Macrophage-conditioned Medium Research Articles

Inhibition of Bruton’s Tyrosine Kinase Alleviates Monocrotaline-Induced Pulmonary Arterial Hypertension by Modulating Macrophage Polarization

Macrophage accumulation and activation contribute to the development of pulmonary arterial hypertension (PAH), while Bruton's tyrosine kinase (BTK) is an important regulator for the activation and polarization of macrophage. However, the role of BTK in PAH remains unknown. In the present study, a selective BTK inhibitor (BTKi) BGB-3111 was applied to investigate the role of BTK in monocrotaline- (MCT-) induced PAH rat and phorbol myristate acetate- (PMA-) differentiated U937 macrophages. Our results showed that BTK was mainly distributed and upregulated in CD68+ macrophages in the lungs of PAH rats. Daily treated with BTKi BGB-3111 alleviated MCT-induced PAH, as indicated by the decrease in right ventricular systolic pressure (RVSP), attenuation in right ventricle hypertrophy and pulmonary vascular remodeling, reduction in perivascular collagen deposition, as well as inhibition of inflammation and endothelial-to-mesenchymal transition (EndMT) in the lung. Moreover, BTK inhibition suppressed MCT-induced recruitment of macrophages, especially the classical activated macrophages (M1) in the lung. In vitro, BGB-3111 significantly suppressed lipopolysaccharide- (LPS-) induced M1 polarization and proinflammatory cytokine production in U937-derived macrophages. The underlying mechanism is associated with the inhibition of NF-κB/MAPK pathways and nucleotide-binding oligomerization domain-like receptor with pyrin domain 3 (NLRP3) inflammasome activation. Furthermore, macrophage conditioned medium (CM) from LPS-induced M1 macrophages promoted migration and EndMT of HPAECs, while CM from BGB-3111-pretreated LPS-induced M1 macrophages failed to induce this response. These findings suggest that BTK inhibition alleviates PAH by regulating macrophage recruitment and polarization and may be a potential therapeutic strategy for the treatment of PAH.

Macrophages Treated with VEGF and PDGF Exert Paracrine Effects on Olfactory Ensheathing Cell Function.

Glial cell transplantation using olfactory ensheathing cells (OECs) holds a promising approach for treating spinal cord injury (SCI). However, integration of OECs into the hostile acute secondary injury site requires interaction and response to macrophages. Immunomodulation of macrophages to reduce their impact on OECs may improve the functionality of OECs. Vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), known for their immunomodulatory and neuroprotective functions, have provided improved outcomes in SCI animal models. Thus, VEGF and PDGF modulation of the SCI microenvironment may be beneficial for OEC transplantation. In this in vitro study, the effect of VEGF and PDGF on macrophages in an inflammatory condition was tested. Combined VEGF + PDGF reduced translocation nuclear factor kappa B p65 in macrophages without altering pro-inflammatory cytokines. Further, the ability of OECs to phagocytose myelin debris was assessed using macrophage-conditioned medium. Conditioned medium from macrophages incubated with PDGF and combined VEGF + PDGF in inflammatory conditions promoted phagocytosis by OECs. The growth factor treated conditioned media also modulated the expression of genes associated with nerve repair and myelin expression in OECs. Overall, these results suggest that the use of growth factors together with OEC transplantation may be beneficial in SCI therapy.

Macrophage induced ERK-TGF-β1 signaling in MCF7 breast cancer cells result in reversible cancer stem cell plasticity and epithelial mesenchymal transition

BackgroundBreast cancer is a heterogenous disease composed of multiple clonal populations and the mechanism by which the tumor microenvironment induces cancer stem cell plasticity is not fully understood. MethodsMCF7 breast cancer cells were treated with macrophage conditioned medium (MɸCM). PD98059 and SB431542 were used for ERK and TGF-βR inhibition respectively. Epithelial-mesenchymal transition (EMT) and cancer stem cell markers (CSC) were studied using qRT-PCR and flowcytometry. SCID mice were used for animal experiments. ResultsMɸCM- induced ERK/TGF-β1 signaling led to enrichment of CSC and EMT in MCF7 cells and mammospheres. These effects were abrogated by both MEK inhibitor PD98059 (TGF-β1 synthesis) and SB431542 (TGF-β1 signaling). The increase in CSC was both hybrid (ALDH1+) and mesenchymal (CD44+ CD24− cells). Increase in hybrid E/M state was at a single cell level as confirmed by the increase in both claudin-1 (E) and vimentin (M). This did not have any growth advantage in SCID mice and monitoring of CSC and EMT markers before and after growth in SCID mice indicated reversal of these markers in tumor cells recovered from mice. Removal of MɸCM and neutralization of TNF-α, IL-6 and IL-1β in MɸCM abrogated ERK phosphorylation, TGF-β and CSC enrichment indicating the requirement of continuous signaling for maintenance. ConclusionsERK signaling plays an important role in MɸCM- induced EMT and CSC plasticity which is completely reversible upon withdrawal of signals. General significanceOur experimental observations support the semi-independent nature of EMT-stemness connection which is very dynamic and reversible depending on the microenvironment.

Effects of the media conditioned by various macrophage subtypes derived from THP-1 cells on tunneling nanotube formation in pancreatic cancer cells

BackgroundTunneling nanotubes (TNTs) are special membrane structures for intercellular communications. Vital cargoes (such as mitochondria) could be delivered from healthy cells to rescue damaged ones through TNTs. The TNTs could be utilized for the purpose of systematic delivery of therapeutic agents between cells. However, there are insufficient studies on the controlled enhancement of TNT formations. The purpose of this study is to understand how macrophages influence the TNT formation in cancer cells.ResultsHere we compared the capabilities of inducing TNTs in human pancreatic cancer cells (PANC-1) of the media conditioned by M0, M1 and M2 macrophages derived from THP-1 cells. The M0 and M1 macrophage conditioned media promoted TNT formation. Using a focused ion beam to cut through a TNT, we observed tunnel-like structures inside dense cytoskeletons with scanning electron microscopy. The TNT formation correlated with raised motility, invasion, and epithelial–mesenchymal transition in the PANC-1 cells. Mitochondria and lysosomes were also found to be transported in the TNTs.ConclusionsThese results suggest that TNT formation could be one of the responses to the immune stress in pancreatic cancer cells caused by M0 and M1 macrophages. This finding is valuable for the development of macrophage-targeting cancer therapy.

Abstract 655: Macrophage mediated resistance to TKI therapy in ALK fusion positive non-small cell lung cancer

Abstract Patients with lung cancer bearing driver oncogenes such as epidermal growth factor receptor (EGFR) mutations or anaplastic lymphoma kinase (ALK) fusions, often experience long term disease control on targeted therapy. Despite excellent initial responses, ultimately these tumors develop resistance to targeted therapy. While several cancer cell intrinsic mechanisms of targeted therapy resistance are well known, they do not account for all cases of resistance. Based on our previous observations, we hypothesized components of the tumor microenvironment, specifically monocytes and macrophages, also contribute to the development of targeted therapy resistance. We generated bone marrow derived macrophages from C57BL/6 mice and polarized them to an M0, M1, or M2 phenotype over the course of 6 days. At day 7, cells were plated in fresh media and after 2 days, the conditioned media (CM) was collected. Macrophage polarization was verified via qPCR. Murine cell lines, EA-1, EA-2, and EA-3 are unique, murine lung adenocarcinomas bearing an oncogenic ALK fusion derived from C57BL/6 mice. These murine cell lines were then cultured in the macrophage CM prior to exposure to ALK fusion specific tyrosine kinase inhibitors (TKI), alectinib or crizotinib. We observed that culture in M2-CM resulted in at least a 10-fold increase in alectinib resistance across all 3 murine cell lines when compared to the same cell lines cultured in M1 or M0-CM. We next tested for the activity of known, clinically relevant bypass signaling pathways as mediator of M2-CM protection. Aftatinib, an inhibitor of EGFR and human epidermal growth factor receptor 2 (HER2) family of proteins, did not alter M2-CM protection in the presence of alectinib. M2-CM did not impart TKI resistance to the murine cell lines in the presence of crizotinib, a TKI which inhibits ALK fusion activity and MET receptor tyrosine kinase activity, suggesting bypass signaling through MET drives the M2-CM resistance. Ongoing animal model studies demonstrate a clear role for innate immune cells in decreasing tumor responsiveness to TKI. In summary, our work demonstrates TKI therapy may be modulated by the presence of and interaction with specific immune cells within the tumor microenvironment. These data support exploring macrophage targeting therapies to improve TKI responsiveness. Citation Format: Melanie Mandell, Katherine Priest, Anh Le, Lynn Heasley, Raphael Nemenoff, Erin L. Schenk. Macrophage mediated resistance to TKI therapy in ALK fusion positive non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 655.

Abstract 1570: Macrophage mediated cell-kill evasion by human prostate cancer cells

Abstract Macrophages can adopt a cytotoxic M1 phenotype which can eliminate cancer cells. However, despite the presence of macrophages in the prostate, prostate cancer (PCa) is a prevalent cancer in men. This suggests that PCa cells can evade M1 macrophage mediated cell-kill. We have shown that human THP-1 derived M1-like macrophages readily kill human prostate cancer LNCap cells, while in testosterone proficient conditions (Fraction Growth over Mono-culture (FGM): 0.1). However, in testosterone deprived conditions LNCap cells survive (FGM: 1.1). In addition, when exposed to M1-like macrophage conditioned medium, LNCap cells are killed in testosterone proficient conditions and not in depleted conditions (FGM: 0.2 and 0.7, respectively). This suggests that M1 macrophage mediated PCa cell-kill depends on Androgen Receptor (AR) signaling and the release of soluble factors by macrophages. This is supported by cell-kill of castration-resistant LNCap-abl cells with an AR amplification (FGM: 0.1) in testosterone deprived conditions and no cell-kill in both testosterone deprived and proficient conditions of castration resistant PC3 cells without AR expression when co-cultured with M1-like macrophages (FGM: 0.7 and 0.7. respectively). To dissect the mechanism of M1 macrophage mediated cell-kill evasion, a genome wide CRISPR knock-out screen was performed in LNCap cells in co-culture with M1-like macrophages. LNCap cells were transfected with the Brunello library and cultured in testosterone proficient conditions. After 72 hours of co-culture, cells were harvested and DNA was isolated and submitted for sequencing. After quality check analysis, five gene knock-outs with a significant (log2FcMedian >1.5) survival advantage were identified, AR, PRKCD (Protein Kinase C Delta), IKBKA (Inhibitor of nuclear factor kappa-B kinase subunit α:), IKBKB (Inhibitor of nuclear factor kappa-B kinase subunit β) and IKBKG (Inhibitor of nuclear factor kappa-B kinase subunit γ). The latter three genes are components of the IkB kinase (IKK) complex, which is essential for activation of members of the nuclear factor-kB (NFkB) family of transcription factors. The hits as identified in the CRISPR knock-out screen, were validated in single guide knock-out LNCap cells in co-culture with M1-like macrophages. Additionally, we have shown that AR signaling enhances PRKCD expression, both at the RNA and protein level. Moreover, expression of PRKCD is significantly lower in prostatectomy specimen of 56 patients treated for 3 months with the AR signalling inhibitor enzalutamide prior to surgery, than untreated matched controls. Furthermore, LNCap cells lacking PRKCD, IKBKB and IKBKG were significantly less affected in their growth by reactive oxygen species (H2O2) than non-targeting LNCap cells. Ongoing studies are aimed to dissect how these critical factors are interconnected in LNCap cells, M1 macrophage mediated cell-kill evasion as a result. Citation Format: Anniek Zaalberg, Emma Minnee, Wilbert Zwart, Andries M. Bergman. Macrophage mediated cell-kill evasion by human prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1570.

Macrophage circadian rhythms are differentially affected based on stimuli.

Macrophages are white blood cells that play disparate roles in homeostasis and immune responses. They can reprogram their phenotypes to pro-inflammatory (M1) or anti-inflammatory (M2) states in response to their environment. About 8-15% of the macrophage transcriptome has circadian oscillations, including genes closely related to their functioning. As circadian rhythms are associated with cellular phenotypes, we hypothesized that polarization of macrophages to opposing subtypes might differently affect their circadian rhythms. We tracked circadian rhythms in RAW 264.7 macrophages using luminescent reporters. Cells were stably transfected with Bmal1:luc and Per2:luc reporters, representing positive and negative components of the molecular clock. Strength of rhythmicity, periods and amplitudes of time series were assessed using multiple approaches. M1 polarization decreased amplitudes and rhythmicities of Bmal1:luc and Per2:luc, but did not significantly affect periods, while M2 polarization increased periods but caused no substantial alterations to amplitudes or rhythmicity. As macrophage phenotypes are also altered in the presence of cancer cells, we tested circadian effects of conditioned media from mouse breast cancer cells. Media from highly aggressive 4T1 cells caused loss of rhythmicity, while media from less aggressive EMT6 cells yielded no changes. As macrophages play roles in tumors, and oncogenic features are associated with circadian rhythms, we tested whether conditioned media from macrophages could alter circadian rhythms of cancer cells. Conditioned media from RAW 264.7 cells resulted in lower rhythmicities and periods, but higher amplitudes in human osteosarcoma, U2OS-Per2:luc cells. We show that phenotypic changes in macrophages result in altered circadian characteristics and suggest that there is an association between circadian rhythms and macrophage polarization state. Additionally, our data demonstrate that macrophages treated with breast cancer-conditioned media have circadian phenotypes similar to those of the M1 subtype, and cancer cells treated with macrophage-conditioned media have circadian alterations, providing insight to another level of cross-talk between macrophages and cancer.

MiR-122–5p regulates the pathogenesis of childhood obesity by targeting CPEB1

BackgroundChildhood obesity is strongly associated with inflammation which contributes to the development of several obesity-related disorders. Accumulating evidence has suggested that microRNAs (miRNAs) are involved in the pathogenesis of multiple human diseases, including childhood obesity. MiR-122–5p was reported to be related to obesity in childhood, however, the detailed function and mechanism of miR-122–5p are still obscure. MethodsSimpson-Golabi-Behmel syndrome (SGBS) adipocytes were cocultured with macrophage cell line THP-1 or macrophage-conditioned medium (MacCM) to promote cytokine expression. Oil Red O staining was used to detect the accumulation of lipid droplets in SGBS cells. The expression of interleukin 6 (IL-6), IL-8, and monocyte chemoattractant protein 1 (MCP-1) at mRNA and protein levels was assessed by RT-qPCR and ELISA, respectively. Western blotting was used for measuring protein levels of target genes of miR-122–5p. The luciferase reporter assay was applied for detecting the binding relation between miR-122–5p and cytoplasmic polyadenylation element binding protein 1 (CPEB1). ResultsCoculture of SGBS adipocytes and THP-1 macrophages/MacCM promoted IL-6, IL-8, and MCP-1 expression at mRNA and protein levels. Overexpression of miR-122–5p inhibited IL-6, IL-8, and MCP-1 expression in SGBS adipocytes, and this inhibitory effect was rescued by CPEB1 upregulation. CPEB1 3′-untranslated region was directly targeted by miR-122–5p. ConclusionMiR-122–5p suppresses cytokine expression in SGBS adipocytes by targeting CPEB1.

Does Macrophage Stimulation by CCL5 Alter Melanoma Migratory Behavior?

BackgroundCCL5 (RANTES, regulated upon activation, normal T‐cell expressed and secreted) is secreted not only by T‐cells, but also by melanoma cells into the tumor microenvironment. The current project is part of a bigger effort by our laboratory to determine how the stimuli present in the tumor microenvironment may contribute to the progression of melanoma. Here, we explore CCL5 receptor expression on monocytes, macrophages, and melanoma cells, and whether endogenous or exogenous CCL5 stimulation of THP‐1 macrophages produce phenotypical changes that affect melanoma migratory behavior.MethodsWe used WM278 and LM‐Mel‐45 melanoma cells and THP‐1 monocytes before and after conversion to macrophages by 2 days exposure to 50 ng/ml PMA. To examine expression of the 3 key CCL5 receptors, we compared median fluorescence intensity (MFI) of antibody‐exposed cells to MFI of unlabeled cells using antibodies for CCR1, CCR3, and CCR5. We gated out debris and multiples, corrected for spillover, and analyzed the samples on a Bio‐Rad S3e cell sorter. We used recombinant CCL5 (100 ng/ml), the CCR1 & CCR3 receptor antagonist, UCB35625 (100 nM) and the CCR5 antagonist, maraviroc (100 ng/ml). Melanoma migration rate was measured by a 16 h wound‐closure assay. Treatments included: Control, CCL5 only, maraviroc only, UCB35625 only, maraviroc and UCB35625 together, and CCL5 plus both blockers. Wound closure of LM‐Mel‐45 melanoma alone, a 1:4 mixture of macrophages to melanoma, or LM‐Mel‐45 melanoma treated with macrophage‐conditioned medium were compared in each treatment condition.ResultsCCL5 receptor expression was not found on WM278 melanoma cells. LM‐Mel‐45 melanoma express CCR1 but not CCR3 or CCR5. CCR1 and CCR5, but not CCR3, are expressed by both THP‐1 monocytes and THP‐1 macrophages. Melanoma cells alone or when co‐cultured with previously treated macrophages migrated at 14.3 ± 0.4 μm/h, but neither CCL5 stimulation nor receptor blockade changed migration significantly. When LM‐Mel‐45 melanoma cells were treated with conditioned macrophage medium, velocity increased to 16.8 ± 0.7 μm/h.ConclusionsThis work suggests that CCL5 does not directly impact melanoma migration either by endogenous expression or by exogenous addition. Even in the presence of CCL5 or antagonist‐exposed macrophages, we found no phenotypical changes that altered the physical interactions of macrophages on melanoma migratory rate. There is a small but significant increase in velocity of melanoma cells when treated with conditioned macrophage medium.

3D printed magnesium-doped β-TCP gyroid scaffold with osteogenesis, angiogenesis, immunomodulation properties and bone regeneration capability in vivo

Bioceramics have been used in orthopedic surgery for several years. Magnesium (Mg) is an essential element in bone tissue and plays an important role in bone metabolism. Mg-doped bioceramics has attracted the attention of researchers recently. However, the optimal doping amount of Mg in β-TCP and the immunomodulatory property of Mg-doped β-TCP (Mg-TCP) have not been determined yet. In this study, β-TCP scaffolds doped with different contents of magnesium oxide (0wt%, 1wt%, 3wt%, and 5wt%) with gyroid structure were printed by digital light processing (DLP) method, and the physicochemical and biological functions were then investigated. Mg-doping improved the physicochemical properties of the β-TCP scaffolds. In vitro experiments confirmed that the doping of Mg in β-TCP scaffolds promoted the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and angiogenic differentiation of endothelial progenitor cells (EPCs), where the 5Mg-TCP has the optimal properties when using the "one cell type" method. It was also found that all Mg-TCP facilitated the polarization of RAW264.7 cells to the M2 phenotype, especially the 3Mg-TCP. However, 3Mg-TCP displayed the optimal osteogenic and angiogenic potential when using a "multiple cell type" method, which referred to culturing the BMSCs or EPCs in the macrophage-conditioned medium. Finally, the in vivo experiments were conducted and the results confirmed that the 3Mg-TCP scaffolds possessed the satisfying bone defect repair capability both after 6 and 12weeks of implantation. This study suggests that 3Mg-TCP scaffolds provide the optimal biological performance and thus have the potential for clinical translation.

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Plaque Evaluation by Ultrasound and Transcriptomics Reveals BCLAF1 as a Regulator of Smooth Muscle Cell Lipid Transdifferentiation in Atherosclerosis.

Understanding the processes behind carotid plaque instability is necessary to develop methods for identification of patients and lesions with stroke risk. Here, we investigated molecular signatures in human plaques stratified by echogenicity as assessed by duplex ultrasound. Lesion echogenicity was correlated to microarray gene expression profiles from carotid endarterectomies (n=96). The findings were extended into studies of human and mouse atherosclerotic lesions in situ, followed by functional investigations in vitro in human carotid smooth muscle cells (SMCs). Pathway analyses highlighted muscle differentiation, iron homeostasis, calcification, matrix organization, cell survival balance, and BCLAF1 (BCL2 [B-cell lymphoma 2]-associated transcription factor 1) as the most significant signatures. BCLAF1 was downregulated in echolucent plaques, positively correlated to proliferation and negatively to apoptosis. By immunohistochemistry, BCLAF1 was found in normal medial SMCs. It was repressed early during atherogenesis but reappeared in CD68+ cells in advanced plaques and interacted with BCL2 by proximity ligation assay. In cultured SMCs, BCLAF1 was induced by differentiation factors and mitogens and suppressed by macrophage-conditioned medium. BCLAF1 silencing led to downregulation of BCL2 and SMC markers, reduced proliferation, and increased apoptosis. Transdifferentiation of SMCs by oxLDL (oxidized low-denisty lipoprotein) was accompanied by upregulation of BCLAF1, CD36, and CD68, while oxLDL exposure with BCLAF1 silencing preserved MYH (myosin heavy chain) 11 expression and prevented transdifferentiation. BCLAF1 was associated with expression of cell differentiation, contractility, viability, and inflammatory genes, as well as the scavenger receptors CD36 and CD68. BCLAF1 expression in CD68+/BCL2+ cells of SMC origin was verified in plaques from MYH11 lineage-tracing atherosclerotic mice. Moreover, BCLAF1 downregulation associated with vulnerability parameters and cardiovascular risk in patients with carotid atherosclerosis. Plaque echogenicity correlated with enrichment of distinct molecular pathways and identified BCLAF1, previously not described in atherosclerosis, as the most significant gene. Functionally, BCLAF1 seems necessary for survival and transdifferentiation of SMCs into a macrophage-like phenotype. The role of BCLAF1 in plaque vulnerability should be further evaluated.

Ketone Body β-Hydroxybutyrate Prevents Myocardial Oxidative Stress in Septic Cardiomyopathy.

Septic cardiomyopathy is a life-threatening complication of severe sepsis and septic shock. Oxidative stress and mitochondrial dysfunction have been identified as significant abnormalities in septic cardiomyopathy. However, specific treatments are rare. This study aims to investigate the impact of β-hydroxybutyrate (β-OHB) on septic cardiomyopathy and explore the underlying mechanism(s). We found that pretreatment of D-β-hydroxybutyrate-(R)-1,3 butanediol monoester (ketone ester, 3 mg/g body weight, once daily) by gavage for three days elevated the levels of ketone bodies, especially that of β-hydroxybutyrate (β-OHB) in the circulation and mouse hearts, which exerted a protective effect against lipopolysaccharide (LPS, 20 mg/kg)-induced septic cardiomyopathy in mice. In addition, an LPS-stimulated macrophage-conditioned medium (MCM) was used to mimic the pathological process of septic cardiomyopathy. Mechanistically, β-OHB alleviated myocardial oxidative stress and improved mitochondrial respiratory function through the antioxidant FoxO3a/MT2 pathway activated via histone deacetylase (HDAC) inhibition, which ultimately enhanced heart performance in septic cardiomyopathy. Our results, therefore, suggested an unappreciated critical role of β-OHB in septic heart protection as well as highlighted the potential of β-OHB as a simple remedy for the septic cardiomyopathy population.

Qishen Granule (QSG) Inhibits Monocytes Released From the Spleen and Protect Myocardial Function via the TLR4-MyD88-NF-κB p65 Pathway in Heart Failure Mice.

Preliminary clinical and basic researches have proved that Qishen granule (QSG) is an effective prescription for treating heart failure (HF) in China, with a characteristic of regulating the ratio of M1/M2 macrophage in the myocardium. However, the regulative mechanism of monocytes targeting the cardio-splenic axis has not been fully elucidated. This study aimed to investigate the effects and mechanism of QSG inhibiting the release of splenic monocytes and the recruitment of myocardial tissue both in vivo and in vitro. Experiments in mice with acute myocardial infarction (AMI)-induced HF demonstrated that QSG could exert anti-inflammatory effects by inhibiting splenic monocytes release and phenotypic changes. Moreover, in vitro experiments indicated QSG could inhibit LPS-stimulated macrophage-conditioned medium (CM)-induced H9C2 cardiomyocyte injury by upregulating the key proteins in TLR4-MyD88-NF-κB p65 pathway. In addition, knockdown or overexpression of TLR4 in H9C2 cells further confirmed that QSG could attenuate inflammatory injury in cardiomyocytes via the TLR4-MyD88-NF-κB p65 pathway. Overall, these data suggested that QSG could improve cardiac function and reduce the inflammatory response in AMI-induced HF by inhibiting splenic monocytes release, and protecting myocardial function via the TLR4-MyD88-NF-κB pathway in heart failure mice.

Therapeutic Effect of Melittin-dKLA Targeting Tumor-Associated Macrophages in Melanoma.

Melanoma is an immunogenic tumor and a serious type of skin cancer. Tumor-associated macrophages (TAMs) express an M2-like phenotype and are involved in all stages of melanomagenesis; it is hence a promising target for cancer immunotherapy. We herein investigated whether melittin–dKLA inhibits the growth of melanoma by inducing apoptosis of M2-like macrophages. For the in vitro study, a conditioned medium of macrophages was prepared from M0, M1, or M2-differentiated THP-1 cells with and without melittin–dKLA. The affinity of melittin for M2 macrophages was studied with FITC (fluorescein isothiocyanate)-conjugated melittin. For the in vivo study, murine melanoma cells were inoculated subcutaneously in the right flank of mice, melittin–dKLA was intraperitoneally injected at 200 nmol/kg every three days, and flow cytometry analysis of TAMs was performed. Since melittin binds preferentially to M2-like macrophages, melittin–dKLA induced more caspase 3 expression and cell death in M2 macrophages compared with M0 and M1 macrophages and melanoma cells. Melittin–dKLA significantly inhibited the proliferation and migration of M2 macrophages, resulting in a decrease in melanoma tumor growth in vivo. The CD206+ M2-like TAMs were reduced, while the CD86+ M1-like TAMs were not affected. Melittin–dKLA is therapeutically effective against melanoma by inducing the apoptosis of M2-like TAMs.

Excess glucocorticoid exposure contributes to adipose tissue fibrosis which involves macrophage interaction with adipose precursor cells

Chronic exposure to elevated glucocorticoid levels, as seen in patients with Cushing’s syndrome, can induce adipose tissue fibrosis. Macrophages play a pivotal role in adipose tissue remodelling. We used the synthetic glucocorticoid analogue dexamethasone to address glucocorticoid effects on adipose tissue fibrosis, in particular involving macrophage to preadipocyte communication. We analysed the direct effects of dexamethasone at a supra-physiological level, 0.3 µM, on gene expression of pro-fibrotic markers in human subcutaneous adipose tissue. The effects of dexamethasone on the differentiation of human SGBS preadipocytes were assessed in the presence or absence of THP1-macrophages or macrophage-conditioned medium. We measured the expression of different pro-fibrotic factors, including α-smooth muscle actin gene (ACTA2) and protein (α-SMA). Dexamethasone increased the expression of pro-fibrotic genes, e.g. CTGF, COL6A3, FN1, in adipose tissue. Macrophages abolished preadipocyte differentiation and increased the expression of the ACTA2 gene and α-SMA protein in preadipocytes after differentiation. Exposure to dexamethasone during differentiation reduced adipogenesis in preadipocytes, and elevated the expression of pro-fibrotic genes. Moreover, dexamethasone added together with macrophages further increased ACTA2 and α-SMA expression in preadipocytes, making them more myofibroblast-like. Cells differentiated in the presence of conditioned media from macrophages pretreated with or without dexamethasone had a higher expression of profibrotic genes compared to control cells. Our data suggest that macrophages promote adipose tissue fibrosis by directly interfering with preadipocyte differentiation and stimulating gene expression of pro-fibrotic factors. Excess glucocorticoid exposure also has pro-fibrotic effect on adipose tissue, but this requires the presence of macrophages.

M2-phenotype tumour-associated macrophages upregulate the expression of prognostic predictors MMP14 and INHBA in pancreatic cancer.

Pancreatic cancer is one of the most lethal gastrointestinal tumours, the most common pathological type is pancreatic adenocarcinoma (PAAD). In recent year, immune imbalanced in tumour microenvironment has been shown to play an important role in the evolution of tumours progression, and the efficacy of immunotherapy has been gradually demonstrated in clinical practice. In this study, we propose to construct an immune‐related prognostic risk model based on immune‐related genes MMP14 and INHBA expression that can assess the prognosis of pancreatic cancer patients and identify potential therapeutic targets for pancreatic cancer, to provide new ideas for the treatment of pancreatic cancer. We also investigate the correlation between macrophage infiltration and MMP14 and INHBA expression. First, the gene expression data of pancreatic cancer and normal pancreatic tissue were obtained from The Cancer Genome Atlas Program (TCGA) and The Genotype‐Tissue Expression public database (GTEx). The differentially expressed immune‐related genes between pancreatic cancer samples and normal sample were screened by R software. Secondly, univariate Cox regression analysis were used to evaluate the relationship between immune‐related genes and the prognosis of pancreatic cancer patients. A polygenic risk score model was constructed by Cox regression analysis. The prognostic nomogram was constructed, and its performance was evaluated comprehensively by internal calibration curve and C‐index. Using the risk model, each patient gets a risk score, and was divided into high‐ or low‐ risk groups. The proportion of 22 types of immune cells infiltration in pancreatic cancer samples was inferred by CIBERSOFT algorithm, correlation analysis (Pearson method) was used to analyse the correlation between the immune‐related genes and immunes cells. Then, we applied macrophage conditioned medium to culture pancreatic cancer cell line PANC1, detected the expression of MMP14 and INHBA by qRT‐PCR and Western blot methods. Knock‐down MMP14 and INHBA in PANC1 cells by transfected with shRNA lentiviruses. Detection of migration ability of pancreatic cells was done by trans‐well cell migration assay. A subcutaneous xenograft tumour model of human pancreatic cancer in nude mice was constructed. In conclusion, an immune‐related gene prognostic model was constructed, patients with high‐risk scores have poorer survival status, M2‐phenotype tumour‐associated macrophages (TAMs) up‐regulate two immune‐related genes, MMP14 and INHBA, which were used to establish the prognostic model. Knock‐down of MMP14 and INHBA inhibited invasion of pancreatic cancer.

Decidual macrophage derived MMP3 contributes to extracellular matrix breakdown in spiral artery remodeling in early human pregnancy

Remodeling of the uterine spiral arteries is required for a successful pregnancy. This process requires the co-ordinated activity of a number of different cell types including uterine natural killer cells, decidual macrophages, extravillous trophoblast cells, vascular smooth muscle cells and endothelial cells. We have previously demonstrated that decidual macrophages facilitate breakdown of fibronectin and laminin in a model of spiral artery remodeling. The aim of the current study was to determine which matrix metalloproteinases (MMPs) decidual macrophages express and play roles in extracellular matrix (ECM) breakdown in vascular remodeling. Decidual macrophages were isolated from first trimester decidua and cultured for 24 h to obtain conditioned medium. MMP secretion was assessed by a membrane based array and immunohistochemistry of decidual sections. In addition, the chorionic plate artery (CPA) model was used with decidual macrophage conditioned medium, with and without a MMP3 inhibitor and ECM protein expression assessed using quickscore. The decidual macrophages secreted a wide range of MMPs, with MMP3 being the most predominant. Co-localization of MMP3 to decidual macrophages was confirmed by immunohistochemistry. Decidual macrophage conditioned medium facilitated breakdown of laminin and fibronectin in the CPA model, an effect that was abrogated by the MMP3 inhibitor. These data further support the role of decidual macrophages in tissue remodeling in the first trimester of pregnancy. An alteration in their numbers or phenotype would impact spiral artery remodeling and contribute to the etiology of a number of complications of pregnancy.

Macrophage C/EBPδ Drives Gemcitabine, but Not 5-FU or Paclitaxel, Resistance of Pancreatic Cancer Cells in a Deoxycytidine-Dependent Manner.

Treatment of pancreatic ductal adenocarcinoma (PDAC), a dismal disease with poor survival rates, is hampered by the high prevalence of chemotherapy resistance. Resistance is accompanied by macrophage infiltration into the tumor microenvironment, and infiltrated macrophages are key players in chemotherapy resistance. In the current manuscript, we identify CCAAT/enhancer-binding protein delta (C/EBPδ) as an important transcription factor driving macrophage-dependent gemcitabine resistance. We show that conditioned medium obtained from wild type macrophages largely diminishes gemcitabine-induced cytotoxicity of PDAC cells, whereas conditioned medium obtained from C/EBPδ-deficient macrophages only minimally affects gemcitabine-induced PDAC cell death. Subsequent analysis of RNA-Seq data identified the pyrimidine metabolism pathway amongst the most significant pathways down-regulated in C/EBPδ-deficient macrophages and size filtration experiments indeed showed that the low molecular weight and free metabolite fraction most effectively induced gemcitabine resistance. In line with a role for pyrimidines, we next show that supplementing macrophage conditioned medium with deoxycytidine overruled the effect of macrophage conditioned media on gemcitabine resistance. Consistently, macrophage C/EBPδ-dependent resistance is specific for gemcitabine and does not affect paclitaxel or 5-FU-induced cytotoxicity. Overall, we thus show that C/EBPδ-dependent deoxycytidine biosynthesis in macrophages induces gemcitabine resistance of pancreatic cancer cells.

HSP90 Inhibitor 17-AAG Attenuates Nucleus Pulposus Inflammation and Catabolism Induced by M1-Polarized Macrophages.

Overactivated inflammation and catabolism induced by proinflammatory macrophages are involved in the pathological processes of intervertebral disc (IVD) degeneration (IVDD). Our previous study suggested the protective role of inhibiting heat shock protein 90 (HSP90) in IVDD, while the underlying mechanisms need advanced research. The current study investigated the effects of HSP90 inhibitor 17-AAG on nucleus pulposus (NP) inflammation and catabolism induced by M1-polarized macrophages. Immunohistochemical staining of degenerated human IVD samples showed massive infiltration of macrophages, especially M1 phenotype, as well as elevated levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α and matrix metalloproteinase (MMP)13. The conditioned medium (CM) of inflamed NP cells (NPCs) enhanced M1 polarization of macrophages, while the CM of M1 macrophages but not M2 macrophages promoted the expression of inflammatory factors and matrix proteases in NPCs. Additionally, we found that 17-AAG could represent anti-inflammatory and anti-catabolic effects by modulating both macrophages and NPCs. On the one hand, 17-AAG attenuated the pro-inflammatory activity of M1 macrophages via inhibiting nuclear factor-κB (NF-κB) pathway and mitogen-activated protein kinase (MAPK) pathways. On the other hand, 17-AAG dampened M1-CM-induced inflammation and catabolism in NPCs by upregulating HSP70 and suppressing the Janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway. Moreover, both in vitro IVD culture models and murine disc puncture models supported that 17-AAG treatment decreased the levels of inflammatory factors and matrix proteases in IVD tissues. In conclusion, HSP90 inhibitor 17-AAG attenuates NP inflammation and catabolism induced by M1 macrophages, suggesting 17-AAG as a promising candidate for IVDD treatment.