CD163-positive M2 Research Articles

A study of the correlation between M2 macrophages and lymph node metastasis of colorectal carcinoma

BackgroundLymph node metastasis is a major prognostic sign of colorectal carcinoma and an important indicator for individualized treatment. M2 macrophages play a key role in carcinogenesis and tumor development by enhancing invasiveness and promoting lymph node metastasis. The purpose of this study was to investigate the effect of CD163-positive M2 macrophages on lymph node metastasis in colorectal carcinoma.MethodsPostoperative lymph node tissues were obtained from 120 patients with colorectal carcinoma who underwent radical surgery in the First Affiliated Hospital of Jinzhou Medical University between December 2019 and May 2020. We detected the expression of the CD163 protein in lymph nodes using immunohistochemistry. Furthermore, the relationships between M2 macrophages identified by expression of CD163 and lymph node metastasis were analyzed using the independent sample t-test and Chi-square test.ResultsM2 macrophages were increased in metastatic lymph nodes and non-metastatic lymph nodes adjacent to the cancer. The M2 macrophage count was higher in patients with macro-metastases than in patients with micro-metastases.ConclusionsThe presence of M2 macrophages represents an important indicator for lymph node metastasis in colorectal carcinoma and may be a potential marker for its prediction. Thus, M2 macrophage localization might offer a new target for the comprehensive treatment of colorectal carcinoma.

Micro-Vibration Stimulation Promotes M2 Polarization of Macrophages Beneficial for Osteogenesis Via Activating FAK/P38MAPK and Suppressing Ca <sup>2+</sup>/ERK/ p65NF-κB Signaling Pathways

The emergence of osteoimmunology has demonstrated that bone system is closely related to immune system. However, most studies have investigated the interaction between immune cells and bone-related cells under a static cell culture, the effects of mechanical stimuli on the inflammatory responses of macrophages remain elusive. In this work, micro-vibration stimulation (MVS) was introduced into regulating macrophage polarization and functional status. The results found that MVS with 40 Hz in frequency and 0.3 g in magnitude could significantly raise the fraction of CD206-positive M2 population, up-regulate the expression of M2 marker genes, and enhance the secretion of M2 marker proteins in RAW 264.7 macrophages. It suggested that calcium ion and focal adhesion played an important role in transmitting MVS signal. Further inhibitor studies and western blotting analyses indicated that MVS-induced M2 polarization might be attributed to the activation of p38MAPK signaling and suppression of the ERK1/2 and p65NF-κB signaling. Moreover, MVS-mediated macrophage secretion enhanced osteoblastic differentiation of MC3T3-E1 pre-osteoblasts via Wnt/β-catenin and ERK1/2 signaling in a paracrine way. These findings highlight a critical role of MVS in inducing M2 polarization of macrophage to create a pro-osteogenic environment, which offers a drug-free modulatory strategy to regulate immune response and promote osteogenesis.

Immunomodulatory effect of dimethyloxallyl glycine/nanosilicates-loaded fibrous structure on periodontal bone remodeling

Background/purposeRelieving immuno-inflammatory responses is the prerequisite step for treating periodontitis. The angiogenic small molecule, dimethyloxalylglycine (DMOG), and osteoinductive inorganic nanomaterial, nanosilicate (nSi) have a powerful effect on bone regeneration, whereas the roles in osteoimmunomodulation have not been totally uncovered. Our study aimed to explore the immunomodulatory effect of DMOG/nSi-loaded fibrous membranes on periodontal bone remodeling. Materials and methodsThe fibrous membranes were prepared by incorporating DMOG and nSi into poly (lactic-co-glycolic acid) (PLGA) with electrospinning. The morphology features, surface chemical property and biocompatibility of DMOG/nSi-PLGA fibrous membranes were characterized. Thereafter, the fibrous membranes were implanted into rat periodontal defects, bone remodeling potential and immunomodulatory effect were evaluated by micro-computed tomography (micro-CT), histological evaluation and immunohistochemical analysis. ResultsDMOG/nSi-PLGA membranes possessed favorable physicochemical properties and biocompatibility. After the fibrous membranes implanted into periodontal defects, DMOG/nSi-PLGA membranes could relieve immuno-inflammatory responses of the defects (reduction of inflammatory cell infiltration, CD40L and CD11b-positive cells), increased CD206-positive M2 macrophages, and eventually facilitated periodontal bone regeneration. ConclusionDMOG/nSi-PLGA fibrous membranes exert protective effects during periodontal bone defect repairing, and steer immune response towards bone regeneration. Consequently, DMOG/nSi-PLGA fibrous membranes may serve as a promising scaffold in periodontal tissue engineering.

CD206-positive M2 Macrophage (MΦ) is an exacerbating factor and a new therapeutic target in endometriosis

Prostaglandin E2 (PGE2), an inflammatory mediator, modulates cytokines, regulates immune responses in reproductive processes and stimulates inflammatory reactions via the prostaglandin E2 receptor 2 (EP2). However, the regulatory effects of EP2 signaling on trophoblasts and its role in unexplained recurrent miscarriage (uRM) remains unclear.A total of 19 placentas from patients with a history of more than two consecutive pregnancy losses of unknown cause (uRM group) and placentas of 19 healthy patients following a legal termination of their pregnancy were used for PGE2 receptor (EP1, EP2 and EP4) expression analyses via immunohistochemistry. Double immunofluorescence was also used to identify EP2 expressing cells in the decidua. Finally, HTR-8/SVneo cells were used to clarify the role of EP2 in in vitro experiments.The expression of EP2 and EP4 was found to be reduced in the syncytiotrophoblast and decidua of uRM patients. A selective EP2 receptor antagonist (PF-04,418,948) reduced the proliferation and secretion of ß-hCG, inhibited interleukin -6 (IL-6) and interleukin-8 (IL-8) and up-regulated the production of the tumor necrosis factor-α (TNF-α) and plasminogen activator inhibitor type 1 (PAI-1) in HTR-8/SVneo cells in vitro.PGE2-EP2 signaling pathway may represent a novel therapy option for uRM. The involvement of EP2 in uRM acts perhaps via inflammatory cytokines and indicates that the PGE2-EP2 signaling pathway might represent an unexplored etiology for uRM.

Hederagenin Modulates M1 Microglial Inflammatory Responses and Neurite Outgrowth

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder. Neurite atrophy and synaptic loss initiate the onset of neuronal death, while the activated M1 microglia-induced neuroinflammatory microenvironment inhibits neurite regeneration and exacerbates neuronal loss. Thus, optimizing the brain microenvironment using small compounds through suppressing activated M1 microglia and promoting neurite regrowth might be an effective therapeutic strategy for AD. We found that hederagenin (HED), a naturally occurring triterpene compound, inhibited lipopolysaccharide-induced nitric oxide generation and downregulated expression of proinflammatory cytokines, such as tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6. Further investigation of primary microglia confirmed that HED inhibited Iba-1 positive M1 microglia. However, no changes were seen in CD206 positive M2 microglia polarization. HED remarkably suppressed phosphorylated nuclear factor kappa-light-chain-enhancer of activated B cells subunit p65 signaling. In addition, HED ameliorated Aβ25-35-induced neuritic atrophy and neuronal death. Therefore, HED might be a therapeutic candidate for AD.

The expression and meaning of CD68, CD163, CD57, and IgG4 in granulomatous lobular mastitis.

The exact etiology and pathogenesis of granulomatous lobular mastitis (GLM) are yet to be illuminated. This study aimed to investigate CD68, CD163-positive M2 macrophages, CD57-positive natural killer (NK) cells, and IgG4 in GLM lesion tissue to explore their correlation with the occurrence and clinical features of GLM. Surgical pathologic specimens of GLM were collected from patients admitted to Hunan Provincial People's Hospital between October, 2014 and October 2015. Based on the postoperative pathological diagnosis, the tissues were divided into 3 groups: the experimental group (GLM, n=36), control group 1 (plasma cell mastitis, PCM, n=17), and control group 2 (breast cystic hyperplasia, n=10). Immunohistochemical staining was carried out using Elivision super testing to detect CD68, CD163, CD57, and IgG4 expression in the pathological tissue samples. The relationship between clinical parameters, including age, reproductive condition, nipple retraction, and tumor size, and the expressions of CD68, CD163, CD57, and IgG4 was analyzed. There was no obvious difference in the levels of CD68, CD163, and CD57 expression between the GLM group and the PCM group, although both groups had higher expression levels of expression than the breast cystic hyperplasia group (P<0.05). In the GLM group, the expression level of CD57 at 2 weeks-3 months was significantly higher than at ≤2 weeks (P<0.05). The expression level of CD57 in PCM patients >2 years after lactation was significantly higher than in patients ≤2 years after lactation (P<0.05). The level of IgG4 expression in GLM patients with nipple retraction was significantly higher than in those without nipple retraction (P<0.05). Inflammatory cells are closely linked to the occurrence of GLM and PCM. In our study, both the GLM and PCM groups had low expression of IgG4, but the expression level of IgG4 in GLM patients with inverted nipples was significantly higher than that in patients without inverted nipples. This suggests that there may be two different clinical subtypes of GLM. Furthermore, our research also found that NK cells can provide a basis for GLM clinical staging.

TMEM119 as a specific marker of microglia reaction in traumatic brain injury in postmortem examination

The aim of the present study was a refined analysis of neuroinflammation including TMEM119 as a useful microglia-specific marker in forensic assessments of traumatic causes of death, e.g., traumatic brain injury (TBI). Human brain tissue samples were obtained from autopsies and divided into cases with lethal TBI (n = 25) and subdivided into three groups according to their trauma survival time and compared with an age-, gender-, and postmortem interval-matched cohort of sudden cardiovascular fatalities as controls (n = 23). Brain tissue samples next to cortex contusions and surrounding white matter as well as samples of the ipsilateral uninjured brain stem and cerebellum were collected and stained immunohistochemically with antibodies against TMEM119, CD206, and CCR2. We could document the highest number of TMEM119-positive cells in acute TBI death with highly significant differences to the control numbers. CCR2-positive monocytes showed a significantly higher cell count in the cortex samples of TBI cases than in the controls with an increasing number of immunopositive cells over time. The number of CD206-positive M2 microglial cells increased survival time-dependent. After 3 days of survival, the cell number increased significantly in all four regions investigated compared with controls. In sum, we validate a specific and robustly expressed as well as fast reacting microglia marker, TMEM119, which distinguishes microglia from resident and infiltrating macrophages and thus offers a great potential for the estimation of the minimum survival time after TBI.

Generation of Novel Diagnostic and Therapeutic Exosomes to Detect and Deplete Protumorigenic M2 Macrophages.

Given their protumorigenic function and prevalence in most malignant tumors with lower survival; early detection, and intervention of CD206-positive M2 macrophages may boost the clinical outcome. To determine in vivo distribution of M2 macrophages, 111In-oxine-based radiolabeling of the targeted exosomes is adopted. When these radiolabeled targeted exosomes are injected into breast tumor-bearing mice, exosomes accumulate at the periphery of the primary tumor, metastatic foci in the lungs, spleen, and liver. Ex vivo quantification of radioactivity also shows similar distribution. Injecting DiI dye-labeled exosomes into the same mice shows adherence of exosomes to the CD206-positive M2 macrophages on ex vivo fluorescent microscopy imaging. In addition, these engineered exosomes are utilized to carry the Fc portion of lgG2b with the intention of augmenting antibody-dependent cell-mediated cytotoxicity. It is demonstrated that M2 macrophage targeting therapeutic exosomes deplete M2 macrophages both in vitro and in vivo, and reduce tumor burden, increasing survival in a metastatic breast cancer model.

Sodium tanshinone IIA sulfonate restrains fibrogenesis through induction of senescence in mice with induced deep endometriosis

Research questionDoes sodium tanshinone IIA sulfonate (STS) induce cellular senescence in endometriotic lesions and thus restrict lesional development and fibrogenesis in a recently established mouse model of deep endometriosis? DesignProspective randomized animal experiment in which deep endometriosis was induced in female Balb/C mice, which were then randomly divided into three groups (low-dose STS, high-dose STS and inert vehicle control) and received treatment for 2 weeks. All mice were then sacrificed and their lesions excised and harvested. Lesion weight was quantified and all lesion samples were subjected to histochemical analysis of the extent of lesional fibrosis by Masson trichrome staining, and of cellular senescence by senescence-associated β-galactosidase (SA-β-gal), along with immunohistochemistry analyses of p53, CCN1, activate Salvador 1 (Sav1), hyaluronan synthase 2 (HAS2), survivin, granulocyte-macrophage colony-stimulating factor (GM-CSF) and CD163-positive M2 macrophages. Plasma P-selectin and hyaluronic acid levels were also quantified. Hotplate testing was also administered before the induction, then before and after treatment. ResultsSTS treatment resulted in significantly reduced lesion weight, stalled lesional fibrogenesis and improved hyperalgesia, seemingly through the induction of cellular senescence by activating p53, Sav1 and CCN1 while suppressing HAS2, survivin and GM-CSF, resulting in increased apoptosis and reduced lesional infiltration of alternatively activated macrophages. In addition, STS treatment significantly reduced the plasma concentration of P-selectin and hyaluronic acid, possibly leading to reduced lesional platelet aggregation. ConclusionsSTS appears to be a promising compound for treating endometriosis. The results suggest that senescence may restrict lesional progression and fibrogenesis, and targeting the senescence pathway may have desirable therapeutic potential.

Immunosuppressive Diversity of Umbilical Cord-Derived Mesenchymal Stromal Cells

Recently, umbilical cord (UC) has become attracted source of mesenchymal stromal cells (MSCs) for immunotherapy such as treatment of severe acute graft versus host disease, because of abundant sources and ease of collection without invasive process. In previous reports on bone marrow-derived MSCs, there is diversity of immunosuppressive mechanisms of MSCs, which have not yet fully clarified in UC-MSCs. Here we studied the immunosuppressive properties of UC-MSCs on the activated immune system in vitro. Methods; UC was collected after obtaining informed consent from the mother. UC-MSCs were obtained from frozen-thawed UC tissue with explant method and expanded in culture medium. Mixed lymphocyte reaction (MLR) consisted with mononuclear cells (MNCs) stained with CFSE and allogeneic dendritic cell (DC) line (PMDC05) co-cultured with or without UC-MSCs was carried out, and the proliferation of CD4 and CD8-positive lymphocytes were analyzed by flowcytometry followed by FlowJo software. Migratory activities of UC-MSCs toward activated lymphocytes in MLR were evaluated by counting the migrating cells stained with DAPI through the insert filter under the microscope. The percentage of CD4+CD25+FOXP3+ regulatory T cells (Treg) before and after co-culture with UC-MSCs was analyzed by flowcytometry. Monocyte phenotypes co-cultured with UC-MSCs directly or separately by insert filter were analyzed with anti-CD14, CD206, and CD168 antibodies. Results: The UC-MSCs were positive for CD105, CD73, CD90, and negative for CD45 and HLA-DR. HLA-DR, CD80, CD86, and CD40 were negative even in the high concentration of IFN-γ, while BM-MSCs became positive for HLA-DR. PD-L2 was constitutively expressed in UC-MSC, while PD-L1 was induced upon the addition of IFN-γ. In MLR, responder T cell proliferation triggered by allogeneic DC was inhibited efficiently by 3rd party derived UC-MSCs. Mean+/- SD distribution index of proliferation in CD4- and CD8- positive cells co-cultured with UC-MSCs showed 7.6+/-4.4% and 8.8+/-3.9% compared to those without UC-MSCs (n=4, as different donor derived MSCs). This inhibition was attenuated by the addition of 1-Methyl-dl-tryptophan, Indoleamine 2, 3-dioxygenase (IDO-1) inhibitor, in a dose-dependent manner. The supernatant of MLR showed the increase of IFN-γ and TNF-α, which were also suppressed in MLR co-cultured with UC-MSCs. UC-MSCs migrated toward activated lymphocytes in MLR compared with those of non-activated lymphocytes significantly, which was inhibited by the addition of AG1296, known as platelet-derived growth factor inhibitor; and PPP as insulin-like growth factor (P&amp;lt;0.05). Cytokine beads array indicated the increase of RANTES, IL-8, and MIP-1. Regulatory T cells in CD4-positive T cells were significantly increased in the MNCs co-cultured with UC-MSCs. Interestingly, CD206-positive M2 monocytes were significantly increased in the MNCs cu-cultured with UC-MSC. Mean +/- SD percentage of CD206+CD80- M2 cells indicated 28.9+/-15.3% in MNCs and 83.3+/-11.2% in MNCs cu-cultured with UC-MSC (n=4, P&amp;lt;0.05). On the other hand, CD206-CD80+ M1 monocytes significantly increased in addition of LPS, which were inhibited by the co-culture with UC-MSCs (P&amp;lt;0.05). Furthermore IFN-γ and TNF-α secreted in the MNCs with LPS were suppressed by the co-culture of UC-MSCs. Discussions and Conclusion: These results demonstrated that UC-MSCs have the diverse immunosuppressive potency through migration toward the activated lymphocytes, suppressing activated T cells proliferation, regulatory T cells induction, and transition of monocyte phenotype. And UC-MSCs are the feasible and abundant source for immunotherapy. Disclosures Nagamura-Inoue: AMED: Research Funding. Nagamura:AMED: Research Funding. Tojo:AMED: Research Funding; Torii Pharmaceutical: Research Funding.

Abstract TP538: Dynamic of Microglial Polarization in Experimental Subarachnoid Hemorrhage

Introduction: Subarachnoid hemorrhage (SAH) is a devastating stroke type. Microglia/Macrophage, in neuroinflammation response, are capable of classic M1 and alternative M2 polarization after activation. However, the profile of microglial activation was not well studied in SAH. In this study, we aimed to unravel the polarization-specific dynamic of microglia after SAH. Methods: Wild-type C57BL/6 mice and Cx3cr1 GFP/GFP Ccr2 RFP/RFP mice were used and SAH was induced by endovascular perforation. The Immunohistochemistry and Immunofluorescence were employed to characterize the profile of microglia activation on day 1, day 3, day 5, day 10 (n=6-8). Results: Iba1-positive microglia accumulated significantly in the cortex adjacent to the bleeding site (CAPS) on day 1 and sustained to increase till the delayed phase in SAH. Microglial accumulation was also observed on motor cortex and hippocampus on the early phase. Immunofluorescence showed the CD16/32-positive M1-predominant phenotype appeared in 3 days, then gradually transformed into the CD206-positive M2 phenotype. The functionally microglial polarization advanced along with the morphological changes through ramified to amoeboid shape. The bipolar microglia appeared through the morphological transformation with the colocalization of M1/M2 markers. In addition, Ccr2 low expressed and Cx3Cr1 highly expressed (CCR2 - /Cx3Cr1 + ) cells are observed on CAPS with a co-existing of a small group of CCR2 + /Cx3Cr1 + cell. The results suggested the preponderant microglial activation rather than the infiltrated macrophage. Conclusions: Microglia demonstrated a preponderant and early accumulation and this accumulation showed a centrifugal spreading through CAPS to remote motor cortex. Pro-inflammatory response occurred early and transform to the anti-inflammation on the delayed phase. The dynamic of microglial morphological changes and polarization implied the function-related neuroinflammatory response and pathophysiologic process of restoration upon the hemorrhage attack.

Colonic M1 macrophage is associated with the prolongation of gastrointestinal motility and obesity in mice treated with vancomycin.

Gut microbiota plays a pivotal role in not only the gastrointestinal (GI) immune system but also GI motility and metabolism. Antibiotic treatments are likely to affect the gut flora and GI immune system, subsequently disturbing GI motility and body metabolism. In the present study, we investigated antibiotic-induced alterations of body metabolism and GI motility in association with the macrophage profile in the colon. Specific pathogen-free (SPF) mice (ICR; 6 weeks old; female) were orally administered vancomycin (0.2 mg/ml) in drinking water for 5 weeks, and subsequent changes in pathophysiology were observed. The expression of CD80 and CD163 was examined by immunohistochemistry and the expression of cytokines in colonic tissues was evaluated by reverse transcription-quantitative polymerase chain reaction. The gastrointestinal transit time (GITT) was measured by administration of carmine red (6% w/v) solution. In the vancomycin-treated SPF mice, significant increases in body weight, cecum weight and GITT were observed compared with the controls. The number of CD80-positive M1 macrophages and the expression of interferon-γ and interleukin-12 were significantly increased, whereas, the numbers of CD163-positive M2 macrophages in the mucosal and muscular layers were decreased in the colon of vancomycin-treated mice. GITT was positively correlated with the number of CD80-positive M1 macrophages in the colonic mucosa; however, was negatively correlated with the number of CD163-positive M2 macrophages in the mucosal and muscular layers. Therefore, it was suggested that antibiotic treatment affects body metabolism and GI motility, accompanied by alterations in macrophage polarization and cytokine profiles in the colon.

Abundant a proliferation-inducing ligand (APRIL)-producing macrophages contribute to plasma cell accumulation in immunoglobulin G4-related disease.

BackgroundThis study aimed to investigate the contribution of a proliferation-inducing ligand (APRIL), a member of the tumor necrosis factor (TNF) superfamily implicated in plasma cell survival, to the development of plasma cell–rich lesions in immunoglobulin G4–related disease (IgG4-RD).MethodsWe performed immunohistochemical staining for APRIL with Stalk-1 and Aprily-8 antibodies specifically recognizing APRIL-producing cells and secreted APRIL, respectively, in renal and submandibular lesions of IgG4-RD in comparison with those of Sjögren’s syndrome and sialolithiasis.ResultsNumerous Stalk-1-positive APRIL-producing cells were detectable in lesions of IgG4-RD. These cells, identified as CD163-positive M2 macrophages, secreted APRIL that distributed close to and even on infiltrating plasma cells. In contrast, APRIL-producing cells and the secreted form of APRIL were rarely detectable in lesions of Sjögren’s syndrome or sialolithiasis. Notably, APRIL expression decreased concomitantly with the level of plasma cell infiltration after successful glucocorticoid treatment.ConclusionsAbundant infiltration into tissue lesions of APRIL-producing M2 macrophages and retention of secreted APRIL in plasma–cell–rich areas support a role for APRIL in the pathogenesis of plasma cell–rich lesions in IgG4-RD.

Macrophage M1/M2 polarization in patients with pregnancy-induced hypertension.

This study aimed to validate whether macrophage polarization imbalance and abnormal cytokines production occurred in pregnancy-induced hypertension (PIH) patients. PIH women (n = 26) were enrolled and the level of biochemical parameters were determined. The percentage of CD86- and CD163-positive cells, representing M1 and M2 macrophages, were determined by flow cytometry. The concentrations of cytokines (TNF-α, IL-1β, IL-4, IL-13, and IL-10) were determined using enzyme-linked immunosorbent assay kit. THP-1 cells were incubated with 10% serum from PIH and control groups, and then macrophage polarization and cytokines production were analyzed. The levels of high-density lipoprotein cholesterol and apolipoprotein A1 were significantly lower, and the levels of fasting blood glucose, total cholesterol, triacylglycerol, low-density lipoprotein cholesterol, apolipoprotein B, and lipoprotein a were significantly higher in the PIH group than that in the control group. The PIH group contained a significant higher percentage of CD86-positive cells (M1) and a significant lower percentage of CD163-positive cells (M2), representing higher M1/M2 ratio, than the control group. The PIH group expressed higher concentrations of TNF-α and IL-1β, and expressed lower concentrations of IL-4, IL-10, and IL-13 than the control group. The in vitro experiment also showed macrophage polarization imbalance and abnormal cytokines production in THP-1 cells treated with PIH serum as compared with that treated with control serum. Macrophage polarization imbalance and abnormal cytokines production occurred in PIH patients and in THP-1 cells treated with PIH serum.

Abstract 5514: Loss of hepatocyte growth factor activator inhibitor type-1 (HAI-1) in human lung adenocarcinomas promotes RON receptor phosphorylation and increased sensitivity to crizotinib

Abstract Introduction: Non-small cell lung cancer (NSCLC) is the top cause of cancer-related mortality. We have identified a role for the tumor suppressor hepatocyte growth factor activator inhibitor type-1 (HAI-1) in human lung adenocarcinoma. HAI-1 loss results in unregulated downstream MET/RON tyrosine kinase receptor signaling. We hypothesized that HAI-1 is lost in human lung adenocarcinomas and could serve as a biomarker for therapy with crizotinib, a tyrosine kinase inhibitor (TKI) with activity against both MET and RON. We also hypothesized that unregulated RON signaling in the tumor microenvironment, via HAI-1 loss, may promote tumor metastasis by skewing tumor associated macrophages (TAMs) from an anti-tumor (M1) to a pro-tumor (M2) phenotype. Methods: Immunohistochemical staining with HAI-1 antibody (1N7) was performed on human tissue microarray containing 20 normal and 20 lung tumor specimens. Intensity grading (0, 1+, 2+, 3+) was performed, and percent cell expression was calculated for each group. For all in vitro assays, H358 lung adenocarcinoma cells, which express high levels of endogenous HAI-1, were stably transfected with mock shRNA or HAI-1 shRNA. Cell protein expression and phosphorylation was assessed via western blot. Cell viability after exposure to crizotinib was assessed by trypan blue staining. For flow cytometric analysis of macrophage phenotype, undifferentiated (M0) macrophages were exposed to conditioned medium from transfected H358 cells and macrophage phenotype was assessed by flow for surface markers CD68 (M0), CCR7 (M1), and CD206 (M2). Results: HAI-1 expression is significantly decreased in human lung adenocarcinoma compared to normal lung with overall HAI-1 IHC positivity ~20% compared to ~60% respectively. HAI-1 knockdown in H358 cells caused increased phosphorylation of RON and increased sensitivity to crizotinib. Culturing M0 macrophages in conditioned media from H358 shHAI-1 knockdown cells caused a decrease in the CCR7 positive M1 fraction from 8.4% to 1.9%, and an increase in the CD206 positive M2 fraction from 0.77% to 2.09%. Conclusion: We have demonstrated that loss of HAI-1 occurs in human lung adenocarcinoma consistent with previous studies in human NSCLC cell lines. Furthermore, we showed significantly increased RON signaling activity after HAI-1 knockdown. We have also demonstrated that HAI-1 loss can skew tumor associated macrophages (TAMs) from an anti-tumor (M1) to a pro-tumor (M2) phenotype. We have shown that loss of HAI-1 in vitro can increase sensitivity to crizotinib, suggesting that loss of HAI-1 may predict for tumor sensitivity to MET/RON inhibition. Our findings show that dysregulation of the HAI-1/MET/RON pathway exerts effects both on lung tumor cells as well as TAMs, making this pathway a potentially powerful therapeutic target. Citation Format: Austin J. McHenry, Gautam Sondarva, Vijayalakshmi Ananthanarayanan, Ashley Hess, Patricia E. Simms, Alhareth Alsayed, Hiroaki Kataoka, Jan Marusarz, Sandeep Kumar, Ravi Salgia, Arkadiusz Dudek, Stanley Borowicz, Ajay Rana. Loss of hepatocyte growth factor activator inhibitor type-1 (HAI-1) in human lung adenocarcinomas promotes RON receptor phosphorylation and increased sensitivity to crizotinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5514.

Mild focal hypothermia regulates the dynamic polarization of microglia after ischemic stroke in mice

ABSTRACTObjectives The protective effects of hypothermia on acute stroke have been demonstrated in many studies. However, its underlying mechanisms have not been thoroughly elucidated. Following an ischemic stroke event, microglia undertakes an early ‘healthy’ M2 phenotype and gradually transform into a ‘sick’ M1 phenotype over time. This transformation of polarity of microglia has influence on the degree of damage following a stroke. This study investigated the effects of mild focal hypothermia on microglia polarization following ischemic stroke.Methods Transient cerebral ischemic models were created by intraluminal filament occlusion of right middle cerebral artery (MCAO) in mice for one hour. By placing an ice box under their skull, hypothermia of mice brain was initiated immediately following MCAO for 2 h. Temporal muscle temperature was recorded and maintained between 32 and 34 °C. Brain tissue loss was assessed by hematoxylin and eosin (H&E) staining 28 days after MCAO. Quantitative real-time polymerase chain reaction (qPCR) and immunostaining were used to assess phenotype of microglia in different ischemic perfusion time.Results Hypothermia reduced brain tissue loss 28 days after ischemic stroke. Hypothermia also reduced the number of CD16-positive M1 microglia and increased the numbers of CD206-positive M2 microglia following ischemic stroke. Moreover, hypothermia also led to the reduction of the M1 markers at the level of transcription, while it increased the expression of mRNA for M2 markers.Conclusions Hypothermia is protective following ischemic stroke and can reduce brain tissue loss. Moreover, hypothermia shifts the polarization of microglia from the M1 to the M2 phenotype in the ischemic mice brain. This observed biological phenomenon may partially explain the protective effects seen due to hypothermia in acute ischemic stroke.

Abberent expression of NOR1 protein in tumor associated macrophages contributes to the development of DEN-induced hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common primary tumor of the liver and the sixth most common lethal cancer worldwide. Recent evidences demonstrated that oxidored nitro domain containing protein 1 (NOR1), a putative tumor suppressor gene, is overexpressed in human HCC tissues. However, the role of NOR1 in HCC development remains unclear. Here, we described that NOR1 protein level is elevated in HCC and is associated with poorer clinical outcome. However, ecotopic overexpression of NOR1 protein in human HCC cell line HepG2 cells had no effect on cells proliferation, migration, and clonality. Immunofluoresence assay indicates NOR1 protein is mainly expressed at CD163 positive M2 tumor associated macrophages (TAMs). To explore the role of NOR1 in the development of HCC, we interrogated the susceptibility of mice lacking the NOR1 gene to DEN-induced hepatocarcinogenesis. NOR1 deficient mice displayed resistance to DEN-induced HCC. We also demonstrate that mNOR1 protein is enriched in F4/80 positive Kupffer cells (KCs) infiltrated in DEN induced murine HCC tissues. Loss of NOR1 led to increase of iNOS whereas decrease of Arg1, Ym1 expression in KCs. Overexpression of NOR1 in THP-1 macrophages led to decrease of iNOS but increase of Arg1. Mechanistic investigations showed that inflammatory cytokines IL-6, TNF-α production, and NF-κB activation were also decreased in NOR1 knockout mice exposed to DEN treatment. Our data suggested that NOR1 is overexpressed in HCC associated TAMs and promotes M2 alternative polarization. Genetic deletion of NOR1 in mice leads to impairment of IL-6 production and NF-κB activation, which in turn attenuates DEN-induced HCC development.

Effects of lobeglitazone, a novel thiazolidinedione, on adipose tissue remodeling and brown and beige adipose tissue development in db/db mice.

We investigated the effect of long-term treatment with lobeglitazone, a novel thiazolidinedione-based activator of peroxisome proliferator-activated receptor gamma, on adipose tissue (AT), focusing on its effects on insulin resistance in obese db/db mice. Seven-week-old male db/db mice were assigned to either a vehicle-treated (n=8) or lobeglitazone-treated (n=8) group. Lobeglitazone (1 mg kg-1 daily) was injected intraperitoneally for 20 weeks. Lobeglitazone treatment for 20 weeks resulted in a remarkably improved glycemic index, including significantly decreased glucose levels, enhanced insulin sensitivity and preserved pancreatic beta cells. Both whole body and subcutaneous AT weight increased in the lobeglitazone-treated group. However, lobeglitazone induced an increase in the number of small adipocyte in both epididymal and subcutaneous AT, with a significant weight decrease in the epididymal AT of db/db mice. Using flow cytometry, the CD11c-positive M1 macrophages and CD206-positive M2 macrophages in the epididymal AT were observed to exhibit a decreased M1-to-M2 ratio in lobeglitazone-treated db/db mice. Furthermore, in the lobeglitazone-treated group, interscapular brown AT was clearly visualized by 18F-fluoro-2-deoxy-D-glucose positron emission tomography combined with computed tomography (18F-FDG-PET/CT) and its mass was significantly greater than that of the vehicle-treated group. In the lobeglitazone-treated group, beige-specific gene expression and the number of mitochondria in white AT were upregulated. Lobeglitazone, with upregulating interferon regulatory factor-4 (a key transcriptional regulator of thermogenesis), promoted the development of brown adipocytes and the differentiation of white adipocytes into beige adipocytes. Long-term lobeglitazone treatment has a beneficial role in remodeling and ameliorating inflammation in white AT and in glycemic control, in relation to insulin sensitivity in obese db/db mice. Moreover, lobeglitazone induced the differentiation of brown and beige adipocytes. Collectively, our data suggest that lobeglitazone treatment provides promising effects on white and brown AT as well as great improvement in glycemic control, as a potent insulin sensitizer.

Elevated serum interleukin-10 level and M2 macrophage infiltration are associated with poor survival in angioimmunoblastic T-cell lymphoma.

Interleukin-10 (IL-10) induces an immunosuppressive microenvironment including M2 macrophages, inhibiting anti-tumor immunity. The aim of this study was to evaluate whether serum IL-10 level at diagnosis and tissue infiltration of M2 macrophages could predict survival outcome of patients with angioimmunoblastic T-cell lymphoma (AITL).We measured serum levels of IL-5, IL-10, IL-12, and interferon-gamma (IFN-γ) at diagnosis in AITL and other common subtypes of nodal T-cell lymphoma including peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), ALK-negative anaplastic large cell lymphoma (ALCL) or ALK-positive ALCL between September 2008 and December 2014. We also analyzed the infiltration of CD68- and CD163-positive macrophages in tumor tissue of AITL. In total, 97 patients with AITL (n=37), PTCL-NOS (n=40), ALK-negative ALCL (n=11), or ALK-positive ALCL (n=9) were treated with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone). Among cytokines, only the serum level of IL-10 was significantly higher in AITL patients than in other subtypes (P < 0.05). Compared to other subtypes, the association of serum IL-10 with overall survival (OS) was only significant in AITL. Accordingly, the response to CHOP chemotherapy was significantly worse in the high IL-10 group, and infiltration of CD163-positive M2 macrophages was significantly associated with OS in AITL. In conclusion, this study demonstrated the prognostic relevance of serum IL-10 and tissue infiltration of M2 macrophages in AITL patients. Our results suggest the possible use of these variables as potential therapeutic targets and novel prognostic indicators in patients with AITL.

Abstract 5011: Tumor associated macrophages promote malignant phenotypes of disseminated human gastric cancer cells in intraperitoneal cancer immune microenvironment

Abstract Gastric cancer is the third leading cause of cancer-related death, and peritoneal dissemination is one of the most frequent site of recurrence. Although the role of tumor microenvironment is recognized, it is still unclear how intraperitoneal cancer-immune microenvironment contributes to peritoneal metastasis. Thus, we investigated the interaction between intraperitoneal cancer and immune cells, especially focusing on tumor-associated macrophages (TAM) in the peritoneal cavity. The peritoneal wash from gastric cancer patients was subjected to conventional cytology, flow cytometry, and immuno-fluorescent assay using antibodies against CD45 (leukocytes), CD14 (monocytes), CD80/163 (M1/M2 macrophages, respectively), in combination with genetically engineered cancer-imaging viral agent ‘TelomeScan’ which specifically expresses GFP in telomerase-positive cells. In cytology-positive samples, CD163-positive macrophages could be detected in approximately 70% of intraperitoneal macrophages with TelomeScan-positive cancer cells, while cytology negative samples contained few CD163-positive macrophages. In addition, peripheral blood mononuclear cells (PBMCs) from healthy volunteers were induced to differentiate into CD163-positive M2 macrophages (TAM-like macrophages), and co-cultured with gastric cancer cells to examine the effect on their malignant phenotype such as migration and invasion. We found that PBMCs-derived CD163-positive M2 macrophages significantly potentiated the ability of migration and invasion in gastric cancer cells. In case of existence of free cancer cells in peritoneal cavity, intraperitoneal macrophages appeared to differentiate into TAM and they potentially accelerate malignant phenotypes of gastric cancer cells. These results suggested that intraperitoneal cancer-immune microenvironment may play an important role to promote peritoneal disseminations in gastric cancer. Citation Format: Shuichi Sakamoto, Shunsuke Kagawa, Kazuya Kuwada, Atene Ito, Tetsuya Kagawa, Satoru Kikuchi, Shinji Kuroda, Ryuichi Yoshida, Hiroshi Tazawa, Toshiyoshi Fujiwara. Tumor associated macrophages promote malignant phenotypes of disseminated human gastric cancer cells in intraperitoneal cancer immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5011. doi:10.1158/1538-7445.AM2017-5011