Levels Of M2 Markers Research Articles

Abstract 2722: Reprogramming of tumor-associated macrophages to M1-like macrophages using M1 macrophage-derived exosomes engineered to target IL-4 receptor and enhance M1 polarization

Abstract Tumor-associated macrophages (TAMs) are M2-polarized protumoral macrophages, whereas inflammatory macrophages are M1-polarized antitumoral macrophages. Interleukin-4 receptor (IL-4R) is expressed in M2 macrophages at higher levels than M1 macrophages. In this study, we aimed to inhibit tumor growth using M1 macrophage-derived exosomes engineered to target IL-4R and reprogram TAMs into M1 macrophages. M1 exosomes were transfected with NF-κB p50 siRNA and miR-511-3p as cargoes to enhance M1 polarization (Exo-si/mi) and surface-modified with IL4RPep-1, an IL-4R-binding peptide, to target IL-4R of M2 macrophages (IL4R-Exo-si/mi). The engineered exosomes showed sizes similar with parental exosomes, and IL4RPep-1 was retained on the exosomes and not degraded in the presence of serum up to 24 h. IL-4R-targeted exosomes were internalized into M2 macrophages and delivered cargoes through IL-4R more efficiently than untargeted exosomes. Whole-body fluorescence imaging showed that IL-4R-targeted exosomes homed to tumors at higher levels than to liver unlike to untargeted exosomes. Intravenous administration of IL4R-Exo-si/mi decreased the levels of M2 markers and immune-suppressive cells including TAMs, while increasing the levels of M1 markers and immune-stimulatory cells including M1 macrophages in tumor and spleen and subsequently inhibited tumor growth more efficiently than Exo-si/mi as well as M1 exosomes. These results suggest that IL-R-targeted exosomes carrying NF-κB p50 siRNA and miR-511-3p inhibit tumor growth by reprogramming TAMs into M1 macrophages and increasing anti-tumor immunity and hold a potential for caner immunotherapy. Citation Format: Byungheon Lee, Gowri Rangaswamy Gunassekaran. Reprogramming of tumor-associated macrophages to M1-like macrophages using M1 macrophage-derived exosomes engineered to target IL-4 receptor and enhance M1 polarization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2722.

TSC1 Affects the Process of Renal Ischemia-ReperfusionInjury by Controlling Macrophage Polarization.

Renal ischemia-reperfusion injury (IRI) contributes to acute kidney injury (AKI), increases morbidity and mortality, and is a significant risk factor for chronic kidney disease (CKD). Macrophage infiltration is a common feature after renal IRI, and infiltrating macrophages can be polarized into the following two distinct types: M1 macrophages, i.e., classically activated macrophages, which can not only inhibit infection but also accelerate renal injury, and M2 macrophages, i.e., alternatively activated macrophages, which have a repair phenotype that can promote wound healing and subsequent fibrosis. The role of TSC1, which is a negative regulator of mTOR signaling that regulates macrophage polarization in inflammation-linked diseases, has been well documented, but whether TSC1 contributes to macrophage polarization in the process of IRI is still unknown. Here, by using a mouse model of renal ischemia-reperfusion, we found that myeloid cell-specific TSC1 knockout mice (termed Lyz-TSC1 cKO mice) had higher serum creatinine levels, more severe histological damage, and greater proinflammatory cytokine production than wild-type (WT) mice during the early phase after renal ischemia-reperfusion. Furthermore, the Lyz-TSC1 cKO mice showed attenuated renal fibrosis during the repair phase of IRI with decreased levels of M2 markers on macrophages in the operated kidneys, which was further confirmed in a cell model of hypoxia-reoxygenation (H/R) in vitro. Mechanistically, by using RNA sequencing of sorted renal macrophages, we found that the expression of most M1-related genes was upregulated in the Lyz-TSC1 cKO group (Supplemental Table 1) during the early phase. However, C/EBPβ and CD206 expression was decreased during the repair phase compared to in the WT group. Overall, our findings demonstrate that the expression of TSC1 in macrophages contributes to the whole process of IRI but serves as an inflammation suppressor during the early phase and a fibrosis promoter during the repair phase.

Ferumoxytol-β-glucan Inhibits Melanoma Growth via Interacting with Dectin-1 to Polarize Macrophages into M1 Phenotype.

Background: Regulating the polarization of macrophages to antitumor M1 macrophages is a promising strategy for overcoming the immunosuppression of the tumor microenvironment for cancer therapy. Ferumoxytol (FMT) can not only serve as a drug deliver agent but also exerts anti-tumor activity. β-glucan has immuno-modulating properties to prevent tumor growth. Thus, a nanocomposite of FMT surface-coated with β-glucan (FMT-β-glucan) was prepared to explore its effect on tumor suppression.Methods: Male B16F10 melanoma mouse model was established to explore the antitumor effect of FMT-β-glucan. The viability and apoptotic rates of B16F10 cells were detected by cell counting kit-8 and Annexin-V/PI experiments. The levels of M1 markers were quantified by quantitative reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay. Phagocytic activity and intracellular reactive oxygen species (ROS) in macrophages were evaluated by the neutral red uptake assay and flow cytometry, respectively. Small interfering RNA (siRNA) transfection was applied to knock down the Dectin-1 gene in RAW 264.7 cells.Results: FMT-β-glucan suppressed tumor growth to a greater extent and induced higher infiltration of M1 macrophages than the combination of FMT and β-glucan (FMT+β-glucan) in vivo. In vitro, supernatant from FMT-β-glucan-treated RAW 264.7 cells led to lower cell viability and induced more apoptosis of B16F10 cells than that from the FMT+β-glucan group. Moreover, FMT-β-glucan boosted the expression of M1 type markers, and increased phagocytic activity and ROS in RAW 264.7 cells. Further research indicated that FMT-β-glucan treatment promoted the level of Dectin-1 on the surface of RAW 264.7 cells and that knockdown of Dectin-1 abrogated the phosphorylation levels of several components in MAPK and NF-κB signaling.Conclusion: The nanocomposite FMT-β-glucan suppressed melanoma growth by inducing the M1 macrophage-activated tumor microenvironment.

Thrombomodulin facilitates peripheral nerve regeneration through regulating M1/M2 switching

BackgroundExcessive inflammation within damaged tissue usually leads to delayed or insufficient regeneration, and nerves in the peripheral nervous system (PNS) generally do not recover fully following damage. Consequently, there is growing interest in whether modulation of the inflammatory response could help to promote nerve regeneration in the PNS. However, to date, there are no practical therapeutic strategies for manipulating inflammation after nerve injury. Thrombomodulin (TM) is a transmembrane glycoprotein containing five domains. The lectin-like domain of TM has the ability to suppress the inflammatory response. However, whether TM can modulate inflammation in the PNS during nerve regeneration has yet to be elucidated.MethodsWe investigated the role of TM in switching proinflammatory type 1 macrophages (M1) to anti-inflammatory type 2 macrophages (M2) in a human monocytic cell line (THP-1) and evaluated the therapeutic application of TM in transected sciatic nerve injury in rats.ResultsThe administration of TM during M1 induction significantly reduced the expression levels of inflammatory cytokines, including TNF-a (p < 0.05), IL-6 (p < 0.05), and CD86 (p < 0.05), in THP-1 cells. Simultaneously, the expression levels of M2 markers, including IL-10 (p < 0.05) and CD206 (p < 0.05), were significantly increased in TM-treated THP-1 cells. Inhibition of IL-4R-c-Myc-pSTAT6-PPARγ signaling abolished the expression levels of IL-10 (p < 0.05) and CD206 (p < 0.05). The conditioned medium (CM) collected from M1 cells triggered an inflammatory response in primary Schwann cells, while CM collected from M1 cells treated with TM resulted in a dose-dependent reduction in inflammation. TM treatment led to better nerve regeneration when tested 6 weeks after injury and preserved effector muscle function. In addition, TM treatment reduced macrophage infiltration at the site of injury and led to potent M1 to M2 transition, thus indicating the anti-inflammatory capacity of TM.ConclusionsCollectively, our findings demonstrate the anti-inflammatory role of TM during nerve regeneration. Therefore, TM represents a potential drug for the promotion and modulation of functional recovery in peripheral nerves that acts by regulating the M1/M2 ratio.

Knockdown of TLR4 Represses the Paraquat-Induced Neuroinflammation and Microglial M1 Polarization.

Paraquat (PQ) is associated with multiple nervous system disorders including Parkinson's disease. Despite the evidence that PQ could induce inflammatory responses in the central nervous system and largely contribute to neurotoxicity, the mechanisms of PQ-induced neuroinflammation are not yet fully understood. Toll-like receptor 4 (TLR4) could recognize various pathogens and initiate inflammation processes. Therefore, we investigated the role of TLR4 in PQ-induced neuroinflammation by using murine microglial immortalized BV-2 cell line. Normal microglia and TLR4-knockdown microglia were treated with PQ to evaluate signal transduction molecular expression, inflammatory responses, and microglial functions. Compared with normal microglia, PQ-induced production of pro-inflammatory cytokines was significantly reduced in TLR4-knockdown microglia. Levels of M1 markers were decreased, while levels of M2 markers were increased upon PQ exposure, confirming that TLR4 depletion inhibited the microglial M1 polarization. Besides, the migration and phagocytosis capability reduced by PQ were to some extent recovered in TLR4-knockdown microglia. Taken together, our results suggested that TLR4 mediated the neuroinflammatory responses in microglia and the depletion of TLR4 protects against PQ neurotoxicity.

Inhibition of TLR4 Induces M2 Microglial Polarization and Provides Neuroprotection via the NLRP3 Inflammasome in Alzheimer's Disease.

Accumulating evidence has indicated that activation of microglia and neuroinflammation reaction play a prominent role in Alzheimer’s disease (AD). Inhibition of toll-like receptor 4 (TLR4) has been shown to be associated with immune responses and brain damage, but its effects on AD remain unclear. This study mainly aimed to investigate the protective effect of TAK-242 (TLR4-specific inhibitor) on microglial polarization and neuroprotection in an AD mouse model and the underlying mechanisms. We found that APP/PS1 transgenic AD mice exhibited a dramatic increase in TLR4 levels concomitant with a significantly higher expression of inflammatory microglia compared to C57BL/6 wild-type mice. Furthermore, inhibition of TLR4 by TAK-242 administration significantly improved neurological function, decreased the level of Bax, and caused a significant reduction in the levels of M1-markers (iNOS and TNFα), while the expressions of M2-phenotype markers (Trem-2 and Arg-1) were increased both in vivo and in vitro. Furthermore, TAK-242 treatment enhanced BV2 microglial phagocytosis. Moreover, Aβ25–35 caused the upregulation of inflammatory cytokine production, MyD88, NF-kappaB-p65, and NLRP3, which could be ameliorated by NLRP3-siRNA or TAK-242. These findings indicated that TLR4 inhibition provided neuroprotection and promoted a microglial switch from the inflammatory M1 phenotype to the protective M2 phenotype in AD. The mechanism involved may be related to modulation of the MyD88/NF-kappaB/NLRP3 signaling pathway.

Resveratrol prevents ISO-induced myocardial remodeling associated with regulating polarization of macrophages through VEGF-B/AMPK/NF-kB pathway

Macrophage expansion and inflammatory responses are involved in induction of cardiac remodeling. Resveratrol has strong anti-inflammatory effects, however its effect on macrophage infiltration and polarization is unknown. This study aimed to investigate the anti-inflammatory effects of RSV on ISO-induced myocardial remodeling in mice and its regulatory role in macrophage polarization. BALB/c mice were orally administered with RSV (100 mg/kg) daily for one week, then were subcutaneously injected with ISO (50 mg/kg) daily for another week. ISO injections to mouse caused cardiac dysfunction evidenced by cardiac hypertrophy and cardiomyocyte fibrosis. Meanwhile, macrophage M1 polarization was found in ISO treated mice, which was evidenced by increased percentage of Ly6Clow macrophages in the heart, levels of M1 cytokines and expression of CD68, and decreased percentage of Ly6Chigh macrophage, levels of M2 cytokines and expression of CD206. All these changes in cardiac and macrophage M1 polarization were ameliorated when mice were pretreated with RSV. The effect of RSV on macrophage polarization was also tested in RAW264.7 cells. It was found that pre-treatment with RSV decreased the levels of M1 marker or proinflammatory cytokines, while increased the levels of M2 markers in ISO treated cells. In addition, it was found that RSV could upregulate the expression of VEGF-B and the activity of AMPK, while it downregulated the expression of phosphorylated NF-κB p65 both in RAW264.7 cells and in mice. Furthermore, pretreatment with VEGF-B siRNA greatly reversed changes in almost all above parameters evoked by RSV in RAW264.7 cells. Therefore, our findings suggest RSV has potential therapeutic effects in ISO-induced myocardial injury, which may be by inhibiting the M1 polarization of macrophages through VEGFB/AMPK/NF-кB pathway.

Riluzole improves functional recovery after acute spinal cord injury in rats and may be associated with changes in spinal microglia/macrophages polarization

Spinal cord injury (SCI) triggers pronounced inflammatory responses that are accompanied by neuronal disruption and functional deficits. SCI treatment remains an unmet clinical need. Emerging evidence suggests that riluzole may exert a neuroprotective effect due to its anti-inflammatory properties. However, details of the underlying mechanisms remain poorly defined. The polarization of microglial/macrophages has an important role in neuroinflammation. Here, we examined whether riluzole can exert a neuroprotective effect after acute SCI, and whether this effect is associated with changes in microglia/macrophages polarization. Riluzole (4 mg/kg) or vehicle were injected intraperitoneally (i.p.) in female rats immediately following SCI and repeated for 7 consecutive days (b.i.d.). Compared with vehicle treatment, riluzole-treated SCI rats showed significant higher locomotor scores (Basso, Beattie, and Bresnahan score, Inclined Plane test score, n = 18/group). Riluzole-treated rats also developed smaller spinal cavities, showed higher levels of myelin basic protein (MBP) and neurofilament (NF)200 immunoreactivities, and lower levels of proinflammatory cytokines in the spinal cord at 7 days post-SCI. Immunofluorescence study revealed more CD206+ cells and less iNOS+ cells in the injured spinal cord of riluzole-treated SCI rats, as compared to vehicle control. Using real-time PCR, we found that riluzole upregulated the mRNA levels of M2 markers, but downregulated that of M1 markers, as compared to the vehicle treatment. Current findings suggest that systemic administration of riluzole after acute SCI facilitated motor function recovery and inhibited inflammatory responses, which may be associated with polarization of M2 microglia/macrophages.

Inhibition of inflammation-mediated DPP-4 expression by linagliptin increases M2 macrophages in atherosclerotic lesions

Background and aimsDipeptidyl peptidase-4 (DPP-4) inhibitors have been reported to suppress atherosclerosis progression in atherosclerotic mouse models through unclear mechanisms. In this study, we investigated the effect of the DPP-4 inhibitor, linagliptin, on macrophage polarization in vitro and in vivo. MethodsMouse bone marrow macrophages (BMMs) were used in in vitro assays. High fat diet (HFD)-fed Apoe−/− mice were treated orally with linagliptin (10 mg/kg−1•day−1) or a vehicle (water) control. ResultsIn in vitro assays using BMMs, treatment with LPS and IFNγ decreased the mRNA-expression levels of alternatively activated macrophage (M2) markers, and linagliptin treatment prevented these reductions. The mRNA levels of M2 markers and the number of M2 macrophages in the aorta were higher in linagliptin groups than in control groups. Linagliptin decreased the size of atherosclerotic lesions in HFD-fed Apoe−/− mice. Interestingly, inflammatory stimulation increased DPP-4 expression, and linagliptin suppressed these effects in BMMs. Treatment with DPP-4 small-interfering RNA (siRNA) reproduced linagliptin-mediated alteration of M2 polarization. ConclusionsLinagliptin increased M2 macrophage polarization by inhibiting DPP-4 expression and activity. These findings may indicate the beneficial effects of DPP-4 inhibitors on the progression of diabetic macrovascular complications.

Scavenging reactive oxygen species selectively inhibits M2 macrophage polarization and their pro-tumorigenic function in part, via Stat3 suppression

Tumor associated macrophages (TAM) enhance the aggressiveness of breast cancer via promoting cancer cell growth, metastasis, and suppression of the patient's immune system. These TAMs are polarized in breast cancer with features more closely resembling the pro-tumorigenic and immunosuppressive M2 type rather than the anti-tumor and pro-inflammatory M1 type. The goal of our study was to examine primary human monocyte-derived M1 and M2 macrophages for key redox differences and determine sensitivities of these macrophages to the redox-active drug, MnTE-2-PyP5+. This compound reduced levels of M2 markers and inhibited their ability to promote cancer cell growth and suppress T cell activation. The surface levels of the T cell suppressing molecule, PD-L2, were reduced by MnTE-2-PyP5+ in a dose-dependent manner. This study also examined key differences in ROS generation and scavenging between M1 and M2 macrophages. Our results indicate that M2 macrophages have lower levels of reactive oxygen species (ROS) and lower production of extracellular hydrogen peroxide compared to the M1 macrophages. These differences are due in part to reduced expression levels of pro-oxidants, Nox2, Nox5, and the non-enzymatic members of the Nox complex, p22phox and p47phox, as well as higher levels of antioxidant enzymes, Cu/ZnSOD, Gpx1, and catalase. More importantly, we found that despite having lower ROS levels, M2 macrophages require ROS for proper polarization, as addition of hydrogen peroxide increased M2 markers. These TAM-like macrophages are also more sensitive to the ROS modulator and a pan-Nox inhibitor. Both MnTE-2-PyP5+ and DPI inhibited expression levels of M2 marker genes. We have further shown that this inhibition was partly mediated through a decrease in Stat3 activation during IL4-induced M2 polarization. Overall, this study reveals key redox differences between M1 and M2 primary human macrophages and that redox-active drugs can be used to inhibit the pro-tumor and immunosuppressive phenotype of TAM-like M2 macrophages. This study also provides rationale for combining MnTE-2-PyP5+ with immunotherapies.

Toll-Like Receptor 2-Mediated Autophagy Promotes Microglial Cell Death by Modulating the Microglial M1/M2 Phenotype.

Toll-like receptor 2 (TLR2) regulates the innate immune response of microglia during infection via autophagy. Microglial M1/M2 phenotypic switching after infection could serve as a novel pathogenic mechanism for cerebral infection. Hence, it has important implications for the damage and restoration of neurological function. However, the effect of TLR2-mediated autophagic signaling on microglial phenotypic transition remains unclear. Therefore, we investigated the mechanisms of TLR2-mediated autophagic signaling in the regulation of microglial M1/M2 phenotypes. Using Western blot analysis and immunofluorescence, increased autophagy was observed in peptidoglycan (PGN)-stimulated BV2 cells, while reduced autophagy was observed in TLR2-KO cells. In contrast to the TLR2 antagonist CU-CPT22 group, increased autophagy was observed in the presence of the TLR2 agonist Pam3CSK4, which was associated with a significant increase in expression levels of M1 phenotype biomarkers (CD86, TNF-α, IL-6), higher levels of apoptosis, and decreased expression levels of M2 markers (CD206, IL-10, Arg-1). In the TLR2-KO mice, the expression levels of autophagy-related proteins in CD11b+ cells were lower than those in CD11b+ cells in the PGN-injected wild-type mice, and neuronal apoptosis was also reduced, but there were no significant differences compared to the control group. Collectively, our study demonstrates that the inhibition of autophagy or the absence of TLR2 induces microglial polarization towards the M2 phenotype, promotes microglial survival alone, and alleviates the development of neuroinflammation. In summary, TLR2-mediated autophagic signaling contributes to regulating the inflammatory response to activate microglial M1/M2 switching, which affects microglial survival after infection.

Hyperglycemia‐Triggered Sphingosine‐1‐Phosphate and Sphingosine‐1‐Phosphate Receptor 3 Signaling Worsens Liver Ischemia/Reperfusion Injury by Regulating M1/M2 Polarization

Hyperglycemia aggravates hepatic ischemia/reperfusion injury (IRI), but the underlying mechanism for the aggravation remains elusive. Sphingosine‐1‐phosphate (S1P) and sphingosine‐1‐phosphate receptors (S1PRs) have been implicated in metabolic and inflammatory diseases. Here, we discuss whether and how S1P/S1PRs are involved in hyperglycemia‐related liver IRI. For our in vivo experiment, we enrolled diabetic patients with benign hepatic disease who had liver resection, and we used streptozotocin (STZ)–induced hyperglycemic mice or normal mice to establish a liver IRI model. In vitro bone marrow–derived macrophages (BMDMs) were differentiated in high‐glucose (HG; 30 mM) or low‐glucose (LG; 5 mM) conditions for 7 days. The expression of S1P/S1PRs was analyzed in the liver and BMDMs. We investigated the functional and molecular mechanisms by which S1P/S1PRs may influence hyperglycemia‐related liver IRI. S1P levels were higher in liver tissues from patients with diabetes mellitus and mice with STZ‐induced diabetes. S1PR3, but not S1PR1 or S1PR2, was activated in liver tissues and Kupffer cells under hyperglycemic conditions. The S1PR3 antagonist CAY10444 attenuated hyperglycemia‐related liver IRI based on hepatic biochemistry, histology, and inflammatory responses. Diabetic livers expressed higher levels of M1 markers but lower levels of M2 markers at baseline and after ischemia/reperfusion. Dual‐immunofluorescence staining showed that hyperglycemia promoted M1 (CD68/CD86) differentiation and inhibited M2 (CD68/CD206) differentiation. Importantly, CAY10444 reversed hyperglycemia‐modulated M1/M2 polarization. HG concentrations in vitro also triggered S1P/S1PR3 signaling, promoted M1 polarization, inhibited M2 polarization, and enhanced inflammatory responses compared with LG concentrations in BMDMs. In contrast, S1PR3 knockdown significantly retrieved hyperglycemia‐modulated M1/M2 polarization and attenuated inflammation. In conclusion, our study reveals that hyperglycemia specifically triggers S1P/S1PR3 signaling and exacerbates liver IRI by facilitating M1 polarization and inhibiting M2 polarization, which may represent an effective therapeutic strategy for liver IRI in diabetes.

Re-Education of Tumor Associated Macrophages by Trabectedin

Immune cells have an important role in the tumor-microenvironment. Macrophages may tune the immune response toward inflammatory or tolerance pathways. Tumor associated macrophages (TAM) have immunosuppressive functions and they are considered a therapeutic target in cancer. The aim of this study was to evaluate the effects of trabectedin, a new class of antitumor agent, on the tumor-microenvironment through the study of electrophysiological and molecular phenotype of macrophages. Experiments were performed using the whole-cell configuration of the patch-clamp technique in resident peritoneal mouse macrophages under different types of polarization. Trabectedin decreased macrophage viability and increased ROS production. Trabectedin does not directly interact with KV1.5 and KV1.3 channels, but treatment (16 h) of macrophages with sub-cytotoxic concentrations (0.1-5 nM) increased their KV current in a concentration-dependent manner due to an upregulation of KV1.3 channels. In vitro generated TAM (TAMiv), by a co-culture of ID8 cells and macrophages, exhibited a M2 phenotype. TAMiv generated a small KV current, similarly to M2 polarized macrophages, and expressed high levels of M2 markers. In this study, we demonstrated that TAMiv polarization could be re-educated by using sub-cytotoxic concentration of trabectedin. TAMiv treated with sub-cytotoxic concentration of trabectedin exhibited an upregulation of KV1.3 channels and their M2 phenotype changed towards M1 pro-inflammatory one. Funded by PharmaMar, CSIC 201820E104, CIBERCV and SAF2016-75021-R.

Hyperglycemia Triggered S1P/S1PR3 Signaling Worsens Liver Ischemia/Reperfusion Injury by Regulating M1/M2 Polarization

Hyperglycemia aggravates hepatic ischemia/reperfusion injury (IRI) but the underlying mechanism remains elusive. Sphingosine 1-phosphate (S1P)/S1P receptors (S1PRs) have been implicated in metabolic and inflammatory diseases. Here, we clarify whether and how S1P/S1PRs are involved in hyperglycemia-related liver IRI.S1P levels were significantly higher in liver tissues from patients with diabetes mellitus and mice with streptozotocin-induced diabetes. S1PR3, but not S1PR1 and S1PR2, was specifically activated in liver tissues and KCs under hyperglycemic conditions. S1PR3 antagonist CAY-10444 attenuated hyperglycemia-related liver IRI based on hepatic biochemistry, histology, and inflammatory responses. Diabetic liver expressed higher levels of M1 markers [NO synthase 2 and phosphorylated signal transducer and activator of transcription (p-STAT)1] but lower levels of M2 markers (arginase, mannose receptor C type 1, p-STAT3, and p-STAT6) at baseline and post-IR. Dual-immunofluorescence staining showed that hyperglycemia promoted M1 (CD68/CD86) differentiation and inhibited M2 (CD68/CD206) differentiation. Importantly, CAY10444 reversed hyperglycemia-modulated M1/M2 polarization. In vitro, high glucose(HG) concentrations also triggered S1P/S1PR3 signaling, promoted M1 polarization, inhibited M2 polarization, and enhanced inflammatory responses compared with low glucose(LG) concentrations in bone marrow-derived macrophages (BMDMs). In contrast, S1PR3 knockdown significantly retrieved hyperglycemia-modulated M1/M2 polarization, and attenuated Toll-like-receptor-4-related inflammation. Administration of HG-cultured BMDMs worsened liver IRI, which was rescued by S1PR3 knockdown. Our study reveals that hyperglycemia specifically triggers S1P/S1PR3 signaling and exacerbates liver IRI by facilitating M1 polarization and inhibiting M2 polarization, which may represent an effective therapeutic strategy for liver IRI in diabetes. Funding: This study was supported by the Foundation of Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, the Priority Academic Program Development of Jiangsu Higher Education Institutions, the National Natural Science Foundation of China (81400650, 814700901, 81273261, and 81270583). Declaration of Interest: All authors declare no conflict of interests. Ethical Approval: All mice received humane care and all procedures were performed according to protocols approved by the Institutional Animal Care and Use Committee of Nanjing Medical University.

Tetrahydroxystilbene glycoside antagonizes β-amyloid-induced inflammatory injury in microglia cells by regulating PU.1 expression.

Inhibiting β-amyloid (Aβ)-induced microglial activation is proposed as an effective strategy for the treatment of Alzheimer’s disease. Tetrahydroxystilbene glycoside (TSG) is the main active ingredient of Polygonum multiflorum and has a wide range of biological properties, including antiinflammation. Here, we focused on the function and regulatory mechanism of TSG in Aβ-induced N9 and BV2 cells. The results showed that Aβ treatment induced the activation of microglia cells and the production of inflammatory molecules, including inducible nitric oxide synthase, nitric oxide, cyclooxygenase 2, and prostaglandin E2, which were significantly inhibited by TSG pretreatment. Furthermore, we found Aβ exposure increased the levels of microglial M1 markers, interleukin (IL)-1β, IL-6, and tumor necrosis factor α, and the pretreatment of TSG suppressed the increase of M1 markers and enhanced the levels of M2 markers, including IL-10, brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, and arginase-1. PU.1 overexpression was found to eradicate the anti-inflammatory effects of TSG in Aβ-induced microglial cells. Taken together, these findings indicate that TSG attenuates Aβ-induced microglial activation and polarizes microglia towards M2 phenotype, which may be closely associated with the regulation of PU.1.

Phenotypic switch in lung interstitial macrophage polarization in an ovalbumin-induced mouse model of asthma.

Macrophage phenotype and function varies according to their polarized state, which in turn is dependent on microenvironmental stimuli. Under normal physiological conditions, lung interstitial macrophages that express interleukin (IL)-10 are considered to serve regulatory roles in the prevention of allergic reactions in the airways. However, the phenotypic profile of lung interstitial macrophages during the pathophysiology of asthma remains unknown. In the current study, the phenotypic characteristics of lung interstitial macrophages were investigated in an ovalbumin (OVA)-induced mouse model of asthma. The patterns of surface markers chemokine ligand and interleukin, and the metabolic enzyme activity of lung interstitial macrophages were investigated using flow cytometry analysis, reverse transcription-quantitative polymerase chain reaction, western blot analysis, and ELISA. It was observed that lung interstitial macrophages derived from OVA-induced asthmatic mice expressed phenotypic markers associated with alternatively activated macrophages (M2), including cluster of differentiation-206, transglutaminase 2, arginase (Arg) 1 and chemokine ligand (CCL)17/CCL22/CCL24 secretion. The M2 macrophages also exhibited increased levels of Arg1 activity and reduced levels of IL-10 expression, relative to macrophages derived from control mice. However, when evaluating the expression of markers associated with classically activated (M1) macrophages, namely inducible nitric oxide synthase and IL-12, it was observed that levels of M1 markers in the interstitial macrophages from asthmatic mice did not differ significantly to those in controls. Collectively, these data suggest that lung interstitial macrophages undergo a phenotypic switch from a regulatory macrophage phenotype under normal conditions to an alternative activation state in OVA-induced asthmatic mice.

Monocyte inflammatory profile is specific for individuals and associated with altered blood lipid levels

Background and aimsAtherogenesis is dependent upon monocyte influx into the vessel wall. In humans, three monocyte subsets exist, the number and function of which are significantly altered in cardiovascular disease (CVD). Whether such alterations arise in individuals with a perturbed lipid profile remains largely unanswered, but is important to delineate, as adoption of a pro-inflammatory state may promote plaque formation. Here, we compared the inflammatory status of monocyte subsets and determined whether monocyte inflammatory changes are evident in individuals with a perturbed lipid profile. MethodsMonocyte subset cytokine production, inflammatory and anti-inflammatory marker expression were determined by whole blood flow cytometry and related to participants' lipid levels. ResultsThe intermediate and non-classical monocytes were more inflammatory than classicals as seen by their higher cytokine production (TNF-α, IL-1β, IL-6) and M1 marker (CD86) expression, but lower levels of M2 markers (CD93, CD163). More importantly, a considerable variation was seen between participants, with all monocytes of one individual being more inflammatory than those of another. Many inter-individual differences were related to participants' lipid levels. IL-1β production correlated negatively with Apo A1 and HDL-C. CD86 and TLR2 correlated positively with Chol:HDL-C but negatively with HDL-C and Apo A1:Apo B. Interestingly, CD163 expression correlated positively with Chol:HDL-C but negatively with Apo A1:Apo B. ConclusionsOur data indicates that priming of all monocytes to an inflammatory state occurs in individuals with a perturbed lipid profile, overriding the normal functional distinction attributed to the different monocyte subsets. As such, all monocytes may be important in CVD.

Phenotype Determines Nanoparticle Uptake by Human Macrophages from Liver and Blood.

A significant challenge to delivering therapeutic doses of nanoparticles to targeted disease sites is the fact that most nanoparticles become trapped in the liver. Liver-resident macrophages, or Kupffer cells, are key cells in the hepatic sequestration of nanoparticles. However, the precise role that the macrophage phenotype plays in nanoparticle uptake is unknown. Here, we show that the human macrophage phenotype modulates hard nanoparticle uptake. Using gold nanoparticles, we examined uptake by human monocyte-derived macrophages that had been driven to a "regulatory" M2 phenotype or an "inflammatory" M1 phenotype and found that M2-type macrophages preferentially take up nanoparticles, with a clear hierarchy among the subtypes (M2c > M2 > M2a > M2b > M1). We also found that stimuli such as LPS/IFN-γ rather than with more "regulatory" stimuli such as TGF-β/IL-10 reduce per cell macrophage nanoparticle uptake by an average of 40%. Primary human Kupffer cells were found to display heterogeneous expression of M1 and M2 markers, and Kupffer cells expressing higher levels of M2 markers (CD163) take up significantly more nanoparticles than Kupffer cells expressing lower levels of surface CD163. Our results demonstrate that hepatic inflammatory microenvironments should be considered when studying liver sequestration of nanoparticles, and that modifying the hepatic microenvironment might offer a tool for enhancing or decreasing this sequestration. Our findings also suggest that models examining the nanoparticle/macrophage interaction should include studies with primary tissue macrophages.

IL-4/IL-13–mediated polarization of renal macrophages/dendritic cells to an M2a phenotype is essential for recovery from acute kidney injury

Cytokines IL-4 and IL-13 play important roles in polarization of macrophages/dendritic cells to an M2 phenotype, which is important for recovery from acute kidney injury. Both IL-4 and IL-13 activate JAK3/STAT6 signaling. In mice with diphtheria toxin receptor expression in proximal tubules (selective injury model), a relatively selective JAK3 inhibitor, tofacitinib, led to more severe kidney injury, delayed recovery from acute kidney injury, increased inflammatory M1 phenotype markers and decreased reparative M2 phenotype markers of macrophages/dendritic cells, and development of more severe renal fibrosis after diphtheria toxin administration. Similarly, there was delayed recovery and increased tubulointerstitial fibrosis in these diphtheria toxin-treated mice following tamoxifen-induced deletion of both IL-4 and IL-13, with increased levels of M1 and decreased levels of M2 markers in the macrophages/dendritic cells. Furthermore, deletion of IL-4 and IL-13 led to a decrease of tissue reparative M2a phenotype markers but had no effect on anti-inflammatory M2c phenotype markers. Deletion of IL-4 and IL-13 also inhibited recovery from ischemia-reperfusion injury in association with increased M1 and decreased M2 markers and promoted subsequent tubulointerstitial fibrosis. Thus, IL-4 and IL-13 are required to effectively polarize macrophages/dendritic cells to an M2a phenotype and to promote recovery from acute kidney injury.

Activation of the niacin receptor HCA2 reduces demyelination and neurofilament loss, and promotes functional recovery after spinal cord injury in mice

After spinal cord injury (SCI), there is an acute phase of alternatively activated (M2) macrophage infiltration, followed by a long-lasting phase of classically activated (M1) macrophage accumulation in the wound, which is believed to derail healing and compromize organ functions. Thus, agents which are able to modulate macrophage phenotypes may provide significant benefits to SCI patients. In the present study, we demonstrate that the niacin receptor HCA2 is specifically expressed on the cell surface of M1 but not M2 macrophages. Treatment of M1 macrophages with niacin (300μM) resulted in down-regulation of the p65 NF-κB phosphorylation, associated with a marked decrease in the levels of M1 markers, including CD86, IL-12, and IL-6, and a significant increase in the expressions of M2 markers, such as CD206, IL-10, and IL-13, suggesting that niacin causes a shift of M1 to M2. Moreover, treatment of the M1-oligodendrocyte precursor cell (OPC) co-cultures with niacin markedly promoted the expression of myelin binding protein (MBP). After SCI in C57/BL6 mice for a week, a marked accumulation of M1 macrophages, which expressed HCA2 receptor, was evident in the wound. Treatment of the SCI mice with niacin (100mg/kg) resulted in a dramatic decrease in the number of M1 macrophages and a significant increase in the number of M2 macrophages in the wound. This was associated with a robust inflammation resolution, attenuation of demyelination and neurofilament loss, and significant improvement of locomotor function. Thus, HCA2 receptor may serve as a therapeutic target to promote post-SCI recovery.