Pro-inflammatory Macrophage Polarization Research Articles

LSD1 Facilitates Pro-Inflammatory Polarization of Macrophages by Repressing Catalase.

The increased level of hydrogen peroxide accompanies some modes of macrophage specification and is linked to ROS-based antimicrobial activity of these phagocytes. In this study, we show that activation of toll-like receptors with bacterial components such as LPS is accompanied by the decline in transcription of hydrogen peroxide decomposing enzyme-catalase, suppression of which facilitates the polarization of human macrophages towards the pro-inflammatory phenotype. The chromatin remodeling at the CAT promoter involves LSD1 and HDAC1, but activity of the first enzyme defines abundance of the two proteins on chromatin, histone acetylation status and the CAT transcription. LSD1 inhibition prior to macrophage activation with LPS prevents CAT repression by enhancing the LSD1 and interfering with the HDAC1 recruitment to the gene promoter. The maintenance of catalase level with LSD1 inhibitors during M1 polarization considerably limits LPS-triggered expression of some pro-inflammatory cytokines and markers such as IL1β, TNFα, COX2, CD14, TLR2, and IFNAR, but the effect of LSD1 inhibitors is lost upon catalase deficiency. Summarizing, activity of LSD1 allows for the CAT repression in LPS stimulated macrophages, which negatively controls expression of some key pro-inflammatory markers. LSD1 inhibitors can be considered as possible immunosuppressive drugs capable of limiting macrophage M1 specialization.

Cannabinoid receptor type 2 is upregulated in synovium following joint injury and mediates anti-inflammatory effects in synovial fibroblasts and macrophages

Joint injury-induced perturbations to the endocannabinoid system (ECS), a regulator of both inflammation and nociception, remain largely uncharacterized. We employed a mouse model of ACL rupture to assess alterations to nociception, inflammation, and the ECS while using in vitro models to determine whether CB2 agonism can mitigate inflammatory signaling in macrophages and fibroblast-like synoviocytes (FLS). Mice underwent noninvasive ACL rupture (ACLR) via tibial compression-based loading. Nociception was measured longitudinally using mechanical allodynia and knee hyperalgesia testing. Synovitis was assessed using histological scoring and histomorphometry. Gene and protein markers of inflammation were characterized in whole joints and synovium. Immunohistochemistry assessed injury-induced alterations to CB1+, CB2+, and F4/80+ cells in synovium. To assess whether CB2 agonism can inhibit pro-inflammatory macrophage polarization, murine bone marrow-derived macrophages (mBMDM) were stimulated with IL-1β or conditioned medium from IL-1β-treated FLS and treated with vehicle (DMSO), the CB2 agonist HU308, or cannabidiol (CBD). Macrophage polarization was assessed as the ratio of M1-associated (IL1b, MMP1b, and IL6) to M2-associated (IL10, IL4, and CD206) gene expression. Human FLS (hFLS) isolated from synovial tissue of OA patients were treated with vehicle (DMSO) or HU308 following TNF-α or IL-1β stimulation to assess inhibition of catabolic/inflammatory gene expression. ACLR induces synovitis, progressively-worsening PTOA severity, and an immediate and sustained increase in both mechanical allodynia and knee hyperalgesia, which persist beyond the resolution of molecular inflammation. Enrichment of CB2, but not CB1, was observed in ACLR synovium at 3d, 14d, and 28d, and CB2 was found to be associated with F4/80 (+) cells, which are increased in number in ACLR synovium at all time points. The CB2 agonist HU308 strongly inhibited mBMDM M1-type polarization following stimulation with either IL-1β or conditioned medium from IL-1β-treated mFLS, which was characterized by reductions in Il1b, Mmp1b, and Il6 and increases in Cd206 gene expression. Cannabidiol similarly inhibited IL-1β-induced mBMDM M1 polarization via a reduction in Il1b and an increase in Cd206 and Il4 gene expression. Lastly, in OA hFLS, HU308 treatment inhibited IL-1β-induced CCL2, MMP1, MMP3, and IL6 expression and further inhibited TNF-α-induced CCL2, MMP1, and GMCSF expression, demonstrating human OA-relevant anti-inflammatory effects by targeting CB2. Joint injury perturbs the intra-articular ECS, characterized by an increase in synovial F4/80(+) cells, which express CB2, but not CB1. Targeting CB2 in murine macrophages and human FLS induced potent anti-inflammatory and anti-catabolic effects, which indicates that the CB2 receptor plays a key role in regulating inflammatory signaling in the two primary effector cells in the synovium. The intraarticular ECS is therefore a potential therapeutic target for blocking pathological inflammation in future disease-modifying PTOA treatments.

Non-Lyn Src Family Kinases Activate SIRPα-SHP-1 to Inhibit PI3K-Akt2 and Dampen Proinflammatory Macrophage Polarization.

Macrophage functional plasticity plays a central role in responding to proinflammatory stimuli. The molecular basis underlying the dynamic phenotypic activation of macrophages, however, remains incompletely understood. In this article, we report that SIRPα is a chief negative regulator of proinflammatory macrophage polarization. In response to TLR agonists, proinflammatory cytokines, or canonical M1 stimulation, Src family kinases (SFK) excluding Lyn phosphorylate SIRPα ITIMs, leading to the preferential recruitment and activation of SHP-1, but not SHP-2. Solely extracellular ligation of SIRPα by CD47 does not greatly induce phosphorylation of SIRPα ITIMs, but it enhances proinflammatory stimuli-induced SIRPα phosphorylation. Examination of downstream signaling elicited by IFN-γ and TLR3/4/9 agonists found that SIRPα-activated SHP-1 moderately represses STAT1, NF-κB, and MAPK signaling but markedly inhibits Akt2, resulting in dampened proinflammatory cytokine production and expression of Ag presentation machinery. Pharmacological inhibition of SHP-1 or deficiency of SIRPα conversely attenuates SIRPα-mediated inhibition and, as such, augments macrophage proinflammatory polarization that in turn exacerbates proinflammation in mouse models of type I diabetes and peritonitis. Our results reveal an SFK-SIRPα-SHP-1 mechanism that fine-tunes macrophage proinflammatory phenotypic activation via inhibition of PI3K-Akt2, which controls the transcription and translation of proinflammatory cytokines, Ag presentation machinery, and other cellular programs.

Berberine remodels adipose tissue to attenuate metabolic disorders by activating sirtuin 3.

Adipose tissue remodelling is considered a critical pathophysiological hallmark of obesity and related metabolic diseases. Berberine (BBR), a natural isoquinoline alkaloid, has potent anti-hyperlipidaemic and anti-hyperglycaemic effects. This study aimed to explore the role of BBR in modulating adipose tissue remodelling and the underlying mechanisms. BBR protected high fat diet (HFD)-fed mice against adiposity, insulin resistance and hyperlipidemia. BBR alleviated adipose tissue inflammation and fibrosis by inhibiting macrophage infiltration, pro-inflammatory macrophage polarization and the abnormal deposition of extracellular matrix, and the effect was mediated by BBR directly binding and activating the deacetylase Sirtuin 3 (SIRT3) and suppressing the activation of the mitogen-activated protein kinases and nuclear factor-κB signalling pathways. Furthermore, BBR decreased microRNA-155-5p secretion by macrophages, which in turn ameliorated liver injury. Moreover, BBR mitigated inflammatory responses in both LPS-stimulated macrophages and TNF-α-treated adipocytes and suppressed macrophage migration towards adipocytes by activating SIRT3. Collectively, this study revealed that BBR improved adipose tissue remodelling, and subsequently inhibited the secretion of microRNA-155-5p by macrophages, which alleviated adiposity, insulin resistance and liver injury in obese mice. The modulation of adipose tissue remodelling by activating SIRT3 could contribute to the anti-hyperlipidemic and anti-hyperglycemic effects of BBR.

CY-09 Inhibits NLRP3 Inflammasome Activation to Relieve Pain via TRPA1.

Peripheral tissue damage leads to inflammatory pain, and inflammatory cytokine releasing is the key factor for inducing the sensitization of nociceptors. As a calcium ion channel, TRPA1 plays an important role in pain and inflammation, thus becoming a new type of anti-inflammatory and analgesic target. However, there is no consensus on the role of this channel in mechanical hyperalgesia caused by inflammation. Here, we aim to explore the role and underlying mechanism of the inflammasome inhibitor CY-09 in two classic inflammatory pain models. We evaluated pain behavior on animal models, cytokine levels, intracellular Ca2+ levels, transient TRPA1 expression, NF-κB transcription, and NLPR3 inflammasome activation. Consistently, CY-09 reduced the production of inflammatory cytokines, intracellular Ca2+ levels, and the activation of TRPA1 by inhibiting the activation of inflammasomes, thereby reducing the proinflammatory polarization of macrophages and alleviating animal pain and injury. Importantly, AITC (TRPA1 agonist) significantly reversed the analgesic effect of CY-09, indicating that TRPA1 was involved in the analgesic effect of CY-09. Our findings indicate that CY-09 relieves inflammation and pain via inhibiting TRPA1-mediated activation of NLRP3 inflammasomes. Thus, NLRP3 inflammasome may be a potential therapeutic target for pain treatment and CY-09 may be a pharmacological agent to relieve inflammatory pain, which needs further research.

EDIL3 deficiency ameliorates adverse cardiac remodelling by neutrophil extracellular traps (NET)-mediated macrophage polarization.

After myocardial infarction (MI), injured cardiomyocytes recruit neutrophils and monocytes/macrophages to myocardium, which in turn initiates inflammatory and reparative cascades, respectively. Either insufficient or excessive inflammation impairs cardiac healing. As an endogenous inhibitor of neutrophil adhesion, EDIL3 plays a crucial role in inflammatory regulation. However, the role of EDIL3 in MI remains obscure. We aimed to define the role of EDIL3 in cardiac remodelling after MI. Serum EDIL3 levels in MI patients were negatively associated with MI biomarkers. Consistently, WT mice after MI showed low levels of cardiac EDIL3. Compared with WT mice, Edil3-/- mice showed improvement of post-MI adverse remodelling, as they exhibited lower mortality, better cardiac function, shorter scar length, and smaller LV cavity. Accordingly, infarcted hearts of Edil3-/- mice contained fewer cellular debris and lower amounts of fibrosis content, with decreased collagen I/III expression and the percentage of α-smooth muscle actin myofibroblasts. Mechanistically, EDIL3 deficiency did not affect the recruitment of monocytes or T cells, but enhanced neutrophil recruitment and following expansion of pro-inflammatory Mertk-MHC-IIlo-int (myeloid-epithelial-reproductive tyrosine kinase/major histocompatibility complex II) macrophages. The injection of neutrophil-specific C-X-C motif chemokine receptor 2 antagonist eliminated the differences in macrophage polarization and cardiac function between WT and Edil3-/- mice after MI. Neutrophil extracellular traps (NETs), which were more abundant in the hearts of Edil3-/- mice, contributed to Mertk-MHC-IIlo-int polarization via Toll-like receptor 9 pathway. The inhibition of NET formation by treatment of neutrophil elastase inhibitor or DNase I impaired macrophage polarization, increased cellular debris and aggravated cardiac adverse remodelling, thus removed the differences of cardiac function between WT and Edil3-/- mice. Totally, EDIL3 plays an important role in NET-primed macrophage polarization and cardiac remodelling during MI. We not only reveal that EDIL3 deficiency ameliorates adverse cardiac healing via NET-mediated pro-inflammatory macrophage polarization but also discover a new crosstalk between neutrophil and macrophage after MI.

USP13-mediated IRAK4 deubiquitination disrupts the pathological symptoms of lipopolysaccharides-induced sepsis

Ubiquitin-specific peptidase 13 (USP13) has been reported to participate in tumorigenesis, cell cycle arrest, endoplasmic reticulum-associated degradation, and immune responses. Here, we explored the function of USP13 in pro-inflammatory cytokine production of macrophages and its role in mouse sepsis model. Primary bone-marrow-derived macrophages (BMDMs) isolated from wild type (WT) and USP13MKO mice were treated by lipopolysaccharides (LPS), IL-4, toll-like receptors (TLRs) agonists, and IRAK4 inhibitor to profile the inflammatory responses with different genotypes. Mouse sepsis model (WT and USP13MKO) created by intraperitoneal injection with LPS plus d-galactosamine was used to assess septic shock-induced survival and lung inflammation. Flow cytometry, qRT-PCT, Western blot, and ELISA were performed to detect pro-inflammatory production and macrophage polarization. USP13 was a key regulator of IRAK4 deubiquitination in BMDMs and its myeloid specific deficiency contributed to LPS-induced pro-inflammatory response and septic symptoms. IRAK4 inhibitor co-administration improved in LPS-induced inflammatory responses in both BMDMs and septic mice. USP13 negatively regulates LPS-induced sepsis shock by targeting IRAK4. In summary, targeting USP13-IRAK4 axis might be a potential therapeutic strategy for the treatment of inflammation in sepsis shock.

IL-17F depletion accelerates chitosan conduit guided peripheral nerve regeneration

Peripheral nerve injury is a serious health problem and repairing long nerve deficits remains a clinical challenge nowadays. Nerve guidance conduit (NGC) serves as the most promising alternative therapy strategy to autografts but its repairing efficiency needs improvement. In this study, we investigated whether modulating the immune microenvironment by Interleukin-17F (IL-17F) could promote NGC mediated peripheral nerve repair. Chitosan conduits were used to bridge sciatic nerve defect in IL-17F knockout mice and wild-type mice with autografts as controls. Our data revealed that IL-17F knockout mice had improved functional recovery and axonal regeneration of sciatic nerve bridged by chitosan conduits comparing to the wild-type mice. Notably, IL-17F knockout mice had enhanced anti-inflammatory macrophages in the NGC repairing microenvironment. In vitro data revealed that IL-17F knockout peritoneal and bone marrow derived macrophages had increased anti-inflammatory markers after treatment with the extracts from chitosan conduits, while higher pro-inflammatory markers were detected in the Raw264.7 macrophage cell line, wild-type peritoneal and bone marrow derived macrophages after the same treatment. The biased anti-inflammatory phenotype of macrophages by IL-17F knockout probably contributed to the improved chitosan conduit guided sciatic nerve regeneration. Additionally, IL-17F could enhance pro-inflammatory factors production in Raw264.7 cells and wild-type peritoneal macrophages. Altogether, IL-17F may partially mediate chitosan conduit induced pro-inflammatory polarization of macrophages during nerve repair. These results not only revealed a role of IL-17F in macrophage function, but also provided a unique and promising target, IL-17F, to modulate the microenvironment and enhance the peripheral nerve regeneration.

Inhibition of KCa3.1 Channels Suppresses Atrial Fibrillation via the Attenuation of Macrophage Pro-inflammatory Polarization in a Canine Model With Prolonged Rapid Atrial Pacing.

Aims: To investigate the role of KCa3. 1 inhibition in macrophage pro-inflammatory polarization and vulnerability to atrial fibrillation (AF) in a canine model with prolonged rapid atrial pacing.Materials and Methods: Twenty beagle dogs (weighing 8–10 kg) were randomly assigned to a sham group (n = 6), pacing group (n = 7) and pacing+TRAM-34 group (n = 7). An experimental model of AF was established by rapid pacing. TRAM-34 was administered to the Pacing+TRAM-34 group by slow intravenous injection (10 mg/kg), 3 times each day. After 7 days of pacing, the electrophysiology was measured in vivo. The levels of interleukin-1β (IL-1β), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), CD68, c-Fos, p38, and NF-κB p65 in both atriums were measured by Western blotting, and the levels of inducible nitric oxide synthase (iNOS) and arginase1 (Arg-1) were measured by real-time PCR. Macrophage and KCa3.1 in macrophage in the atrium were quantized following double labeled immunofluorescent.Results: Greater inducibility of AF, an extended duration of AF and lower atrial effective refractory period (AERP) were observed in the pacing group compared with those in the sham group. Both CD68-labeled macrophage and the expression of KCa3.1 in macrophage were elevated in the pacing group and inhibited by TRAM-34, led to higher iNOS expression, lower Arg-1 expression, elevated levels of IL-1β, MCP-1, and TNF-α in the atria, which could be reversed by TRAM-34 treatment (all P < 0.01). KCa3.1 channels were possibly activated via the p38/AP-1/NF-κB signaling pathway.Conclusions: Inhibition of KCa3.1 suppresses vulnerability to AF by attenuating macrophage pro-inflammatory polarization and inflammatory cytokine secretion in a canine model with prolonged rapid atrial pacing.

Wireless Localized Electrical Stimulation Generated by an Ultrasound-Driven Piezoelectric Discharge Regulates Proinflammatory Macrophage Polarization.

Proinflammatory (M1) macrophages play a vital role in antitumor immunity, and regulation of proinflammatory macrophage polarization is critical for immunotherapy. The polarization of macrophages can be regulated by biological or chemical stimulation, but investigations of the regulatory effect of physical stimulation are limited. Herein, regulating macrophage polarization with localized electrical signals derived from a piezoelectric β‐phase poly(vinylidene fluoride) (β‐PVDF) film in a wireless mode is proposed. Charges released on the surface of the β‐PVDF film driven by ultrasonic irradiation can significantly enhance the M1 polarization of macrophages. Mechanistic investigation confirms that electrical potentials rather than reactive oxygen species and mechanical forces enable Ca2+ influx through voltage‐gated channels and establishment of the Ca2+‐CAMK2A‐NF‐κB axis to promote the proinflammatory macrophage response during ultrasound treatment. Piezoelectric material‐mediated electrical signal‐activated proinflammatory macrophages significantly inhibit tumor cell proliferation. A method for electrogenetic regulation of immune cells as well as a powerful tool for engineering macrophages for immunotherapy is provided here.

Adipocyte-Derived Lactate Potentiates Obesity-Evoked Adipose Macrophage Inflammation

Introduction: Obesity is characterized by mobilization of macrophage inflammation, which represents the major events of obesity-associated adipose tissue inflammation. . On the other hand, lactate accumulation in adipose tissue long been observed. However, whether elevation of lactate plays an essential role in adipose inflammation is not known. In this study, we sought to examine the intermediary role of lactate in macrophage polarization and adipose inflammation upon obesity. Method: Lactate level and activity of lactate dehydrogense (LDH), the key enzyme of lactate production, were measured by biochemical assays. Adipocyte- and macrophage- specific Ldha knock out mice were constructed by cre-LoxP system to study the physiological role of lactate in diet induced obesity. Macrophage polarization and inflammation were examined by western blotting and Q-PCR. Results: Lactate and LDH activity were selectively upregulated in adipose tissues of obese mice. Adipocyte-, but not macrophage-selective deletion of LDHA, led to a significant improvement of adipose inflammation and metabolic dysfunctions. In vitro experiments showed that the lactate promoted M1 polarization through direct interation and inhibition of the PHD2, which subsequently stabilizes HIF-1alpha. In addition, a positive correlation between adipose lactate level and adipose tissue inflammation was found in obese patients. Conclusion: In obese condition, increased production of lactate from adipocytes enhances adipose tissue inflammation by promoting the proinflammatory polarization of adipose macrophages.

Control of Macrophage Activation by Coenzyme A

Macrophages are innate immune cells responsible for processes as varied as killing pathogens, initiating wound-healing programs, and presenting antigens to T cells. To achieve this broad range of functions, they acquire pro- or anti-inflammatory programs (or ‘polarize’) by integrating intrinsic, extrinsic, and environmental signals. It is now well established that metabolic changes are critical for this process, though it remains unclear precisely which metabolic alterations are essential for (rather than merely being associated with) activation. Pro-inflammatory macrophage polarization with lipopolysaccharide (LPS) collapses mitochondrial ATP production. This profound decrease in oxidative phosphorylation is associated with increases in glycolysis, signaling metabolites from the TCA cycle, and mitochondrially derived redox signals. Conversely, anti-inflammatory activation with interleukin-4 (IL-4) is associated with substantial increases in oxidative phosphorylation and fatty acid oxidation (FAO). Previous work in our laboratory has shown that although FAO and mitochondrial ATP production are strongly associated with the IL-4 response, these are surprisingly dispensable, whereas coenzyme A homeostasis is obligatory [PMCID: PMC6125190]. This presentation will cover our current understanding of the essential metabolic requirements for anti-inflammatory macrophage activation, with a focus on the potential mechanisms by which coenzyme A may control the IL-4 response.

The E3 ubiquitin ligase Itch limits the progression of post-traumatic osteoarthritis in mice by inhibiting macrophage polarization

Osteoarthritis (OA) is characterized by articular cartilage loss, associated with synovial inflammation. We recently reported increased pro-inflammatory macrophages in murine post-traumatic OA (PTOA) joints, and blockade of the ubiquitin-proteasome system alleviates PTOA progression. However, the mechanisms whereby protein ubiquitination influences PTOA pathology are not well studied. We hypothesized that loss of the negative regulator of inflammation, E3 ligase Itch, in macrophages contributes to joint OA tissue damage by promoting pro-inflammatory polarization of macrophages. Mice deficient Itch in macrophages (MΔItch) were generated by crossing Itchfl/fl mice with LysM-Cre mice. PTOA surgery was performed on global Itch knockout, Itch-/-, mice and MΔItch mice. Joint tissue damage and synovial macrophages were examined. Itch-/- cells were treated with IL-1 and pro-inflammatory polarization was determined. Expression of Itch protein and mRNA in PTOA synovium were assessed at different time points post PTOA. Similar to Itch-/- mice, MΔItch mice developed more severe joint damage than control mice following PTOA surgery (mean difference of OARSI score: 1.17 (95% CI 0.31-2.03) between MΔItch and Itchfl/fl mice), accompanied by increased the inflammatory macrophage infiltration in the synovium (mean difference of % F4/80+CD86+CD36-inflammatory macrophages: 14.81 (95% CI 8.90-20.73) between MΔItch and Itchfl/fl mice). Itch-/- macrophages exerted pro-inflammatory phenotype in response to IL-1β treatment. Itch protein, but not mRNA levels decreased during PTOA progression. The negative regulator of inflammation, Itch, limits PTOA progression by inhibiting macrophage pro-inflammatory polarization. Itch protein degradation may contribute to PTOA pathology.

A NOVEL NOX/PHOX-CD38-NAADP-TFEB AXIS IMPORTANT FOR MACROPHAGE ACTIVATION DURING BACTERIAL PHAGOCYTOSIS

ABSTRACT Macrophage activation in the presence of bacterial cells and molecules entails complex programs of gene expression. How such triggers elicit specific gene expression programs is incompletely understood. We previously discovered that TFEB (transcription factor EB) is a key contributor to macrophage activation during bacterial phagocytosis. However, the mechanism linking phagocytosis of bacterial cells to TFEB activation and downstream pro-inflammatory cytokine induction remained unknown. We found that macrophages lacking both TFEB and TFE3 (transcription factor E3) were unable to mount a pro-inflammatory phenotype in response to bacterial infection. The NOX/PHOX (NADPH oxidase)-dependent oxidative burst was required for nuclear translocation of TFEB during phagocytosis of Gram-positive or -negative bacteria, and reactive oxygen species (ROS) were sufficient to trigger TFEB activation in a CD38- and NAADP (nicotinic acid adenine dinucleotide phosphate)-dependent manner. Consistent with the Ca2+-releasing activity of NAADP, intracellular Ca2+ chelation and PPP3/calcineurin inhibition prevented TFEB activation by phagocytosis and ROS (reactive oxygen species), impairing the induction of pro-inflammatory cytokines such as IL6 and TNF/TNFα. Therefore, here we describe a previously unknown pathway that links phagocytosis with macrophage pro-inflammatory polarization via TFEB and related transcription factor TFE3. These findings reveal that activation of TFEB and TFE3 is a key regulatory event for the activation of macrophages, and have important implications for infections, inflammation, cancer, obesity, and atherosclerosis.

Hypothermia Protects Mice Against Ischemic Stroke by Modulating Macrophage Polarization Through Upregulation of Interferon Regulatory Factor-4.

BackgroundTherapeutic hypothermia (TH) has been proven to be protective in ischemic stroke (IS) due to its anti-inflammatory capacity. Recently, the interferon regulatory factor 4 (IRF4) has been characterized as a central regulator of neuroinflammation in IS. Here we aim to determine whether IFR4 contributes to the neuroprotective effects of TH in IS.MethodsIn the present study, IRF4 knockout (IRF4−/-) and wild-type (IRF4+/+) mice were treated with or without TH after IS. Cerebral IRF4 expression, the production of pro-inflammatory and anti-inflammatory cytokines and macrophage polarization were determined at 8 hours after reperfusion. In addition, cerebral infarct volume and neurological function were evaluated at 7 days after IS.ResultsTH attenuates IS together with enhanced IRF4 expression as well as reduced production of pro-inflammatory cytokines. In addition, TH increased M2 macrophage polarization while inhibited M1 macrophage polarization. However, IRF4 knockout worsens neurological outcomes of stoke mice. The expression of pro-inflammatory cytokines were markedly increased in IRF4−/- mice as compared with IRF4+/+ mice at 8 h after stroke. Moreover, IRF4 knockout driven the macrophage polarization toward M1phenotype at 8 h after stroke. Most importantly, IRF4 knockout abolished the neuroprotective and anti-inflammatory effects of TH in IS.ConclusionTogether, we report for the first time that TH attenuates neuroinflammation following IS by modulating M1/M2 macrophage polarization through the upregulation of IRF4 expression.

Mineralocorticoid Receptor in Myeloid Cells Mediates Angiotensin II-Induced Vascular Dysfunction in Female Mice

Enhanced mineralocorticoid receptor (MR) signaling is critical to the development of endothelial dysfunction and arterial stiffening. However, there is a lack of knowledge about the role of MR-induced adipose tissue inflammation in the genesis of vascular dysfunction in women. In this study, we hypothesize that MR activation in myeloid cells contributes to angiotensin II (Ang II)-induced aortic stiffening and endothelial dysfunction in females via increased pro-inflammatory (M1) macrophage polarization. Female mice lacking MR in myeloid cells (MyMRKO) were infused with Ang II (500 ng/kg/min) for 4 weeks. This was followed by determinations of aortic stiffness and vasomotor responses, as well as measurements of markers of inflammation and macrophage infiltration/polarization in different adipose tissue compartments. MyMRKO mice were protected against Ang II-induced aortic endothelial stiffening, as assessed via atomic force microscopy in aortic explants, and vasorelaxation dysfunction, as measured by aortic wire myography. In alignment, MyMRKO mice were protected against Ang II-induced macrophage infiltration and M1 polarization in visceral adipose tissue (VAT) and thoracic perivascular adipose tissue (tPVAT). Collectively, this study demonstrates a critical role of MR activation in myeloid cells in the pathogenesis of vascular dysfunction in females associated with pro-inflammatory macrophage polarization in VAT and tPVAT. Our data have potential clinical implications for the prevention and management of cardiovascular disease in women, who are disproportionally at higher risk for poor outcomes.

Minocycline alleviates peripheral nerve adhesion by promoting regulatory macrophage polarization via the TAK1 and its downstream pathway

AimsInflammation plays a key role in peripheral nerve adhesion and often leads to severe pain and nerve dysfunction. Minocycline was reported to have potent anti-inflammatory effects and might be a promising drug to prevent or attenuate peripheral nerve adhesion. The present study aimed to clarify whether minocycline contributes to nerve adhesion protection and its underlying mechanism. Materials and methodsRats with sciatic nerve adhesion induced by glutaraldehyde glue (GG) were intraperitoneally injected with minocycline or saline every 12 h for 7 consecutive days. After that, the adhesion score, Ashcroft score, demyelination, macrophage polarization and inflammatory factors in peripheral nerve adhesion tissues or tissues in sham group were determined with histological staining, western blot and real time-PCR. Murine macrophage RAW264.7 cells were stimulated by LPS alone or together with minocycline at different concentrations and time duration to study the mechanism of minocycline in alleviating nerve adhesion. Key findingsWe found that minocycline treatment reduced the adhesion score, Ashcroft score, the growth of scar tissue, demyelination, and macrophage recruitment. Moreover, minocycline significantly and dose-dependently promoted regulatory macrophage polarization but decreased pro-inflammatory macrophage polarization. Furthermore, mechanism studies showed that TAK1 and its downstream pathway p38/JNK/ERK1/2/p65 were inhibited by minocycline, which led to lower IL-1β and TNFα expression, but increased IL-10 expression. SignificanceAltogether, these results suggest that minocycline is highly effective against peripheral nerve adhesion through anti-fibrosis, anti-inflammation, and myelination protection, making it a highly promising candidate for treating adhesion-related disorders.

Metallothioneins alter macrophage phenotype and represent novel therapeutic targets for acetaminophen-induced liver injury.

Acetaminophen (APAP) intoxication is the foremost cause of drug-induced liver failure in developed countries. The only pharmacologic treatment option, N-acetylcysteine (NAC), is not effective for patients who are admitted too late and/or who have excessive liver damage, emphasizing the need for alternative treatment options. APAP intoxication results in hepatocyte death and release of danger signals, which further contribute to liver injury, in part by hepatic monocyte/macrophage infiltration and activation. Metallothionein (MT) 1 and 2 have important danger signaling functions and might represent novel therapeutic targets in APAP overdose. Therefore, we evaluated hepatic MT expression and the effect of anti-MT antibodies on the transcriptional profile of the hepatic macrophage population and liver injury following APAP overdose in mice. Hepatic MT expression was significantly induced in APAP-intoxicated mice and abundantly present in human livers. APAP intoxication in mice resulted in increased serum transaminase levels, extended necrotic regions on liver histology and induced expression of proinflammatory markers, which was significantly less pronounced in mice treated with anti-MT antibodies. Anti-MT antibody therapy attenuated proinflammatory macrophage polarization, as demonstrated by RNA sequencing analyses of isolated liver macrophages and in LPS-stimulated bone marrow-derived macrophages. Importantly, NAC and anti-MT antibodies were equally effective whereas administration of anti-MT antibody in combination with NAC exceeded the efficiency of both monotherapies in APAP-induced liver injury (AILI). We conclude that the neutralization of secreted MTs using a monoclonal antibody is a novel therapeutic strategy as mono- or add-on therapy for AILI. In addition, we provide evidence suggesting that MTs in the extracellular environment are involved in macrophage polarization.

MiR-30b-5p releases HMGB1 via UBE2D2/KAT2B/HMGB1 pathway to promote pro-inflammatory polarization and recruitment of macrophages

Background and aimsOxidized low-density lipoprotein (ox-LDL) is a key risk factor for atherosclerosis, but there are few reports on ox-LDL-mediated inflammation after injury. In this study, we investigated the effects of ox-LDL on endothelial injury and macrophage polarization and recruitment using human aortic endothelial cell line HAEC cells. MethodsChanges in miRNA levels after ox-LDL treatment were assessed with qRT-PCR. Luciferase experiments were performed to verify the interaction between miRNA and protein, and co-IP and ubiquitination experiments to detect proteins interactions. Cell phenotype was assessed by cytometry and Western blot. ResultsqRT-PCR data indicated that ox-LDL treatment up-regulates the expression of miR-30b-5p. Luciferase test and ubiquitination assay showed miR-30b-5p can bind to UBE2D2 and reduce its ubiquitination ability to degrade KAT2B. The up-regulated KAT2B promotes the acetylation of HMGB1, acetylated HMGB1 dissociates from SIRT1, exit the nucleus, and it is secreted from the cell. Flow cytometry and transwell experiments showed that HMGB1 secreted from HAEC can induce pro-inflammatory (M1-like) polarization and recruitment of RAW264.7 cells. ConclusionsOur results indicate that ox-LDL activates the UBE2D2/KAT2B pathway by upregulating miR-30b-5p, thus acetylating HMGB1, which is then secreted from the cell, thereby promoting pro-inflammatory cell polarization and recruitment of macrophage.

GM-CSF Expression and Macrophage Polarization in Joints of Undifferentiated Arthritis Patients Evolving to Rheumatoid Arthritis or Psoriatic Arthritis.

Background and AimsGM-CSF-dependent macrophage polarization has been demonstrated in rheumatoid arthritis (RA). Our aim was to seek diagnostic/prognostic biomarkers for undifferentiated arthritis (UA) by analyzing GM-CSF expression and source, macrophage polarization and density in joints of patients with UA evolving to RA or PsA compared with established RA or PsA, respectively.MethodsSynovial tissue (ST) from patients with UA evolving to RA (UA>RA, n=8), PsA (UA>PsA, n=9), persistent UA (UA, n=16), established RA (n=12) and PsA (n=10), and healthy controls (n=6), were analyzed. Cell source and quantitative expression of GM-CSF and proteins associated with pro-inflammatory (GM-CSF-driven) and anti-inflammatory (M-CSF-driven) macrophage polarization (activin A, TNFα, MMP12, and CD209, respectively) were assessed in ST CD163+ macrophages by multicolor immunofluorescence. GM-CSF and activin A levels were also quantified in paired synovial fluid samples. CD163+ macrophage density was determined in all groups by immunofluorescence.ResultsSynovial stromal cells (FAP+ CD90+ fibroblast, CD90+ endothelial cells) and CD163+ sublining macrophages were the sources of GM-CSF. ST CD163+ macrophages from all groups expressed pro-inflammatory polarization markers (activin A, TNFα, and MMP12). Expression of the M-CSF-dependent anti-inflammatory marker CD209 identified two macrophage subsets (CD163+ CD209high and CD163+ CD209low/-). CD209+ macrophages were more abundant in ST from healthy controls and PsA patients, although both macrophage subtypes showed similar levels of pro-inflammatory markers in all groups. In paired synovial fluid samples, activin A was detected in all patients, with higher levels in UA>RA and RA, while GM-CSF was infrequently detected. ST CD163+ macrophage density was comparable between UA>RA and UA>PsA patients, but significantly higher than in persistent UA.ConclusionsGM-CSF is highly expressed by sublining CD90+ FAP+ synovial fibroblasts, CD90+ activated endothelium and CD163+ macrophages in different types of arthritis. The polarization state of ST macrophages was similar in all UA and established arthritis groups, with a predominance of pro-inflammatory GM-CSF-associated markers. CD163+ macrophage density was significantly higher in the UA phases of RA and PsA compared with persistent UA. Taken together, our findings support the idea that GM-CSF is a strong driver of macrophage polarization and a potential therapeutic target not only in RA but also in PsA and all types of UA.