Facilitates Macrophage Polarization Research Articles

NGF facilitates ICAM-1-dependent monocyte adhesion and M1 macrophage polarization in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disorder in which monocytes adhering to synovial tissue differentiate into the pro-inflammatory M1 macrophage phenotype. Nerve growth factors (NGF) referred to as neurotrophins have been associated with inflammatory events; however, researchers have yet to elucidate the role of NGF in RA. Our examination of clinical tissue samples and analysis of data sourced from the Gene Expression Omnibus dataset unveiled elevated expression levels of M1 macrophage markers in human RA synovial tissue samples compared to normal tissue, with no such distinction observed for M2 markers. Furthermore, immunofluorescence data depicted increased expression levels of NGF and M1 macrophages in RA mice in contrast to normal mice. It appears that NGF stimulation facilitates macrophage polarization from the M0 to the M1 phenotype. It also appears that NGF promotes ICAM-1 production in human RA synovial fibroblasts, which enhances monocyte adhesion through the TrkA, MEK/ERK, and AP-1 signaling cascades. Our findings indicate NGF/TrkA axis as a novel target for the treatment of RA.

A guanosine/konjac glucomannan supramolecular hydrogel with antioxidant, antibacterial and immunoregulatory properties for cutaneous wound treatment

Developing naturally-derived wound dressing materials with intrinsic therapeutic effects is desirable for the clinical applications. Recently, guanosine-based supramolecular G-quadruplex (G4) hydrogel exhibited great potential in preparing biological materials due to its simple fabrication method and responsive gel networks. However, the weak mechanical properties and the consequent burst release of bioactive molecules restrict its clinical applications. Herein, we found that konjac glucomannan (KGM) with immunoregulatory effect did not affect the self-assembly of G-quadruplexes and thus effectively enhancing the mechanical properties of G4 hydrogel. Aloin, as a model drug, was in situ loaded into gel networks, finally obtaining the G4/Aloin-KGM hydrogel. This hydrogel exhibited porous morphology, swelling ability and hemostatic capability. Boronate bonds in G4 networks and aloin collectively endowed the hydrogel with excellent antioxidant performance. Meanwhile, aloin also provided outstanding in vitro and in vivo bactericidal ability. The wounds treated with this biocompatible hydrogel demonstrated faster regeneration of epithelial and dermal tissues, and the whole wound healing stages were accelerated by promoting collagen deposition, facilitating macrophage polarization towards M2 phenotype, down-regulating the expression level of IL-6, and up-regulating the expression level of IL-10, CD31 and α-SMA.

Moluodan promotes DSS-induced intestinal inflammation involving the reprogram of macrophage function and polarization

Ethnopharmacological relevanceMoluodan (MLD) is a traditional Chinese medicine that is composed of 18 herbal medicines based on traditional Chinese medicine theory and practice. It has long been used in treating chronic gastritis and its components were traditionally used in dealing with intestinal inflammation. However, its specific pharmacological mechanism is still unclear. Aim of the studyThe upper and lower digestive tract diseases are correlated. In clinical practice, some chronic gastritis patients are also accompanied by intestinal inflammation. Due to the unclear pharmacological mechanism of MLD and its effect on intestinal inflammation, there is doubt whether MLD is still suitable for this type of patient. Therefore, this study aims to elucidate the pharmacological mechanism of MLD and identify its effect in the mouse model of intestinal inflammation. Materials and methodsMice intestinal inflammation model was induced by 2.5% dextran sulfate sodium (DSS). The mice were given different concentrations of MLD via oral gavage (0.25, 0.5 g/kg b.w.). Pharmacodynamic indicators were assessed including body weight, colon length, disease activity index (DAI), bloody stool score, inflammatory factors, histological change, etc. RAW264.7 macrophage cells were used for in vitro experiments that illuminated the role of MLD in reprogramming macrophage function and polarization. RT-qPCR and western blots were performed to measure the mRNA and protein levels of macrophage polarization marker and effector molecules. The functions of polarized macrophages were tested using ROS detection probes, Edu assay and wound healing assay. ResultsThe administration of MLD exhibited obvious hemostatic effects, while unexpectedly accentuating various aspects of the DSS-induced intestinal inflammation in mice, including increased body weight loss and colon shortening, elevated disease activity index, and intensified colonic tissue damage. Additionally, MLD treatment induced more severe inflammatory cell infiltration and higher proinflammatory cytokines expression in colon tissue. Further results showed that MLD promoted M1 macrophage polarization and stimulated its proinflammatory cytokines expression, while only slightly affecting the function of M2 macrophage. Western blot analysis revealed that MLD induced the phosphorylation of AKT and NF-κB. The polarization of M1 macrophages induced by MLD was inhibited by either an Akt inhibitor or a NF-κB inhibitor. ConclusionsAlthough MLD has an obvious hemostatic effect, it generally promoted the severity of DSS-induced colitis in mice by facilitating macrophage polarization toward the M1 phenotype through the AKT/NF-κB pathway. Our study suggested that MLD may not be suitable for colitis, especially during the acute inflammation stage.

Stereotactic ablative radiotherapy and FAPα-based cancer vaccine suppresses metastatic tumor growth in 4T1 mouse breast cancer

Background and purposeThis study tested the hypothesis that a novel combination of stereotactic ablation radiotherapy (SABR) and a cancer vaccine against fibroblast activation protein-alpha (FAPα) can suppress established tumor growth and impede potential metastasis. MethodsThe poorly immunogenic metastatic mouse mammary carcinoma 4T1 was used as a model. Mice were randomly assigned to five treatment groups: (1) untreated control, (2) FAPα-based cancer vaccine, (3) SABR, (4) SABR + pCDH (lentiviral control vector), (5) SABR + FAPα-based cancer vaccine (SABR/FAPα-Vax). FAPα-based cancer vaccine were administered subcutaneously every week for a total of three treatments. SABR was delivered to the primary tumor by 3 × 8 Gy after the first vaccination. ResultsConsistent with the poorly immunogenic nature of 4T1, tumor-bearing mice receiving FAPα-based cancer vaccine or SABR monotherapy showed a modest reduction in tumor volume and increased animal lifespan. In contrast, SABR/FAPα-Vax was well-tolerated, significantly reduced tumor burden, and increased survival compared to monotherapy. The increased survival correlated with inhibition of extracellular matrix (ECM) production, tumor vascularization and lymphangiogenesis. SABR/FAPα-Vax also resulted in an abscopal effect capable of eliminating lung metastases. SABR/FAPα-Vax recruited and activated CD8 + T cells to attack tumor cells and FAPα + stromal cells, and initiated suppressor cell reprogramming, including facilitating macrophage polarization toward an anti-tumor (M1) state, as well as depleting myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). ConclusionThese findings provide a novel therapeutic combination of radiation and FAPα-based cancer vaccine with promising results against poorly immunogenic metastatic cancer. This study may pave the way to overcome the therapeutic resistance caused by FAPα + CAFs.

Biocompatible Cryogel with Good Breathability, Exudate Management, Antibacterial and Immunomodulatory Properties for Infected Diabetic Wound Healing.

Due to the complex microenvironment and healing process of diabetic wounds, developing wound dressing with good biocompatibility, mechanical stability, breathability, exudate management, antibacterial ability, and immunomodulatory property is highly desired but remains a huge challenge. Herein, a multifunctional cryogel is designed and prepared with bio-friendly bacterial cellulose, gelatin, and dopamine under the condition of sodium periodate oxidation. Bacterial cellulose can enhance the mechanical stability of the cryogel by improving the skeleton supporting effect and crosslinking degree. The cryogel shows outstanding breathability and exudate management capability thanks to the interpenetrated porous structures. I2 and sodium iodides produced in situ by reduction of sodium periodate provide efficient antibacterial properties for the cryogel. The cryogel facilitates macrophage polarization from M1 to M2, thus regulating the immune microenvironment of infected diabetic wounds. With these advantages, the multifunctional cryogel effectively promotes collagen deposition and neovascularization, thus accelerating the healing of infected diabetic wounds.

Hydroxyapatite nanoparticles promote TLR4 agonist-mediated anti-tumor immunity through synergically enhanced macrophage polarization.

Macrophages represent the most prevalent immune cells in the tumor micro-environment, making them an appealing target for tumor immunotherapy. One of our previous studies showed that hydroxyapatite nanoparticles (HANPs) enhanced Toll-like receptor 4 (TLR4) signal transduction in macrophages. This study was proposed to investigate how HANPs manipulated the phenotype and function of macrophage against 4T1 breast tumors in the presence or absence of MPLA, a low toxic Toll-like receptor 4 (TLR4) agonist. The results demonstrated that the addition of HANPs to MPLA significantly promoted cytokine secretion and macrophage polarization toward a tumoricidal M1 phenotype. Further, the resulting supernatant from HANPs/MPLA co-stimulated macrophages enhanced 4T1 tumor cells apoptosis compared to that from macrophages treated with a single component or PBS control. In particular, we found HANPs elicited immunogenic cell death (ICD) indicated by the increased expression of "danger signals", including HMGB1, CRT and ATP in 4T1 cells. Subsequently, the ICD derivatives-containing supernatant from HANPs-treated 4T1 cells activated macrophage and shifted the phenotype of the cells toward M1 type. Moreover, in a tumor-bearing mice model, HANPs and MPLA synergistically delayed tumor growth compared to PBS control, which was positively associated with the promoted macrophage polarization and ICD induction. Therefore, our findings demonstrated a potential platform to modulate the function of macrophages, and shed a new insight into the mechanism involving the immunomodulatory effect of HANPs for tumor therapy. STATEMENT OF SIGNIFICANCE: Polarizing macrophage toward tumoricidal phenotype by harnessing Toll-like receptor (TLR) agonists has been proven effective for tumor immunotherapy. However, the immunomodulatory potency of TLR agonists is limited due to immune suppression or tolerance associated with TLR activation in immune cells. Herein, we introduced hydroxyapatite nanoparticles (HANPs) to MPLA, a TLR4 agonist. The results demonstrated that the addition of HANPs to MPLA promoted macrophage shift toward tumoricidal M1 phenotype, supported a "hot" tumor transformation, and delayed 4T1 tumor growth. Moreover, we found that HANPs elicited immunogenic cell death that produced "danger" signals from cancer cells thereby further facilitated macrophage polarization. This work is significant to direct the rational design of HANPs coupled with or without TLR agonists for tumor immunotherapy.

Inhibition of Toll-Like Receptor 4 Signaling Pathway Accelerates the Repair of Avascular Necrosis of Femoral Epiphysis through Regulating Macrophage Polarization in Perthes Disease.

Legg-Calvé-Perthes disease (LCPD) is still a refractory disease in children's orthopedics. With the introduction of the concept of "osteoimmunology", the immune-inflammatory mechanisms between bone and immune system have become a research focus of LCPD. However, few studies have reported on the pathological role of inflammation-related receptors such as toll-like receptors (TLRs) as well as immune cells such as macrophages in LCPD. This study was for investigating the mechanism of TLR4 signaling pathway on the direction of macrophage polarization and the repair of avascular necrosis of femoral epiphysis in LCPD. With GSE57614 and GSE74089, differentially expressed genes were screened. Through enrichment analysis and protein-protein interaction network, the functions of TLR4 were explored. Furthermore, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), hematoxylin & eosin (H&E) staining, micro-CT, tartrate-resistant acid phosphatase (TRAP) dyeing and western blotting were performed for determining the influences of TAK-242 (a TLR4 inhibitor) on the repair of avascular necrosis of femoral epiphysis in rat models. Totally 40 co-expression genes were screened as well as enriched in TLR4 signaling pathway. Immunohistochemistry and ELISA analyses certified that TLR4 facilitated macrophage polarization toward the M1 phenotype and prevented macrophage polarization toward the M2 phenotype. Besides, the results of H&E and TRAP staining, micro-CT, and western blotting showed that TAK-242 can inhibit osteoclastogenesis and promote osteogenesis. Inhibition of TLR4 signaling pathway accelerated the repair of avascular necrosis of femoral epiphysis by regulating macrophage polarization in LCPD.

Hypoxic bone marrow mesenchymal stromal cells-derived exosomal miR-182-5p promotes liver regeneration via FOXO1-mediated macrophage polarization.

Mesenchymal stromal cells (MSCs) are attractive candidates for treating hepatic disorders given their potential to enhance liver regeneration and function. The paracrine paradigm may be involved in the mechanism of MSC-based therapy, and exosomes (Exo) play an important role in this paracrine activity. Hypoxia significantly improves the effectiveness of MSC transplantation. However, whether hypoxia preconditioned MSCs (Hp-MSCs) can enhance liver regeneration, and whether this enhancement is mediated by Exo, are unknown. In this study, mouse bone marrow-derived MSCs (BM-MSCs) and secreted Exo were injected through the tail vein. We report that Hp-MSCs promote liver regeneration after partial hepatectomy in mice through their secreted exosomes. Interestingly, MSC-Exo were concentrated in liver 6h after administration and mainly taken up by macrophages, but not hepatocytes. Compared with normoxic MSC-Exo (N-Exo), hypoxic MSC-Exo (Hp-Exo) enhanced M2 macrophage polarization both in vivo and in vitro. Microarray analysis revealed significant enrichment of microRNA (miR)-182-5p in Hp-Exo compared with that in N-Exo. In addition, miR-182-5p knockdown partially abolished the beneficial effect of Hp-Exo. Finally, Hp-MSC-derived exosomal miR-182-5p inhibited theprotein expression of forkhead box transcription factor 1 (FOXO1) in macrophages, which inhibited toll-like receptor 4 (TLR4) expression and subsequently induced an anti-inflammatory response. These results highlight the therapeutic potential of Hp-Exo in liver regeneration and suggest that miR-182-5p from Hp-Exo facilitates macrophage polarization during liver regeneration by modulating the FOXO1/TLR4 signaling pathway.

Abstract 10391: Elevated Plasma Branched-Chain Amino Acids Level Promotes Atherosclerosis Progression by Facilitating Macrophage Polarization Toward M1 Phenotype That Results in the Increase of Inflammation

Introduction: Few studies have reported that whether elevated plasma branched-chain amino acids (BCAA) level is causally related to atherosclerosis (AS) and the mechanism involved. Methods: First, the plasma BCAA level of 258 atherosclerotic cardiovascular disease (ASCVD) patients and 188 normal people was measured. Second, plaque volume, stability and inflammation of AS mice were evaluated. Third, we assayed BCAA level, catabolism-promoting genes and inflammation in the circulating monocytes of ASCVD patients and abdominal macrophages of AS mice. Fourth, inflammation and macrophage polarization were evaluated in BCAA catabolic deficient RAW 264.7 cells. Finally, mitochondrial hydrogen peroxide (mtH 2 O 2 ), nuclear H 2 O 2 and High mobility group box 1 (HMGB1) levels in RAW 264.7 cells were measured and RAW 264.7 cells-overexpressing catalase targeted to mitochondria (MCAT), nucleus (NCAT) and HMGB1KD RAW 264.7 cells were built to detect their effect on macrophage polarization. Results: First, we found that plasma BCAA level was an independent risk factor of ASCVD. Second, we also found plasma BCAA level was elevated in AS mice, which led to the increase of plaque volume and instability, as well as inflammation. The growth of plaque instability was characterized by increased macrophage amounts and decreased smooth muscle cells (SMCs) and collagen contents. Conversely, when plasma BCAA level was lowered, the above phenomena could be reversed. Third, elevated plasma BCAA level resulting from BCAA catabolic defect occurred in the monocytes of ASCVD patients and the abdominal macrophages of AS mice, which increased inflammatory response by promoting the polarization of macrophages toward M1 phenotype. Fourth, elevated BCAA level-mediated macrophage polarization toward M1 phenotype was regulated by increasing mtH 2 O 2 level to induce secretion of HMGB1 by increasing nuclear H 2 O 2 level. Finally, the improvement of BCAA catabolism of macrophages alleviated AS progression in mice. Conclusions: Our data demonstrates that elevated plasma BCAA level is an independent risk factor of ASCVD and promotes AS progression, indicating that plasma BCAA level might be a therapeutic target for ASCVD.

Abstract 1384: Selective PI3Kγ inhibitor ZX-4081 reprograms the tumor microenvironment to facilitate anti-cancer immunity

Abstract PI3Kγ is highly expressed in leukocytes and also known to play a critical role in activation of myeloid cells at sites of inflammation and tumorigenesis, T cell recruitment into tumor microenvironment, and chemokine-mediated chemotaxis as well as growth factor signaling. Previous studies reported that blockade of PI3Kγ-AKT signaling is able to inhibit tumor growth and metastasis by increasing CD8+T cell activation and M1(anti-tumoral)-like macrophage appearance in the tumor milieu, and thereby overcome resistance to immune checkpoint inhibitors (e.g., anti-PD1 antibody). In this report, we present a highly selective PI3Kγ inhibitor ZX-4081 with an IC50 of 1.5 nM for PI3Kγ- and over 1000-fold selectivity over the other isoform (α, β, and δ) in biochemical assay. To confirm inhibitory effect of ZX-4081 on PI3Kγ-AKT activation, LPS-stimulated THP-1 cells (monocytic cells) were treated with 10 nM or 500 nM ZX-4081, and their AKT phosphorylation (at T308 and S473 sites) were examined by immunoblotting. The immunoblot result indicated that both T308 and S473 phosphorylation was down-regulated by ZX-4081 in a time-dependent and dose-dependent manner. Next, to verify whether ZX-4081 can alter macrophage polarization, M-CSF-stimulated human peripheral blood mononuclear cells (PBMCs) were subsequently either induced into M1-like macrophages with LPS and IFN-γ or induced into M2-like macrophages with IL-4 and IL-13 in the absence (vehicle) or presence of ZX4081 (from 1.52 to1000 nM). The flow cytometry result exhibited that ZX-4081 effectively facilitated macrophage polarization toward M1-like phenotype, evidenced by an increase (a 2.5-fold increase at 10 nM compared to vehicle) in iNOS and CD86 expressions of M1-like macrophages; however, CD206 expression (M2-like phenotype) was not changed by ZX-4081 treatment in M2-like macrophages. Additionally, ZX-4081 could reduce both IL-10 protein and mRNA levels in M2-like macrophages (2-5 times lower than vehicle). The cell proliferation assessment showed that ZX-4081(< 5μM) did not exert potent growth inhibitory effects on solid tumor cell lines including HT-29, HT-1197, 4T1, B16-F10, and CT-26 cells. In the 4T1 syngeneic mouse model, ZX-4081 treatment at 25 mg/kg (p.o, BID), 50 mg/kg (p.o., QD), and 50 mg/kg (p.o., BID) for 28 days gave rise to a reduction in tumor growth by 34.6%, 21.2%, and 44.1% in size, respectively (p< 0.0001). Therefore, the in vivo inhibitory effect of ZX-4081 on 4T1 tumor growth was attributed to its modulation on the mouse anti-tumor immunity. Overall, our data suggest that ZX-4081 possesses ability of tumor growth inhibition by reprogramming tumor microenvironment via impairing PI3Kγ-AKT-mediated macrophage polarization to enhance anti-cancer immunity. Citation Format: Ying-Ying Li, Kun Guo, Zhiyuan Peng, Deming Kong, Xiaolin Hao, Jinfu Yang, Xiaoli (Shelley) Qin. Selective PI3Kγ inhibitor ZX-4081 reprograms the tumor microenvironment to facilitate anti-cancer immunity [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 1384.

Mesenchymal Stem Cells Enhance Therapeutic Effect and Prevent Adverse Gastrointestinal Reaction of Methotrexate Treatment in Collagen-Induced Arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by articular destruction and functional loss. Methotrexate (MTX) is effective in RA treatment. However, MTX induces several adverse events and 20%-30% of patients do not respond to MTX. Thus, it is urgent to enhance the therapeutic effects and reduce the side effects of MTX. Recent studies showed that mesenchymal stem cells (MSCs) were participants in anti-inflammation, immunoregulation, and tissue regeneration. However, whether the combined application of MSCs and MTX promotes the therapeutic effects and reduces the side effects of MTX has not been studied. In this study, we used bovine type II collagen to induce rheumatoid arthritis in mice (collagen-induced arthritis, CIA). Then, CIA mice were subjected to MTX or MSC treatment, or both. The therapeutic effect and adverse events of different treatments on RA were evaluated with micro-CT, HE staining, and immunohistochemistry in vivo. Apoptosis and proliferation of MODE-K cells were measured after treated with MTX or/and cocultured with UCs. To test M2 polarization, Raw264.7 macrophages were stimulated by MTX with different concentrations or cocultured with UCs. We found that the combined application of MSCs and MTX increased the therapeutic effects on RA, as evidenced by decreased arthritis score, inflammatory responses, and mortality. Moreover, in this combination remedy, MTX prefers to suppress inflammation by facilitating macrophage polarization to M2 type while UCs prefer to eliminate gastrointestinal side effects of MTX via mitigating the apoptosis of intestinal epithelial cells. Thus, a combination of MTX and UCs is a promising strategy for RA treatment.

Fibrinogen-like protein 2 deficiency aggravates renal fibrosis by facilitating macrophage polarization

Renal fibrosis has no effective target for its prevention or reversal. Fibinogen-like protein 2 (Fgl2) is a novel prothrombinase exhibiting coagulation activity and immunomodulatory effects. Although Fgl2 is known to play a vital role in the development of liver and interstitial fibrosis, its function in renal fibrosis remains unclear. In this study, Fgl2 expression was found to be markedly increased in kidney tissues from mice with unilateral ureteral obstruction (UUO)-induced renal fibrosis and patients with chronic kidney disease. However, Fgl2 deficiency aggravated UUO-induced renal fibrosis, as evidenced by the significantly increasing collagen I, fibronectin, and α-SMA expression, extracellular matrix deposition, and profibrotic factor (TGF-β1) secretion. Administration of rmFgl2 (recombinant mouse Fgl2) significantly alleviated UUO-induced renal fibrosis in mice, suggesting that the increased fibrosis can be reversed by supplementing rmFgl2. Although there was no difference in the percentages of total macrophages between Fgl2+/+ and Fgl2–/– mice, Fgl2 deficiency remarkably facilitated M2 macrophage polarization and accelerated M1 macrophage polarization to a low degree, during UUO-induced renal fibrosis development in mice. Similar results were observed when Fgl2+/+ and Fgl2–/– mice bone marrow-derived macrophages were treated for M1 or M2 polarization. Moreover, Fgl2 deficiency significantly increased the phosphorylation of STAT6, a critical mediator of M2 polarization, in both UUO-induced fibrotic kidney tissues and bone marrow-derived M2 macrophages. In conclusion, the aggravation of renal fibrosis by Fgl2 deficiency is facilitated by the p-STAT6-dependent upregulation of macrophage polarization, especially of M2.

Piperine from Black Pepper Decreased the Expression of Intercellular Adhesion Molecule-1 in Macrophages.

Macrophages are the main players involved in inflammation. Intercellular adhesion molecule-1 (ICAM-1) facilitates macrophage polarization prior to extravasation into inflamed tissue. Piperine, a natural product derived from black pepper, possesses useful biological and pharmacological activities. In the current study, the possible anti-inflammatory effect of piperine on the expression of ICAM-1 on J774.1 murine macrophage cell line was investigated. Lipopolysaccharide (LPS)-stimulated J774.1 cells were cultured in the presence of different concentrations of piperine to examine the changes in ICAM-1 expression by real-time PCR and flow cytometry. We found that piperine decreased ICAM-1 gene expression level from 2.4 ± 0.25 RFC (relative fold change) in LPS-only treated cells to 0.85 ± 0.525 RFC at 1µg/ml (p<0.05), 0.43 ± 0.27 RFC at 10µg/ml (p<0.01), and 0.26 ± 0.25 RFC at 20µg/ml (p<0.01). In flow cytometry, piperine at all concentrations significantly decreased ICAM-1 surface expressions (P<0.05). The geometric mean fluorescence intensity (g-MFI) in LPS-only treated cells (792 ± 57.3) decreased to 482±70 g-MFI at 20 μg/ml piperine. According to the results of this study, by decreasing the expression of ICAM-1, piperine has been suggested to reduce inflammation and have the potential to provide therapeutic benefits for immune-mediated diseases.

LincRNA-p21 knockdown reversed tumor-associated macrophages function by promoting MDM2 to antagonize* p53 activation and alleviate breast cancer development.

Tumor-associated macrophages (TAMs) are important regulators of the complex interplay between immune system and breast cancer. TAMs fuel the cancer progression and metastasis by reprogramming their specific functional phenotype in cancer settings. Therefore, it is important to clarify the mechanisms of shaping specific functional phenotype of macrophages in tumor milieu. LncRNA profiles of TAMs were identified by LncRNA microarray. Flow cytometry was used to detect the surface markers of TAMs. The co-localization among lincRNA-p21, p53 and Mouse Double Minute 2 (MDM2) was identified by FISH probe and immunofluorescence. PyVT-MMTV and BALB/c mice were used for in vivo analysis. In the present work, we found that lincRNA-p21 significantly up-regulated in 4T1 educated macrophages. LincRNA-p21 knockdown facilitated macrophage polarization into pro-inflammatory M1 in tumor microenvironment, which might be caused by MDM2 eliciting proteasome-dependent degradation to p53 and activated NF-κB and STAT3 pathway. TAMs with lincRNA-p21 knockdown induced cancer cell apoptosis, inhibited tumor cell migration and invasion. In vivo, lincRNA-p21 knockdown macrophage adoptive transfer could alleviate breast cancer progression. Our results indicated that lincRNA-p21 was a key regulator of TAMs function in tumor milieu. Our data also shed a light on novel therapeutic targets of tumors characterized by monocytes/macrophages infiltration.

Antiinflammatory activity of ANGPTL4 facilitates macrophage polarization to induce cardiac repair.

Mesenchymal stem cells (MSCs) can suppress pathological inflammation. However, the mechanisms underlying the association between MSCs and inflammation remain unclear. Under coculture conditions with macrophages, MSCs highly expressed angiopoietin-like 4 (ANGPTL4) to blunt the polarization of macrophages toward the proinflammatory phenotype. ANGPTL4-deficient MSCs failed to inhibit the inflammatory macrophage phenotype. In inflammation-related animal models, the injection of coculture medium or ANGPTL4 protein increased the antiinflammatory macrophages in both peritonitis and myocardial infarction. In particular, cardiac function and pathology were markedly improved by ANGPTL4 treatment. We found that retinoic acid-related orphan receptor α (RORα) was increased by inflammatory mediators, such as IL-1β, and bound to ANGPTL4 promoter in MSCs. Collectively, RORα-mediated ANGPTL4 induction was shown to contribute to the antiinflammatory activity of MSCs against macrophages under pathological conditions. This study suggests that the capability of ANGPTL4 to induce tissue repair is a promising opportunity for safe stem cell-free regeneration therapy from a translational perspective.

The myeloid mineralocorticoid receptor controls inflammatory and fibrotic responses after renal injury via macrophage interleukin-4 receptor signaling

Acute kidney injury induced by ischemia/reperfusion is an independent risk factor for chronic kidney disease. Macrophage recruitment plays an essential role during the injury and repair phases after an ischemic episode in the kidney. Here we show that the novel non-steroidal mineralocorticoid receptor antagonist finerenone or selective myeloid mineralocorticoid receptor ablation protects against subsequent chronic dysfunction and fibrosis induced by an episode of bilateral kidney ischemia/reperfusion in mice. This protection was associated with increased expression of M2-antiinflamatory markers in macrophages from finerenone-treated or myeloid mineralocorticoid receptor-deficient mice. Moreover, the inflammatory population of CD11b+, F4/80+, Ly6Chigh macrophages was also reduced. Mineralocorticoid receptor inhibition promoted increased IL-4 receptor expression and activation in the whole kidney and in isolated macrophages, thereby facilitating macrophage polarization to an M2 phenotype. The long-term protection conferred by mineralocorticoid receptor antagonism was also translated to the Large White pig pre-clinical model. Thus, our studies support the rationale for using mineralocorticoid receptor antagonists in clinical practice to prevent transition of acute kidney injury to chronic kidney disease.

Magnesium isoglycyrrhizinate positively affects concanavalin A-induced liver damage by regulating macrophage polarization

ABSTRACT The deficient functional polarization of macrophages is implicated in the disease progression of autoimmune hepatitis (AIH). This study aims to evaluate the impact of Magnesium isoglycyrrhizinate (MgIG) on concanavalin A (Con A)-induced hepatitis in a mouse model, thereby clarifying the molecular mechanisms with which it is associated. MgIG was periodically administered to C57BL/6 mice before one intravenous injection of Con A (20 mg/kg). The MgIG treatment demonstrated a protective function in mice for Con A-induced AIH, the expression of proinflammatory cytokines, and the serum levels of alanine aminotransferase and aspartate aminotransferase. In addition, the MgIG pre-treatment had a significant effect on the number of F4/80+ cells entering the liver. MgIG efficiently facilitated macrophage polarization toward an M2 phenotype. The results indicate that a relationship may exist between the protective impacts of MgIG with respect to Con A-induced liver injury and the capability of the hepatoprotective agent to regulate macrophage polarization.

Abstract 112: Myeloid Mineralocorticoid Receptor Controls Inflammatory and Fibrotic Responses After Renal Ischemic Injury via Macrophage Interleukin-4 Receptor

Introduction: Patients who survive an episode of acute kidney injury (AKI) are at high risk of de novo chronic kidney disease (CKD) development. Pharmacological mineralocorticoid receptor (MR) antagonism is useful to prevent CKD after a single episode of ischemic AKI in the rat. Objective: Test the involvement of myeloid MR in the development of kidney fibrosis after an ischemic AKI episode. Methods: We included 18 male C57/B6 mice that were divided in: sham, renal ischemia for 22.5 min and IR plus treatment with the non-steroidal MR antagonist finerenone (10 mg/kg) at -48, -24 and -1 h before IR. MR inactivation in myeloid cells (MR MyKO ) was achieved by crossing mice with the MR alleles flanked by loxP sites (MR f/f ) with mice expressing the Cre recombinase under the LysM promoter activity. In MR f/f and MR MyKO mice we induced renal IR of 22.5 min or sham surgery. The mice were followed-up during 4 weeks to test for AKI to CKD transition. In another set of mice, the macrophages were sorted from kidneys after 24 h of reperfusion and flow cytometry characterization or mRNA extraction was performed. Thyoglycolate elicited peritoneal macrophages were used for in vitro studies. Results: The progression of AKI to CKD after 4 weeks of renal ischemia in the untreated C57/B6 and MR f/f mice was characterized by a 50% increase in plasma creatinine, a 2-fold increase in the mRNA levels of TGF-β and fibronectin as well as by severe tubule-interstitial fibrosis. The mice that received finerenone or MR MyKO mice were protected against these alterations. Increased expression of M2-anti-inflamatory markers in kidney-isolated macrophages from finerenone-treated or MR MyKO mice was observed. The inflammatory population of Ly6C high macrophages was reduced by 50%. In peritoneal macrophages in culture, MR inhibition promoted increased IL-4 receptor expression and activation, facilitating macrophage polarization to an M2 phenotype. Conclusion: MR antagonism or myeloid MR deficiency facilitates macrophage polarization to a M2, anti-inflammatory phenotype after kidney IR, preventing maladaptive repair and chronic kidney fibrosis and dysfunction. MR inhibition acts through the modulation of IL-4 receptor signaling to facilitate macrophage phenotype switching.