MΦ Infiltration Research Articles

P6297Genetic mutation of galectin-3 altered the temporal evolution of macrophage polarization and healing affecting the post myocardial infarct remodeling in mice

Abstract Background Myocardial infarction (MI) is a dynamic process that leads to ventricular remodeling (VR) and largely to heart failure (HF). Previous studies established that Galectin-3 (Gal-3) is highly increased in the infarct zone from the beginning of MI and also that it is a prognostic marker of HF. Purpose We aimed to study the effects of genetic deletion of Gal-3 on macrophage (MΦ) infiltration, cytokines expression, fibrosis and MMP-2 activity as well as VR and function after MI in mice. Methods Male C57BL/6J and Gal-3 KO mice were subjected to permanent coronary ligature or sham. At 1 week post-MI LV function and VR were studied by echocardiography. We also studied in the infarct zone: 1) F4/80+ MΦ infiltration by flow cytometry; 2) M2 macrophage polarization by detection of mannose receptor (MR) and chitinase-3-like protein-3 (YM1) phenotype markers by rt-qPCR; 3) mRNA expression of TNF-α, IL-6, IL-10 and TGF-β; 4) MMP-2 activity by zymography and 5) fibrosis by histology. Results Results are expressed as X±SEM; *p<0.05 C57 MI vs Gal-3 KO MI. After 1 week post-MI, the pulmonary congestion assessed by the lung weight/body weight ratio (mg/g) was 9±0.4, 8±0.5 and 9±0.4 in C57 sham, Gal-3 KO sham and C57 MI, respectively, nevertheless it was severely increased to 15±1.2* in infarcted Gal-3 KO mice. MΦ infiltration, cytokine expression and MMP-2 activity in the infarct zone are shown in the table. Table 1 Groups F4/80+ MR YM1 TNF-α IL-6 IL-10 TGF-β MMP-2 MΦ (%) (A.U.) (A.U.) (A.U.) (A.U.) (A.U.) (A.U.) (A.U.) C57 MI 5.6±0.9 (8) 0.5±0.2 (3) 1.7±0.4 (3) 26±0.3 (3) 1.7±0.5 (3) 0.7±0.03 (3) 1.8±0.5 (3) 1±0.1 (5) Gal-3 KO MI 2.6±0.4* (8) 1.8±0.4*(8) 4.2±0.4* (8) 0.4±0.5* (8) 6.7±0.9 * (8) 2.8±0.3 * (8) 0.5±0.2 * (8) 2±0.4* (6) A.U.: Arbitrary units. In MR, YM1, TNF-α, IL-6, IL-10 and TGF-β represent mRNA expression. In MMP-2 represent gelatinolytic activity. Number of samples is shown between parentheses. After 1 week post-MI, LV end diastolic dimension was increased from 4.4±0.1 to 4.8±0.2* at the same time that ejection fraction (%) was significantly reduced from 47±2 to 38±3* in C57+MI (13) and Gal-3 KO+MI (16) respectively. Collagen concentration in the infarct zone was significantly reduced from 30±1.2% (6) to 17±0.5* % (8) in C57 and Gal-3 KO respectively. Conclusion(s) Gal-3 is an essential regulatory factor for the early wound healing since it regulates the dynamics of the reparative process through the phenotypic profile of MΦ, the pro- and anti-inflammatory cytokines expression and fibrosis along the temporal evolution of MI in mice. The deficit of Gal-3 diminished the infiltration of MΦ altering its phenotypic polarization and consequently, the dynamics of the wound healing as well as aggravating the functional and structural evolution of cardiac remodeling. Acknowledgement/Funding Argentine Agency for Promotion of Science and Technology (PICT 2014-2320), University of Buenos Aires (UBACyT 20020170100619BA)

1999P - Impact of tumor reoxygenation by nanoparticles on tumor associated macrophages (TAMs)

BackgroundGlioblastoma (GB), a highly hypoxic brain tumor (Bekaert et al. 2017), is characterized by a massive macrophage (MΦ) infiltration (Lapa et al. 2015). Hypoxia triggers a shift to a pro-tumoral M2 phenotype in GB (Leblond et al. 2016). Thus, strategies aiming to reduce hypoxia could promote an anti-tumoral M1 phenotype. Among these reoxygenation strategies, we recently developed a new approach with zeolites nanoparticles. These zeolites are able to carry hyperoxic/hypercapnic gases and release them according to a hypoxic gradient. We have demonstrated that the charge balancing cation changes affinity to the gases but also the ability to track zeolite with MRI. Our objective is to study the reoxygenation efficacy of zeolites specifically in the GB and to evaluate their impact on tumor associated MΦ with in vitro and in vivo studies. MethodsFaujasite zeolites (FAU, ∼20nm of diameter) were used and modified by ion exchange with various cations (Fe, Gd, Cu, Ag). GB model was obtained by orthotopic glioblastoma cells implantation (U251) in nude rats (ONCOModels/Unicaen). 7T MRI (Bruker/Cyceron) was used to follow zeolites after intravenous injection and for oxygen measurement. Murine bone marrow derived MΦ were prepared and polarized to M1 and M2 using LPS/IFNg or IL4 as previously described (Leblond et al. 2016). Zeolites were added in MΦ medium and their impact on MΦ were evaluated by crystal violet dye assay, flow cytometry (Plateau ICORE/Unicaen) and polarization assays. ResultsOur results show that zeolites are able to accumulate and release the carried gases specifically in the brain tumor leading to tumor reoxygenation. Regarding the effect on MΦ, our preliminary results show, in vitro, the safety of as-prepared zeolites or Fe, Gd or Cu dopped zeolites on M0, M1 and M2 MΦ cultures. Similarly, no alteration of the cell cycle was observed. As a positive control of cell death, the presence of Ag-dopped zeolites dramatically decreased M0 and M1 MΦ viability. ConclusionsZeolites can deliver oxygen to the brain tumor and may improve the effectiveness of conventional treatments. Zeolites do not exhibit toxicity on primary cultures of MΦ. Additional studies are underway to evaluate the effect of zeolites on the polarization of MΦ, both in vitro and in vivo. Legal entity responsible for the studyThe authors. FundingRégion Normandie, CNRS, Université de Caen Normandie, Ministère de l’Enseignement Supérieur et de la Recherche, European Union-Fonds Européen de Développement Régional (FEDER), HABIONOR European project, co-funded by the Normandy County Council, the French State in the framework of the interregional development Contract “Vallée de la Seine” 2015-2020, ARCHADE, Fédération pour la Recherche sur le Cerveau (FRC) et INCa (INCA-11699). DisclosureAll authors have declared no conflicts of interest.

319-OR: Deficit of Retrograde Axonal Transportation Was Mediated via Activation of Macrophage RAGE Signaling in Experimental Diabetic Polyneuropathy

Activation of AGEs-RAGE signal at macrophage (Mφ) aggravates local inflammation in diabetic polyneuropathy (DPN). Mφ skewing toward pro-inflammatory phenotype (M1) is implicated in insulin resistance (IR) in fatty tissue, suggesting activation of RAGE at Mφ may similarly trigger IR in DPN. Generally, IR leads to axonal transport deficit in peripheral nerves. The aim of this study is to clarify the role of RAGE in the inflammation of DPN and its relationship to IR and axonal transport. Male C57BL/6 mice (W) and RAGE null mice (R) were recruited and rendered diabetic with streprozotocin (STZ) (D). DPN was confirmed by delay of nerve conduction velocities (NCVs) 8 weeks later of STZ injection. Polarization of Mφ in the sciatic nerve (SN) was determined by immunofluorescence (IF) and qPCR. IR of SN was evaluated by phospho (p)-AKT expression with stimulation of 5 unit insulin. Cell size of the dorsal root ganglia (DRG) neuron was morphometrically measured. For the retrograde axonal transport, DRG was quantitatively evaluated by IF for Fluoro-Gold (FG) 7 days after subcutaneous injection of FG into the hindlimb footpad. WD showed significant delay of NCVs (p<0.01, vs. W), while those were maintained in RD. Significant increase in the infiltration M1 in SN of WD (p<0.05, vs. W), whereas robust infiltration of anti-inflammatory Mφ (M2) was seen in RD (p<0.01, vs. R and WD). Increased nuclear translocation of NF-κB p65 and decreased pAKT expression were seen in SN of WD, but not in RD. The cell size in DRG neuron was significantly decreased in WD (p<0.05, vs. W), but that was similar in R and RD. The frequency of transported FG in DRG were significantly decreased in WD (p<0.05, vs. W), while maintained in RD. Taken together, our results suggest that RAGE induces inflammation in SN with skewing toward M1, which associates with IR and attenuation of the retrograde axonal transport in STZ DPN model. Those changes may contribute to the atrophic change of neuronal body in DRG of DPN. Disclosure S. Osonoi: None. H. Mizukami: None. K. Takahashi: None. K. Kudo: None. K. Sango: None. S. Yagihashi: None. Funding Japan Society for the Promotion of Science

The Role of Monocytes and Macrophages in Autoimmune Diseases: A Comprehensive Review.

Monocytes (Mo) and macrophages (Mϕ) are key components of the innate immune system and are involved in regulation of the initiation, development, and resolution of many inflammatory disorders. In addition, these cells also play important immunoregulatory and tissue-repairing roles to decrease immune reactions and promote tissue regeneration. Several lines of evidence have suggested a causal link between the presence or activation of these cells and the development of autoimmune diseases. In addition, Mo or Mϕ infiltration in diseased tissues is a hallmark of several autoimmune diseases. However, the detailed contributions of these cells, whether they actually initiate disease or perpetuate disease progression, and whether their phenotype and functional alteration are merely epiphenomena are still unclear in many autoimmune diseases. Additionally, little is known about their heterogeneous populations in different autoimmune diseases. Elucidating the relevance of Mo and Mϕ in autoimmune diseases and the associated mechanisms could lead to the identification of more effective therapeutic strategies in the future.

On the role of macrophages in the control of adipocyte energy metabolism.

The crosstalk between macrophages (MΦ) and adipocytes within white adipose tissue (WAT) influences obesity-associated insulin resistance and other associated metabolic disorders, such as atherosclerosis, hypertension and type 2 diabetes. MΦ infiltration is increased in WAT during obesity, which is linked to decreased mitochondrial content and activity. The mechanistic interplay between MΦ and mitochondrial function of adipocytes is under intense investigation, as MΦ and inflammatory pathways exhibit a pivotal role in the reprogramming of WAT metabolism in physiological responses during cold, fasting and exercise. Thus, the underlying immunometabolic pathways may offer therapeutic targets to correct obesity and metabolic disease. Here, I review the current knowledge on the quantity and the quality of human adipose tissue macrophages (ATMΦ) and their impact on the bioenergetics of human adipocytes. The effects of ATMΦ and their secreted factors on mitochondrial function of white adipocytes are discussed, including recent research on MΦ as part of an immune signaling cascade involved in the ‘browning’ of WAT, which is defined as the conversion from white, energy-storing adipocytes into brown, energy-dissipating adipocytes.

Iron nanoparticle-labeled mesenchymal stromal cells persist and demonstrate anti-inflammatory mechanism of action in an osteoarthritis mouse model

Background & Aim Osteoarthritis (OA) is characterized by cartilage degradation and chronic joint inflammation. Mesenchymal stromal cells (MSCs) have shown promising results in OA, but their mechanism of action is not fully understood. We hypothesize that MSCs polarize macrophages (MΦ) to more homeostatic sub-types. We tracked ferumoxytol (iron-nanoparticle)-loaded MSCs in mouse osteoarthritic joints, and investigated mechanisms of action of injected MSCs via immunohistochemical staining of homeostatic MΦ populations. Methods, Results & Conclusion 10-week-old C57/BL6 male mice were induced to undergo OA by destabilization of medical meniscus (DMM) or SHAM surgery. 3 weeks post-surgery, mice were injected intra-articularly with saline or ferumoxytol-labeled murine MSCs (50 × 103 MSCs/mouse) to yield the following groups (n=5/group): i) DMM+MSC with ferumoxytol; ii) DMM+MSC without ferumoxytol iii) DMM+Saline; iv) SHAM+MSC; and v) SHAM+Saline. MRIs of mice were taken at 1, 3 days and 1, 2, or 4 weeks after injection. At 4 weeks post-injection, mice were scored for i) OARSI to determine cartilage damage and synovitis and ii) immunohistochemical changes in CD206, F/480, Prussian Blue/Sca-1, and cleaved caspase-3 to detect homeostatic MΦ, total MΦ, ferumoxytol-labeled MSCs, and apoptotic cells respectively. Ferumoxytol-loaded MSCs persisted to time of euthanization (4 weeks) at greater levels in DMM- vs. SHAM-joints. Prussian Blue/Sca-1 co-staining confirmed the iron label resides in MSCs (at 1, 2, and 4 weeks), although some iron label was also detected in F4/80+ MΦ, likely due to phagocytosis of apoptotic MSCs. MRI analyses demonstrated a significant decrease in joint swelling at day 3 and 4 week groups in MSCs vs saline treated animals. However, OARSI synovitis scores did not capture any differences between MSCs vs saline treated animals. In depth examination by immunohistochemical staining showed for the first time that MSCs vs saline treated had increased proportions of CD206+ homeostatic MΦ (p We demonstrate that ferumoxytol-MSC can be imaged in OA mice by MRI for 4 weeks, and these mice have augmented MΦ infiltration (p

Momordica charantia (bitter melon) modulates adipose tissue inflammasome gene expression and adipose-gut inflammatory cross talk in high-fat diet (HFD)-fed mice

Systemic and tissue-specific inflammation has a profound influence on regulation of metabolism, and therefore, strategies to reduce inflammation are of special interest in prevention and treatment of obesity and type 2 diabetes (T2D). Antiobesity and antidiabetic properties of Momordica charantia (bitter melon, BM) have been linked to its protective effects on inflammation and gut microbial dysbiosis. We investigated the mechanisms by which freeze-dried BM juice reduces adipose inflammation in mice fed a 60% high-fat diet (HFD) for 16 weeks. Although earlier studies indicated that BM inhibited recruitment of macrophages (Mφ) infiltration in adipose tissue of rodents and reduced NF-kB and IL-1β secretions, the mechanisms remain unknown. We demonstrate that freeze-dried BM juice inhibits recruitment of Mφ into adipose tissue and its polarization to inflammatory phenotype possibly due to reduction of sphingokinase 1 (SPK1) mRNA in HFD-fed mice. Furthermore, reduction of IL-1β secretion by freeze-dried BM juice in the adipose tissue of HFD-fed mice is correlated to alleviation of NLRP3 inflammasome components and their downstream signaling targets. We confirm previous observations that BM inhibited inflammation of colon and gut microbial dysbiosis in HFD-fed mice, which in part may be associated with the observed anti-inflammatory effects in adipose tissue if HFD-fed mice. Overall, functional foods such as BM may offer potential dietary interventions that may impact sterile inflammatory diseases such as obesity and T2D.

The Clinical Significance of Tumor Associated Macrophages in Myelodysplastic Syndromes

Objective:The purpose of this study is to investigate the relationship between the infiltration of tumor associated macrophages (TAMs) and the disease risk stratification and survival of patients with myelodysplastic syndrome (MDS) and to explore the role of TAMs in the clinical prognosis.Methods:We retrospectively collected and analyzed 115 patients initially diagnosed wih myelodysplastic syndromes from January, 2010 to July, 2017 in West China Hospital of Sichuan University. Both bone marrow biopsy specimens and clinical data of the patients enrolled were collected. All patients were assessed prognosis by international prognostic scoring system (IPSS). We quantified the involvement of macrophage (MΦ), alternatively activated macrophage (M2 MΦ) and classic activated macrophage (M1 MΦ) in bone marrow specimen by staining with anti-CD68 monoclonal antibody, anti-CD163 monoclonal antibody, and anti-iNOS monoclonal antibody respectively. Log-rank test was used to evaluate the difference of overall survival (OS) among different subgroups. Logistic regression was used to evaluate the effect of clinical parameters on patients' OS. Cox proportional-hazards models were used to estimate the independent risk factors influencing the prognosis of patients.Results:1 In the high risk group of MDS, the composition of CD163+MΦ was higher than that of the low risk group(p < 0.001). In the low risk group of MDS, the composition of iNOS+ MΦ was higher than that of the group with high risk(p < 0.001).2 The median survival time and 3-year survival rate in the high CD163+MΦinfiltration group were significantly lower than those in the low infiltration group (13 months vs 43 months, 27% vs 64%, Log-rank test, P<0.001). The median survival time and 3-year survival rate in high iNOS+MΦ infiltration group were significantly higher than those in low infiltration group (42. 5 months vs 11 months, 65% vs 27% Log-rank test, P< 0.001).3 According to the infiltration degree of iNOS+MΦ and CD163+MΦ, the M1 (iNOShigh/CD 163low)and M2 TA(iNOSlow/CD163high) MΦ were further distinguished (The median OS were 53m and 15m respectively, p< 0.001).4 In multivariate analysis, high infiltration of CD163+MΦ and CD68+MΦ, low infiltration of iNOS+MΦ and low PLT level were independent risk factors for patients' OS .Conclusion and Discuss:The degree of bone marrow TAMs subtype infiltration in MDS patients was associated with disease risk group. CD163+M Φ was the main TAMs in the high risk group of MDS,while iNOS+M Φ was the main TAMs in the low risk group. There was a positive correlation between the infiltration of M1-TAMs and OS. On the contrary, there was a negative correlation between the infiltration of M2-TAMs and OS. Analyzing TAMs subsets in bone marrow of patients with MDS is helpful to evaluate disease risk and prognosis. DisclosuresNo relevant conflicts of interest to declare.

Lack of the pH-sensing Receptor TDAG8 [GPR65] in Macrophages Plays a Detrimental Role in Murine Models of Inflammatory Bowel Disease.

Tissue inflammation in inflammatory bowel diseases [IBD] is associated with local acidification. Genetic variants in the pH-sensing G protein-coupled receptor 65, also known as T cell death-associated gene 8 [TDAG8], have been implicated in IBD and other autoimmune diseases. Since the role of TDAG8 in intestinal inflammation remains unclear, we investigated the function of TDAG8 using murine colitis models. The effects of TDAG8 deficiency were assessed in dextran sodium sulphate [DSS], IL-10-/-, and T cell transfer colitis murine models. RNA sequencing of acidosis-activated TDAG8-/- and wild-type [WT] peritoneal macrophages [MΦs] was performed. mRNA expression of IFN-γ, TNF, IL-6, and iNOS in TDAG8-/- mice increased significantly in colonic lymphoid patches and in colonic tissue in acute and chronic DSS colitis, respectively. In transfer colitis, there was a trend towards increased IFN-γ, iNOS, and IL-6 expression in mice receiving TDAG8-/- T cells. However, absence of TDAG8 did not lead to changes in clinical scores in the models tested. Increased numbers of infiltrating MΦs and neutrophils, but not CD3+ T cells, were observed in DSS-treated TDAG8-/- mice. No differences in infiltrating CD3+ T cells were observed between mice receiving TDAG8-/- or WT naïve T cells in transfer colitis. RNA sequencing showed that acidosis activation of TDAG8 in MΦs modulated the expression of immune response genes. TDAG8 deficiency triggers colonic MΦ and neutrophil infiltration, and expression of pro-inflammatory mediators in DSS colitis models. In transfer colitis, mice receiving TDAG8-/- T cells presented a significantly higher spleen weight and a tendency towards increased expression of pro-inflammatory markers of monocyte/MΦ activity.

Suppression of Neuropathy Development in Diabetic Rage-Deficient Mice Is Associated with Absence of M1/M2 Macrophage Skewing in the Sciatic Nerve

Infiltrating macrophage (Mφ) into sciatic nerve (SN) is assumed one of the pathogenesis of diabetic polyneuropathy (DPN). In particular, skewing infiltrating Mφ toward pro-inflammatory (M1) Mφ, but not anti-inflammatory (M2) Mφ, possibly contributes to dysfunction of SN in diabetic state. However, the impacts of Mφ into dorsal root ganglia (DRG) in DPN are not yet well explored. For this purpose, male C57BL/6 mice (W) and receptor for AGEs (RAGE) null mice (R) were recruited and rendered diabetic (D) with STZ injection. After 8 weeks, nerve conduction velocity (NCV) and tail flick test were performed. Subsequently, mice were sacrificed to dissect SN and DRG, which were evaluated in the pathology and mRNA expression by qPCR. WD showed significant delay of NCV and increase in threshold for thermal sensation (p&amp;lt;0.01), while those were maintained in RD. Pathological analysis elucidated decrease in the average size of cell bodies in the DRG neuron of WD compared to W (p&amp;lt;0.01), as well as the expression of CGRP in DRG neurons (p&amp;lt;0.01). RD maintained neuronal cell size and CGRP expression. Significant increase in infiltration of Iba-I positive Mφ emerged in SN of WD compared to W (p&amp;lt;0.05). The increase was more robust, composed mostly of M2 in RD than WD (p&amp;lt;0.01). No increase of Mφ infiltration was identified in DRG of diabetic groups. The expression of iNOS (a marker for M1) was increased in SN of WD compared to W (p &amp;lt; 0.01), but not in that of RD. The expression of Arg1 (a marker for M2) was significantly increased in SN of RD compared to WD (p &amp;lt; 0.01). However, no changes of iNOS and Arg1 expression were detected in DRG. Our results suggest that proinflammatory changes in distal nerve elicits systematic sensory neuropathy in diabetes and correction of M1φ/M2φ imbalance may be beneficial for its prevention. Disclosure S. Osonoi: None. H. Mizukami: Research Support; Self; Sanwa Kagaku Kenkyusho Co., Ltd., Mitsubishi Tanabe Pharma Corporation. S. Ogasawara: None. K. Takahashi: None. K. Sango: None. S. Yagihashi: None.

Immunohistochemical characterization of gastrointestinal macrophages/phagocytes in dogs with inflammatory bowel disease (IBD) and non-IBD dogs

Intestinal Mϕ play a pivotal role in the maintenance of gut homeostasis, but can also contribute to inflammation such as inflammatory bowel disease (IBD). In contrast to human tissues, little is known about phenotypes of Mϕ in the canine gastrointestinal tract. Therefore, an immunohistochemical study was performed using Abs against Mϕ-associated molecules (Cluster of differentiation (CD)64, CD163, CD204, ionized calcium-binding adaptor molecule 1, L1 Ag, and MHC II) on stomach, duodenum, jejunum, ileum and colon from non-IBD dogs. In addition, marker-expression in the stomach, duodenum and colon of the non-IBD dogs was compared to that in dogs with IBD. Results revealed predominance of resident Mϕ displaying an anti-inflammatory phenotype represented by expression of CD163 as well as CD204 in the gut of non-IBD dogs with high Mϕ numbers especially present in the small intestinal villus area. Compared to non-IBD tissue counterparts, stomach, duodenum, and colon from dogs with IBD showed reduced Mϕ numbers with the exception of slightly increased numbers of CD64+ Mϕ. Correlation analyses between marker-expression of Mϕ and the Canine Inflammatory Bowel Disease Activity Index as well as histological scores failed to reveal relevant relationships. The present study provides evidence of the canine steady state gastrointestinal tract being dominated by Mϕ with anti-inflammatory properties maintaining gut homeostasis. A significant reduction in these resident Mϕ may reflect disturbances in homeostatic capacity that could contribute to the development of canine IBD. In contrast to human IBD and murine disease models, infiltration of pro-inflammatory Mϕ does not significantly contribute to the inflammatory process of canine IBD, which may illustrate possible species-specific differences in IBD pathogenesis.

Abstract 21026: Macrophages Preferentially Endocytose Systemically-Delivered Exosomes at Steady State or During Acute Inflammation

Introduction: Cardiosphere-derived cells (CDCs), which are in advanced clinical testing for heart failure, Duchenne muscular dystrophy and pulmonary hypertension, secrete exosomes (CDC exo ) which are highly anti-inflammatory. CDC exo , but not fibroblast (FB) exosomes, lead to cardioprotection through polarization of macrophages (Mφ) following ischemia/reperfusion injury (de Couto et al., Circulation, 2017). Here we demonstrate that intravenous delivery of CDC exo in mice (healthy or acutely inflamed) leads to preferential uptake by resident Mφ via phagocytosis. Methods &amp; Results: To assess biodistribution, we labeled CDC exo with the fluorescent liposomal dye DiD (Invitrogen). Healthy mice (C57Bl6; 8-12 weeks) were treated with an intraperitoneal injection of either LPS (5mg/kg) or saline prior to intravenous infusion of DiD-labeled CDC exo (in 100μL saline). Four hours later, organs were collected for ex vivo analysis. Biofluorescence imaging (Xenogen) revealed strong DiD signal in Mφ-rich organs including the liver, spleen, and lung, while immunohistochemistry and flow cytometry resolved preferential uptake of CDC exo by tissue Mφ within the liver (60-80% DiD + CD11b + ), spleen (70-80% DiD + F4/80 + ), lung (60-90% DiD + F4/80 + ), and peritoneal cavity (40-60% DiD + CD11b + ). The heart also exhibited measurable fluorescence signals. To determine how avidly distinct cardiovascular cell types preferentially take up CDC exo in vitro , Mφ, FB, endothelial cells (EC), and cardiomyocytes (CM) were exposed to DiD-labeled CDC exo and examined over time. Within one hour, Mφ demonstrated the greatest uptake of CDC exo (Mφ: 81%; FB: 60%; EC: 65%; CM: 51%; p&lt;0.05). The addition of both phagocytosis (Cytochalasin D) and clathrin (Pitstop 2) inhibitors completely blocked CDC exo uptake by Mφ. Conclusions: Intravenous delivery of CDC exo in mice (healthy or acutely inflamed) leads to preferential uptake by Mφ within organs that are rich in such cells (i.e., liver, spleen, lung, and peritoneal cavity). In vitro , Mφ take up CDC exo via phagocytosis and endocytosis more readily than other cardiac cell types. These data suggest the potential utility of CDC exo treatment in cardiovascular diseases where Mφ infiltration is prominent (e.g., atherosclerosis).

Netrin-1 Promotes Inflammation Resolution to Achieve Endothelialization of Small-Diameter Tissue Engineering Blood Vessels by Improving Endothelial Progenitor Cells Function In Situ.

The transplant of small‐diameter tissue engineering blood vessels (small‐diameter TEBVs) (<6 mm) in vascular replacement therapy often fails because of early onset thrombosis and long‐standing chronic inflammation. The specific inflammation state involved in small‐diameter TEBVs transplants remains unclear, and whether promoting inflammation resolution would be useful for small‐diameter TEBVs therapy need study. The neural protuberant orientation factor 1 (Netrin‐1) is found present in endothelial cells of natural blood vessels and has anti‐inflammatory effects. This work generates netrin‐1‐modified small‐diameter TEBVs by using layer‐by‐layer self‐assembly to resolve the inflammation. The results show that netrin‐1 reprograms macrophages (MΦ) to assume an anti‐inflammatory phenotype and promotes the infiltration and subsequent efflux of MΦ from inflamed sites over time, which improves the local microenvironment and the function of early homing endothelial progenitor cells (EPCs). Small‐diameter TEBVs modified by netrin‐1 achieve endothelialization after 30 d and retain patency at 14 months. These findings suggest that promoting the resolution of inflammation in time is necessary to induce endothelialization of small‐diameter TEBVs and prevent early thrombosis and problems associated with chronic inflammation. Furthermore, this work finds that the MΦ‐derived exosomes can target and regulate EPCs, which may serve as a useful treatment for other inflammatory diseases.

Abstract 624: Characterization of PD-L1 expression and the immune cell microenvironment in hepatocellular carcinoma (HCC) and non-cirrhotic liver tissue adjacent to HCC

Abstract Still-emerging evidence suggests that HCC is at least moderately responsive to therapeutic agents that target tumor immune suppression pathways, specifically the programmed death 1/programmed death ligand 1 (PD-L1) pathway. Prior studies raise the possibility that PD-L1 is a prognostic biomarker in HCC, where its expression levels have been reported to correlate with tumor aggressiveness and recurrence following surgical resection. The purpose of this study was to evaluate the expression of PD-L1 in non-cirrhotic liver tissue adjacent to HCC and HCC itself in 68 procured tissue samples, as well as elucidate the immune cell composition of the HCC tumor microenvironment. All cases showed the typical morphology of HCC and were classified as low- to high-grade trabecular, pseudoglandular, or solid with the common cytoplasmic features. Immunohistochemical (IHC) staining for PD-L1 in tumor-free liver (TFL) revealed PD-L1 staining in sinusoidal lining cells (macrophages [Mϕ] and endothelial cells [EC], as confirmed by double labeling for CD68 and CD31), although there was considerable heterogeneity in the extent of PD-L1 staining. More specifically, in TFL, some but not all CD68+ Mϕ were also PD-L1+, whereas most CD31+ EC were also PD-L1+. Furthermore, we evaluated PD-L1 staining qualitatively and semiquantitatively in HCC. Only few tumor cells displayed membranous PD-L1 staining, and 62/68 of the cases (91.2%) were categorized as PD-L1- vs 6/68 (8.8%) PD-L1+ (≥1% and &amp;lt;25% = PD-L1+); instead, PD-L1 expression within the tumor microenvironment predominantly emanated from immune cell infiltrates (as confirmed by PD-L1/pan-cytokeratin double labeling). Then we assessed the immune milieu in HCC tissue specimens using quantitative IHC. We found considerable interspecimen variation in the number of CD68+ Mϕ, CD8+ cytotoxic T lymphocytes, and FoxP3+ regulatory T lymphocytes. Comparing the number of immune cells in different tumor compartments showed that the prevalence of CD68+ cells (p=.0005), CD8+ cells (p=.0004), and FoxP3+ cells (p=.05) was significantly higher within the invasive margin vs the center of the tumor. Quantitative analysis of the immune cell content also showed that there was no correlation between T lymphocyte infiltration (CD8+/FoxP3+) and Mϕ infiltration in any compartment. Taken together, our findings confirm the intertumoral heterogeneity of the immune cell microenvironment in HCC; however, future studies are needed to correlate these findings to the clinical setting with immunooncologic treatments. Citation Format: Christian Ihling, Sienna Yoast, Yue Zhang, Bartholomew Naughton, Miriam Urban, P. Alexander Rolfe, Eveline Frick-Krieger, Isabelle Dussault. Characterization of PD-L1 expression and the immune cell microenvironment in hepatocellular carcinoma (HCC) and non-cirrhotic liver tissue adjacent to HCC [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 624. doi:10.1158/1538-7445.AM2017-624

Innate Immune Memory Contributes to Host Defense against Recurrent Skin and Skin Structure Infections Caused by Methicillin-Resistant Staphylococcus aureus.

Staphylococcus aureus is the leading cause of skin and skin structure infections (SSSI). The high frequency of recurring SSSI due to S. aureus, including methicillin-resistant S. aureus (MRSA) strains, despite high titers of specific antibodies and circulating T cells, implies that traditional adaptive immunity imparts incomplete protection. We hypothesized that innate immune memory contributes to the protective host defense against recurring MRSA infection. To test this hypothesis, SSSI was induced in wild-type and rag1-/- mice in the BALB/c and C57BL/6 backgrounds. Prior infection (priming) of wild-type and rag1-/- mice of either background afforded protection against repeat infection, as evidenced by reduced abscess severities and decreased CFU densities compared to those in naive controls. Interestingly, protection was greater on the previously infected flank than on the naive flank for wild-type and rag1-/- mice. For wild-type mice, protective efficacy corresponded to increased infiltration of neutrophils (polymorphonuclear leukocytes [PMN]), macrophages (MΦ), Langerin+ dendritic cells (LDC), and natural killer (NK) cells. Protection was associated with the induction of interleukin-17A (IL-17A), IL-22, and gamma interferon (IFN-γ) as well as the antimicrobial peptides CRAMP and mβD-3. Priming also protected rag1-/- mice against recurring SSSI, with increased MΦ and LDC infiltration and induction of IL-22, CRAMP, and mβD-3. These findings suggest that innate immune memory, mediated by specific cellular and molecular programs, likely contributes to the localized host defense in recurrent MRSA SSSI. These insights support the development of targeted immunotherapeutic strategies to address the challenge of MRSA infection.

Potentiation of Anti-Myeloma Activity of Daratumumab with Combination of Cyclophosphamide, Lenalidomide or Bortezomib Via a Tumor Secretory Response That Greatly Augments Macrophage-Induced ADCP

IntroductionDaratumumab (Dara) is the first CD38-directed monoclonal antibody (moAb) approved anywhere in the world as well as the first moAb approved for the treatment of Multiple Myeloma (MM). While currently approved as monotherapy, recent results from randomized trials have shown dramatic activity in relapsed MM patients in combination with both lenalidomide (Len) and bortezomib (Bor) and Dara is likely to become an intrinsic component of MM regimens in the future. Based on pre-clinical data Dara is thought to have pleiotropic mechanisms of activity, killing myeloma cells via a direct anti-CD38 effect, induction of complement dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Therefore, we focused our attention on potential mechanisms of enhancing macrophage (Mϕ)-mediated ADCP. Previously, Pallasch reported that resistance to Mϕ-mediated killing in lymphoid malignancy can be overcome by combination regimens involving therapeutic antibodies and chemotherapy. Specifically, Cyclophosphamide (Cy) was shown to induce an acute secretory activating phenotype from treated tumor cells, which led to Mϕ infiltration and phagocytic activity in the bone marrow. Given this background we sought to establish the existence of this phenomenon in MM in the context of drugs likely to be used in combination with Dara, i.e. Cy, Len and Bor, respectively.Materials and Methods:MM1S MM cells were conditioned with low dose single agents, Cy, Len and Bor for 24 hours. Media containing single agent chemotherapy was removed and replaced with fresh media for 24 hours (TCM). THP-1 Mϕ were conditioned with TCM for up to 48 hours and then incubated at 2:1 effector target ratio with CFSE labelled MM1S cells for 18 hours in the presence or absence of Dara or isotype control antibody. The % of Dara-specific tumour cell clearance was calculated as the number of live CFSE labelled tumour cells in Dara-treated cells/Isotype-treated cells. Additionally, Mϕ were pre-incubated with Cytochalasin D (CytoD), an inhibitor of actin polymerization, to determine if the mechanism of tumour cell clearance is mediated by Mϕ mediated ADCP. MM1S expression of CD47, as well as Mϕ expression of FC receptor expression, CD32 and CD64 was analysed by flow cytometry. Mϕ migration was assessed using transwell assays assessment of fluorescently labelled Mϕ migration to Cy, Len and /or Bor TCM.Results:Exposure of Mϕ to TCM from Cy, Len and Bor treated MM1S cells significantly enhanced % Dara specific clearance of MM1S cells (p<0.01). The effect was also maintained when combinations of the Cy, Len and Bor were used. This effect was partially reversed by incubation of Mϕ co-cultures with CytoD (p<0.01), indicating ADCP as one of the likely mechanisms of tumour cell clearance. We observed reduced expression of the "don’t eat me" antigen, CD47, on chemotherapy treated MMIS cells after 24 hours, which could enhance phagocytosis. In addition, we detected induction of CD32 and CD64 Fc gamma receptor expression on conditioned Mϕ as well as altered migratory capacity, which was more evident in Cy and Bor TCM conditioned Mϕ.Conclusions:Exposure of MM cells to both low doses of Cy, Len and Bor leads to a secretory response, which, along with downregulation of CD47, greatly augments Mϕ induced ADCP of Dara coated MM cells. This effect may explain in part the dramatic synergy observed between Len, Bor and Dara in clinical trials and provides a rationale for combining Cy with Dara in the clinic. Future research, including use of patient samples, will focus on understanding the mechanisms responsible for this anti-tumour effect.Reference:Pallasch CP et al. Sensitizing protective tumor microenvironments to antibody-mediated therapy. Cell. 2014 Jan 30;156(3):590-602. DisclosuresChiu:Janssen: Employment. Sasser:Janssen Pharma: Employment. O'Dwyer:Glycomimetics: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding.

Abstract 015: Immunosuppression Attenuates Intrarenal Angiotensinogen Augmentation in Angiotensin II Dependent Hypertension

Augmented intrarenal angiotensinogen (AGT) is a critical contributor to activation of intrarenal renin-angiotensin system (RAS) leading to the development of hypertension and associated kidney injury. It has been shown that treatment with mycophenolate mofetil (MMF), an immunosuppressive drug, mitigates the increased intrarenal angiotensin (Ang) II levels and blood pressure in hypertensive animal models, suggesting that an activated immune system mediates intrarenal RAS activation and consequent hypertension. Associated macrophage (MΦ) infiltration augments pro-inflammatory cytokine levels including interleukin-6 (IL-6), which plays a crucial role in augmentation of AGT expression in cultured renal proximal tubular cells. Accordingly, this study was performed to establish pathophysiological relevance for the effects of stimulated MΦ and IL-6 on intrarenal AGT augmentation in Ang II-dependent hypertension. Ang II (80 ng/min) was infused with/without daily MMF administration (50 ng/kg) to Sprague-Dawley rats for 2 weeks. Mean arterial pressure (MAP) in Ang II infused rats was slightly higher (169.7±6.1 mmHg) than MAP in Ang II+MMF group (154.7±2.0 mmHg) which was not statistically different than in control group. The augmentation of urinary AGT and urinary protein by Ang II infusion was attenuated by MMF treatment (AGT, control: 89.3±25.2, Ang II: 1,194±305.1, and Ang II+MMF: 389±192.0 ng/day). Importantly, the augmentation of urinary AGT by Ang II infusion was observed before the onset of proteinuria. Urinary 8-isoprostane levels were not altered by Ang II and/or MMF during the 2-week treatments. MMF treatment suppressed Ang II-induced renal MΦ infiltration and IL-6 elevation (IL-6 mRNA, Ang II: 32.4±7.5 and Ang II+MMF: 3.6±1.7, ratio to control). qRT-PCR, western blot and immunohistochemistry revealed elevated intrarenal AGT mRNA and protein levels in Ang II infused rats which were normalized by the MMF treatment (AGT mRNA, Ang II: 2.5±0.2 and Ang II+MMF: 1.5±0.1, ratio to control). These results indicate that stimulated IL-6 production in infiltrated MΦ contributes to intrarenal AGT augmentation in early stages of Ang II-dependent hypertension, which contributes to the development of kidney injury.

PS 01-26] THE ROLE OF ACTIVATED RENIN-ANGIOTENSIN SYSTEM FOR THE MRP8/TLR4 SIGNALING IN DIABETIC NEPHROPATHY

Objective: We previously reported that myeloid-related protein 8 (MRP8: S100A8) / toll-like receptor 4 (TLR4) signaling activated by macrophage (Mϕ)-mediated glucolipotoxicity might play an important role in the progression of diabetic nephropathy. Although local activation of the renin-angiotensin system (RAS) in the kidney has been observed in concurrence with the MRP8/TLR4 activation in the diabetic-hyperlipidemic model mice, the relationship between these signals is unknown. Design and Method: In vivo, diabetic-hyperlipidemic mice by STZ plus high-fat diet (STZ-HFD mice) or db/db mice were treated with an angiotensin-receptor blocker olmesartan (Olm, 0.5 microg/kg/min, 4wk) or angiotensin II (AngII, 0.3 microg/kg/min, 2wk), respectively. In vitro, Mϕ was stimulated with mesangial cell-conditioned medium (Mes-sup) with or without a TLR4 antagonist. Expression of MRP8 mRNA and inflammatory pathway in detail were evaluated with TaqMan real-time PCR and THP1-dual reporter cell line, respectively. Results: Angiotensinogen (Agt) mRNA was locally upregulated in the glomeruli of STZ-HFD mice, whose changes also occurred in TLR4 knockout mice. Olm effectively suppressed upregulation of glomerular MRP8/TLR4 and Agt in STZ-HFD mice, which were associated with reduction of albuminuria, Mϕ infiltration and proinflammatory/profibrotic gene expressions in the kidney. Besides, a subpressor dose of AngII markedly worsened albuminuria and increased glomerular infiltrated MRP8-positive cells in db/db mice. Since glomerular infiltrated Mϕ showed obviously high MRP8 positivity compared to tubulointerstitial Mϕ, we speculated the intraglomerular crosstalk between Mϕ and glomerular intrinsic cells. Mes-sup stimulation, rather than proximal tubular cell-conditioned medium, potently induced MRP8 expression and also interferon regulatory factor (IRF)-pathway, which could cause the ER stress, in Mϕ. This induction was partially abrogated by the TLR4 antagonist. Conclusions: RAS activation in diabetic kidney could contribute to progression of nephropathy through promoting intraglomerular crosstalk, which may trigger MRP8 production in glomerular infiltrated Mϕ.

Short-term high-fat feeding induces islet macrophage infiltration and β-cell replication independently of insulin resistance in mice.

Short-term high-fat consumption stimulates mouse islet β-cell replication through unknown mechanisms. Resident macrophages (MΦs) are capable of secreting various factors involved in islet development and tissue remodeling. We hypothesized that a short-term high-fat diet (HFD) promotes MΦ infiltration in pancreatic islets and that MΦs serve as a regulator of β-cell replication. To test these hypotheses and dissect mechanisms involved in HFD-induced β-cell replication, adult C57BL/6J mice were fed a HFD for 7 days with or without administration of clodronate-containing liposomes, an MΦ-depleting agent. Mouse body and epididymal fat pad weights, and nonfasting blood glucose and fasting serum insulin levels were measured, and pancreatic islet β-cell replication, oxidative stress, and MΦ infiltration were examined. Short-term HFD promoted an increase in body and epididymal fat pad weight and blood glucose levels, along with an increased fasting serum insulin concentration. β-Cell replication, islet MΦ infiltration, and the percentage of inducible NO synthase positive MΦs in the islets increased significantly in mice fed the HFD. Immunofluorescence staining for 8-oxo-2'-deoxyguanosine or activated caspase-3 revealed no significant induction of DNA damage or apoptosis, respectively. In addition, no change in stromal-derived factor 1-expressing cells was found induced by HFD. Despite continuous elevation of nonfasting blood glucose and fasting serum insulin levels, depletion of MΦs through treatments of clodronate abrogated HFD-induced β-cell replication. These findings demonstrated that HFD-induced MΦ infiltration is responsible for β-cell replication. This study suggests the existence of MΦ-mediated mechanisms in β-cell replication that are independent of insulin resistance.

Cross-talk between intestinal epithelial cells and immune cells in inflammatory bowel disease.

Inflammatory bowel disease (IBD) involves functional impairment of intestinal epithelial cells (IECs), concomitant with the infiltration of the lamina propria by inflammatory cells. We explored the reciprocal paracrine and direct interaction between human IECs and macrophages (MΦ) in a co-culture system that mimics some aspects of IBD. We investigated the expression of intercellular junctional proteins in cultured IECs under inflammatory conditions and in tissues from IBD patients. IECs establish functional gap junctions with IECs and MΦ, respectively. Connexin (Cx26) and Cx43 expression in cultured IECs is augmented under inflammatory conditions; while, Cx43-associated junctional complexes partners, E-cadherin, ZO-1, and β-catenin expression is decreased. The expression of Cx26 and Cx43 in IBD tissues is redistributed to the basal membrane of IEC, which is associated with decrease in junctional complex proteins’ expression, collagen type IV expression and infiltration of MΦ. These data support the notion that the combination of paracrine and hetero-cellular communication between IECs and MΦs may regulate epithelial cell function through the establishment of junctional complexes between inflammatory cells and IECs, which ultimately contribute to the dys-regulation of intestinal epithelial barrier.