Anti-inflammatory M2 Phenotype Research Articles

IL-4 functionalized titanium dioxide nanotubes modulate the inflammatory response of macrophages

Inflammatory response is an essential part of optimal tissue-implant integration and the regeneration process. Due to their highly plastic properties, macrophages display phenotypic changes during inflammatory signaling. Investigating these changes on implant surfaces is essential for evaluating implant stability and longevity. In order to control macrophage polarization, IL-4 was conjugated to titanium dioxide nanotubes (TNTs) through polydopamine, and successful fabrication was checked by scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and contact angle, respectively. In vitro experiments including immunofluorescence staining, cell proliferation, the expression of genes associated with pro-inflammatory M1 phenotype (tumor necrosis factor-alpha (TNF-α), Interleukin-18 (IL-18)) and cytokines related to the anti-inflammatory M2 phenotype (IL-4 and IL-10), and the production of nitric oxide (NO) and cytokines TNF-α, IL-10 were detected. Macrophage response showed that IL-4 functionalized TNTs favored macrophage polarization towards an anti-inflammatory M2-phenotype. This study provides a new strategy for use in medical devices and the development of advanced nano-biomaterials in immunotherapy applications.

Allogeneic adipose-derived stem cells promote ischemic muscle repair by inducing M2 macrophage polarization via the HIF-1α/IL-10 pathway.

Adipose‐derived mesenchymal stem cells (ASCs) are multipotent stromal cells that possess considerable therapeutic potential for tissue remodeling. However, their protective mechanism in critical limb ischemia has not been fully defined. After the occlusion of blood vessels, hypoxia becomes a prominent feature of the ischemic limb. This study investigated the immunomodulatory effect of ASCs on ischemic muscle repair and explored the specific mechanism. We found that the ability of RAW264.7 cells to migrate was impaired in hypoxia, whereas coculturing with ASCs could enhance the migration capacity. In addition, under hypoxic conditions, the paracrine effect of ASCs was enhanced and ASCs could induce RAW264.7 macrophages toward the anti‐inflammatory M2 phenotype. We further demonstrated that ASCs‐derived interleukin 10 (IL‐10), mediated by hypoxia inducible factor‐1α (HIF‐1α), played a crucial role in the induction of M2 macrophages by activating the signal transducer and activator of transcription 3 (STAT3)/Arginase (Arg‐1) pathway. Our in vivo experiments revealed that transplanted ASCs exhibited an immunomodulatory effect by recruiting macrophages to ischemic muscle and increasing the density of M2 macrophages. The transplantation of ASCs into ischemic limbs induced increased blood flow reperfusion and limb salvage rate, whereas the depletion of tissue macrophages or transplanting HIF‐1α‐silenced ASCs inhibited the therapeutic effect. These findings elucidated the critical role of macrophages in ASCs‐mediated ischemic muscle repair and proved that allogeneic ASCs could exert the protective effect by enhancing the recruitment of macrophages and inducing macrophages toward M2 phenotype through HIF‐1α/IL‐10 pathway.

DHZCP Modulates Microglial M1/M2 Polarization via the p38 and TLR4/NF-κB Signaling Pathways in LPS-Stimulated Microglial Cells.

Intracerebral hemorrhage (ICH) is a disease with a significantly high rate of morbidity, mortality and disability. Inhibition of inflammation is considered a potential strategy for improving the clinical symptoms induced by ICH. The hallmark of neuroinflammation is microglial activation. Microglia can polarize into either the classically activated M1 (proinflammatory) phenotype, exacerbating neuronal damage, or the alternatively activated M2 (antiinflammatory) phenotype, exerting neuroprotection and promoting neuronal recovery. Promoting microglial polarization to the M2 phenotype may be a viable strategy for treating neuroinflammation. Several studies have indicated that promoting blood circulation and removing blood stasis exhibits therapeutic effects on intracerebral hemorrhage. Dahuang Zhechong Pill (DHZCP), a classical recipe that promotes blood circulation and removes blood stasis, has been reported to improve the clinical outcome of ICH. DHZCP has been shown to exert antiinflammatory effects. However, the detailed antiinflammatory mechanism of DHZCP in ICH has rarely been investigated. In this study, DHZCP inhibited lipopolysaccharide (LPS)-induced M1 microglial activation. DHZCP exerted antiinflammatory effects, by inhibiting LPS-induced M1 proinflammatory cytokine (TNF-α and IL-6), and iNOS production and increasing M2 antiinflammatory cytokine (IL-10) production. DHZCP also switched microglial polarization from M1 to M2, as indicated by significantly increased expression of M2 polarization markers (CD209, and CD206) and markedly decreased expression of an M1 polarization marker (CD54). In addition, DHZCP inhibited p38 and TLR4/NF-κB signaling activation, as demonstrated by inhibition of LPS-induced increases in p-p38, TLR4 and nuclear factor kappa B p-65 (NF-κB p-65) protein expression. Taken together, DHZCP modulates microglial M1/M2 polarization via the p38 and TLR4/NF-κB signaling pathways to confer antiinflammatory effects.

Injectable Gelatin Hydrogel Suppresses Inflammation and Enhances Functional Recovery in a Mouse Model of Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) is a devastating subtype of stroke with high morbidity and mortality. However, there is no effective therapy method to improve its clinical outcomes to date. Here we report an injectable gelatin hydrogel that is capable of suppressing inflammation and enhancing functional recovery in a mouse model of ICH. Thiolated gelatin was synthesized by EDC chemistry and then the hydrogel was formed through Michael addition reaction between the thiolated gelatin and polyethylene glycol diacrylate. The hydrogel was characterized by scanning electron microscopy, porosity, rheology, and cytotoxicity before evaluating in a mouse model of ICH. The in vivo study showed that the hydrogel injection into the ICH lesion reduced the neuron loss, attenuated the neurological deficit post-operation, and decreased the activation of the microglia/macrophages and astrocytes. More importantly, the pro-inflammatory M1 microglia/macrophages polarization was suppressed while the anti-inflammatory M2 phenotype was promoted after the hydrogel injection. Besides, the hydrogel injection reduced the release of inflammatory cytokines (IL-1β and TNF-α). Moreover, integrin β1 was confirmed up-regulated around the lesion that is positively correlated with the M2 microglia/macrophages. The related mechanism was proposed and discussed. Taken together, the injectable gelatin hydrogel suppressed the inflammation which might contribute to enhance the functional recovery of the ICH mouse, making it a promising application in the clinic.

EP4 signalling is essential for controlling islet inflammation by causing a shift in macrophage polarization in obesity/type 2 diabetes.

Activation of the prostaglandin E2 receptor EP4 alters polarization of adipose tissue macrophages towards the anti-inflammatory M2 phenotype to suppress chronic inflammation. However, the role of EP4 signalling in pancreatic macrophages that affect insulin secretion is unclear. We examined the role of EP4 signalling in islet inflammation in vitro and in vivo. Obese diabetic db/db mice were treated with an EP4-selective agonist or vehicle for 4 weeks. Islet morphology did not significantly differ and glucose-stimulated insulin secretion was increased, whereas the pancreatic M1/M2 ratio was decreased in the EP4 agonist-treated group compared to the vehicle group. Because EP4 activation in MIN6 cells did not affect insulin secretion, we used a MIN6/macrophage co-culture system to evaluate the role of EP4 signalling in islet inflammation and subsequent inhibition of insulin release. Co-culture with M1-polarized macrophages markedly suppressed insulin expression in MIN6 cells; however, modulation of M1 polarization by the EP4 agonist significantly reversed the negative impact of co-cultivation on insulin production. The enhanced expression levels of pro-inflammatory cytokines in co-cultured MIN6 cells were markedly inhibited by EP4 agonist treatment of M1 macrophages. Thus, EP4 activation may suppress islet inflammation and protect β-cell function by altering inflammatory macrophages in the diabetic pancreas.

Suppressors of Cytokine Signaling (SOCS)1 and SOCS3 Proteins Are Mediators of Interleukin-10 Modulation of Inflammatory Responses Induced by Chlamydia muridarum and Its Major Outer Membrane Protein (MOMP) in Mouse J774 Macrophages.

The immunopathology of chlamydial diseases is exacerbated by a broad-spectrum of inflammatory mediators, which we reported are inhibited by IL-10 in macrophages. However, the chlamydial protein moiety that induces the inflammatory mediators and the mechanisms by which IL-10 inhibits them are unknown. We hypothesized that Chlamydia major outer membrane protein (MOMP) mediates its disease pathogenesis, and the suppressor of cytokine signaling (SOCS)1 and SOCS3 proteins are mediators of the IL-10 inhibitory actions. Our hypothesis was tested by exposing mouse J774 macrophages to chlamydial stimulants (live Chlamydia muridarum and MOMP) with and without IL-10. MOMP significantly induced several inflammatory mediators (IL-6, IL-12p40, CCL5, CXCL10), which were dose-dependently inhibited by IL-10. Chlamydial stimulants induced the mRNA gene transcripts and protein expression of SOCS1 and SOCS3, with more SOCS3 expression. Notably, IL-10 reciprocally regulated their expression by reducing SOCS1 and increasing SOCS3. Specific inhibitions of MAPK pathways revealed that p38, JNK, and MEK1/2 are required for inducing inflammatory mediators as well as SOCS1 and SOCS3. Chlamydial stimulants triggered an M1 pro-inflammatory phenotype evidently by an enhanced nos2 (M1 marker) expression, which was skewed by IL-10 towards a more M2 anti-inflammatory phenotype by the increased expression of mrc1 and arg1 (M2 markers) and the reduced SOCS1/SOCS3 ratios. Neutralization of endogenously produced IL-10 augmented the secretion of inflammatory mediators, reduced SOCS3 expression, and skewed the chlamydial M1 to an M2 phenotype. Inhibition of proteasome degradation increased TNF but decreased IL-10, CCL5, and CXCL10 secretion by suppressing SOCS1 and SOCS3 expressions and dysregulating their STAT1 and STAT3 transcription factors. Our data show that SOCS1 and SOCS3 are regulators of IL-10 inhibitory actions, and underscore SOCS proteins as therapeutic targets for IL-10 control of inflammation for Chlamydia and other bacterial inflammatory diseases.

Macrophage Modification Strategies for Efficient Cell Therapy.

Macrophages, important cells of innate immunity, are known for their phagocytic activity, capability for antigen presentation, and flexible phenotypes. Macrophages are found in all tissues and therefore represent an attractive therapeutic target for the treatment of diseases of various etiology. Genetic programming of macrophages is an important issue of modern molecular and cellular medicine. The controllable activation of macrophages towards desirable phenotypes in vivo and in vitro will provide effective treatments for a number of inflammatory and proliferative diseases. This review is focused on the methods for specific alteration of gene expression in macrophages, including the controllable promotion of the desired M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotypes in certain pathologies or model systems. Here we review the strategies of target selection, the methods of vector delivery, and the gene editing approaches used for modification of macrophages.

THU0199 ABX464, A NOVEL DRUG IN THE FIELD OF INFLAMMATION, INCREASES MIR-124 AND MODULATES MACROPHAGES AND T-CELL FUNCTIONS.

Background:ABX464 is a small oral molecule with a novel mode of action. It binds the Cap Binding Complex, involved in the biogenesis of RNAs and predominantly upregulates the expression of a microRNA miR-124 in PBMCs and T cells (1). miR-124 has been widely described for its anti-inflammatory properties, with many confirmed targets i.e. monocyte chemoattractant protein 1 (MCP-1, CXCL-1, SERPIN-E1, STAT-3, IL-6 receptor. It post-transcriptionally regulates the expression of MCP-1 in rheumatoid arthritis (RA) synoviocytes and decreases their proliferation (2). While miR-124 is decreased in synoviocytes of RA patients, its injection in joint improved arthritis in rats (3). miR-124 expression in macrophages leads to the induction and maintenance of anti-inflammatory M2 phenotype (4). Its effect in T cells remains controversial.Objectives:(i) To assess the effect of ABX464 on miR-124 expressionin vitroin macrophages andin vivoin patients; (ii) to assess the effect of ABX464 on arthritis in mice and (iii) to decipher the effect of ABX464 on human macrophages and T cells.Methods:miR-124 was measured in human monocyte-derived macrophages (huMDM) treated with ABX464 for 4 days and in patients with ulcerative colitis included in a phase IIa RCT in blood and rectal biopsies at day 56 by TaqMan qPCR. Collagen-induced arthritis (CIA) was induced using usual protocol and ABX464 was given by gavage 2 weeks at 40 mg/kg after the 2ndinjection of collagen and Freund adjuvant. HuMDM were exposed to 5 µM of ABX464 or DMSO (control) for 4 days, during a M1-polarization. Cytokines and chemokines were assessed in supernatants using both Proteome Profiler Array and Luminex. PBMCs were exposed to ABX464 (5 µM) for 6 days. Th1 (IFN-g+), Th17 (CCR6+IL-17+), Th2 (CRTH2+ IL-4+) and Tregs (CD25+CD125-/loFoxP3+) were assessed by flow cytometry. IL-6 receptor was assessed in CD4+ supernatant using ELISA.Results:ABX464 increased miR-124 in vitro by 3.41 folds in huMDM (p=0.001) compared to DMSO. The phase IIa RCT conducted in 32 patients with moderate to severe active ulcerative colitis showed a good safety profile and significant clinical efficacy. A strong increase of miR-124 was observed both in blood and rectal biopsies of patients treated with ABX464 (637 and 7.69 folds respectively, compared to placebo, p<0.05). The use of ABX464 drastically decreased the incidence of arthritis from 52% (15/ 29 mice) to 10% (3/30 mice) in a CIA model. Macrophages treated with ABX464 produced significantly less MCP-1 (median decrease -67%, p=0.004), CXCL-1 (-18%, p=0.004) and SERPIN-E1 (-53%, p=0.004), as confirmed by the two technics (n=9). ABX464 significantly decreased Th17 (-56%, p=0.02), while increasing Th2 (+21%, p=0.01). IL-6 soluble receptor was significantly decreased in supernatant of PBMCs treated with ABX464 (-43%, p=0.04).Conclusion:We demonstrated that ABX464 increases miR-124 bothin vitroand in ulcerative colitis patients.In vitro, ABX464 decreased the expression of miR-124 target genes, that is MCP-1, CXCL-1, SERPIN-E1 in macrophages and decreases the number of Th17 as well as IL-6 soluble receptor in CD4+ T cells. A phase IIa RCT is currently ongoing in patients with rheumatoid arthritis and inadequate response to methotrexate and/or TNF-inhibitors (n=60). Results are expected during 2020 summer.

MO064TISSUE-RESIDENT B CELLS DETERMINE SUSCEPTIBILITY TO URINARY TRACT INFECTION BY ORCHESTRATING MACROPHAGE POLARISATION

Abstract Background Urinary tract infection (UTI) is an important clinical problem. More than half of women and 1 in 10 men will be affected during their lifetime. Many of these affect the lower urinary tract but recurrent pyelonephritis can lead to scarring and chronic kidney disease. There is an increasing appreciation that tissue-resident immune cells, such as macrophages, play an important role in defence against infection, but only little is known about B lymphocytes in this context. Here we sought to address the question of whether B cells reside in the kidney and bladder in homeostasis and to determine their phenotype and contribution to local organ immunity. Methods and Results Using intravenous labelling and parabiosis, we identified a population of bona-fide self-renewing, tissue-resident B cells that included non-naïve and innate-like CD5+ B-1 cells, in murine kidneys and urinary bladder (but also in liver and lung). The size and phenotype of this B cell subset was influenced by genetic background, age, and microbiome, with an expanded population evident after co-housing with pet-store mice. Although kidney B cells had less diverse Igh repertoire compared to blood, their seeding was largely independent of their B-cell receptor specificity. In human kidneys we found a similar enrichment for non-naïve B cells compared to blood and spleen. Using two strains of genetically modified mice with higher (PI3KδE1020K-B) or lower (μMT-) numbers of tissue-resident B cells, we tested the function of these cells during UTI. Surprisingly, the number of tissue-resident B cells inversely correlated with bacterial clearance. We found that these B cells were spatially co-localised with kidney macrophages and skewed their polarization towards an anti-inflammatory M2 phenotype, leading to reduced anti-microbial responses. This effect was, at least in part, driven via IL-10. Conclusion In conclusion, our data identify a critical role for tissue-resident B cells in modulating local immunity in the urinary tract, determining the inflammatory ‘set-point’ of resident and recruited myeloid cells, with important clinical implications for the use of B-cell depleting therapies and conditions such as infection, transplant rejection, fibrosis or autoimmunity. Graphical Abstract

Hippocampal overexpression of TREM2 ameliorates high fat diet induced cognitive impairment and modulates phenotypic polarization of the microglia

Type 2 diabetes mellitus (T2DM) and Alzheimer's disease (AD) share several common pathophysiological features. Rare variants of triggering receptor expressed on myeloid cells 2 (TREM2) increase the risk of developing AD, suggesting the involvement of TREM2 and innate immunity in AD development. It is still unknown whether TREM2 is related to cognitive impairment in T2DM. Here, we investigated the effects of the hippocampal overexpression of TREM2 on cognitive in long-term high-fat diet (HFD)-fed mice. Male C57BL/6J mice were maintained on HFD for 50 weeks. TREM2 was overexpressed in the hippocampus 36 weeks after HFD feeding using adeno-associated virus vector (AAV)-mediated gene delivery. The results showed that the HFD feeding induced rapid and persistent weight gain, glucose intolerance and significant impairments in learning and memory. Compared with AAV-con, AAV-TREM2 significantly ameliorated cognitive impairment without altering body weight and glucose homeostasis in HFD mice. The overexpression of TREM2 upregulated the synaptic proteins spinophilin, PSD95 and synaptophysin, suggesting the improvement in synaptic transmission. Dendritic complexity and spine density in the CA1 region were rescued after TREM2 overexpression. Furthermore, TREM2 markedly increased the number of iba-1/Arg-1-positive microglia in the hippocampus, suppressed neuroinflammation and microglial activation. In sum, hippocampal TREM2 plays an important role in improving HFD-induced cognitive dysfunction and promoting microglial polarization towards the M2 anti-inflammatory phenotype. Our study also suggests that TREM2 might be a novel target for the intervention of obesity/diabetes-associated cognitive decline.

Anti-inflammatory effects of haptoglobin on LPS-stimulated macrophages: Role of HMGB1 signaling and implications in chronic wound healing.

Nonhealing wounds possess elevated numbers of pro-inflammatory M1 macrophages, which fail to transition to anti-inflammatory M2 phenotypes that promote healing. Hemoglobin (Hb) and haptoglobin (Hp) proteins, when complexed (Hb-Hp), can elicit M2-like macrophages through the heme oxygenase-1 (HO-1) pathway. Despite the fact that nonhealing wounds are chronically inflamed, previous studies have focused on non-inflammatory systems, and do not thoroughly compare the effects of complexed vs individual proteins. We aimed to investigate the effect of Hb/Hp treatments on macrophage phenotype in an inflammatory, lipopolysaccharide (LPS)-stimulated environment, similar to chronic wounds. Human M1 macrophages were cultured in vitro and stimulated with LPS. Concurrently, Hp, Hb, or Hb-Hp complexes were delivered. The next day, 27 proteins related to inflammation were measured in the supernatants. Hp treatment decreased a majority of inflammatory factors, Hb increased many, and Hb-Hp had intermediate trends, indicating that Hp attenuated overall inflammation to the greatest extent. From this data, Ingenuity Pathway Analysis software identified high motility group box 1 (HMGB1) as a key canonical pathway-strongly down-regulated from Hp, strongly up-regulated from Hb, and slightly activated from Hb-Hp. HMGB1 measurements in macrophage supernatants confirmed this trend. In vivo results in diabetic mice with biopsy punch wounds demonstrated accelerated wound closure with Hp treatment, and delayed wound closure with Hb treatment. This work specifically studied Hb/Hp effects on macrophages in a highly inflammatory environment relevant to chronic wound healing. Results show that Hp-and not Hb-Hp, which is known to be superior in noninflammatory conditions-reduces inflammation in LPS-stimulated macrophages, and HMGB1 signaling is also implicated. Overall, Hp treatment on M1 macrophages in vitro reduced the inflammatory secretion profile, and also exhibited benefits in in silico and in vivo wound-healing models.

IL-37 inhibits M1-like macrophage activation to ameliorate temporomandibular joint inflammation through the NLRP3 pathway.

IL-37 has been identified as an important anti-inflammatory and immunosuppressive factor. This study was undertaken to explore how IL-37 affects M1/M2-like macrophage polarization and thus contributes to anti-inflammatory processes in the temporomandibular joint. Western blotting, quantitative real-time PCR (qRT-PCR) and immunofluorescence were used to verify the IL-37-induced polarization shift from the M1 phenotype to the M2 phenotype, and the related key pathways were analysed by western blotting. Human chondrocytes were stimulated with M1-conditioned medium (CM) or IL-37-pretreated M1-CM, and inflammatory cytokines were detected. siRNA-IL-1R8 and MCC-950 were used to investigate the mechanism underlying the anti-inflammatory effects of IL-37. Complete Freund's adjuvant-induced and disc perforation-induced inflammation models were used for in vivo studies. Haematoxylin and eosin, immunohistochemical and safranin-O staining protocols were used to analyse histological changes in the synovium and condyle. Western blotting, qRT-PCR and immunofluorescence showed that IL-37 inhibited M1 marker expression and upregulated M2 marker expression. Western blotting and qRT-PCR showed that pretreatment with IL-37 suppressed inflammatory cytokine expression in chondrocytes. IL-37 inhibited the expression of NLRP3 and upregulated the expression of IL-1R8. Si-IL-1R8 and MCC-950 further confirmed that the anti-inflammatory properties of IL-37 were dependent on the presence of IL-1R8 and NLRP3. In vivo, IL-37 reduced synovial M1 marker expression and cartilage degeneration and increased M2 marker expression. IL-37 shifting of the polarization of macrophages from the pro-inflammatory M1 phenotype to the beneficial anti-inflammatory M2 phenotype seems to be a promising therapeutic strategy for treating temporomandibular joint inflammation.

Injection of mscs mitigates particle associated chronic inflammation of bone

Background & Aim Chronic inflammation is a common feature in many diseases of different organ systems, including bone. Unfortunately, there are few interventions to mitigate chronic inflammation and preserve host tissue. We have used diverse strategies to regulate chronic inflammation including polarization of local macrophages to an M2 anti-inflammatory phenotype and modulation of the transcription factor NF-kB. In the current study, we investigate local injection of unmodified and pre-conditioned MSCs (pMSCs) in a model of chronic inflammation of bone: the murine continuous femoral intramedullary polyethylene particle infusion model. Methods, Results & Conclusion Methods: Osmotic pumps were connected to vinyl catheter tubing and a hollow titanium rod was press-fit into the distal femur through the intercondylar region of 11-12 week BALB/c mice. At primary surgery, the pumps were filled with 10% BSA/PBS +/- 1.25% polyethylene (PE) particles and 10 ng/ml of LPS (contaminated polyethylene particles, cPE). For preparation of pMSCs, murine MSCs were seeded at 5,000 cells/cm2 and treated with 20 ng/ml TNF-a and 20 mg/ml LPS for 3 days. Three weeks after primary surgery, pumps were changed to new ones for 3 more weeks. In specific treatment groups, 5 × 105 murine primary MSCs or pMSCs were injected intramedullary at the second surgery. There were six groups +/- PE/cPE and +/- treatment with MSCs or pMSCs (7-9 mice/group). MicroCT analysis was carried out 6 weeks after primary surgery. A three-dimensional region of interest (ROI: 4 × 4 × 3 mm) was created in the distal femur. Bone mineral density (BMD, mg/mm3) was quantified. Histomorphological analysis was performed of the area of osteoclasts (TRAP staining) and alkaline phosphatase (ALP) staining; immunohistochemistry was used to calculate the total number of macrophages (F4/80+), and the number of M1 (iNOS+) and M2 (arginase+) phenotypes. A one-way ANOVA with Tukey's post-hoc test was performed to quantify the results. Results Contaminated polyethylene particles increased total macrophages, M1 macrophage phenotype, and the percentage of TRAP positive area in the ROI, and decreased M2 macrophage phenotype (Figures 1 and 2). Injection of un-modified MSCs or pMSCs reversed these findings. There were no significant effects on BMD for the treatments in the polyethylene particle groups. Conclusion Local injection of MSCs may be a potential intervention to mitigate the adverse effects of chronic inflammation of bone due to contaminated polyethylene particles.

Clinical Implications

Clinical Implications

Profilin 1 knockdown prevents ischemic brain damage by promoting M2 microglial polarization associated with the RhoA/ROCK pathway.

Microglial polarization to the anti-inflammatory M2 phenotype is essential in resolving neuroinflammation, making it a promising therapeutic strategy for stroke intervention. The actin cytoskeleton is known to be important for the physiological functions of microglia, including migration and phagocytosis. Profilin 1 (PFN1), an actin-binding protein, is involved in the dynamic transformation and reorganization of actin. However, the role of PFN1 in microglial polarization and ischemia/reperfusion injury is unclear. The role of PFN1 on microglial polarization was examined in vitro in BV2 microglial cells subjected to oxygen-glucose deprivation/reoxygenation (OGDR) and in vivo in male mice after transient middle cerebral artery occlusion (MCAO). Knockdown of PFN1 inhibited M1 microglial polarization and promoted M2 microglia polarization 48hr after OGDR stimulation in BV2 cells and 7days after MCAO-induced injury in male mice. RhoA/ROCK pathway was involved in the regulation of PFN1 during microglial polarization. Knockdown of PFN1 also significantly attenuated brain infarcts and edema, improved cerebral blood flow and neurological deficits in MCAO-injured mice. Inhibition of PFN1 effectively protected the brain against ischemia/reperfusion injuries by promoting M2 microglial polarization in vitro and in vivo.

Zinc-aggravated M1 microglia regulate astrocytic engulfment via P2×7 receptors

BackgroundGlial cells such as astrocytes and microglia play an important role in the central nervous system via communication between these glial cells. Activated microglia can exhibit either the inflammatory M1 phenotype or the anti-inflammatory M2 phenotype, which influences astrocytic neuroprotective functions, including engulfment of cell debris. Recently, extracellular zinc has been shown to promote the inflammatory M1 phenotype in microglia through intracellular zinc accumulation and reactive oxygen species (ROS) generation. PurposeHere, we investigated whether the zinc-enhanced inflammatory M1 phenotype of microglia affects the astrocytic engulfing activity. MethodsEngulfing activity was assessed in astrocytes treated with microglial-conditioned medium (MCM) from lipopolysaccharide (LPS)-activated or from ZnCl2-pretreated LPS-activated M1 microglia. The effect of zinc on microglia phenotype was also validated using the zinc chelator N,N,N’,N’-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and the ROS scavenger Trolox. ResultsAlthough treatment of astrocytes with LPS showed no significant effect on the engulfing activity, MCM from LPS-induced M1 microglia increased the beads uptake by astrocytes. This increased uptake activity was suppressed when MCM from LPS-induced M1 microglia pretreated with ZnCl2 was applied to astrocytes, which was further abolished by the intracellular zinc chelator TPEN and the ROS scavenger Trolox. In addition, expression of P2×7 receptors (P2×7R) was increased in astrocytes treated with MCM derived from M1 microglia but not in the M1 microglia pretreated with ZnCl2. ConclusionThese findings suggest that zinc pre-treatment abolishes the ability of LPS-induced M1 microglia to increase the engulfing activity in astrocytes via alteration of astrocytic P2×7R.

2-Methoxyestradiol inhibits high fat diet-induced obesity in rats through modulation of adipose tissue macrophage infiltration and immunophenotype

Recently, experimental studies demonstrated that 2-methoxyestradiol (2ME2) ameliorates high fat diet (HFD)-induced obesity and restores insulin sensitivity. However, the mechanisms underlying these effects are unveiled yet. The current study was undertaken to test the hypothesis that 2ME2 exerts its effects by modulating adipose tissue macrophages (ATMs) accumulation, polarization and immunophenotypes. The experiment was carried out in males Wistar rats (n = 28) for 13 consecutive weeks. In HFD-fed group; body weight, glucose intolerance, serum insulin, HOMA-IR, lipid profile and adipose tissue (AT) weight were significantly higher compared to normal standard diet (NSD)- fed rats. However, treatment of HFD-fed rats with 2ME2 (200 μg/kg/day; i.p. from the beginning of the 9th week) resulted in significant enhancements in all these parameters as compared to HFD-fed rats. Treatment with 2ME2 was associated with a significant reduction in macrophage infiltration in the AT, shifting macrophage polarization towards M2 phenotype as indicated by significant decrease in the expression of pro-inflammatory M1 macrophages markers (IL-6, IL-1β, CD11c and iNOS) and concurrent significant increase in the M2 anti-inflammatory macrophage markers (Arginase 1 and IL-10). 2ME2 ameliorates HFD-induced obesity and glucose intolerance through inhibition of ATM infiltration in AT and shifting macrophage polarization from pro-inflammatory M1 to M2 anti-inflammatory phenotypes.

Healing the Broken Heart; The Immunomodulatory Effects of Stem Cell Therapy.

Cardiovascular Disease (CVD) is a leading cause of mortality within the United States. Current treatments being administered to patients who suffered a myocardial infarction (MI) have increased patient survival, but do not facilitate the replacement of damaged myocardium. Recent studies demonstrate that stem cell-based therapies promote myocardial repair; however, the poor engraftment of the transferred stem cell populations within the infarcted myocardium is a major limitation, regardless of the cell type. One explanation for poor cell retention is attributed to the harsh inflammatory response mounted following MI. The inflammatory response coupled to cardiac repair processes is divided into two distinct phases. The first phase is initiated during ischemic injury when necrosed myocardium releases Danger Associated Molecular Patterns (DAMPs) and chemokines/cytokines to induce the activation and recruitment of neutrophils and pro-inflammatory M1 macrophages (MΦs); in turn, facilitating necrotic tissue clearance. During the second phase, a shift from the M1 inflammatory functional phenotype to the M2 anti-inflammatory and pro-reparative functional phenotype, permits the resolution of inflammation and the establishment of tissue repair. T-regulatory cells (Tregs) are also influential in mediating the establishment of the pro-reparative phase by directly regulating M1 to M2 MΦ differentiation. Current studies suggest CD4+ T-lymphocyte populations become activated when presented with autoantigens released from the injured myocardium. The identity of the cardiac autoantigens or paracrine signaling molecules released from the ischemic tissue that directly mediate the phenotypic plasticity of T-lymphocyte populations in the post-MI heart are just beginning to be elucidated. Stem cells are enriched centers that contain a diverse paracrine secretome that can directly regulate responses within neighboring cell populations. Previous studies identify that stem cell mediated paracrine signaling can influence the phenotype and function of immune cell populations in vitro, but how stem cells directly mediate the inflammatory microenvironment of the ischemic heart is poorly characterized and is a topic of extensive investigation. In this review, we summarize the complex literature that details the inflammatory microenvironment of the ischemic heart and provide novel insights regarding how paracrine mediated signaling produced by stem cell-based therapies can regulate immune cell subsets to facilitate pro-reparative myocardial wound healing.

Follistatin like protein-1 modulates macrophage polarization and aggravates dextran sodium sulfate-induced colitis

Follistatin-like protein 1 (FSTL1) is a pleiotropic cytokine involved in multiple processes including organ development, carcinogenesis, metastasis and so on. Some recent studies have suggested a possible role of FSTL1 in the inflammatory diseases. We for the first time tried to unravel its effect on the colitis, and explore the possible mechanisms. Here we found that FSTL1 was upregulated in active human and murine colitis. It facilitated proinflammatory M1 polarization of macrophages and inhibited the M2 anti-inflammatory phenotype, leading to excessive production of multiple inflammatory cytokines in vitro and in vivo. Haplodeletion of FSTL1 in mice significantly reduced the clinical and histological activity of colitis. Most importantly, macrophage depletion diminished the difference between DSS-treated WT and FSTL1+/− mice. Altogether, our results suggested that FSTL1 may also serve as an important contributor in the colonic inflammation. The possible mechanism may be related to its modulation on macrophage polarization.

Abstract 114: A Novel Hydrogel-based Nanofiber Drug Delivery System For Sustained Release Of Igf-1 Nanoparticles To Improve Functional Outcomes After Nerve Repair

Purpose: Despite extensive research efforts, no therapeutic agents are currently clinically indicated for the treatment of peripheral nerve injuries. Insulin-like growth factor 1 (IGF-1) is an ideal therapeutic candidate as it can accelerate axonal regeneration and also minimize the deleterious effects of prolonged denervation on muscle and Schwann cells. However, given its short half-life, a practical delivery system is needed to stabilize the protein and provide sustained release to target tissues. Using a novel encapsulation method, we demonstrated sustained release of bioactive IGF-1 from nanoparticles, in vitro, and improved nerve regeneration and functional recovery, in vivo. An optimized carrier system to maintain the nanoparticles at target tissue sites for the duration of drug release and avoid frequent re-dosing is now needed. We therefore developed a biocompatible nanofiber hydrogel composite that could be loaded with IGF-1 nanoparticles; fine-tuned its drug release kinetics in vitro and in vivo; and applied it in a chronic denervation median nerve model to assess its impact on functional recovery. Methods: An injectable nanofiber-hydrogel composite system (made of PCL nanofibers covalently bonded to hyaluronic acid) was developed by electrospinning. Its 3-D structure was formulated to mimic that of fat extracellular matrix (ECM). The release kinetics of this delivery system were then optimized in vitro and in vivo (using ELISA and immunofluorescent staining) to achieve controlled release of IGF-1 at therapeutic levels (~10 times EC50) for a prolonged period. Finally, using a chronic median nerve denervation model, we tested the effects of this modality on axonal regeneration, Schwann cell senescence, muscle atrophy and muscle force. Results: The level of synthetic mimicry between our drug-delivery system and ECM fat was noted to confer high levels of biocompatibility as evidenced by a minimal inflammatory response 25 days post injection. The composite system polarized the invading macrophages into an anti-inflammatory and pro-regenerative M2 phenotype. The release kinetics of IGF-1 from the nanofiber system were superior to other carriers (fibrin glue and saline), both in vitro and in vivo. When injected into the target muscle and deposited at the nerve coaptation site in a chronic median nerve denervation model, functional outcomes were improved (although not reaching a customary level of statistical significance. Conclusion: We introduce a novel drug delivery system in which IGF-1 nanoparticles are combined with a nanofiber hydrogel carrier to provide sustained local concentrations of bioactive IGF-1within target nerve and muscle. This therapeutic approach has the potential to improve functional outcomes via enhanced axonal regeneration and maintenance of denervated muscle and Schwann cells. IGF-1 and the polymer components of the engineered delivery system are currently used in FDA-approved formulations and devices, which will facilitate clearance of regulatory hurdles.