Polarization Of Bone Marrow-derived Macrophages Research Articles

CPT1A-IL-10-mediated macrophage metabolic and phenotypic alterations ameliorate acute lung injury.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a common acute respiratory failure due to diffuse pulmonary inflammation and oedema. Elaborate regulation of macrophage activation is essential for managing this inflammatory process and maintaining tissue homeostasis. In the past decades, metabolic reprogramming of macrophages has emerged as a predominant role in modulating their biology and function. Here, we observed reduced expression of carnitine palmitoyltransferase 1A (CPT1A), a key rate-limiting enzyme of fatty acid oxidation (FAO), in macrophages of lipopolysaccharide (LPS)-induced ALI mouse model. We assume that CPT1A and its regulated FAO is involved in the regulation of macrophage polarization, which could be positive regulated by interleukin-10 (IL-10). After nasal inhalation rIL-10 and/or LPS, wild type (WT), IL-10-/-, Cre-CPT1Afl/fl and Cre+CPT1Afl/fl mice were sacrificed to harvest bronchoalveolar lavage fluid, blood serum and lungs to examine cell infiltration, cytokine production, lung injury severity and IHC. Bone marrow-derived macrophages (BMDMs) were extracted from mice and stimulated by exogenous rIL-10 and/or LPS. The qRT-PCR, Seahorse XFe96 and FAO metabolite related kits were used to test the glycolysis and FAO level in BMDMs. Immunoblotting assay, confocal microscopy and fluorescence microplate were used to test macrophage polarization as well as mitochondrial structure and function damage. In in vivo experiments, we found that mice lacking CPT1A or IL-10 produced an aggravate inflammatory response to LPS stimulation. However, the addition of rIL-10 could alleviate the pulmonary inflammation in mice effectively. IHC results showed that IL-10 expression in lung macrophage decreased dramatically in Cre+CPT1Afl/fl mice. The in vitro experiments showed Cre+CPT1Afl/fl and IL-10-/- BMDMs became more "glycolytic", but less "FAO" when subjected to external attacks. However, the supplementation of rIL-10 into macrophages showed reverse effect. CPT1A and IL-10 can drive the polarization of BMDM from M1 phenotype to M2 phenotype, and CPT1A-IL-10 axis is also involved in the process of maintaining mitochondrial homeostasis. CPT1A modulated metabolic reprogramming and polarisation of macrophage under LPS stimulation. The protective effects of CPT1A may be partly attributed to the induction of IL-10/IL-10 receptor expression.

CGRP promotes osteogenic differentiation by regulating macrophage M2 polarization through HDAC6/AKAP12 signaling pathway.

Aim: To determine the mechanism of Calcitonin gene-related peptide (CGRP) in bone healing. Materials & methods: Alkaline phosphatase (ALP) activity and inflammatory-factor levels were detected usingELISA. Osteogenic differentiation was assessed using Alizarin red staining technique. The interaction between histone deacetylase 6 (HDAC6) and A-kinase anchoring protein 12 (AKAP12) was investigated through Co- immunoprecipitation. Results: CGRP treatment promoted rat bone marrow-derived macrophages (BMDMs) M2 polarization. CGRP facilitated osteogenic differentiation by enhancing M2 polarization of BMDMs. Mechanistically, CGRP promoted AKAP12 acetylation to activate the extracellular regulated protein kinases pathway by HDAC6 inhibition. Conclusion: CGRP promoted M2 polarization of rat BMDMs and facilitated osteogenic differentiation through the HDAC6/AKAP12/extracellular regulated protein kinases signaling pathway, thereby promoting bone healing.

Matrine reduces traumatic heterotopic ossification in mice by inhibiting M2 macrophage polarization through the MAPK pathway

In this study, the role of matrine, a component derived from traditional Chinese medicine, in modulating macrophage polarization and its effects on traumatic heterotopic ossification (HO) in mice was investigated. Traumatic HO is a pathological condition characterized by abnormal bone formation in nonskeletal tissues, often following severe trauma or surgery. The mechanisms underlying HO involve an enhanced inflammatory response and abnormal bone formation, with macrophages playing a crucial role. Our study demonstrated that matrine effectively inhibits the polarization of bone marrow-derived macrophages (BMDMs) toward the M2 phenotype, a subtype associated with anti-inflammatory processes and implicated in the progression of HO. Using in vitro assays, we showed that matrine suppresses key M2 markers and inhibits the MAPK signaling pathway in BMDMs. Furthermore, in vivo experiments revealed that matrine treatment significantly reduced HO formation in the Achilles tendons of mice and downregulated the expression of markers associated with M2 macrophages and the MAPK pathway. Our findings suggest that the ability of matrine to modulate macrophage polarization and inhibit the MAPK pathway has therapeutic potential for treating traumatic HO, providing a novel approach to managing this complex condition.

Ganlu formula ethyl acetate extract (GLEE) blocked the development of experimental arthritis by inhibiting NLRP3 activation and reducing M1 type macrophage polarization

Ethnopharmacological relevanceThe Tibetan medicine Ganlu Formula, as a classic prescription, is widely used across the Qinghai-Tibet Plateau area of China, which has a significant effect on relieving the course of rheumatoid arthritis (RA). However, the active compounds and underlying mechanisms of Ganlu Formula in RA treatment remain largely unexplored. Aim of the studyThis study aimed to elucidate the active substances and potential mechanisms of the ethyl acetate extract of Ganlu Formula ethyl acetate extract (GLEE) in the treatment of RA. Materials and methodsUltra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was utilized to analyze and identify the chemical constituents within GLEE. Discovery Studio molecular virtual docking technology was utilized to dock the interaction of GLEE with inflammation-related pathway proteins. The GLEE gene library was obtained by transcriptome sequencing. Collagen-induced arthritic（CIA) rats were utilized to assess the antiarthritic efficacy of GLEE. Micro-CT imaging was employed to visualize the rat paw, and ultrasound imaging revealed knee joint effusion. Evaluation of synovial tissue pathological changes was conducted through hematoxylin-eosin staining and saffranine solid green staining, while immunohistochemical staining was employed to assess NLRP3 expression along with inflammatory markers. Immunofluorescence staining was utilized to identify M1 macrophages. ResultsMetabolomic analysis via UPLC-Q-TOF-MS identified 28 potentially bioactive compounds in GLEE, which interacted with the active sites of key proteins such as NLRP3, NF-κB, and STAT3 through hydrogen bonds, C–H bonds, and electrostatic attractions. In vitro analyses demonstrated that GLEE significantly attenuated NLRP3 inflammasome activation and inhibited the polarization of bone marrow-derived macrophages (BMDMs) towards the M1 phenotype. In vivo, GLEE not only prevented bone mineral density (BMD) loss but also reduced ankle swelling in CIA rats. Furthermore, it decreased the expression of the NLRP3 inflammasome and curtailed the release of inflammatory mediators within the knee joint. ConclusionGLEE effectively mitigated inflammatory responses in both blood and knee synovial membranes of CIA rats, potentially through the down-regulation of the NLRP3/Caspase-1/IL-1β signaling pathway and reduction in M1 macrophage polarization.

LncRNA MEG3 Reduces the Ratio of M2/M1 Macrophages Through the HuR/CCL5 Axis in Hepatocellular Carcinoma.

Tumor-associated macrophages play a crucial role in the development of hepatocellular carcinoma (HCC). Our study aimed to investigate the relationship between long coding RNA (lncRNA) maternally expressed gene 3 (MEG3), RNA-binding protein human antigen R (HuR), and messenger RNA C-C motif chemokine 5 (CCL5) in the modulation of M1 and M2 macrophage polarization in HCC. To induce M1 or M2 polarization, LPS/IFNγ- or IL4/IL13 were used to treat bone marrow derived macrophages (BMDMs). The localization of MEG3 in M1 and M2 macrophages was assessed using fluorescence in situ hybridization assay. Expression levels of MEG3, HuR, CCL5, M1, and M2 markers were measured by RT-qPCR or immunofluorescence staining. Flow cytometry was performed to determine the proportion of F4/80+CD206+ and F4/80+CD68+ cells. RNA pulldown assay was performed to detect the binding of lncRNA MEG3 and HuR. The impacts of HuR on CCL5 stability and activity of CCL5 promoter were evaluated using actinomycin D treatment and luciferase reporter assay. Cell migration, invasiveness, and angiogenesis were assessed using transwell migration and invasion assays and a tube formation assay. A mixture of Huh-7 cells and macrophages were injected into nude mice to explore the effect of MEG3 on tumorigenesis. MEG3 promoted M1-like polarization while dampening M2-like polarization of BMDMs. MEG3 bound to HuR in M1 and M2 macrophages. HuR downregulated CCL5 by inhibiting CCL5 transcription in macrophages. In addition, overexpression of MEG3 suppressed cell metastasis, invasion, and angiogenesis by obstructing macrophage M2 polarization. MEG3 inhibited tumorigenesis in HCC via promotion of M1-like polarization and inhibition of M2-like polarization. Rescue experiments showed that depletion of CCL5 in M2 macrophages reversed MEG3-induced suppressive effect on cell migration, invasion, and tube formation. MEG3 suppresses HCC progression by promoting M1-like while inhibiting M2-like macrophage polarization via binding to HuR and thus upregulating CCL5.

Mogroside Ⅴ Inhibits M1 Polarization and Inflammation of Diabetic Mouse Macrophages via p38 MAPK/NF-Κb Signaling Pathway

ABSTRACT Background Mogroside V (MV) has anti-inflammatory properties. However, its impact on macrophage polarization under diabetic condition is yet unclear. This study aimed to investigate effects and underlying mechanisms of MV on inflammatory response and M1 polarization of bone marrow-derived macrophages (BMDMs) from diabetic mice. Methods BMDMs were isolated from normal and diabetic C57BL/6 mice. LPS and IFN-γwere used to produce M1-polarized BMDMs. MV treatment was administered throughout the M1 polarization process with or without SB203580 or PDTC. Surface markers CD11b, F4/80 and CD86 of macrophages were identified using flow cytometry or immunofluorescence staining. Inflammatory cytokines IL-1β and IL-6 and phosphorylation levels of p65 and p38 were examined by western blot. Results High glucose increased proportion of CD11b+F4/80+CD86+ cells, protein levels of inflammatory cytokines IL-1β and IL-6 and phosphorylation levels of p65 and p38 in LPS+IFN-γ-induced BMDMs, while they were decreased upon MV treatment. Additionally, these effects were further downregulated when MV was co-added with SB203580 or PDTC. Conclusions MV suppressed M1 macrophage polarization and inflammatory response, which was partially through NF-κB and p38 MAPK in LPS+IFN-γ induced BMDMs under high glucose condition, implying the potential of MV in treatment for inflammatory complications of diabetes.

Preliminary study on the effect of Echinococcus multilocaris on phenotypic transformations of glucose metabolism and polarization types in macrophages

To investigate the effects of Echinococcus multilocularis on the phenotypic transformations of glucose metabolism, polarization types and inflammatory responses in macrophages, so as to provide insights into elucidation of echinococcosis pathogenesis. Bone marrow cells were isolated from C57BL/6J mice at ages of 6 to 8 weeks, and induced into bone marrow-derived macrophages (BMDMs) with mouse macrophage colony-stimulating factor (M-CSF), which served as controls (BMDMs-M0). BMDMs-M0 induced M2 macrophages by interleukin-4 for 24 hours served as the IL-4 induction group, and BMDMs-M0 co-cultured with 2.4 ng/mL E. multilocularis cystic fluid (CF) served as the BMDM-CF co-culture group, while BMDMs-M0 co-cultured with E. multilocularis protoscolex (PSC) at a ratio of 500:1 served as the BMDM-PSC co-culture group. The types of polarization of BMDMs co-cultured with E. multilocularis CF and PSC were analyzed using flow cytometry, and the expression of macrophage markers, inflammatory factors, and glucose metabolism-related enzymes was quantified using fluorescent quantitative real-time PCR (qPCR) and Western blotting assays. There were significant differences among the four groups in terms of Arginase-1 (Arg1) (F = 1 457.00, P < 0.000 1), macrophages-derived C-C motif chemokine 22 (Ccl22) (F = 22 203.00, P < 0.000 1), resistin-like α (Retnla) (F = 151.90, P < 0.000 1), inducible nitric oxide synthase (iNOS) (F = 107.80, P < 0.001), hexokinase (HK) (F = 9 389.00, P < 0.000 1), pyruvate kinase (PK) (F = 641.40, P < 0.001), phosphofructokinase 1 (PFK1) (F = 43.97, P < 0.01), glucokinase (GK) (F = 432.50, P < 0.000 1), pyruvate dehydrogenase kinases1 (PDK1) (F = 737.30, P < 0.000 1), lactic dehydrogenase (LDH) (F = 3 632.00, P < 0.000 1), glucose transporter 1 (GLUT1) (F = 532.40, P < 0.000 1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (F = 460.00, P < 0.000 1), citrate synthase (CS) (F = 5 642.00, P < 0.01), glycogen synthase1 (GYS1) (F = 273.30, P < 0.000 1), IL-6 (F = 1 823.00, P < 0.000 1), IL-10 (F = 291.70, P < 0.000 1), IL-1β (F = 986.60, P < 0.000 1), and tumor necrosis factor (TNF)-α (F = 334.80, P < 0.000 1) and transforming growth factor (TGF)-β mRNA expression (F = 163.30, P < 0.001). The proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-PSC co-culture group [(22.87% ±1.48%) vs. (1.70% ±0.17%); t = 24.61, P < 0.001], and the proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-CF co-culture group [(20.07% ±0.64%) vs. (1.93% ±0.25%); t = 45.73, P < 0.001]. The mRNA expression of M2 macrophages markers Arg1, Ccl22 and Retnla was significantly higher in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01), and no significant difference was seen in the mRNA expression of the M1 macrophage marker iNOS among the three groups (P > 0.05), while qPCR assay quantified higher mRNA expression of key glycolytic enzymes HK, PK and PFK, as well as inflammatory factors IL-10, IL-1β, TNF-α and TGF-β in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01). Western blotting assay determined higher HK, PK and PFK protein expression in the BMDM-PSC co-culture group than in the control group (all P values < 0.05), and qPCR quantified higher GLUT1, GAPDH and IL-6 mRNA expression in the BMDM-CF co-culture group than in the control group (all P values < 0.05), while higher HK, PK and PFK protein and mRNA expression (all P values < 0.01), as well as lower IL-6 and TNF-α and higher TGF-β mRNA expression (both P values < 0.05) was detected in the IL-4 induction group than in the control group. Glycolytic stress test showed no significant difference in the extracellular acidification rate (ECAR) of mouse BMDM among the control group, IL-4 induction group and BMDM-PSC co-culture group (F = 124.4, P < 0.05), and a higher ECAR was seen in the BMDM-PSC co-culture group and a lower ECAR was found in the IL-4 induction group than in the control group (both P values < 0.05). Treatment of E. multilocularis CF or PSC mainly causes polarization of BMDM into M2 macrophages, and phenotypic transformation of glucose metabolism into high-energy and high-glycolytic metabolism, and affects inflammatory responses in BMDM.

MicroRNA-146a-loaded magnesium silicate nanospheres promote bone regeneration in an inflammatory microenvironment

Reconstruction of irregular oral-maxillofacial bone defects with an inflammatory microenvironment remains a challenge, as chronic local inflammation can largely impair bone healing. Here, we used magnesium silicate nanospheres (MSNs) to load microRNA-146a-5p (miR-146a) to fabricate a nanobiomaterial, MSN+miR-146a, which showed synergistic promoting effects on the osteogenic differentiation of human dental pulp stem cells (hDPSCs). In addition, miR-146a exhibited an anti-inflammatory effect on mouse bone marrow-derived macrophages (BMMs) under lipopolysaccharide (LPS) stimulation by inhibiting the NF-κB pathway via targeting tumor necrosis factor receptor-associated factor 6 (TRAF6), and MSNs could simultaneously promote M2 polarization of BMMs. MiR-146a was also found to inhibit osteoclast formation. Finally, the dual osteogenic-promoting and immunoregulatory effects of MSN+miR-146a were further validated in a stimulated infected mouse mandibular bone defect model via delivery by a photocuring hydrogel. Collectively, the MSN+miR-146a complex revealed good potential in treating inflammatory irregular oral-maxillofacial bone defects.

Myeloid-derived Wnts play an indispensible role in macrophage and fibroblast activation and kidney fibrosis.

Wnt/β-catenin signaling plays a pivotal role in the pathogenesis of chronic kidney diseases (CKD), which is associated with macrophage activation and polarization. However, the relative contribution of macrophage-derived Wnts in the evolution of CKD is poorly understood. Here we demonstrate a critical role of Wnts secreted by macrophages in regulating renal inflammation and fibrosis after various injuries. In mouse model of kidney fibrosis induced by unilateral ureteral obstruction (UUO), macrophages were activated and polarized to M1 and M2 subtypes, which coincided with the activation of Wnt/β-catenin signaling. In vitro, multiple Wnts were induced in primary cultured bone marrow-derived macrophages (BMDMs) after polarization. Conversely, Wnt proteins also stimulated the activation and polarization of BMDMs to M1 and M2 subtype. Blockade of Wnt secretion from macrophages in mice with myeloid-specific ablation of Wntless (Wls), a cargo receptor that is obligatory for Wnt trafficking and secretion, blunted macrophage infiltration and activation and inhibited the expression of inflammatory cytokines. Inhibition of Wnt secretion by macrophages also abolished β-catenin activation in tubular epithelium, repressed myofibroblast activation and reduced kidney fibrosis after either obstructive or ischemic injury. Furthermore, conditioned medium from Wls-deficient BMDMs exhibited less potency to stimulate fibroblast proliferation and activation, compared to the controls. These results underscore an indispensable role of macrophage-derived Wnts in promoting renal inflammation, fibroblasts activation and kidney fibrosis.

Tramadol suppresses growth of orthotopic liver tumors via promoting M1 macrophage polarization in the tumor microenvironment.

Tumor-associated macrophages (TAMs) are major infiltrating immune cells in liver cancer. They are polarized to anti-tumor M1 type or tumor-supporting M2 type in a dynamic changing state. Tramadol, a synthetic opioid, exhibits tumor-suppressing effect in several cancers, but whether it plays a role in TAMs polarization is uncertain. In the present study, the potential influence of tramadol on TAMs polarization was explored in liver cancer. An orthotopic murine Hepa 1-6 liver cancer model was constructed. The potential function of tramadol was evaluated by cell viability assay, EdU incorporation assay, flow cytometry, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) assay, T cell proliferation and suppression assays and western blot. We found that tramadol suppressed proliferation and tumor formation of murine Hepa 1-6 cells in vitro and in vivo. Tramadol reprogramed the immune microenvironment to favor M1 macrophage polarization in orthotopic Hepa 1-6 tumors. Moreover, tramadol facilitated M1 macrophage polarization and inhibited M2 macrophage polarization of bone marrow-derived macrophages (BMDMs) and human THP-1 macrophages in vitro. Furthermore, tramadol-treated BMDMs promoted proliferation and activation of splenic CD4+ and CD8+ T cells. Tramadol induced cellular ROS production and mitochondrial dysfunction of BMDMs. Finally, tramadol activated NF-κB signaling in BMDMs and THP-1 macrophages, while inhibition of NF-κB signaling by JSH-23 attenuated the influence of tramadol on macrophage polarization. In conclusion, these data elucidated a novel anti-tumor mechanism of tramadol in liver cancer. Tramadol might be a promising treatment strategy for liver cancer patients.

Irisin modulates bone marrow derived macrophage polarization towards M2 profile in high fat fed mice

Background and Aims: The chronic inflammatory process in the adipose tissue during obesity suggests constant polarization of classically activated macrophages (M1) and low polarization of alternately activated ones (M2). Since irisin has anti-inflammatory and antidiabetogenic properties our aim is to evaluate its effects on macrophage polarization in mice subjected to high fat diet. Methods and Results: C57Bl/6 mice were fed a high fat diet for 30 weeks. Bone marrow derived macrophages (BMDM) were isolated and incubated with 50 nM of irisin. Cellular phenotype, nitrite production, arginase-1 expression and cell viability were analyzed. Irisin was able to increase F480+CD206+ and F480+CD301+ cell number, and arginase1 expression. Irisin was not able to induce nitrite production and did not affect cell viability. Conclusion: Irisin was able to elicit an M2-like profile in macrophages in mice subjected to high fat diet, suggesting an anti-inflammatory action of this cytokine with potential therapeutic use in the control of imflammation during obesity.

Beneficial Role of Erythrocyte Sphingosine 1-Phosphate in Preventing Renal Macrophage Polarization and Fibrosis through Combating HIF-1α-Dependent Creatine Phosphate Shuttle

Introduction: Erythrocytes, the major reservoir and supplier for plasma sphingosine 1 phosphate (S1P), is a potent biolipid signaling via its specific receptors responsible for immune cell trafficking and function. Current evidence has revealed that erythrocyte intracellular S1P has its own function to promote oxygen (O 2) delivery to counteract tissue hypoxia. Aim: To understand whether O 2 delivery mediated by erythrocyte S1P is involved in tissue macrophage polarization and fibrosis. Methods and Results: Genetic ablation of erythrocyte SphK1 (the only enzyme to generate S1P in erythrocyte, eSphK1 -/-) resulted in less O 2 delivery from erythrocytes, thus leading to severe renal hypoxia, damage and fibrosis in angiotensin II induced hypertensive kidney disease mouse model. Surprisingly, although plasma S1P level was reduced to 50% in Ang II-infused eSphK1 -/- mice, these mice displayed preferentially elevated pro-fibrosis M2 macrophages surrounding the hypoxic renal tubules. Surprisingly, there was no significant difference of S1P level in either kidney or renal macrophages between Ang II-infused controls and eSphK1 -/- mice, ruling out the possibility of difference of kidney or macrophage S1P levels responsible for renal M2 macrophage polarization. Metabolically, untargeted high-throughput metabolic profiling in macrophages purified from mouse kidney revealed a substantial reduction of amino acids (AAs) and fatty acids (FAs) but increased creatine. We further validate this in vitro by 13C 6, 15N 4-labeled arginine tracer. Hypoxia (1~2% O 2) promoted increased 13C 2-labeled creatine (arginine downstream metabolite) in murine bone marrow derived macrophages (BMDMs) time dependently. At cellular and molecular level, we further demonstrated that hypoxia signaling via HIF-1a directly enhanced IL-4 induced M2 polarization of BMDMs as well as expression of Ckb (a gene encoding creatine shuttle), which can be reversed by Chrysin (an HIF-1α inhibitor). Preclinical studies revealed that chrysin rescued the severe renal damage in eSphK1 -/-mice manipulated with unilateral ureteral obstruction (UUO) and prevented development of chronic kidney disease (CKD) in the control mice. Translational research validated mouse findings in kidney tissue of CKD patients. Conclusion: Our work has revealed that erythrocyte S1P combats HIF1α-dependent creatine shuttle, renal macrophage polarization and fibrosis and highlighted potential diagnostic and therapeutic possibilities for macrophage polarization and renal fibrosis.

Abstract P3066: TSC2-mTOR Axis Exerts Biased Control Over Macrophage Infiltration Following Myocardial Infarction

Introduction: The mechanistic target of rapamycin complex 1 (mTORC1) is an evolutionarily conserved serine/threonine kinase, which regulates pivotal physiological processes. mTORC1 activity is tightly regulated upstream by the tuberous sclerosis complex (TSC) which consists of TSC1 and TSC2. In macrophages as throughout the immune system, mTORC1 is a potent regulator of cell activation and function. Myocardial infarction (MI) is associated with stimulation of mTORC1; however, studies vary as to whether this activation is beneficial or detrimental, and its influence on macrophage differentiation, and infiltration after MI is unknown. Hypothesis: We tested the hypothesis that cardiac infiltration of macrophages following MI is regulated by their expression of TSC2. Methods/Results: Macrophage-specific TSC2 knock-out mice (TSC2-LyzM) were made by crossing mice with floxed-TSC2 and LysM-Cre that display a significant reduction of macrophage TSC2 protein. TSC2-LyzM bone marrow-derived macrophages (BMDMs) have constitutive mTORC1 activation reflected by phosphorylation of downstream targets S6K1 and 4E-BP1 and were correspondingly larger. To assess macrophage polarization, cells were stimulated with LPS to promote an M1-like phenotype or IL-4 to induce an M2-like phenotype. LPS- treated TSC2-LyzM BMDMs secreted more of the proinflammatory cytokine TNF-a, while IL-4 stimulation resulted in less of an M2 program, with significantly less Arg1, Mrc1, and Rentla expression. These findings show TSC2 is required for normal BMDM polarization. We next tested if TSC2 impacts macrophage myocardial infiltration after MI. Macrophages were identified as CD45+CD11b+CD64+ and further parsed by CCR2 and MHC-II. In concordance with prior studies, we found substantial infiltration of CCR2+ (BMDMs) macrophages in both genotype groups after MI. However, more TSC2-LyzM had increased CCR2+ MHC-II+ phenotype (infiltrating macrophages) versus wild-type. Conclusion: Overall, our findings highlight a key role for the TSC2-mTORC1 pathway in regulating macrophage fate specification and cardiac infiltration following a MI. This supports novel signaling control over BMDMs by TSC2-phosphorylation mediated mTORC1 activation or inhibition.

Oncostatin M regulates macrophages polarization in osseointegration via yes-associated protein

ObjectivesOncostatin M(OSM), secreted by monocytes and macrophages, has been noted to participate in bone homeostasis and macrophage polarization, which might be regulated by yes-associated protein (YAP). This study aimed to elucidate the influence and mechanisms of OSM-YAP on macrophages polarization in osseointegration. Material and methodsIn vitro, flow cytometry, real-time PCR, and Elisa were performed to evaluate inflammatory function in bone marrow-derived macrophages (BMDMs) with OSM, siOSMR, and YAP inhibitor verteporfin (VP). In vivo, macrophage-specific YAP-deficient mice were generated to investigate the role of OSM via YAP signaling in osseointegration. ResultsThis study demonstrated that OSM could inhibit the M1 polarization, promote the M2 polarization, and induce the expression of osteogenic-related factors via VP. The conditional knock-out of YAP inhibited the osseointegration in mice, and promoted the inflammatory reaction around the implants, while OSM could restore the effect. ConclusionsOur results demonstrated that OSM might play an important role in the polarization of BMDMs, and bone formation around dental and femoral implants. This effect was closely conducted by Hippo-YAP pathway. Clinical SignificanceUnderstanding the role and mechanism of OSM in macrophage polarization around dental implants could improve comprehension of signal network of osseointegration, and it might offer a potential target of therapies to accelerate osseointegration and reduce inflammatory reactions.

Regulation of Macrophage Polarization on Chiral Potential Distribution of CFO/P(VDF-TrFE) Films

Surface potentials of biomaterials have been shown to regulate cell fate commitment. However, the effects of chirality-patterned potential distribution on macrophage polarization are still only beginning to be explored. In this work, we demonstrated that the chirality-patterned potential distribution of CoFe2O4/poly(vinylidene fluoride-trifluoroethylene) (CFO/P(VDF-TrFE)) films could significantly down-regulate the M1 polarization of bone marrow-derived macrophages (BMDMs). Specifically, the dextral-patterned surface potential distribution simultaneously up-regulated the expression of M2-related markers of BMDMs. The results were attributed to the sensitive difference of integrin subunits (α5β1 and αvβ3) to the dextral- and sinistral-patterned surface potential distribution, respectively. The interaction difference between the integrin subunits and surface potential distribution altered the cell adhesion and cytoskeletal structure and thereby the polarization behavior of BMDMs. This work, therefore, emphasizes the importance of chirality of potential distribution on cell behavior and provides a new strategy to regulate the immune response of biomaterials.

Abstract 5176: CDDO-methyl ester redirects macrophage polarization and reduces lung tumor burden in a Nrf2-dependent manner

Abstract The cytoprotective Nrf2 pathway impacts immune cell function and has been proposed as a target for many inflammation-related diseases, but the effects of Nrf2 activation on immune cells in the cancer context are not well characterized. With Nrf2 activators under development and in clinical trials, it is critical to understand the influence of these drugs on cancer progression. While the anti-inflammatory nature of Nrf2 activation protects healthy cells from malignant transformation, cancer cells can utilize the pathway to promote resistance to anti-cancer drugs and increase tumor cell survival. Up to 30% of human lung adenocarcinomas acquire mutations in the Nrf2 pathway which result in constitutive activation. However, triterpenoids, including CDDO-methyl ester (CDDO-Me, also known as bardoxolone methyl), are potent pharmacological Nrf2 activators with demonstrated anti-cancer activity in preclinical models. Lung cancer is the leading cause of cancer-related mortality worldwide, and macrophages are the most common immune cell type in the lung tumor microenvironment. To investigate Nrf2 activation in macrophages in the context of lung cancer, bone marrow-derived macrophages (BMDMs) were isolated from wild type (WT) and Nrf2 knock out (KO) mice and cultured in conditioned media from lung cancer cells to produce a tumor-educated phenotype. The triterpenoid CDDO-Me had anti-inflammatory effects in BMDMs stimulated with the conventional cytokines IFN-γ and LPS. Conversely, CDDO-Me increased (p &amp;lt; 0.05) the M1 macrophage markers TNFα, IL-6, and MHC-II and decreased the M2 macrophage markers VEGF, CCL2, and CD206 in tumor-educated BMDMs in a Nrf2-dependent manner. The phenotypic changes were observed on transcription, protein, and surface marker levels. This context-dependent reversal of BMDM polarization suggests that Nrf2 activation has different outcomes in different environments. To test CDDO-Me in vivo, lung tumors were initiated with vinyl carbamate in A/J WT and Nrf2 KO mice, and animals were fed either control diet or CDDO-Me (12.5-50 mg/kg of diet) for 16 weeks. CDDO-Me significantly (p &amp;lt; 0.05) decreased tumor number, size, and overall burden and reduced the histopathological severity of tumors in a Nrf2- and dose-dependent manner. Additionally, CDDO-Me increased the infiltration of CD64+ macrophages but decreased CD206+ expression in macrophages in the lungs of WT tumor-bearing mice. Interestingly, CD206 expression was higher on macrophages in the spleen of WT mice treated with CDDO-Me, suggesting a cancer context dependency. Future studies will evaluate the dependency of Nrf2 activation in macrophages for the anti-tumor activity of CDDO-Me. Citation Format: Jess Ann Moerland, Karen T. Liby. CDDO-methyl ester redirects macrophage polarization and reduces lung tumor burden in a Nrf2-dependent manner. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5176.

Effects of porcine urinary bladder matrix on motility and polarization of bone marrow-derived macrophages in mice

Objective: To explore the effects of porcine urinary bladder matrix (UBM) on the motility and polarization of bone marrow-derived macrophages in mice, so as to provide evidence for the rational selection of stent in clinical wound repair. Methods: The method of experimental research was used. The microstructure of porcine UBM and absorbable dressing was observed under scanning electron microscope. Polyacrylamide gel electrophoresis was used to observe the protein distribution of the two stent extracts. The primary macrophages were induced from bone marrow-derived cells isolated from six 6-8-week-old male C57BL/6J mice (mouse age, sex, and strain, the same below) and identified. Three batches of macrophages were divided into porcine UBM extract group and absorbable dressing extract group. The cells in each group were cultured with Dulbecco's modified Eagle medium/F12 medium containing the corresponding extracts. The cell migration rate was detected and calculated on 1, 3, and 7 d after scratching by scratch test. The number of migrated cells at 12 and 24 h of culture was detected by Transwell experiment. The percentages of CD206 and CD86 positive cells at 24 h of culture was detected by flow cytometer. The numbers of sample in the above cell experiments were all 3. An incision was prepared on the left and right back of twelve mice, respectively. The left incision of each mouse was included in porcine UBM group and the right incision was included in absorbable dressing group, and the corresponding stents were implanted into the incisions respectively. On post operation day (POD) 7 and 14, the number of inflammatory cells infiltrated in the stent was detected by hematoxylin-eosin staining; the number of F4/80, transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), and matrix metalloprotein-9 (MMP-9) positive cells and type Ⅰ collagen deposition in stents were observed by immunohistochemistry; the percentages of F4/80, CD86, and CD206 positive cells were observed by immunofluorescence staining. The numbers of sample in the above animal experiments were all 6. Data were statistically analyzed with analysis of variance for factorial design, analysis of variance for repeated measurement, and independent sample t test. Results: Porcine UBM has a dense basement membrane structure on one side and porous propria containing a fibrous structures on the other. Both sides of the absorbable dressing had three-dimensional porous structure. In the molecular weight range of (50-70)×103, multiple non-type Ⅰ collagen bands appeared in the lanes of porcine UBM extract, while no obvious bands appeared in the lanes of absorbable dressing extract. It had been identified that mouse bone marrow-derived cells had been successfully induced into macrophages. The cell migration rates in porcine UBM extract group were significantly higher than those in absorbable dressing extract group on 1, 3, and 7 d after scratching (with t values of 15.31, 19.76, and 20.58, respectively, P<0.05). The numbers of migrated cells in porcine UBM extract group were significantly more than those in absorbable dressing extract group at 12 and 24 h of culture (with t values of 12.20 and 33.26, respectively, P<0.05). At 24 h of culture, the percentage of CD86 positive cells in porcine UBM extract group ((1.27±0.19)%) was significantly lower than (7.34±0.14)% in absorbable dressing extract group (t=17.03, P<0.05);the percentage of CD206 positive cells in porcine UBM extract group was (73.4±0.7)%, significantly higher than (32.2±0.5)% in absorbable dressing extract group (t=119.10, P<0.05). On POD 7 and 14, the numbers of inflammatory cells infiltrated in the stents in porcine UBM group was significantly more than those in absorbable dressing group (with t values of 6.58 and 10.70, respectively, P<0.05). On POD 7 and 14, the numbers of F4/80, TGF-β1, VEGF, and MMP-9 positive cells in the stents in porcine UBM group were significantly more than those in absorbable dressing group (with t values of 46.11, 40.69, 13.90, 14.15, 19.79, 32.93, 12.16, and 13.21, respectively, P<0.05); type Ⅰ collagen deposition in the stents in porcine UBM group was more pronounced than that in absorbable dressing group; the percentages of CD206 positive cells in the stents in porcine UBM group were significantly higher than those in absorbable dressing group (with t values of 5.05 and 4.13, respectively, P<0.05), while the percentages of CD86 positive cells were significantly lower than those in absorbable dressing group (with t values of 20.90 and 19.64, respectively, P<0.05), and more M2-type macrophages were seen in the stents in porcine UBM group and more M1-type macrophages were seen in the stents in absorbable dressing group. Conclusions: Porcine UBM can enhance macrophage motility, induce M2 polarization and paracrine function, create a microenvironment containing growth factors such as TGF-β1 and MMP-9 tissue remodeling molecules, and promote tissue regeneration and extracellular matrix remodeling in mice.

Photobiomodulation promotes spinal cord injury repair by inhibiting macrophage polarization through lncRNA TUG1-miR-1192/TLR3 axis

BackgroundSecondary spinal cord injury (SCI) often causes the aggravation of inflammatory reaction and nerve injury, which affects the recovery of motor function. Bone-marrow-derived macrophages (BMDMs) were recruited to the injured area after SCI, and the M1 polarization is the key process for inducing inflammatory response and neuronal apoptosis. We previously showed that photobiomodulation (PBM) can inhibit the polarization of M1 phenotype of BMDMs and reduce inflammation, but the underlying mechanisms are unclear. The purpose of this study is to explore the potential target and mechanism of PBM in treating SCI.MethodsTranscriptome sequencing and bioinformatics analysis showed that long noncoding RNA taurine upregulated gene 1 (lncRNA TUG1) was a potential target of PBM. The expression and specific mechanism of lncRNA TUG1 were detected by qPCR, immunofluorescence, flow cytometry, western blotting, fluorescence in situ hybridization, and luciferase assay. The Basso mouse scale (BMS) and gait analysis were used to evaluate the recovery of motor function in mice.ResultsResults showed that lncRNA TUG1 may be a potential target of PBM, regulating the polarization of BMDMs, inflammatory response, and the axial growth of DRG. Mechanistically, TUG1 competed with TLR3 for binding to miR-1192 and attenuated the inhibitory effect of miR-1192 on TLR3. This effect protected TLR3 from degradation, enabling the high expression of TLR3, which promoted the activation of downstream NF-κB signal and the release of inflammatory cytokines. In vivo, PBM treatment could reduce the expression of TUG1, TLR3, and inflammatory cytokines and promoted nerve survival and motor function recovery in SCI mice.ConclusionsOur study clarified that the lncRNA TUG1/miR-1192/TLR3 axis is an important pathway for PBM to inhibit M1 macrophage polarization and inflammation, which provides theoretical support for its clinical application in patients with SCI.

Tumor cell-derived conditioned medium induced pro-tumoral phenotypes in macrophages through calcium-nuclear factor κB interaction

BackgroundThe malignant behaviors of lung cancers are affected by not only cancer cells but also many kinds of stromal cells in tumor microenvironment (TME), including macrophages. Macrophages have been proven to extensively influence tumor progression through several mechanisms, among which switching of macrophages from pro-inflammatory phenotypes (M1-like) to anti-inflammatory phenotypes (M2-like) mediated by transcription factors such as nuclear factor κB (NF-κB) is the most crucial event. The regulation of NF-κB has been well studied, however some details remain fuzzy.MethodsMouse primary bone marrow-derived macrophages (BMDMs) were cultured in Lewis lung carcinoma cell line LL-2-derived conditioned medium (LL-2-CM). Proliferation, migration, and polarization of BMDMs were tested by CCK8, scratch test, transwell, and flow cytometry. Secretion of several cytokines were detected by ELISA or cytometric bead array. To further explore the underlying mechanisms, BMDMs cultured in LL-2-CM were harvested for RNA-seq. Cytosolic calcium was detected by calcium probe Fluo-4-AM. Western blot was applied to exam the activation of NF-κB signal. BAPTA-AM was applied to sequestrate cytosolic calcium to further investigate the relationship between calcium and NF-κB signal. The polarization, calcium alteration, and NF-κB signal activation were further validated in BMDMs treated by CMT-64-derived conditioned medium (CMT-64-CM).ResultsLL-2-CM promoted proliferation, migration, and M2-like polarization of BMDMs and inhibited M1-like polarization of BMDMs. However two pro-inflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor-mathrm{alpha } (TNF-mathrm{alpha }) were secreted. RNA-seq indicated that LL-2-CM activated both canonical and non-canonical NF-κB signal in BMDMs. Western blot showed that canonical NF-κB was temporarily elicited and attenuated at 24 h, while non-canonical NF-κB was consistently activated. At the same time, expression of genes that regulate cytosolic calcium ion concentration were down regulated, which caused diminution of cytosolic calcium in BMDMs treated with LL-2-CM. The decreased cytosolic calcium, M2-like polarization, and NF-κB activation was also observed in CMT-64-CM treated BMDMs. On the contrary, elevated cytosolic calcium was observed during M1-like polarization of BMDMs elicited by lipopolysaccharide (LPS). Interestingly, administration of calcium chelator, BAPTA-AM, impeded activation of canonical NF-κB and expression of M1-like marker induced by LPS, which further confirmed the relationship between cytosolic calcium and canonical NF-κB signal.ConclusionsIn summary, lung cancer cell-derived conditioned medium promoted migration, proliferation, and M2-like polarization of BMDMs. The suppressed M1-like polarization was achieved through mitigating canonical NF-κB pathway via diminishing cytosolic calcium concentration. As far as we know, our work firstly revealed that cytosolic calcium is the key during inhibition of canonical NF-κB and M1-like polarization in macrophages by tumor cells.

Protein phosphatase 2A regulates xanthine oxidase-derived ROS production in macrophages and influx of inflammatory monocytes in a murine gout model.

Background: Gout is a common arthritis, due to deposition of monosodium urate (MSU) crystals which results in IL-1β secretion by tissue-resident macrophages. Xanthine oxidase (XO) catalyzes uric acid (UA) production and in the process, reactive oxygen species (ROS) are generated which contributes to NLRP3 inflammasome activation. Protein phosphatase 2A (PP2A) may be involved in regulating inflammatory pathways in macrophages. The objective of this study was to investigate whether PP2A regulates gout inflammation, mediated by XO activity modulation. We studied UA and ROS generations in MSU stimulated murine bone marrow derived macrophages (BMDMs) in response to fingolimod phosphate, a PP2A activator, and compared its anti-inflammatory efficacy to that of an XO inhibitor, febuxostat. Methods: BMDMs were stimulated with MSU, GM-CSF/IL-1β or nigericin ± fingolimod (2.5μM) or febuxostat (200μM) and UA levels, ROS, XO, and PP2A activities, Xdh (XO) expression and secreted IL-1β levels were determined. PP2A activity and IL-1β in MSU stimulated BMDMs ± N-acetylcysteine (NAC) (10μM) ± okadaic acid (a PP2A inhibitor) were also determined. M1 polarization of BMDMs in response to MSU ± fingolimod treatment was assessed by a combination of iNOS expression and multiplex cytokine assay. The in vivo efficacy of fingolimod was assessed in a murine peritoneal model of acute gout where peritoneal lavages were studied for pro-inflammatory classical monocytes (CMs), anti-inflammatory nonclassical monocytes (NCMs) and neutrophils by flow cytometry and IL-1β by ELISA. Results: Fingolimod reduced intracellular and secreted UA levels (p < 0.05), Xdh expression (p < 0.001), XO activity (p < 0.001), ROS generation (p < 0.0001) and IL-1β secretion (p < 0.0001), whereas febuxostat enhanced PP2A activity (p < 0.05). NAC treatment enhanced PP2A activity and reduced XO activity and PP2A restoration mediated NAC's efficacy as co-treatment with okadaic acid increased IL-1β secretion (p < 0.05). Nigericin activated caspase-1 and reduced PP2A activity (p < 0.001) and fingolimod reduced caspase-1 activity in BMDMs (p < 0.001). Fingolimod reduced iNOS expression (p < 0.0001) and secretion of IL-6 and TNF-α (p < 0.05). Fingolimod reduced CMs (p < 0.0001), neutrophil (p < 0.001) and IL-1β (p < 0.05) lavage levels while increasing NCMs (p < 0.001). Conclusion: Macrophage PP2A is inactivated in acute gout by ROS and a PP2A activator exhibited a broad anti-inflammatory effect in acute gout in vitro and in vivo.