytometry in Irf7-/- mouse testis the CD64loMHChi TM subpopulation was found to be decreased. Together with lower IL-10 protein levels in Irf7-/- TM this indicates a shift towards an M1-like macrophage profile. In summary, our data indicates that M-CSF could act as an inducer of high Irf7 expression levels in the mouse testis. However, the exact role of the high 25HC concentration in the testis in maintaining the local immune milieu still needs further study.

Reversible SAHH inhibitor ameliorates MIA-induced osteoarthritis of rats through suppressing MEK/ERK pathway.

Osteoarthritis (OA) is characterized by gradual articular cartilage degradation, accompanied by persistent low-grade joint inflammation, correlating with radiographic and pain-related progression. The latent therapeutic potential of DZ2002, a reversible inhibitor of S-adenosyl-L-homocysteine hydrolase (SAHH), holds promise for OA intervention. This study endeavored to examine the therapeutic efficacy of DZ2002 within the milieu of OA. The cytotoxicity of DZ2002 was evaluated using the MTT assay on bone marrow-derived macrophages. The inhibitory impact of DZ2002 during the process of osteoclastogenesis was assessed using TRAP staining, analysis of bone resorption pits, and F-actin ring formation. Mechanistic insights were derived from qPCR and Western blot analyses. Through the intra-articular injection of monosodium iodoacetate (MIA), an experimental rat model of OA was successfully instituted. This was subsequently accompanied by a series of assessments including Von Frey filament testing, analysis of weight-bearing behaviors, and micro-CT imaging, all aimed at assessing the effectiveness of DZ2002. The findings emphasized the effectiveness of DZ2002 in mitigating osteoclastogenesis induced by M-CSF/RANKL, evident through a reduction in TRAP-positive OCs and bone resorption. Moreover, DZ2002 modulated bone resorption-associated gene and protein expression (CTSK, CTR, Integrin ß3) via the MEK/ERK pathway. Encouragingly, DZ2002 also alleviates MIA-induced pain, cartilage degradation, and bone loss. In conclusion, DZ2002 emerges as a potential therapeutic contender for OA, as evidenced by its capacity to hinder in vitro M-CSF/RANKL-induced osteoclastogenesis and mitigate in vivo osteoarthritis progression. This newfound perspective provides substantial support for considering DZ2002 as a compelling agent for osteoarthritis intervention.

The nuclear receptor Nurr1 is preferentially expressed in human pro-inflammatory macrophages and limits their inflammatory profile.

Nurr1 is a member of the orphan nuclear receptor family NR4A (nuclear receptor subfamily 4 group A) that modulates inflammation in several cell lineages, both positively and negatively. Macrophages are key regulators of inflammatory responses, yet information about the role of Nurr1 in human macrophages is scarce. Here we examined Nurr1 expression and activity in steady state and activated human macrophages. Pro- and anti-inflammatory macrophages were generated in vitro by culture of blood monocytes with granulocyte/macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), respectively. Nurr1 expression was predominant in macrophages with the pro-inflammatory phenotype. Nurr1 activation with the agonists 1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane (C-DIM12) or isoxazolo-pyridinone 7e (IP7e) did not globally modify the polarization status of pro-inflammatory macrophages, but they decreased their production of TNF, IL-1ß, IL-6, IL-8, IL-12 p40, CCL2, IFN-ß, and reactive oxygen species, with variable potencies. Conversely, Nurr1 deficient macrophages increased the expression of transcripts encoding inflammatory mediators, particularly that of IL6, IFNB1, and CCL2. Mechanistically, endogenous Nurr1 interacted with NF-κB p65 in basal conditions and upon lipopolysaccharide (LPS)-mediated activation. C-DIM12 stabilized those complexes in cells exposed to LPS and concurrently decreased NF-κB transcriptional activity and p65 nuclear translocation. Expression of high levels of Nurr1 was associated with a subset of dermal macrophages that display enhanced levels of TNF and lower expression of the anti-inflammatory marker CD163L1 in skin lesions from patients with bullous pemphigoid (BP), a chronic inflammatory autoimmune blistering disorder. These results suggest that Nurr1 expression is linked with the pro-inflammatory phenotype of human macrophages, both in vivo and in vitro, where it may constitute a brake to attenuate the synthesis of inflammatory mediators.

Reversible expansion of tissue macrophages in response to macrophage colony-stimulating factor (CSF1) transforms systemic lipid and carbohydrate metabolism.

Macrophages regulate metabolic homeostasis in health and disease. Macrophage colony-stimulating factor (CSF1)-dependent macrophages contribute to homeostatic control of the size of the liver. This study aimed to determine the systemic metabolic consequences of elevating circulating CSF1. Acute administration of a CSF1-Fc fusion protein to mice led to monocytosis, increased resident tissue macrophages in the liver and all major organs, and liver growth. These effects were associated with increased hepatic glucose uptake and extensive mobilization of body fat. The impacts of CSF1 on macrophage abundance, liver size, and body composition were rapidly reversed to restore homeostasis. The effects of CSF1 on metabolism were independent of several known endocrine regulators and did not impact the physiological fasting response. Analysis using implantable telemetry in metabolic cages revealed progressively reduced body temperature and physical activity with no change in diurnal food intake. These results demonstrate the existence of a dynamic equilibrium between CSF1, the mononuclear phagocyte system, and control of liver-to-body weight ratio, which in turn controls systemic metabolic homeostasis. This novel macrophage regulatory axis has the potential to promote fat mobilization, without changes in appetence, which may have novel implications for managing metabolic syndrome.NEW & NOTEWORTHY CSF1 administration expands tissue macrophages, which transforms systemic metabolism. CSF1 drives fat mobilization and glucose uptake to support liver growth. The effects of CSF1 are independent of normal hormonal metabolic regulation. The effects of CSF1 are rapidly reversible, restoring homeostatic body composition. CSF1-dependent macrophages and liver size are coupled in a dynamic equilibrium.

Cucumber seed polypeptides regulate RANKL-induced osteoclastogenesis through OPG/RANKL/RANK and NF-κB.

Postmenopausal osteoporosis (PMOP) is a common disease that endangers the health of elderly women. Cucumber seeds have shown excellent therapeutic effects on PMOP, but the mechanism of cucumber seed peptide (CSP) remains unclear. The expression levels of NF-κB and osteoclast-related genes were detected by RT-qPCR. The levels of apoptosis-related proteins were detected by Western blotting. Nuclear translocation of NF-κB p65 and osteoclast formation were detected by immunofluorescence and tartrate-resistant acid phosphatase (TRAP) staining, respectively. ELISA was used to detect the expression levels of OPG, M-CSF, and RANKL. Hematoxylin-eosin (H&E) and TRAP staining were used to observe the effects of CSP on bone formation. In RAW264.7 cells, CSP (0.4 mg/L, 4 mg/L, and 40 mg/L) effectively inhibited the expression of osteoclast-related genes (Cathepsin-K, MT1-MMP, MMP-9, and TRAP). TRAP-positive multinucleated giant cells gradually decreased. Furthermore, NF-κB pathway activation downstream of RANK was inhibited. In bone marrow stromal cells (BMSCs), the expression levels of M-CSF and RANKL gradually decreased, and OPG gradually increased with increasing CSP concentrations. Treatment of RAW264.7 cells with pyrrolidine dithiocarbamate (PDTC, an inhibitor of NF-κB) prevented the formation of osteoclasts. Treatment with different concentrations of CSP effectively decreased the levels of RANKL and M-CSF in rat serum and increased the expression of OPG in the oophorectomy (OVX) rat model. Furthermore, different concentrations of CSP could ameliorate the loss of bone structure and inhibit the formation of osteoclasts in rats. CSP inhibits osteoclastogenesis by regulating the OPG/RANKL/RANK pathway and inhibiting the NF-kB pathway.

Adenine is an anti-inflammatory metabolite found to be more abundant in M-CSF over GM-CSF-differentiated human macrophages.

Immunometabolism has been unveiled in the last decade to play a major role in controlling macrophage metabolism and inflammation. There has been a constant effort to understand the immunomodulating properties of regulated metabolites during inflammation with the aim of controlling and re-wiring aberrant macrophages in inflammatory diseases. M-CSF and GM-CSF-differentiated macrophages play a key role in mounting successful innate immune responses. When a resolution phase is not achieved however, GM-CSF macrophages contribute substantially more towards an adverse inflammatory milieu than M-CSF macrophages, consequently driving disease progression. Whether there are specific immunometabolites that determine the homoeostatic or inflammatory nature of M-CSF and GM-CSF-differentiated macrophages is still unknown. As such, we performed metabolomics analysis on LPS and IL-4-stimulated M-CSF and GM-CSF-differentiated human macrophages to identify differentially accumulating metabolites. Adenine was distinguished as a metabolite significantly higher in M-CSF-differentiated macrophages after both LPS or IL-4 stimulation. Human macrophages treated with adenine before LPS stimulation showed a reduction in inflammatory gene expression, cytokine secretion and surface marker expression. Adenine caused macrophages to become more quiescent by lowering glycolysis and OXPHOS which resulted in reduced ATP production. Moreover, typical metabolite changes seen during LPS-induced macrophage metabolic reprogramming were absent in the presence of adenine. Phosphorylation of metabolic signalling proteins AMPK, p38 MAPK and AKT were not responsible for the suppressed metabolic activity of adenine-treated macrophages. Altogether, in this study we highlight the immunomodulating capacity of adenine in human macrophages and its function in driving cellular quiescence.

NOTCH2 promotes osteoclast maturation and metabolism and modulates the transcriptome profile during osteoclastogenesis.

Notch signaling plays a key regulatory role in bone remodeling and NOTCH2 enhances osteoclastogenesis, an effect that is mostly mediated by its target gene Hes1. In the present study, we explored mechanisms responsible for the enhanced osteoclastogenesis in bone marrow-derived macrophages (BMM) from Notch2tm1.1Ecan, harboring a NOTCH2 gain-of-function mutation, and control mice. Notch2tm1.1Ecan mice are osteopenic and have enhanced osteoclastogenesis. Bulk RNA-Seq and gene set enrichment analysis of Notch2tm1.1Ecan BMMs cultured in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand revealed enrichment of genes associated with enhanced cell metabolism, aerobic respiration, and mitochondrial function, all associated with osteoclastogenesis. These pathways were not enhanced in the context of a Hes1 inactivation. Analysis of single cell RNA-Seq data of pooled control and Notch2tm1.1Ecan BMMs treated with M-CSF or M-CSF and receptor activator of NF-κB ligand for 3 days identified 11 well-defined cellular clusters. Pseudotime trajectory analysis indicated a trajectory of clusters expressing genes associated with osteoclast progenitors, osteoclast precursors, and mature cells. There were an increased number of cells expressing gene markers associated with the osteoclast and with an unknown, albeit related, cluster in Notch2tm1.1Ecan than in control BMMs as well as enhanced expression of genes associated with osteoclast progenitors and precursors in Notch2tm1.1Ecan cells. In conclusion, BMM cultures display cellular heterogeneity, and NOTCH2 enhances osteoclastogenesis, increases mitochondrial and metabolic activity of osteoclasts, and affects cell cluster allocation in BMMs.

Extracellular Signal-Regulated Kinases Play Essential but Contrasting Roles in Osteoclast Differentiation.

Bone homeostasis is regulated by the balanced actions of osteoblasts that form the bone and osteoclasts (OCs) that resorb the bone. Bone-resorbing OCs are differentiated from hematopoietic monocyte/macrophage lineage cells, whereas osteoblasts are derived from mesenchymal progenitors. OC differentiation is induced by two key cytokines, macrophage colony-stimulating factor (M-CSF), a factor essential for the proliferation and survival of the OCs, and receptor activator of nuclear factor kappa-B ligand (RANKL), a factor for responsible for the differentiation of the OCs. Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERKs), p38, and c-Jun N-terminal kinases, play an essential role in regulating the proliferation, differentiation, and function of OCs. ERKs have been known to play a critical role in the differentiation and activation of OCs. In most cases, ERKs positively regulate OC differentiation and function. However, several reports present conflicting conclusions. Interestingly, the inhibition of OC differentiation by ERK1/2 is observed only in OCs differentiated from RAW 264.7 cells. Therefore, in this review, we summarize the current understanding of the conflicting actions of ERK1/2 in OC differentiation.

Integrated transcriptomic and metabolomic analysis reveals the metabolic programming of GM-CSF- and M-CSF- differentiated mouse macrophages.

Macrophages play a critical role in the inflammatory response and tumor development. Macrophages are primarily divided into pro-inflammatory M1-like and anti-inflammatory M2-like macrophages based on their activation status and functions. In vitro macrophage models could be derived from mouse bone marrow cells stimulated with two types of differentiation factors: GM-CSF (GM-BMDMs) and M-CSF (M-BMDMs), to represent M1- and M2-like macrophages, respectively. Since macrophage differentiation requires coordinated metabolic reprogramming and transcriptional rewiring in order to fulfill their distinct roles, we combined both transcriptome and metabolome analysis, coupled with experimental validation, to gain insight into the metabolic status of GM- and M-BMDMs. The data revealed higher levels of the tricarboxylic acid cycle (TCA cycle), oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO), and urea and ornithine production from arginine in GM-BMDMs, and a preference for glycolysis, fatty acid storage, bile acid metabolism, and citrulline and nitric oxide (NO) production from arginine in M-BMDMs. Correlation analysis with the proteomic data showed high consistency in the mRNA and protein levels of metabolic genes. Similar results were also obtained when compared to RNA-seq data of human monocyte derived macrophages from the GEO database. Furthermore, canonical macrophage functions such as inflammatory response and phagocytosis were tightly associated with the representative metabolic pathways. In the current study, we identified the core metabolites, metabolic genes, and functional terms of the two distinct mouse macrophage populations. We also distinguished the metabolic influences of the differentiation factors GM-CSF and M-CSF, and wish to provide valuable information for in vitro macrophage studies.

WNT4 promotes macrophage polarization via granulosa cell M-CSF and reduces granulosa cell apoptosis in endometriosis.

BACKGROUND: WNT4 gene polymorphism are common in endometriosis and may functionally link estrogen and estrogen receptor signaling. Previous study confirmed estrogen and estrogen receptor signaling recruit macrophage to promote the pathogenesis of endometriosis. To investigate the effect of WNT4 in endometriosis involved in macrophage polarization and whether WNT4 could reduce the apoptosis of granulosa cells. METHODS: An observational study consisting of 8 cases of women with endometriosis (diagnosed by surgery and histology) and 22 mice of endometriosis animal model was conducted. Granulosa cells were isolated from 16 patients with endometriosis and co-cultured with macrophage under WNT4 treatment using TUNEL assay, quantitative reverse transcription PCR, flow cytometry and ELISA analysis. 22 mice of endometriosis animal model confirmed the WNT4 treatment effects using histology and immunohistochemistry, Western blot and flow cytometry. RESULTS: We observed that the apoptotic proportion of granulosa cells was significantly decreased and M2 macrophage was significantly increased after WNT4 treatment during the granulosa cell and macrophage co-culture system. To reveal the underlying mechanism for this, we conducted a series of experiments and found that high expression of granulosa cell M-CSF led to the M2 polarization of macrophages. The animal model also suggested that the anti-apoptotic effect of WNT4 on granulosa cells were conducted by the M2 polarized macrophage. CONCLUSIONS: WNT4 could reduce granulosa cell apoptosis and improve ovarian reserve by promoting macrophage polarization in endometriosis. M-CSF secreted by granulosa cell after WNT4 treatment was the main mediator of macrophage polarization.

p53 Gain-of-Function Mutation Induces Metastasis via BRD4-Dependent CSF-1 Expression.

TP53 mutations are frequent in esophageal squamous cell carcinoma (ESCC) and other SCCs and are associated with a proclivity for metastasis. Here, we report that colony-stimulating factor-1 (CSF-1) expression is upregulated significantly in a p53-R172H-dependent manner in metastatic lung lesions of ESCC. The p53-R172H-dependent CSF-1 signaling, through its cognate receptor CSF-1R, increases tumor cell invasion and lung metastasis, which in turn is mediated in part through Stat3 phosphorylation and epithelial-to-mesenchymal transition (EMT). In Trp53R172H tumor cells, p53 occupies the Csf-1 promoter. The Csf-1 locus is enriched with histone 3 lysine 27 acetylation (H3K27ac), which is likely permissive for fostering an interaction between bromodomain-containing domain 4 (BRD4) and p53-R172H to regulate Csf-1 transcription. Inhibition of BRD4 not only reduces tumor invasion and lung metastasis but also reduces circulating CSF-1 levels. Overall, our results establish a novel p53-R172H-dependent BRD4-CSF-1 axis that promotes ESCC lung metastasis and suggest avenues for therapeutic strategies for this difficult-to-treat disease. SIGNIFICANCE: The invasion-metastasis cascade is a recalcitrant barrier to effective cancer therapy. We establish that the p53-R172H-dependent BRD4-CSF-1 axis is a mediator of prometastatic properties, correlates with patient survival and tumor stages, and its inhibition significantly reduces tumor cell invasion and lung metastasis. This axis can be exploited for therapeutic advantage. This article is featured in Selected Articles from This Issue, p. 2489.