M2 Differentiation Research Articles

Modulating poststroke inflammatory mechanisms: Novel aspects of mesenchymal stem cells, extracellular vesicles and microglia.

Inflammation plays an important role in the pathological process of ischemic stroke, and systemic inflammation affects patient prognosis. As resident immune cells in the brain, microglia are significantly involved in immune defense and tissue repair under various pathological conditions, including cerebral ischemia. Although the differentiation of M1 and M2 microglia is certainly oversimplified, changing the activation state of microglia appears to be an intriguing therapeutic strategy for cerebral ischemia. Recent evidence indicates that both mesenchymal stem cells (MSCs) and MSC-derived extracellular vesicles (EVs) regulate inflammation and modify tissue repair under preclinical stroke conditions. However, the precise mechanisms of these signaling pathways, especially in the context of the mutual interaction between MSCs or MSC-derived EVs and resident microglia, have not been sufficiently unveiled. Hence, this review summarizes the state-of-the-art knowledge on MSC- and MSC-EV-mediated regulation of microglial activity under ischemic stroke conditions with respect to various signaling pathways, including cytokines, neurotrophic factors, transcription factors, and microRNAs.

African Trypanosomiasis: Extracellular Vesicles Shed by Trypanosoma brucei brucei Manipulate Host Mononuclear Cells.

African trypanosomiasis or sleeping sickness is a zoonotic disease caused by Trypanosoma brucei, a protozoan parasite transmitted by Glossina spp. (tsetse fly). Parasite introduction into mammal hosts triggers a succession of events, involving both innate and adaptive immunity. Macrophages (MΦ) have a key role in innate defence since they are antigen-presenting cells and have a microbicidal function essential for trypanosome clearance. Adaptive immune defence is carried out by lymphocytes, especially by T cells that promote an integrated immune response. Like mammal cells, T. b. brucei parasites release extracellular vesicles (TbEVs), which carry macromolecules that can be transferred to host cells, transmitting biological information able to manipulate cell immune response. However, the exact role of TbEVs in host immune response remains poorly understood. Thus, the current study examined the effect elicited by TbEVs on MΦ and T lymphocytes. A combined approach of microscopy, nanoparticle tracking analysis, multiparametric flow cytometry, colourimetric assays and detailed statistical analyses were used to evaluate the influence of TbEVs in mouse mononuclear cells. It was shown that TbEVs can establish direct communication with cells of innate and adaptative immunity. TbEVs induce the differentiation of both M1- and M2-MΦ and elicit the expansion of MHCI+, MHCII+ and MHCI+MHCII+ MΦ subpopulations. In T lymphocytes, TbEVs drive the overexpression of cell-surface CD3 and the nuclear factor FoxP3, which lead to the differentiation of regulatory CD4+ and CD8+ T cells. Moreover, this study indicates that T. b. brucei and TbEVs seem to display opposite but complementary effects in the host, establishing a balance between parasite growth and controlled immune response, at least during the early phase of infection.

Fibroblasts Control Macrophage Differentiation during Pulp Inflammation

IntroductionDuring pulp inflammation, recruited macrophages can differentiate into 2 phenotypes: proinflammatory M1 and anti-inflammatory M2. Pulp fibroblasts have previously been shown to regulate pulp inflammation via cytokine and growth factor secretion. We hypothesized that upon carious injury, pulp fibroblasts interact with macrophages and modulate their differentiation. MethodsCultures of pulp fibroblasts were physically injured and incubated with lipoteichoic acid (LTA) to mimic the pulp environment underlying a carious lesion. Physical injuries without LTA were performed on cultured fibroblasts to simulate the surrounding pulp tissue. Fibroblast supernatants were collected and added to undifferentiated macrophages to study their differentiation into M1 or M2 phenotypes by investigating cytokine secretion profiles and phagocytosis capacity. Histologic staining and immunofluorescence were performed on healthy and carious human tooth sections to localize the 2 macrophage phenotypes. ResultsLTA-stimulated fibroblasts induced macrophage differentiation into the M1 phenotype with a significant increase both in tumor necrosis factor alpha secretion and phagocytosis capacity. By contrast, injured fibroblasts without LTA led to M2 differentiation with a significant increase in interleukin 10 secretion and low phagocytosis capacity. In carious teeth, M1 macrophages were detected mainly in the pulp zone underlying caries, whereas M2 macrophages were detected in the peripheral inflammatory zone. ConclusionsFibroblasts induced macrophage differentiation to proinflammatory M1 with high bacteria phagocytosis capacity to control infection at the carious front. Fibroblasts located at the periphery of the inflammatory zone induced macrophage differentiation to anti-inflammatory M2. The fine balance between the 2 phenotypes may represent a prerequisite for initiating the healing process.

PI3K-Delta Inhibitors Induce Primary Monocyte Cytotoxicity but Do Not Alter Monocyte Differentiation

It is well appreciated that an altered tumor microenvironment is a hallmark of cancer, including in chronic lymphocytic leukemia (CLL) where monocyte-derived cells known as “nurse-like cells” (NLC) in the lymph node microenvironment support CLL cell viability, survival, and resistance to drug-induced apoptosis. CLL directed therapy may include conventional chemotherapy, immunotherapy, targeted kinase inhibitors, and/or Bcl2 inhibitors. Of these options, kinase inhibitors such as PI3K-delta inhibitors are an effective therapy that have been found to disrupt NLC-CLL cell interaction in vitro and induce initial lymphocytosis thought to be related to this disrupted cellular interaction. We previously demonstrated that the PI3K-delta inhibitors idelalisib and umbralisib (TGR-1202) induce cytotoxicity, apoptosis, and inhibition of pAKT in primary CLL cells. We now investigate the effect of idelalisib and umbralisib on monocytes by evaluating cytotoxicity and cytokine-induced differentiation. We isolated monocytes from blood obtained from normal donors using negative selection (RosetteSep monocyte). For cytotoxicity experiments, primary purified monocytes were incubated ± M-CSF (10 ng/mL) ± PI3K-delta inhibitor (at 1.25 to 20 μM) for three days, and cell viability was measured with the MTS reagent after three hours. For differentiation experiments, primary purified monocytes were incubated first with M-CSF (10 ng/mL) for three days, then washed and incubated ± IL-10 (20 ng/mL) ± PI3K-delta inhibitor (10 μM) for three days. To judge monocyte differentiation, we used flow cytometry to measure expression of CD14, CD206, CD163, CD124, CD80, and CD86. We also visually assessed monocyte appearance using phase microscopy with these conditions. Both idelalisib and umbralisib induced concentration dependent cytotoxicity (N = 3), but cytotoxicity was higher with idelalisib (62% cytotoxicity at 1.25 μM) compared to umbralisib (24% cytotoxicity at 1.25 μM). The addition of M-CSF reduced PI3K-delta inhibitor-induced cytotoxicity of primary monocytes. For example, 1.25 μM idelalisib induced 62% cytotoxicity without M-CSF, compared with 5% cytotoxicity with M-CSF. Staged cytokine incubation of monocytes with M-CSF and IL-10 induced the expression of M2 differentiation markers (CD163 and CD124) and reduced expression of the M1 differentiation marker (CD86). Addition of idelalisib or umbralisib did not significantly affect monocyte surface marker expression compared to no drug (N = 4). Even though idelalisib and umbralisib were cytotoxic for monocytes, these drugs did not affect morphologic changes induced by IL-10 (marked spreading and shape change). Thus, PI3K-delta inhibitors do not appear to affect cytokine-induced monocyte differentiation, but they do induce monocyte cytotoxicity (idelalisib > umbralisib). The addition of M-CSF, which contributes to M2 differentiation, blocks these effects. Together, our results suggest that depleting monocyte or NLC number may contribute to the anti-CLL therapeutic effects of PI3K-delta inhibitors. In addition, a higher level of monocyte cytotoxicity with idelalisib compared to umbralisib could potentially contribute to the higher infectious side effect profile with idelalisib seen in clinical use, as monocyte derived cells are a key component of the innate immune system. The anti-monocyte effect of PI3K-delta inhibitors could provide rationale for using these agents in other malignancies in which monocyte-derived cells are an integral part of the tumor microenvironment. DisclosuresFriedman:TG Therapeutics: Research Funding. Maryanski:TG Therapeutics, Inc.: Employment, Equity Ownership. Miskin:TG Therapeutics, Inc.: Employment, Equity Ownership.

NSE Promotes Macrophage Towards to M2 Polarization in Diffuse Large B-Cell Lymphoma

AbstractBackground: Diffuse large B-cell lymphoma (DLBCL) is a highly common malignant and heterogeneous non-Hodgkin lymphoma (NHL). Tumor-associated macrophages, predominantly the M2-type, promote tumorigenesis and tumor resistance. Clinical outcome of patients with high neuron-specific enolase (NSE) expression was poorer than that of patients with low NSE level. NSE in tumor-promoting mechanism is currently unclear.Methods: We explored the role of NSE in macrophages polarization associated with DLBCL immune microenvironment.Results: We demonstrated that overexpression of NSE in co-cultured lymphoma cells and THP1 cells could promote M2-type polarization and increase P50 nuclear translocation and reduce p65 nuclear translocation, thus inhibiting NF-κB function by increasing p50/p50 homodimer. Moreover, overexpression of NSE in vivo promoted the proliferation of tumor. However, NSE knockdown significantly reduced the tumor volume. Overexpression NSE promoted macrophage towards M2-type polarization.Conclusion: Our results illustrated that NSE plays a pivotal role in promoting M2 differentiation and reversing M1 to M2 polarization, thereby contributing to lymphoma proliferation and progression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Non-Invasive Differentiation of M1 and M2 Activation in Macrophages Using Hyperpolarized 13C MRS of Pyruvate and DHA at 1.47 Tesla.

Macrophage activation, first generalized to the M1/M2 dichotomy, is a complex and central process of the innate immune response. Simply, M1 describes the classical proinflammatory activation, leading to tissue damage, and M2 the alternative activation promoting tissue repair. Given the central role of macrophages in multiple diseases, the ability to noninvasively differentiate between M1 and M2 activation states would be highly valuable for monitoring disease progression and therapeutic responses. Since M1/M2 activation patterns are associated with differential metabolic reprogramming, we hypothesized that hyperpolarized 13C magnetic resonance spectroscopy (HP 13C MRS), an innovative metabolic imaging approach, could distinguish between macrophage activation states noninvasively. The metabolic conversions of HP [1-13C]pyruvate to HP [1-13C]lactate, and HP [1-13C]dehydroascorbic acid to HP [1-13C]ascorbic acid were monitored in live M1 and M2 activated J774a.1 macrophages noninvasively by HP 13C MRS on a 1.47 Tesla NMR system. Our results show that both metabolic conversions were significantly increased in M1 macrophages compared to M2 and nonactivated cells. Biochemical assays and high resolution 1H MRS were also performed to investigate the underlying changes in enzymatic activities and metabolite levels linked to M1/M2 activation. Altogether, our results demonstrate the potential of HP 13C MRS for monitoring macrophage activation states noninvasively.

Role of Indole-3-Acetic Acid in NAFLD Amelioration After Sleeve Gastrectomy.

This study aimed to investigate the weight-independent mechanism of sleeve gastrectomy on the relief of nonalcoholic fatty liver disease (NAFLD). A total of 58 obese patients who had undergone sleeve gastrostomy (SG) were recruited. Plasma levels of indole-3-acetic acid (I3A), a metabolite from gut microbiota before and after SG were investigated. In addition, we had 78 C57BL/6J mice included in the study. High-fat diet (HFD) was used to induce obesity in mice. Sleeve gastrectomy (SG) was then performed. The liver of the mice was analyzed by HE and oil red staining to study lipid accumulation. Fluorescence-activated cell sorting (FACS) analysis was performed to study the phenotype of macrophages in the liver. The levels of I3A in serum, stool, and liver were tested by ELISA. Macrophages and hepatocytes were cultured in vitro and stimulated with I3A to study the effects on differentiation and proliferation/apoptosis. In human samples, I3A increased after SG and plasma I3A levels were positively correlated with liver CT values and negatively correlated with liver fat attenuation. In mice models, after surgery, the percentage of M2 macrophages significantly increased in the liver. Both oral gavage and in vitro stimulation of I3A could promote M2 differentiation and did not significantly affect the state of hepatocytes. This study suggested that increased I3A from the intestine after SG could reduce the M1/M2 ratio in the liver and thus promote relief of NAFLD in obese individuals. Graphical Abstract.

Suppressors of signaling protein 3 inhibits glucocorticoid‐induced M2 polarization of monocytic cells by attenuation of ROS/p38 and GILZ‐mediated anti‐inflammatory cytokine production

Macrophages respond to diverse environmental cues to acquire different phenotypes, ranging from pro-inflammatory (M1) to anti-inflammatory (M2) types. Suppressors of signaling protein (SOCS) have emerged as critical regulators of inflammation participating in distinct phases of macrophage activation and differentiation. We have recently reported that both SOC1 and SOCS3 attenuated LPS/TLR4-induced inflammatory signaling via down-regulation of ROS levels and inflammasome activation associated with M1 differentiation. We have subsequently observed that SOCS3 also suppresses M2 polarization of THP1 monocytic cells induced by glucocorticoid (GC) and investigated the signaling mechanisms regulated by SOCS3.Treatment of PMA-differentiated THP1 cells with dexamethasone (Dex) induced M2 polarization by reducing TNF-alpha, IL-1 beta and IL-6 and by promoting IL-10 and TGF-beta production with the increased expression of Arg1 and CD163.SOCS3 over-expression reduced, while SOCS3 ablation increased IL-10 and TGF-beta in ROS and p38-dependent manners.A potential mediator of Dex-induced M2 response, glucocorticoid-induced leucine zipper (GILZ) was down-regulated by SOCS3 and up-regulated by shSOCS3. GILZ ablation blocked ROS generation and p38 activation leading to the inhibition of Dex-induced anti-inflammatory cytokines and the expression of M2 markers. Together the data suggest that SOCS3 target ROS/p38-dependent GILZ expression to suppress Dex-induced M2 polarization of THP1 cells. The critical role and detailed regulation mechanism of GILZ as a target of SOCS3 action during GC-induced M2 polarization is under investigation.

The Impact of Clear Speech on the Perceptual and Acoustic Properties of Fricative-Vowel Sequences in Speakers With Dysarthria.

Purpose Studies have reported that clear speech has the potential to influence suprasegmental and segmental aspects of speech, in both healthy and dysarthric speakers. While the impact of clear speech has been studied on the articulation of individual segments, few studies have investigated its effects on coarticulation with multisegment sequences such as fricative-vowel. Objectives The goals of this study are to investigate, in healthy and dysarthric speech, the impact of clear speech on (a) the perception of anticipatory vowel coarticulation in fricatives and (b) the acoustic characteristics of this effect. Method Ten speakers with dysarthria secondary to idiopathic Parkinson's disease were recruited as well as 10 age- and sex-matched healthy speakers. A sentence reading task was performed in natural and clear speaking conditions. The sentences contained words with the initial fricatives /s/ and /ʃ/ preceded by /ə/ and followed by the vowels /i/, /y/, /u/, or /a/. For the perceptual measurements, five listeners were recruited and were asked to predict the upcoming word by listening only to the isolated fricative. Acoustic analyses consisted of spectral moment analysis (M1-M4) on averaged time series. Results Perceptual findings report that identification rates were improved with clear speech for the speakers with dysarthria, but only for the fricative-/i/ sequences. Error pattern analysis indicates that this improvement is associated with an increase in the roundness parameter (lip spreading) identification. Acoustic results are unclear for M1 and M3 but suggest that M2 and M4 differentiation between the rounded versus unrounded vowel contexts is increased with clear speech for the speakers with dysarthria. Discussion Taken together, these findings suggest that clear speech may improve lip coordination in dysarthric speakers with Parkinson's disease. However, the impact of clear speech on the acoustic measures of fricative spectral moments is somewhat limited. This suggests that these metrics, when taken individually, do not capture the entire complexity of fricative-vowel coarticulation.

Tissue-Specific Hydrogels Ameliorate Hepatic Ischemia/Reperfusion Injury in Rats by Regulating Macrophage Polarization via TLR4/NF-κB Signaling.

Injectable acellular matrix hydrogels are proven to be potential translational materials to facilitate the repairment in various tissues. However, their potential to repair hepatic ischemia/reperfusion injury (IRI) has not been explored. In this work, we made hepatic acellular matrix (HAM) hydrogels based on the decellularized process and evaluated the biocompatibility and hepatoprotective effects in a rat IRI model. HAM hydrogels supported viability, proliferation, and attachment of hepatocytes in vitro. Treatment with HAM hydrogels significantly attenuated hepatic damage caused by IRI, as evidenced by hepatic biochemistry, histology, and inflammatory responses. Importantly, HAM hydrogels inhibited macrophage M1 (CD68/CCR7) differentiation but promoted M2 (CD68/CD206) differentiation. Additionally, TLR4/NF-κB signaling was found to be involved in the hepatoprotective effect of HAM hydrogels. Collectively, our study reveals that HAM hydrogels ameliorate hepatic IRI by facilitating M2 polarization via TLR4/NF-κB signaling.

Lycium barbarum Polysaccharide Antagonizes LPS-Induced Inflammation by Altering the Glycolysis and Differentiation of Macrophages by Triggering the Degradation of PKM2.

Lipopolysaccharide (LPS)-induced inflammation is the leading cause of multiple organ failure in sepsis. Pyruvate kinase 2 (PKM2) is a protein kinase and transcriptional coactivator that plays an important role in glycolysis. Recent studies have confirmed that glycolysis maintains the M1 differentiation and induces immune activation in macrophages. Lycium barbarum polysaccharide (LBP), the main bioactive component of Chinese wolfberry, suppresses glycolysis and inflammation. Here, RAW264.7 macrophages were treated with LBP for evaluating its effects against LPS-induced inflammation. The differentiation of M1/M2 macrophages was assessed by flow cytometry for assessing the cell surface markers, CD86 and CD206. The enrichment of hypoxia inducible factor (HIF)-1α and ubiquitin in the PKM2 protein complex was determined by co-immunoprecipitation. LBP suppressed LPS-induced glycolysis, differentiation of M1 macrophages, and the production of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and high mobility group (HMG) 1 proteins. The suppressive effects of LBP were similar to those of PKM2 knockdown, but were abolished by the overexpression of PKM2. LPS elevated the mRNA and protein levels of PKM2. LBP reduced the LPS-induced expression of PKM2 protein, but had no effects on the expression of PKM2 mRNA. LPS inhibited the ubiquitination of PKM2, probably by downregulating the expression of ubiquitin ligases, including Nedd4L, Nedd4, and Gnb2. LBP interfered with the inhibition of PKM2 ubiquitination by upregulating the expression of Nedd4L, Nedd4, and Gnb2. In conclusion, LBP suppressed the LPS-induced inflammation by altering glycolysis and the M1 differentiation of macrophages. The effects of LBP were mediated by the downregulation of PKM2 via enhanced ubiquitination.

Evaluation of the inflammatory responses to sol-gel coatings with distinct biocompatibility levels.

The immune system plays a crucial role in determining the implantation outcome, and macrophages are in the frontline of the inflammatory processes. Further, cellular oxidative stress resulting from the material recognition can influence how cell responses develop. Considering this, the aim of this study was to study oxidative stress and macrophages phenotypes in response to sol-gel materials with distinct in vivo outcomes. Four materials were selected (70M30T and 35M35G30T, with high biocompatibility, and 50M50G and 50V50G, with low biocompatibility). Gene expression, immunocytochemistry and cytokine secretion profiles for M1 and M2 markers were determined. Moreover, oxidative stress markers were studied. Immunocytochemistry and ELISA showed that 50M50G and 50V50G lead to a higher differentiation to M1 phenotype, while 70M30T and 35M35G30T promoted M2 differentiation. In oxidative stress, no differences were found. These results show that the balance between M1 and M2, more than individual quantification of each phenotype, determines a biomaterial outcome.

Abstract PR005: Highly potent fully human anti-VISTA antibodies – A new target checkpoint inhibitor against immunosuppressive myeloid cells

Abstract V-domain Immunoglobulin Suppressor of T cell Activation (VISTA/PD-1H) is a B7 family ligand expressed on circulating and intratumoural myeloid cells as well as Treg and NK cells. It has been shown to inhibit T cell responses in vitro and in preclinical models. In patients, VISTA is also a potential mediator of resistance to anti-CTLA-4 and anti-PD1 therapies and therefore is a valuable new target for cancer immunotherapy. Kineta has analyzed 107 fully human ScFv antibodies directed against VISTA. Our lead candidates exhibit high potencies in the subnanomolar range and are also characterized by a long kDis. They specifically target human and cynomolgus monkey VISTA on a singular unique epitope. In a Staphylococcus Enterotoxin B T-cell activation assay, Kineta’s anti-VISTA antibodies efficiently induce IFNg secretion. They also promote strong maturation of Antigen Presenting Cells with an increase of CD80 and HLA-DR surface expression as well as CXCL10 secretion. The mechanism of action is mediated in part by NK cells. We demonstrated that myeloid cells acquire a high level of VISTA expression during MDSC or M2 differentiation in vitro and that Kineta’s anti-VISTA antibodies prevent the differentiation of MDSC as well as their immunosuppressive activity against T cells. Anti-VISTA antibodies mediate single-agent antitumor effects in syngeneic tumor models in wild-type mice and show enhanced activity in combination with anti-PD1 and anti-CTLA-4 treatment. Candidate anti-VISTA antibodies have also been evaluated in exploratory tolerability and PK studies in cynomolgus monkey. These studies demonstrated that multiple weekly doses of antibodies are well-tolerated with appropriate PK for lead selection and optimization. Our results strongly favor further characterization and continued development of selected lead antibodies for the potential treatment of colder, less immunogenic tumors. This abstract is also being presented as PO035. Citation Format: Thierry Guillaudeux, Eric Tarcha, Robert Bader, Benjamin Dutzar, Nathan Eyde, Emily Frazier, David Jurchen, Remington Lance, Cristina Loomis, Kurt Lustig, Yulia Ovechkina, David Peckham, Jeff Posakony, Shaarwari Sridhar, Mei Xu, Shawn Iadonato. Highly potent fully human anti-VISTA antibodies – A new target checkpoint inhibitor against immunosuppressive myeloid cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR005.

A Novel Standardized Inflammatory Cell-Modulated 3D Tumor Tissue Model for Analysis of Tumor-Stroma Interaction and Drug Discovery

The last decades were marked by substantial progress in understanding the role of tumor-supporting inflammatory reactions in tumor growth and progression. While <i>in vivo</i> data substantiate the contribution of the inflammatory infiltrate and of tumor associated fibroblasts in promoting tumor growth and progression, little is known about the dynamic interaction of these two stromal cell types and their reciprocal influence on each other and on the tumor cells. Mechanistical analyses of these crucial interactions require a standardized and easy to manipulate environment. We therefore established a 3D organotypic <i>in vitro</i> model for epithelial tumors to analyze the interaction of macrophages, neutrophils and fibroblasts in the tumor microenvironment of malignant tumors. In the 3D model, epithelial tumor cells are grown on a collagen type I gel containing fibroblasts, macrophages and neutrophils. Comparable to the <i>in vivo </i>setting, the cytokine driven interaction between macrophages and fibroblasts markedly influences invasion and enhances M2 differentiation in the presence of tumor cells. Addition of neutrophils further leads to a strikingly enhanced tumor invasion associated with an increased expression of MMP-9 and a N2 differentiation of neutrophils. Thus, this novel 3D model provides an <i>in vivo</i> like tissue context to analyze tumor stroma interactions and presents an excellent tool for targeted interference. As such, the model is highly suitable for pharmaceutical screening of novel therapeutics. However, the use of collagen type 1 with its known batch to batch variability as ECM equivalent prohibits the model-standardization that is needed for pharmaceutical testing. Therefore, the 3D <i>in vitro</i> tumor-stroma model was adapted to the use of a bioinert dextran-hydrogel providing a highly standardized and easily modifiable scaffold material that allows the recovery of cells after pharmaceutical experiments. Comparable to the collagen-based model, cells maintained their physiological proliferation, migration and differentiation. Utilizing this standardized model, the efficacy and the tissue impact of novel pharmaceuticals can be investigated in detail with respect to cell morphology, behavior, viability as well as gene expression profiles thereby providing a 3D hydrogel tumor stroma a model that is of great interest for the pharmaceutical industry.

Telocytes Enhances M1 Differentiation and Phagocytosis While Inhibits Mitochondria-Mediated Apoptosis Via Activation of NF-κB in Macrophages

Telocytes (TCs), which are a recently discovered interstitial cell type present in various organs and tissues, perform multiple biological functions and participate in extensive crosstalk with neighboring cells. Endometriosis (EMs) is a gynecological disease characterized by the presence of viable endometrial debris and impaired macrophage phagocytosis in the peritoneal environment. Here, CD34/vimentin-positive TCs were co-cultured with RAW264.7 cells in vitro. M1/M2 differentiation-related markers were detected; phagocytosis, energy metabolism, proliferation, apoptosis, and pathway mechanisms were studied; and the mitochondrial membrane potential (ΔΨm) was measured. Furthermore, in an EMs mouse model, the differentiation of macrophages in response to treatment with TC-conditioned medium (TCM) in vivo was studied. The results showed that upon in vitro co-culture with TCM, RAW264.7 cells differentiated more toward the M1 phenotype with enhancement of phagocytosis, increase in energy metabolism and proliferation owing to reduced the loss of ΔΨm, and suppression of dexamethasone-induced apoptosis. Further, along with the activation of NF-κB, Bcl-2 and Bcl-xl, the expression of Bax, cleaved-caspase9, and cleaved-caspase3 reduced in RAW264.7 cells. In addition, the M1 subtype was found to be the dominant phenotype among tissue and peritoneal macrophages in the EMs model subjected to in vivo TCM treatment. In conclusion, TCs enhanced M1 differentiation and phagocytosis while inhibiting apoptosis via the activation of NF-κB in macrophages, which potentially inhibited the onset of EMs. Our findings provide a potential research target and the scope for developing a promising therapeutic strategy for EMs.

Differentiation of Monocytes into Phenotypically Distinct Macrophages After Treatment with Human Cord Blood Stem Cell (CB-SC)-Derived Exosomes.

Stem Cell Educator (SCE) therapy is a novel clinical approach for the treatment of type 1 diabetes and other autoimmune diseases. SCE therapy circulates the isolated patient's blood mononuclear cells (e.g., lymphocytes and monocytes) through an apheresis machine, co-cultures the patient's blood mononuclear cells with adherent cord blood-derived stem cells (CB-SC) in the SCE device, and then returns these "educated" immune cells to the patient's blood. Exosomes are nano-sized extracellular vesicles between 30‒150 nm existing in all biofluid and cell culture media. To further explore molecular mechanisms underlying SCE therapy and determine the actions of exosomes released from CB-SC, we investigate which cells phagocytize these exosomes during the treatment with CB-SC. By co-culturing Dio-labeled CB-SC-derived exosomes with human peripheral blood mononuclear cells (PBMC), we found that CB-SC-derived exosomes were predominantly taken up by human CD14-positive monocytes, leading to the differentiation of monocytes into type 2 macrophages (M2), with spindle-like morphology and expression of M2-associated surface molecular markers. Here, we present a protocol for the isolation and characterization of CB-SC-derived exosomes and the protocol for the co-culture of CB-SC-derived exosomes with human monocytes and the monitoring of M2 differentiation.

Inhibition of let-7b-5p contributes to an anti-tumorigenic macrophage phenotype through the SOCS1/STAT pathway in prostate cancer

BackgroundDysfunction of microRNAs (miRNAs) is a major cause of aberrant expression of inflammatory cytokines and contributes to macrophage polarization. Proinflammatory M1 macrophages promote T helper (Th) 1 responses and show tumoricidal activity, whereas M2 macrophages display regulatory functions in tissue repair and remodeling and promote Th2 immune responses. Previous studies have shown that miRNA let-7 is associated with cellular differentiation and that the expression of let-7b-5p is significantly augmented in M2 macrophages. However, the mechanism by which let-7b-5p regulates macrophage differentiation in prostate cancer (PCa) remains largely unknown.MethodsHuman macrophages were induced by blood monocytes from healthy male donors, and M1 macrophages were polarized by stimulating them overnight with 100 ng/ml of lipopolysaccharides and 100 ng/ml of IFN-γ. Conditioned medium from PC-3 cells was used to induce prostatic macrophages (M-CMs) in vitro, and we then transfected let-7b-5p mimics or inhibitors into M1 and M-CMs for 72 h. The expression of cluster of differentiation 206 (CD206) in each group was detected with the High-Throughput Connotation of Imaging System. We used quantitative real-time polymerase chain reaction (qRT-PCR) to examine the expression of the inflammatory cytokines IL-10, IL-12, IL-13, TNF-alpha, and let-7b in macrophages. SOCS1 protein levels were evaluated by ELISA, and the phosphorylation difference in STAT family member proteins was analyzed using CST signal-pathway chip. Phagocytosis by macrophages and the effect of macrophages on the proliferation of prostate cancer PC-3 cells were evaluated with phagocytosis assay or the Cell Counting Kit-8 (CCK-8) and colony formation assay. The relationship between SOCS1 and let-7b-5p was confirmed with a dual-luciferase reporter.ResultsThe expression of cluster of differentiation 206 (CD206, a M2-like macrophage surface molecule) was significantly increased in M1 macrophages treated with let-7b-5p mimics, while CD206 expression was decreased in M-CMs treated with let-7b-5p inhibitors. Overexpression or knockdown of let-7b-5p significantly affected the expression of inflammatory factors in macrophages—including interleukin 10 (IL-10), IL-12, IL-13, and tumor necrosis factor alpha. Let-7b-5p downregulated the expression of suppressor of cytokine signaling 1 (SOCS1) and increased the phosphorylation of signal transducer and activator of transcription 1 (STAT1), STAT3, and STAT5a proteins in M-CMs and M1 macrophages with let-7b-5p mimics relative to the other groups. In addition, with the elevated expression of let-7b-5p, the phagocytosis by macrophages showed a commensurate and significant decrease. As a result, M-CMs treated with let-7b-5p inhibitors reduced the proliferation of PC-3 PCa cells.ConclusionsCollectively, these data indicated that let-7b-5p may regulate M2 polarization through the SOCS1/STAT pathway and that reversal of M2 differentiation by let-7b-5p inhibitors enhanced macrophage phagocytosis, ultimately inhibiting the proliferation of PCa cells.

MiR-148a Affects Polarization of THP-1-Derived Macrophages and Reduces Recruitment of Tumor-Associated Macrophages via Targeting SIRPα.

PurposeThe objective of this study was to investigate the effect of miR-148a on the polarization and recruitment of tumor-associated macrophages (TAMs).MethodsIn human monocyte THP-1 cells, M1 or M2 differentiation was induced by phorbol 12-myristate 13-acetate (PMA) with specific induction supplements and identified using flow cytometry and ELISA. To alter cellular miR-148a expression level, THP-1 cells were transfected with miR-148a mimics or inhibitors. A dual-luciferase assay was used to determine whether miR-148a could directly regulate the expression of signal regulatory protein α (SIRPα). Expression of miR-148a and SIRPα was detected with RT-PCR or Western blot. A co-culture system of THP-1 cells and colorectal cancer SW480 cells was used for TAM induction. The recruitment of macrophage to SW480 cells was measured using chemotaxis assay. In SW480 cells, apoptosis induced by macrophages was detected using flow cytometry and a xenograft assay. Macrophage infiltration was detected by immunofluorescence assay in tumor tissues.ResultsmiR-148a over-expression increased M1-related CD86 expression in THP-1 cells and promoted differentiation to M1-like macrophages. Inhibition of miR-148a increased M2-related CD206 expression and promoted differentiation to M2-like macrophages. In the co-culture system, THP-1 cells were induced to the M2-like state by SW480 cells. The level of miR-148a negatively correlated with the levels of M2-related cytokines. Additionally, miR-148a expression level was negatively associated with macrophage recruitment by colorectal cancer cells. Furthermore, in miR-148a over-expression, the number of macrophages recruited by SW480 cells was reduced. Meanwhile, cancer cell apoptosis induced by macrophages was enhanced. Thus, miR-148a expression was beneficial for the transformation of macrophages, from an immune-suppressive status to an immune-promoting status. These anti-cancer effects of miR-148a were related to the down-regulation of SIRPα in macrophages, directly targeted by miR-148a. A xenograft assay showed that the co-inoculation of macrophages over-expressing miR-148a reduced subcutaneous tumorigenesis and M2 macrophage infiltration.ConclusionmiR-148a promoted the differentiation of M0 macrophages into anti-tumor classical activation type M1, and reduced TAM recruitment by targeting SIRPα to inhibit colorectal cancer cell viability.

Abstract 5103: Bidirectional EMT-dependent modulation of cancer cell and M1/M2 macrophage differentiation

Abstract Background. Head and neck (H&amp;N) and liver carcinomas are considered as the most deadly cancers in the world. Despite advances in diagnosis and treatment, survival rates remain low mainly due to locoregional relapse, possibly triggered by the activation of epithelial to mesenchymal transition (EMT). EMT is a critical step for tumor progression, increasing motility and invasiveness of tumor cells. M1 and M2 macrophages, major components of the tumor microenvironment, were recently described as involved in EMT. Moreover, proteins expressed by cancer cells, such as CCL2 and CSF1, and promoting M2 differentiation, have been correlated with poor prognosis, and tumor aggressiveness. The aim of this study is to understand the interactions between M1/M2 macrophages and H&amp;N or liver cancer cells considering their EMT status. Materials and Methods. M1 and M2 macrophages were obtained from THP-1 cell after exposure to PMA followed by LPS/IFNg for M1 or IL4/IL13 for M2. Differentiation was validated by immunofluorescence (IF) using CD14 for monocyte, CD68 for macrophage, CD80 for M1, and CD163 for M2. H&amp;N, cholangiocarcinoma (CK), and hepatocellular carcinoma (HCC) cells were characterized for their EMT status (E-cadherin/vimentin expressions) by western blot. In each tumor type, 1 epithelial and 1 mesenchymal cells were selected to analyze the effects of conditioned medium (CM) from cancer cells on macrophages differentiation, using IF. Furthermore, CCL2 and CSF1 mRNA levels were evaluated in the selected cancer cell lines. In addition, the effect of M1 and M2 CM on cancer cell proliferation, migration, and invasion were also analyzed by MTT, wound-healing, and Boyden chamber assays. Results. Using our two steps methods, monocytes were first differentiated into macrophages (decrease of CD14 and increase of CD68 expression), and then into M1 and M2 (increase of CD80 or CD163 expression, respectively). Based on cell lines EMT status, SQ20B, SNU1196, and C3A (epithelial) and Hep2, SNU1079, and SKHep1 (mesenchymal) cells were selected. Macrophages exposed to CM from epithelial cells displayed a M1 phenotype, while macrophages exposed to CM from mesenchymal cells exhibited a M2 phenotype. Interestingly, higher mRNA expression of CCL2 and CSF1 mRNA were detected in mesenchymal cells compared to epithelial cells. In H&amp;N and CK cell lines, M1 CM displayed increased antiproliferative effects on epithelial cells, whereas M1 CM displayed antiproliferative effects only on mesenchymal HCC cells. M1 and M2 CM increased migration and invasion in all selected cell lines, except for migration in the epithelial HCC cell line. Further characterization of M1 and M2 macrophages will be displayed at the conference. Conclusions. This study showed that EMT status of cancer cells modulates macrophages differentiation. In fact, mesenchymal cells showed elevated levels of CCL2 and CSF1 expression, promoting M2 differentiation. Conversely, M1 and M2 displayed differential effects on H&amp;N and liver carcinomas cell lines. These results open up new perspectives on the role of M1/M2 macrophages in EMT-dependent tumors, with the aim of developing new therapeutic approaches for patients with EMT-dependent tumors. Citation Format: Lucile Astorgues-Xerri, Matthieu Martinet, Eric Raymond, Sandrine Faivre, Annemilaï Tijeras-Raballand. Bidirectional EMT-dependent modulation of cancer cell and M1/M2 macrophage differentiation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5103.

MEK5/ERK5 signaling mediates IL-4-induced M2 macrophage differentiation through regulation of c-Myc expression.

Macrophages are highly plastic cells, responding to diverse environmental stimuli to acquire different functional phenotypes. Signaling through MAPKs has been reported to regulate the differentiation of macrophages, but the role of ERK5 in IL-4-mediated M2 macrophage differentiation is still unclear. Here, we showed that the ERK5 signaling pathway plays a critical role in IL-4-induced M2 macrophage differentiation. Pharmacologic inhibition of MEK5, an upstream activator of ERK5, markedly reduced the expression of classical M2 markers, such as Arg-1, Ym-1, and Fizz-1, as well as the production of M2-related chemokines and cytokines, CCL22, CCL17, and IGF-1 in IL-4-stimulated macrophages. Moreover, pharmacologic inhibition of ERK5 also decreased the expression of several M2 markers induced by IL-4. In accordance, myeloid cell-specific Erk5 depletion (Erk5∆mye ), using LysMcre /Erk5f/f mice, confirmed the involvement of ERK5 in IL-4-induced M2 polarization. Mechanistically, the inhibition of ERK5 did not affect STAT3 or STAT6 phosphorylation, suggesting that ERK5 signaling regulates M2 differentiation in a STAT3 and STAT6-independent manner. However, genetic deficiency or pharmacologic inhibition of the MEK5/ERK5 pathway reduced the expression of c-Myc in IL-4-activated macrophages, which is a critical transcription factor involved in M2 differentiation. Our study thus suggests that the MEK5/ERK5 signaling pathway is crucial in IL-4-induced M2 macrophage differentiation through the induction of c-Myc expression.