Activated M2 Macrophages Research Articles

Mathematical modeling and bifurcation analysis of pro- and anti-tumor macrophages

More and more evidences show that macrophages can induce both pro-tumor and anti-tumor effects. The study on genetically engineered macrophages to kill tumor cells has become a new direction for anti-cancer therapy in recent years. In this paper, we propose a mathematical model of interactions between tumor cells, M1 and M2 macrophages, and study the stability and bifurcations around the interior equilibrium. It is shown that the model exhibits stable periodic oscillations bifurcating from Hopf bifurcation, which indicates the long-term coexistence of tumor cells, M1 and M2 macrophages as well as long-term tumor relapse. Furthermore, sensitivity analysis is performed to illustrate the effect of parameters on tumor size. Our results show that an increase in ξ (the ratio of activation rates by tumor cells of M2/M1 macrophages) leads to a decrease of tumor cells, which can be explained that ξ has significant effects on γ1 (the transition rate from M1 to M2 macrophages) and γ2 (the transition rate from M2 to M1 macrophages). Hence, the decay of tumor cells is not only the result of direct activation of M1 and M2 macrophages by tumor cells, but also the combined effect of the transition between M1 and M2 macrophages.

Abstract B07: Ovarian cancer stem-like cells and alternately activated macrophages reciprocally interact through the WNT pathway to promote protumoral and malignant phenotypes in 3D engineered microenvironments

Abstract Background: Innate immune cells such as macrophages are abundantly present within malignant ascites, where they share the microenvironment with ovarian cancer stem cells (CSC). Methods: To mimic this malignant ascites microenvironment, we created a hanging-drop hetero-spheroid model to bring CSCs and macrophages in close association. Within these hetero-spheroids, CD68+ macrophages (derived from U937 or peripheral blood monocytes) make up ~20% of the population, while the rest are ovarian cancer cells and ovarian cancer stem cells (derived from the high-grade serous ovarian cancer cell line, OVCAR3). Results: Our results indicate that CSCs drive the upregulation of M2 macrophage marker CD206 within hetero-spheroids, compared to bulk ovarian cancer cells, implying an inherently more immunosuppressive program. Moreover, an increased maintenance of elevated aldehyde dehydrogenase (ALDH) activity is noted within hetero-spheroids that include prepolarized CD206+ M2 macrophages, implying a reciprocal interaction that drives protumoral activation as well as CSC self-renewal. Consistent with enriched CSCs, we also observe increased levels of protumoral IL-10 and IL-6 cytokines in the CSC/M2-macrophage hetero-spheroids. CSC/M2-macrophage hetero-spheroids are also less sensitive to the chemotherapeutic agent carboplatin and are subsequently more invasive in transwell assays. Using inhibitors of WNT secretion in both CSCs and macrophages, we found that CSC-derived WNT ligands drove CD206+ M2 macrophage activation, and that, conversely, macrophage-derived WNT ligands enriched ALDH+ cells within the CSC compartment of hetero-spheroids. Upon examination of specific WNT ligand expression within the monocyte-derived macrophage system, we observed a significant elevation in gene expression for WNT5B. In CSCs co-cultured with macrophages within hetero-spheroids, increases in several WNT ligands were observed, and this increase was significantly inhibited when WNT5B was knocked down in macrophages. Conclusions: Our data imply that macrophage-initiated WNT signaling could play a significant role in the maintenance of stemness, and the resulting phenotypes of chemoresistance and invasiveness. Our results indicate paracrine WNT activation during CSC/M2 macrophages interaction constitutes a positive feedback loop that likely contributes to the more aggressive phenotype, which makes the WNT pathway a potential target to reduce the CSC and M2 macrophage compartments in the tumor microenvironment. Acknowledgments: This work was supported by the DOD OCRP W81XWH-13-1-0134 (GM), Michigan Ovarian Cancer Alliance (MIOCA, GM), Rivkin Center for Ovarian Cancer (GM), NIH/NIDCR DE026728 (YLL) and the NIH/NIDCR T32DE00007057-41, 42 (SR). Flow cytometry facilities used to perform experiments were partially funded by NIH/NCI P30CA046592. Citation Format: Shreya Raghavan, Pooja Mehta, Yuying Xie, Yu L Lei, Geeta Mehta. Ovarian cancer stem-like cells and alternately activated macrophages reciprocally interact through the WNT pathway to promote protumoral and malignant phenotypes in 3D engineered microenvironments [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B07.

Localized Immunosuppression With Tannic Acid Encapsulation Delays Islet Allograft and Autoimmune-Mediated Rejection.

Type 1 diabetes (T1D) is an autoimmune disease of insulin-producing β-cells. Islet transplantation is a promising treatment for T1D, but long-term graft viability and function remain challenging. Oxidative stress plays a key role in the activation of alloreactive and autoreactive immunity toward the engrafted islets. Therefore, targeting these pathways by encapsulating islets with an antioxidant may delay immune-mediated rejection. Utilizing a layer-by-layer approach, we generated nanothin encapsulation materials containing tannic acid (TA), a polyphenolic compound with redox scavenging and anti-inflammatory effects, and poly(N-vinylpyrrolidone) (PVPON), a biocompatible polymer. We hypothesize that transplantation of PVPON/TA-encapsulated allogeneic C57BL/6 islets into diabetic NOD mice will prolong graft function and elicit localized immunosuppression. In the absence of systemic immunosuppression, diabetic recipients containing PVPON/TA-encapsulated islets maintained euglycemia and delayed graft rejection significantly longer than those receiving nonencapsulated islets. Transplantation of PVPON/TA-encapsulated islets was immunomodulatory because gene expression and flow cytometric analysis revealed significantly decreased immune cell infiltration, synthesis of reactive oxygen species, inflammatory chemokines, cytokines, CD8 T-cell effector responses, and concomitant increases in alternatively activated M2 macrophage and dendritic cell phenotypes. Our results provide evidence that reducing oxidative stress following allotransplantation of PVPON/TA-encapsulated islets can elicit localized immunosuppression and potentially delay graft destruction in future human islet transplantation studies.

Activation of M1 Macrophages in Response to Recombinant TB Vaccines With Enhanced Antimycobacterial Activity.

Pulmonary tuberculosis (TB) is a difficult-to-eliminate disease. Although the Bacille Calmette–Guérin (BCG) vaccine against Mycobacterium tuberculosis (MTB) has been available for decades, its efficacy is variable and has lessened over time. Furthermore, the BCG vaccine no longer protects against newly emerged Beijing strains which are responsible for many current infections in adults. Development of a novel vaccine is urgently needed. In this study, we first tested the efficacy of our recombinant BCG vaccines rBCG1 and rBCG2, compared to parental BCG, against MTB strain H37Ra in mice. Both the bacterial load and the level of lymphocyte infiltration decreased dramatically in the three groups treated with vaccine, especially rBCG1 and rBCG2. Furthermore, the Th1 and Th17 responses increased and macrophage numbers rose in the vaccination groups. Th1-mediated production of cytokines TNF-α, IFN-γ, and MCP-1 as well as M1-polarized cells all increased in lung tissue of the rBCG1 and rBCG2 groups. Clodronate-induced depletion of macrophages reduced the level of protection. Based on these results, we conclude that rBCG vaccines induce a significant increase in the number of M1 macrophages, which augments their potential as TB vaccine candidates.

Combined anti-fibrotic and anti-inflammatory properties of JAK-inhibitors on macrophages in vitro and in vivo: Perspectives for scleroderma-associated interstitial lung disease

Janus kinase (JAK) inhibitors (also termed Jakinibs) constitute a family of small drugs that target various isoforms of JAKs (JAK1, JAK2, JAK3 and/or tyrosine kinase 2 (Tyk2)). They exert anti-inflammatory properties linked, in part, to the modulation of the activation state of pro-inflammatory M1 macrophages. The exact impact of JAK inhibitors on a wider spectrum of activation states of macrophages is however still to be determined, especially in the context of disorders involving concomitant activation of pro-inflammatory M1 macrophages and profibrotic M2 macrophages. This is especially the case in autoimmune pulmonary fibrosis like scleroderma-associated interstitial lung disease (ILD), in which M1 and M2 macrophages play a key pathogenic role. In this study, we directly compared the anti-inflammatory and anti-fibrotic effects of three JAK inhibitors (ruxolitinib (JAK2/1 inhibitor); tofacitinib (JAK3/2 inhibitor) and itacitinib (JAK1 inhibitor)) on five different activation states of primary human monocyte-derived macrophages (MDM). These three JAK inhibitors exert anti-inflammatory properties towards macrophages, as demonstrated by the down-expression of key polarization markers (CD86, MHCII, TLR4) and the limited secretion of key pro-inflammatory cytokines (CXCL10, IL-6 and TNFα) in M1 macrophages activated by IFNγ and LPS or by IFNγ alone. We also highlighted that these JAK inhibitors can limit M2a activation of macrophages induced by IL-4 and IL-13, as notably demonstrated by the down-regulation of the M2a associated surface marker CD206 and of the secretion of CCL18. Moreover, these JAK inhibitors reduced the expression of markers such as CXCL13, MARCO and SOCS3 in alternatively activated macrophages induced by IL-10 and dexamethasone (M2c + dex) or IL-10 alone (M2c MDM). For all polarization states, Jakinibs with inhibitory properties over JAK2 had the highest effects, at both 1 μM or 0.1 μM. Based on these in vitro results, we also explored the effects of JAK2/1 inhibition by ruxolitinib in vivo, on mouse macrophages in a model of HOCl-induced ILD, that mimics scleroderma-associated ILD. In this model, we showed that ruxolitinib significantly prevented the upregulation of pro-inflammatory M1 markers (TNFα, CXCL10, NOS2) and pro-fibrotic M2 markers (Arg1 and Chi3L3). These results were associated with an improvement of skin and pulmonary involvement. Overall, our results suggest that the combined anti-inflammatory and anti-fibrotic properties of JAK2/1 inhibitors could be relevant to target lung macrophages in autoimmune and inflammatory pulmonary disorders that have no efficient disease modifying drugs to date.

Mechanism of Inhibition of M1 Macrophage Expression and Activation of MIP-1α/CCL3 Signaling Pathway on Vascular Remodeling

Macrophages mediate the activation of MIP-1α/CCL3 signaling and participate in inflammatory response. In our study, M1 gene-deficient mice were used to assess the effect and mechanism of specific inhibition of macrophage M1 expression on angiotensin II-induced hypertensive vascular remodeling. Fifty 8-week-old male Lyz2-cre M1 gene-deficient mice and 50 Cre mice were divided into control group (saline group) and experimental group (angiotensin II group) followed by HE staining, collagen or elastin staining as well as measuring wall thickness. DHE staining was performed to observe the density of coronary artery and aortic oxidative stress. PCR Array was used to detect cytokine changes which were then verified by RT-PCR. The changes of aortic inflammatory factors were assessed by immunofluorescence and Western blotting. IHC was to measure MIP-1α level. Specific inhibition of M1 expression aggravated AngII-induced hypertensive vascular remodeling; Ang II stimulation increased vascular superoxide production by aggravation of ROS; specific inhibition of macrophage M1 expression significantly up-regulated vascular inflammatory cytokines and the expression and activation of MIP-1α/CCL3 in vascular smooth muscle cells to aggravate vascular remodeling. Further, in vitro cell experiments showed that MIP-1α significantly up-regulated CD31 tube staining. M1 is involved in AngII-induced hypertensive vascular remodeling by mediating the release of macrophage inflammatory factor MIP-1α/CCL3.

Targeting the vascular endothelial growth factor receptor-1 by the monoclonal antibody D16F7 to increase the activity of immune checkpoint inhibitors against cutaneous melanoma

The vascular endothelial growth factor receptor-1 (VEGFR-1) is a membrane receptor for VEGF-A, placenta growth factor (PlGF) and VEGF-B that plays a crucial role in melanoma invasiveness, vasculogenic mimicry and tumor-associated angiogenesis. Furthermore, activation of VEGFR-1 is involved in the mobilization of myeloid progenitors from the bone marrow that infiltrate the tumor. Myeloid-derived suppressor cells and tumor-associated macrophages have been involved in tumor progression and resistance to cancer treatment with immune checkpoint inhibitors (ICIs). We have recently demonstrated that the anti-VEGFR-1 monoclonal antibody (mAb) D16F7 developed in our laboratories is able to inhibit melanoma growth in preclinical in vivo models and to reduce monocyte/macrophage progenitor mobilization and tumor infiltration by myeloid cells. Aim of the study was to investigate whether the anti-VEGFR-1 mAb D16F7 affects the activity of protumoral M2 macrophages in vitro in response to PlGF and inhibits the recruitment of these cells to the melanoma site in vivo. Finally, we tested whether, through its multi-targeted action, D16F7 mAb might increase the efficacy of ICIs against melanoma. The results indicated that VEGFR-1 expression is up-regulated in human activated M2 macrophages compared to activated M1 cells and exposure to the D16F7 mAb decreases in vitro chemotaxis of activated M2 macrophages. In vivo treatment with the anti-VEGFR-1 mAb D16F7 of B6D2F1 mice injected with syngeneic B16F10 melanoma cells resulted in tumor growth inhibition associated with the modification of tumor microenvironment that involves a decrease of melanoma infiltration by M2 macrophages and PD-1+ and FoxP3+ cells. These alterations result in increased M1/M2 and CD8+/FoxP3+ ratios, which favor an antitumor and immunostimulating milieu. Accordingly, D16F7 mAb increased the antitumor activity of the ICIs anti-CTLA-4 and anti-PD-1 mAbs. Overall, these data reinforce the role of VEGFR-1-mediated-signalling as a valid target for reducing tumor infiltration by protumoral macrophages and for improving the efficacy of immunotherapy with ICIs.

TFEB-NF-\u03baB inflammatory signaling axis: a novel therapeutic pathway of Dihydrotanshinone I in doxorubicin-induced cardiotoxicity

BackgroundDoxorubicin is effective in a variety of solid and hematological malignancies. Unfortunately, clinical application of doxorubicin is limited due to a cumulative dose-dependent cardiotoxicity. Dihydrotanshinone I (DHT) is a natural product from Salvia miltiorrhiza Bunge with multiple anti-tumor activity and anti-inflammation effects. However, its anti-doxorubicin-induced cardiotoxicity (DIC) effect, either in vivo or in vitro, has not been elucidated yet. This study aims to explore the anti-inflammation effects of DHT against DIC, and to elucidate the potential regulatory mechanism.MethodsEffects of DHT on DIC were assessed in zebrafish, C57BL/6 mice and H9C2 cardiomyocytes. Echocardiography, histological examination, flow cytometry, immunochemistry and immunofluorescence were utilized to evaluate cardio-protective effects and anti-inflammation effects. mTOR agonist and lentivirus vector carrying GFP-TFEB were applied to explore the regulatory signaling pathway.ResultsDHT improved cardiac function via inhibiting the activation of M1 macrophages and the excessive release of pro-inflammatory cytokines both in vivo and in vitro. The activation and nuclear localization of NF-κB were suppressed by DHT, and the effect was abolished by mTOR agonist with concomitant reduced expression of nuclear TFEB. Furthermore, reduced expression of nuclear TFEB is accompanied by up-regulated phosphorylation of IKKα/β and NF-κB, while TFEB overexpression reversed these changes. Intriguingly, DHT could upregulate nuclear expression of TFEB and reduce expressions of p-IKKα/β and p-NF-κB.ConclusionsOur results demonstrated that DHT can be applied as a novel cardioprotective compound in the anti-inflammation management of DIC via mTOR-TFEB-NF-κB signaling pathway. The current study implicates TFEB-IKK-NF-κB signaling axis as a previously undescribed, druggable pathway for DIC.

Immunosuppressive Functions of M2 Macrophages Derived from iPSCs of Patients with ALS and Healthy Controls.

SummaryAmyotrophic lateral sclerosis (ALS) is a disorder with immune alterations that augment disease severity. M2 macrophages benefit diabetic and nephrotic mice by suppressing the pro-inflammatory state. However, neither have M2 cells been investigated in ALS nor have human induced pluripotent stem cell (iPSC)-derived M2 cells been thoroughly studied for immunosuppressive potentials. Here, iPSCs of C9orf72 mutated or sporadic ALS patients were differentiated into M2 macrophages, which suppressed activation of pro-inflammatory M1 macrophages as well as proliferation of ALS CD4+CD25- Tc (Teffs). M2 cells converted ALS Teffs into CD4+CD25+Foxp3+ regulatory T cells (Tregs) and rescued Tregs of ALS patients from losing CD25 and Foxp3. Furthermore, Tregs induced or rescued by iPSC-derived M2 had strong suppressive functions. ALS iPSC-derived M2 cells including those with C9orf72 mutation had similar immunomodulatory activity as control iPSC-derived M2 cells. This study demonstrates that M2 cells differentiated from iPSCs of ALS patients are immunosuppressive, boost ALS Tregs, and may serve as a candidate for immune-cell-based therapy to mitigate inflammation in ALS.

Lipotoxic Hepatocyte-Derived Exosomal MicroRNA 192-5p Activates Macrophages Through Rictor/Akt/Forkhead Box Transcription Factor O1 Signaling in Nonalcoholic Fatty Liver Disease.

Hepatic macrophages can be activated by many factors such as gut-derived bacterial components and factors released from damaged hepatocytes. Macrophage polarization toward a proinflammatory phenotype (M1) represents an important event in the disease progression of nonalcoholic fatty liver disease (NAFLD). However, the underlying molecular mechanisms remain incompletely understood. Exosomes have been identified as important mediators for cell-cell communication by transferring various biological components such as microRNAs (miRs), proteins, and lipids. The role of exosomes in crosstalk between hepatocytes and macrophages in disease progression of NAFLD is yet to be explored. In the present study, we reported that lipotoxic injury-induced release of hepatocyte exosomes enriched with miR-192-5p played a critical role in the activation of M1 macrophages and hepatic inflammation. Serum miR-192-5p levels in patients with NAFLD positively correlated with hepatic inflammatory activity score and disease progression. Similarly, the serum miR-192-5p level and the number of M1 macrophages, as well as the expression levels of the hepatic proinflammatory mediators, were correlated with disease progression in high-fat high-cholesterol diet-fed rat models. Lipotoxic hepatocytes released more miR-192-5p-enriched exosomes than controls, which induced M1 macrophage (cluster of differentiation 11b-positive [CD11b+ ]/CD86+ ) activation and increase of inducible nitric oxide synthase, interleukin 6, and tumor necrosis factor alpha expression. Furthermore, hepatocyte-derived exosomal miR-192-5p inhibited the protein expression of the rapamycin-insensitive companion of mammalian target of rapamycin (Rictor), which further inhibited the phosphorylation levels of Akt and forkhead box transcription factor O1 (FoxO1) and resulted in activation of FoxO1 and subsequent induction of the inflammatory response. Hepatocyte-derived exosomal miR-192-5p plays a critical role in the activation of proinflammatory macrophages and disease progression of NAFLD through modulating Rictor/Akt/FoxO1 signaling. Serum exosomal miR-192-5p represents a potential noninvasive biomarker and therapeutic target for nonalcoholic steatohepatitis.

Natural antiviral compound silvestrol modulates human monocyte-derived macrophages and dendritic cells.

Outbreaks of infections with viruses like Sars‐CoV‐2, Ebola virus and Zika virus lead to major global health and economic problems because of limited treatment options. Therefore, new antiviral drug candidates are urgently needed. The promising new antiviral drug candidate silvestrol effectively inhibited replication of Corona‐, Ebola‐, Zika‐, Picorna‐, Hepatis E and Chikungunya viruses. Besides a direct impact on pathogens, modulation of the host immune system provides an additional facet to antiviral drug development because suitable immune modulation can boost innate defence mechanisms against the pathogens. In the present study, silvestrol down‐regulated several pro‐ and anti‐inflammatory cytokines (IL‐6, IL‐8, IL‐10, CCL2, CCL18) and increased TNF‐α during differentiation and activation of M1‐macrophages, suggesting that the effects of silvestrol might cancel each other out. However, silvestrol amplified the anti‐inflammatory potential of M2‐macrophages by increasing expression of anti‐inflammatory surface markers CD206, TREM2 and reducing release of pro‐inflammatory IL‐8 and CCL2. The differentiation of dendritic cells in the presence of silvestrol is characterized by down‐regulation of several surface markers and cytokines indicating that differentiation is impaired by silvestrol. In conclusion, silvestrol influences the inflammatory status of immune cells depending on the cell type and activation status.

Regulation of human macrophage phenotype and GPBAR1 expression by cell seeding density and glucocorticosteroids

Abstract Macrophages are key mediators of tissue homeostasis and inflammation and do so by virtue of differential expression of secreted and cell surface proteins. Cytokines such as IFNγ or IL-4 and IL-13 are known to generate classical proinflammatory M1 and alternatively activated M2 macrophages, respectively, but it is less clear how cell density, cell-cell contact and corticosteroids work together to affect macrophage function. Corticosteroids such as cortisol are known to downregulate the inflammatory signaling but they can also alter the expression of cell surface proteins, such as CD163, on steady-state macrophages. Here we examined the effect of monocyte seeding density and cortisol on the expression of cell surface proteins on human macrophages. The expression of the macrophage markers CD16, CD163, CD204 and VSIG4 were found to correlate with monocyte seeding density such that at low seeding densities, monocyte-derived macrophages were found to express minimal amounts of CD16, CD163, CD204 and VSIG4 while at high seeding densities, expression of these markers was restored. When cultured in the presence of cortisol, macrophages seeded at low densities were found to upregulate expression of these markers, but not CD204, to levels comparable to macrophages seeded at high densities grown in the presence of MCSF. Additionally, in the presence of cortisol, macrophages expressed higher levels of GPBAR1 which has also been shown to reduce classical proinflammatory macrophage activation in colitis models. This data suggests that corticosteroids may regulate macrophage function by modulating the expression of cell surface proteins such as GPBAR1 and not just interfering with morer conventional inflammatory cell signaling pathways..

Increased Vascular Adhesion Protein 1 (VAP-1) Levels are Associated with Alternative M2 Macrophage Activation and Poor Prognosis for Human Gliomas.

Glioma is characterized by a high heterogeneity in the brain tumor. Abundant tumor-associated macrophages (TAMs) exist as neoplastic tissues, implicating tumor plasticity and thus leading to therapeutic challenges. Vascular adhesion protein (VAP-1) potentially serves as a mediator for TAM immunity in tumor milieu. We previously demonstrated that VAP-1 could contribute to tumor malignancy, but its characteristics in TAM immunity of glioma progression are still unclear. This study explored the association of VAP-1 expression with TAM distribution as well as the resulting clinical significance and prognostic value in human gliomas. An in-depth analysis of AOC3 (VAP-1) gene expression was performed using 695 glioma samples derived from the cancer genome atlas (TCGA)-lower grade glioma and glioblastoma (GBMLGG) cohort. Bioinformatic analysis confirmed that VAP-1 expression is associated with poor prognosis of glioma patients (p = 0.0283). VAP-1 and TAM biomarkers (CD68, iNOS, and CD163) were evaluated by immunohistochemistry in 108 gliomas from Kaohsiung Medical University Hospital. VAP-1+ was expressed in 56 (51.85%) cases and this phenotype revealed a significant association with overall survival in Kaplan–Meier analysis (p < 0.0001). Immunohistochemical double staining showed that VAP-1 immunoreactivity was present around CD163+ M2 infiltration location, including aggressive lesions and neighboring neovasculature. We demonstrated that high VAP-1 expression levels positively correlated with CD163+ M2 activation and coexpression of these two proteins was associated with worse survival in gliomas (p < 0.0001). Multivariate analysis indicated that VAP-1 alone and co-expressed with CD163 were the significantly independent indicators (both p < 0.0001). Furthermore, VAP-1/CD163 coexpression exhibited excellent diagnostic accuracy in gliomas (AUC = 0.8008). In conclusion, VAP-1 and TAM CD163 M2 coexpression was found in glioma tissues belonging to a highly malignant subgroup that was associated with poor prognosis. These results implied VAP-1 abundance is closely linked to alternative M2 activation during glioma progression. From the aforementioned data, a reasonable inference is that VAP-1 combined with targeting M2 immunity might be an effective therapeutic target for human gliomas.

Inhibiting Monocyte Recruitment to Prevent the Pro-Tumoral Activity of Tumor-Associated Macrophages in Chondrosarcoma.

Chondrosarcomas (CHS) are malignant cartilaginous neoplasms with diverse morphological features, characterized by resistance to chemo- and radiation therapies. In this study, we investigated the role of tumor-associated macrophages (TAM)s in tumor tissues from CHS patients by immunohistochemistry. Three-dimensional organotypic co-cultures were set up in order to evaluate the contribution of primary human CHS cells in driving an M2-like phenotype in monocyte-derived primary macrophages, and the capability of macrophages to promote growth and/or invasiveness of CHS cells. Finally, with an in vivo model of primary CHS cells engrafted in nude mice, we tested the ability of a potent peptide inhibitor of cell migration (Ac-d-Tyr-d-Arg-Aib-d-Arg-NH2, denoted RI-3) to reduce recruitment and infiltration of monocytes into CHS neoplastic lesions. We found a significant correlation between alternatively activated M2 macrophages and intratumor microvessel density in both conventional and dedifferentiated CHS human tissues, suggesting a link between TAM abundance and vascularization in CHS. In 3D and non-contact cu-culture models, soluble factors produced by CHS induced a M2-like phenotype in macrophages that, in turn, increased motility, invasion and matrix spreading of CHS cells. Finally, we present evidence that RI-3 successfully prevent both recruitment and infiltration of monocytes into CHS tissues, in nude mice.

Growth differentiation factor 15 facilitates lung fibrosis by activating macrophages and fibroblasts

Lung fibrosis is a devastating disease characterized by fibroblast accumulation and extracellular matrix deposition in lungs. However, its molecular and cellular pathogenesis is not fully understood and the current therapeutic strategies are ineffective. Bleomycin-induced lung fibrosis is the most widely used experimental model for research aimed at in-depth analysis of lung fibrosis mechanisms. The present study aimed to analyse the effects of growth differentiation factor 15 (GDF15), which is associated with many diseases, in lung fibrosis. GDF15 mRNA expression was elevated in the lungs of bleomycin-treated mice, revealed by comprehensive gene analysis. Its protein levels were also increased in the lungs, bronchoalveolar lavage fluid, and plasma obtained from bleomycin-treated mice as compared to those in saline-treated mice. Bleomycin administration in mice resulted in a marked increase in senescence-associated β-galactosidase-positive and p16INK4a-positive lung structural cells including alveolar epithelial cells and macrophages. Immunohistochemical staining using anti-GDF15 antibody and increased mRNA expression of GDF15 in bleomycin-induced senescent A549 cells indicated that GDF15 is produced from alveolar epithelial cells undergoing bleomycin-induced cellular senescence. GDF15 was also implicated in the augmentation of interleukin-4/interleukin-13-induced mRNA expression of M2 markers including arginase 1 and chitinase-3-like protein and was also responsible for increased α-smooth muscle actin expression through the ALK5-Smad2/3 pathway in WI-38 lung fibroblasts. Therefore, GDF15 secreted from senescent alveolar epithelial cells might act as a profibrotic factor through activation of M2 macrophages and fibroblasts. This implies that GDF15 could be a potential therapeutic target and a predictor of lung fibrosis progression.

Immunosuppressive Functions of M2 Macrophages Derived from iPSCs of ALS Patients

Amyotrophic lateral sclerosis (ALS), an adult-onset neurodegenerative disease, is a systemic inflammatory disorder that augments disease burden and rates of disease progression, and is pathologically characterized by activated leukocytes. Activated macrophages are members of this family and can be simplistically separated into M1 and M2 cells. M2 macrophages (M2) have been shown to provide therapeutic benefits in mouse models of diabetes and Adriamycin nephrosis by suppressing the pro-inflammatory state. However, the suppressive functions of M2 have not been investigated in ALS, nor have M2 derived from human iPSCs (Induced Pluripotent Stem Cells) been studied for their immunosuppressive potential. In this study, human iPSCs from ALS patients with C9orf72 (C9) mutation or from sporadic ALS patients were differentiated into M2 macrophages, which suppressed activation of pro-inflammatory M1 macrophages as-well-as proliferation of pro-inflammatory CD4+CD25- T lymphocytes (Teffs) of ALS patients. These M2 macrophages also converted ALS Teffs into CD4+CD25+Foxp3+ regulatory T cells (Tregs), and rescued ALS patients’ Tregs from the loss of functional markers CD25 and Foxp3. Furthermore, the ALS Tregs induced or rescued by iPSC-derived M2 cells had strong suppressive functions. Without influence of C9 mutation or disease insult, immunomodulatory effects of ALS iPSC-derived M2 cells behaved similarly to control iPSC-derived M2 cells. This study demonstrates that M2 cells differentiated from ALS patients’ iPSCs have immunosuppressive functions and boost ALS Tregs, and may serve as a candidate for immune-cell-based therapy to mitigate the systemic inflammation occurring in patients with ALS.

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.

MP01-16 IL-10 AND CXCL10 URINE QUANTIFICATION AS USEFUL BIOMARKERS TO PREDICT BCG RESPONSE IN BLADDER CANCER PATIENTS

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I (MP01)1 Apr 2020MP01-16 IL-10 AND CXCL10 URINE QUANTIFICATION AS USEFUL BIOMARKERS TO PREDICT BCG RESPONSE IN BLADDER CANCER PATIENTS Felix Guerrero-Ramos*, Pablo Abad-Lopez, Cristian Suarez-Cabrera, Marta Rodriguez-Izquierdo, Carmen Gomez-Cañizo, Federico de la Rosa-Kehrmann, Victor Garcia-Martinez, Alejandra Bernardini, Iris Lodewijk, Guillermo Paramio, Alfredo Rodriguez-Antolin, Jesus M Paramio, and Marta Dueñas Felix Guerrero-Ramos*Felix Guerrero-Ramos* More articles by this author , Pablo Abad-LopezPablo Abad-Lopez More articles by this author , Cristian Suarez-CabreraCristian Suarez-Cabrera More articles by this author , Marta Rodriguez-IzquierdoMarta Rodriguez-Izquierdo More articles by this author , Carmen Gomez-CañizoCarmen Gomez-Cañizo More articles by this author , Federico de la Rosa-KehrmannFederico de la Rosa-Kehrmann More articles by this author , Victor Garcia-MartinezVictor Garcia-Martinez More articles by this author , Alejandra BernardiniAlejandra Bernardini More articles by this author , Iris LodewijkIris Lodewijk More articles by this author , Guillermo ParamioGuillermo Paramio More articles by this author , Alfredo Rodriguez-AntolinAlfredo Rodriguez-Antolin More articles by this author , Jesus M ParamioJesus M Paramio More articles by this author , and Marta DueñasMarta Dueñas More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000815.016AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Identifying biomarkers to predict BCG response before beginning the treatment is an unsolved need in the clinical practice for patients with high risk non-muscle invasive bladder cancer. Nowadays, there is no method to predict BCG response in our patients. In this context, protumor activity of M2 macrophages could be mediating this treatment response, so evaluation of cytokines as IL-10 and CXCL10 in urine could be an indicator of their activity on patient’s urothelium. The main aim of this work is to evaluate if expression levels of IL-10 and CXCL10 in urine samples obtained before treatment are able to identify BGC responders and non-responders. We also aimed to find out the correlation of these levels in urine with the presence of M2 macrophages in tumor tissue samples obtained after transurethral resection of the bladder tumor (TURBT) and before the onset of BCG instillations. METHODS: Tumor tissue samples as well as urine samples were collected from a paired sample set (n= 20) from patients before BCG treatment at our Urology Department. Response to BCG instillations was defined as no recurrence after 2 years. Informed consent was obtained from all patients. The expression levels of IL-10 and CXCL10 were measured by ELISA and RT-qPCR. The percentage of M2 polarized macrophages was calculated by immunohistochemistry staining using CD163 as M2 surface marker. We also carried out nanostring profiling with IO360 panel to characterize immune cell population in tumor tissue. RESULTS: We found a good correlation between the levels of IL-10 and CXCL10 measured by ELISA in urine samples and the levels determined by RT-qPCR in urine and tumor tissue. In order to define if the presence of M2 macrophages before BCG instillation correlates with the BCG response rate, we quantified CD163-positive cells in tissue samples from responders and non-responders. We found that patients with higher count of CD163+ infiltrating cells show worse response to BCG. We found that IL-10 quantification is a good predictor for BCG response. CONCLUSIONS: The presence of M2 polarized macrophages in TURBT tissue as well as M2 markers in urine samples of bladder cancer patients appear to be predictive for BCG response. However, validation in a larger cohort is needed to confirm our data. Source of Funding: None. © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e8-e8 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Felix Guerrero-Ramos* More articles by this author Pablo Abad-Lopez More articles by this author Cristian Suarez-Cabrera More articles by this author Marta Rodriguez-Izquierdo More articles by this author Carmen Gomez-Cañizo More articles by this author Federico de la Rosa-Kehrmann More articles by this author Victor Garcia-Martinez More articles by this author Alejandra Bernardini More articles by this author Iris Lodewijk More articles by this author Guillermo Paramio More articles by this author Alfredo Rodriguez-Antolin More articles by this author Jesus M Paramio More articles by this author Marta Dueñas More articles by this author Expand All Advertisement PDF downloadLoading ...

Abstract A69: Elucidating the cell type-specific mechanisms of the Ron receptor in the prostate tumor microenvironment

Abstract The incidence of advanced prostate cancer (PCa) in American men has grown by 72% within the last decade, yet current treatments fail to effectively treat advanced disease. We study the Ron receptor tyrosine kinase as a novel target for PCa treatment. Ron is primarily expressed in macrophages and epithelial cells and is found overexpressed in PCa. Ron expression increases with disease severity in humans and mouse models, suggesting Ron drives progression to advanced PCa. Our laboratory has shown that loss of Ron signaling in prostate epithelial cells or in myeloid cells markedly reduces prostate tumor growth and modulates the tumor microenvironment, particularly the functions of macrophages and CD8+ T cells. Thus, we hypothesize that the Ron receptor utilizes cell type-specific mechanisms to suppress the antitumor immune response. However, our previous studies were limited because they were performed separately in different orthotopic models. Therefore, we employed the well-established Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) PCa mouse model to study loss of Ron signaling in either cell type in a single autochthonous model. We used Cre-Lox technology to generate TRAMP mice containing conditional deletion of Ron signaling in prostate epithelial cells or in myeloid cells. At 30 weeks of age, prostate tumors are harvested and analyzed by weight, immunohistochemical staining, quantitative PCR, Western blot, and flow cytometry. To assess the effects of Ron modulation in each cell type and interrogate the interaction between tumor cells and macrophages, we perform in vitro co-culture assays utilizing an established prostate cancer epithelial cell line derived from the TRAMP model and bone marrow-derived macrophages. We observe that Ron signaling in either cell type is critical for prostate tumor growth in TRAMP mice. Furthermore, loss of Ron in either cell type leads to increased macrophage and CD8+ T-cell tumor infiltration, and our in vitro data show that loss of Ron in either cell type suppresses antitumor M1 macrophage activation and promotes tumor-supporting M2 macrophage activation. Additionally, preliminary data suggest loss of Ron in either cell type leads to increased activation of CD8+ tumor-infiltrating lymphocytes. Overall, our data suggest the Ron receptor coordinates immunosuppressive mechanisms in both the malignant tumor cells and the host macrophages to dampen antitumor immunity and promote prostate cancer. These studies implicate Ron targeting as a powerful therapeutic tool with broad utility for understanding and refining therapeutic strategies to treat advanced PCa. Citation Format: Camille Sullivan, Nicholas E. Brown, Susan E. Waltz. Elucidating the cell type-specific mechanisms of the Ron receptor in the prostate tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A69.

The Combination of Phytonutrients in Spectra7 Provide Synergistic Inhibition of M1‐Macrophage Activation

Unbalanced or prolonged inflammation contributes to the pathogenesis of many conditions such as diabetes, arthritis, atherosclerosis and cancer to name a few. At the cellular level, macrophages of the innate immune system are central players that become inflamed in an M1‐state upon encountering pathogen, and, following clearance, shift in activation to an anti‐inflammatory (M2) state to aid in resolution of inflammation. Unfortunately, chronic diseases are promoted and exacerbated by M1‐macrophages that have become “stuck” in the on position.Spectra7 (S7) is a novel phytonutrient supplement containing extracts rich in curcuminoids, epigallocatechin gallate (EGCG), catechins, chlorogenic acid and anthocyanins. While these phytonutrients have individually demonstrated anti‐inflammatory properties in previous research, it is not known if the combination of these in S7 is effective at reducing M1‐macrophage activation. Here we tested the hypothesis that S7 would reduce LPS‐induced M1‐macrophage activation, and that the combination of these phytonutrients would provide greater inhibition than the sum of the individual components. Hypothesizing that the curcuminoids would exert the greatest effect on M1 activation, we further tested two formulations of S7 with either curcumin (S7‐C) or its metabolite tetrahydrocurcumin (S7‐THC).Phytonutrients individually or in combination were tested for their ability to alter LPS‐induced M1 activation in RAW264.7 macrophages. We found that both S7‐THC and S7‐C exhibited strong dose‐dependent inhibition of nitric oxide (NO) production as assessed by the Griess assay. Moreover, this effect was largely attributed to EGCG in the green tea extract (GTE) and curcuminoids in the turmeric extract (TE). Combinations of phytonutrients containing 5–10 μM curcumin exerted the greatest effect. Interestingly, while the other five components of S7 (green coffee bean extract, blueberries, whole tart cherries, broccoli and kale) did not inhibit NO production, they did enhance the ability of GTE and TE in inhibiting M1‐activation. This suggests that the combination of individual ingredients together in S7‐THC/C exhibit synergistic interactions. Determination of inflammatory gene expression revealed differential regulation between S7‐C, S7‐THC and the corticosteroid budesonide. While all three inhibited LPS‐induced NO production, only S7‐C and budesonide decreased expression of inducible nitric oxide synthase (iNOS), suggesting posttranscriptional regulation was also a factor. IFN‐β and IL‐6 expression were also inhibited by S7‐C and budesonide, but S7‐THC paradoxically prolonged LPS‐induced expression at 12 and 24 hours, suggesting delayed negative feedback inhibition. Lastly, we also found that S7‐C and budesonide, but not S7‐THC, were able to prevent cellular and mitochondrial reactive oxygen species (ROS) as assessed by DCFDA and MitoSOX fluorescence respectively. Together, these data support continued investigation of natural alternatives, like S7‐THC/C, to pharmacologic agents for their ability to resolve M1‐inflammation, and thus contribute to managing and even resolving disease progression.Support or Funding InformationPence‐Boyce Undergraduate Summer Research Experience