M2 Monocytes Research Articles

CX3CR1 Is Protective in the Development of Myocardial Fibrosis-Dependent Heart Failure

There are no established therapies for myocardial fibrosis, which is known to be associated with the development of heart failure. Recent evidence would suggest that alternatively activated (M2) monocytes may be involved in promoting myocardial fibrosis by producing pro-fibrotic factors such as TGF-β. In the present study, we sought to block the migration of M2 monocytes using KO animals to the fractalkine receptor, CX3CR1, which M2 monocytes are thought to use for chemotaxis.

Glioma Grade Is Associated with the Accumulation and Activity of Cells Bearing M2 Monocyte Markers

This study is directed at identifying the cell source(s) of immunomodulatory cytokines in high-grade gliomas and establishing whether the analysis of associated markers has implications for tumor grading. Glioma specimens classified as WHO grade II-IV by histopathology were assessed by gene expression analysis and immunohistochemistry to identify the cells producing interleukin (IL)-10, which was confirmed by flow cytometry and factor secretion in culture. Finally, principal component analysis (PCA) and mixture discriminant analysis (MDA) were used to investigate associations between expressed genes and glioma grade. The principle source of glioma-associated IL-10 is a cell type that bears phenotype markers consistent with M2 monocytes but does not express all M2-associated genes. Measures of expression of the M2 cell markers CD14, CD68, CD163, and CD204, which are elevated in high-grade gliomas, and the neutrophil/myeloid-derived suppressor cell (MDSC) subset marker CD15, which is reduced, provide the best index of glioma grade. Grade II and IV astrocytomas can be clearly differentiated on the basis of the expression of certain M2 markers in tumor tissues, whereas grade III astrocytomas exhibit a range of expression between the lower and higher grade specimens. The content of CD163(+) cells distinguishes grade III astrocytoma subsets with different prognosis.

Pro‐inflammatory capacity of classically activated monocytes relates positively to muscle mass and strength

In mice, monocytes that exhibit a pro-inflammatory profile enter muscle tissue after muscle injury and are crucial for clearance of necrotic tissue and stimulation of muscle progenitor cell proliferation and differentiation. The aim of this study was to test if pro-inflammatory capacity of classically activated (M1) monocytes relates to muscle mass and strength in humans. This study included 191 male and 195 female subjects (mean age 64.2 years (SD 6.4) and 61.9 ± 6.4, respectively) of the Leiden Longevity Study. Pro-inflammatory capacity of M1 monocytes was assessed by ex vivo stimulation of whole blood with Toll-like receptor (TLR) 4 agonist lipopolysaccharide (LPS) and TLR-2/1 agonist tripalmitoyl-S-glycerylcysteine (Pam₃Cys-SK₄), both M1 phenotype activators. Cytokines that stimulate M1 monocyte response (IFN-γ and GM-CSF) as well as cytokines that are secreted by M1 monocytes (IL-6, TNF-α, IL-12, and IL-1β) were measured. Analyses were adjusted for age, height, and body fat mass. Upon stimulation with LPS, the cytokine production capacity of INF-γ, GM-CSF, and TNF-α was significantly positively associated with lean body mass, appendicular lean mass and handgrip strength in men, but not in women. Upon stimulation with Pam₃Cys-SK₄, IL-6; TNF-α; and Il-1β were significantly positively associated with lean body mass and appendicular lean in women, but not in men. Taken together, this study shows that higher pro-inflammatory capacity of M1 monocytes upon stimulation is associated with muscle characteristics and sex dependent.

M1 monocyte induction from advanced HCC-derived monocytes by CCL1 antisense oligodeoxynucleotides (P2129)

Abstract We have reported that patients with advanced stages of HCC were shown to be carriers of M2b monocytes and these monocytes did not have a tumorcidal activity. In this study, we tried to induce M1 monocytes from M2b monocytes in advanced HCC patients. Five advanced HCC patients admitted to Osaka Medical Collage were enrolled. CD14+monocytes were isolated from peripheral blood from patients by Ficoll-Hyperque sedimentation and magnetic beads. These monocytes (1 x 106 cells/ml) were cultured for 48 hrs with 6 μg/ml of CCL1 antisense oligodeoxynucleotides (ODN). Cells harvested were re-cultured with fresh medium supplemented with CpG DNA (10 μg/ml) for 24 hrs and Culture fluids were assayed for IL-12. These cells were co-cultured with syngeneic HCC cells (2 x 10 6 cells/ml) for 48 hrs. In the results, M2b monocytes treated with CCL1 antisense ODN were produced IL-12 after stimulation with CpG DNA. And these cells have a tumorcidal activity against syngeneic HCC cells. These results indicate that M2b monocytes treated with CCL1 antisense ODN were converted to M1 monocytes after stimulation with CpG DNA. And M2b monocytes detected in advanced HCC patients play a role of the suppressing host antitumor surveillance. M2b monocytes/Mϕ may be intervened by the elimination of CCL1, a product of M2b monocytes themselves.

Susceptibility of NOD/scid IL-2Rγnull mice inoculated with alcoholic patient white blood cells to Pseudomonas aeruginosa infection (P3029)

Abstract We have previously demonstrated that CCL2+ neutrophils (PMN-II) and IL-12−IL-10+ monocytes (M2 monocytes) are predominated in WBC of alcoholic patients. Since these cells are inhibitory on the generation of major antibacterial effector cells, in this study, we investigated the susceptibility of alcoholic patients to P. aeruginosa infections in a humanized murine model. To create an experimental model of alcoholic patients, 5 x 106 cells of WBC of alcoholic patients were adoptively transferred to X-irradiated-NOD/scid IL-2Rγnull mice. As a control, these mice were inoculated with healthy donor WBC. Then, they were infected s.c. with P. aeruginosa (10 CFU/chimera) and bacteria in liver and kidneys of these chimeras were quantified. In the results, bacteria grew (> 103 CFU/g organ, 24 hrs after infection) in liver and kidneys of the chimeras created with alcoholic patient WBC, while bacteria disappeared within 24 hrs after P. aeruginosa infection from these organs of the control chimeras. CD14+ cells isolated from WBC of alcoholic patients were shown to be IL-10+IL-12−CCL1+ cells, while IL-10−IL-12+CCL1- cells were isolated from healthy donor WBC stimulated with heat-killed P. aeruginosa. These results indicate that chimeras created with alcoholic patient WBC are carriers of M2b monocytes and susceptible to P. aeruginosa infection, while chimeras created with healthy donor WBC are carriers of M1MΦ and resistant against these infections.

91 Sustained Therapeutic Benefit of Vedolizumab Throughout 1 Year in Crohn's Disease in Gemini II, a Randomized, Placebo-Controlled, Double-Blind, Multicenter Study

Introduction: Laquinimod (LAQ) is a novel oral immunomodulator in development for the treatment of Crohn's disease (CD). LAQ has an immune modulatory effect on antigen presenting cells (mainly M2 monocytes), directing T cells towards an antiinflammatory phenotype& resulting in down regulation of proinflammatory cytokines. This study evaluated the safety & efficacy of LAQ in patients with active moderate-severe CD and also assessed the impact of the drug on fecal calprotectin as an objective biomarker of intestinal inflammation. Methods: Phase IIa, multicenter, sequential-cohorts RCT with LAQ doses of 0.5, 1, 1.5, or 2 mg/day or placebo (45 patients per cohort randomized 2:1) for 8 weeks with 4-weeks follow-up. CD patients with a CD Activity Index (CDAI) of 220-450 & serum CRP .5mg/ L or mucosal ulcerations evident on endoscopy were included. Stable concomitant therapies (corticosteroids, immunosuppressive drugs [AZT, 6MP, MTX], 5-ASA, antibiotics) & prior anti-TNF use were allowed. Elevated fecal calprotectin was not required. Comprehensive safety monitoring and assessments were performed. Efficacy analyses included the proportions of patients in remission (CDAI ,150 & no treatment failure [TF]), those with a response 100 (i.e. 100 point CDAI reduction & no TF) and those who had a reduction in fecal calprotectin defined as a decrease from ≥250 μg/g to below 250 μg/g & ≥50% reduction. This study was exploratory & no hypothesis testing was planned. Results: Overall 117 patients received LAQ & 63 pts received placebo. AEs occurred in 86-97% of LAQ groups vs. 83% in pooled placebo group; most common AEs were headache, abdominal pain, nausea & vomiting, and musculoskeletal pain. For pooled LAQ and placebo groups, mean (SD) baseline CDAI were 298(58) & 311(75), and median (IQR) fecal calprotectin levels were 389(749) & 475(802) μg/g, respectively. At week 8, effects on remission and response were observed with the 0.5 mg LAQ dose; 1mg dose showed lower magnitude effects and higher LAQ doses showed similar effects to placebo (Table). All doses reduced fecal calprotectin. Conclusions: This study indicates that treatment with LAQ is well tolerated and the 0.5 and 1 mg doses may have clinically relevant effects on remission & response. All doses (LAQ 0.5-2 mg) were found to reduce objective measures of intestinal inflammation in active CD.

An unbalanced monocyte polarisation in peripheral blood and bone marrow of patients with type 2 diabetes has an impact on microangiopathy

Monocytes/macrophages play important roles in adipose and vascular tissues and can be polarised as inflammatory M1 or anti-inflammatory M2. We sought to analyse monocyte polarisation status in type 2 diabetes, which is characterised by chronic inflammation. We enrolled 60 individuals without diabetes and 53 patients with type 2 diabetes. We quantified standard monocyte subsets defined by cluster of differentiation (CD)14 and CD16. In addition, based on the phenotype of polarised macrophages in vitro, we characterised and quantified more definite M1 (CD68(+)CCR2(+)) and M2 (CX3CR1(+)CD206(+)/CD163(+)) monocytes. We also analysed bone marrow (BM) samples and the effects of granulocyte-colony stimulating factor (G-CSF) stimulation in diabetic and control individuals. We found no alterations in standard monocyte subsets (classical, intermediate and non-classical) when comparing groups. For validation of M1 and M2 phenotypes, we observed that M2 were enriched in non-classical monocytes and had lower TNF-α content, higher LDL scavenging and lower transendothelial migratory capacity than M1. Diabetic patients displayed an imbalanced M1/M2 ratio compared with the control group, attributable to a reduction in M2. The M1/M2 ratio was directly correlated with waist circumference and HbA1c and, among diabetic patients, M2 reduction and M1/M2 increase were associated with microangiopathy. A decrease in M2 was also found in the BM from diabetic patients, with a relative M2 excess compared with the bloodstream. BM stimulation with G-CSF mobilised M2 macrophages in diabetic but not in healthy individuals. We show that type 2 diabetes markedly reduces anti-inflammatory M2 monocytes through a dysregulation in bone-marrow function. This defect may have a negative impact on microangiopathy.

Abstract A20: Investigating gene expression alterations in the tumor microenvironment based on receptor status in breast cancer using a microfluidic multiculture platform

Abstract Numerous cellular influences regulate the behavior of cancer cells, contributing to an orchestration of responses to stimuli within the tumor microenvironment. Development of novel therapies and drug targets specific to tumor receptor status in breast cancer necessitate the understanding of tumor microenvironment interactions. The current four tumor subgroups based on receptor status differ in tumor grade, recurrence and metastasis making these tumor types unique to one another in their microenvironments. Of particular interest, the HER2+ subgroup, is associated with higher recurrence, metastasis and poor prognosis. Microfluidic multi-culture techniques provide the opportunity to manipulate multiple cell types in a highly-controlled fashion, permitting evaluation of the tumor microenvironment that is not possible in traditional culture techniques. Utilizing a microfluidic multi-culture platform, where we have the ability to culture more than two cell types in compartmentalized culture chambers separated by diffusion ports to allow for soluble factor exchange, the HER2+ breast carcinoma cell lines SKBR3 (HER2+/ER-/PR-), and BT474 (HER2+/ER+/PR+) were cultured in co- and tri-culture combinations with bone marrow stroma, human mammary fibroblast, and the human M2 monocyte lines to examine signaling interactions between cancer, fibroblast and immune cell types. Gene expression changes of tumor progression markers were then evaluated using RT-qPCR for a constellation of growth factors, receptors and cytokines. Importantly, this new device allows isolation and gene expression analysis of individual cell types subsequent to culture without the need for downstream cell separation techniques. Significant changes in gene expression of the two breast cancer cell lines were detected in multi-culture that was dependent on the tumor microenvironment. Differences in gene expression between the cancer cells based on receptor status in multi-culture were also shown. These findings suggest that both the tumor microenvironment and receptor status may be interrelated and together influence tumor progression. The use of a micro multi-culture platform allows one to easily recapitulate individual complex tumor microenvironments. Even in as simple a model as two or three cell types, shapes tumor responses, which can now be rapidly dissected using this model system. Micro multi-culture techniques have the potential to provide insight on essential tumor microenvironment interactions that would otherwise be difficult to detect in traditional culture methods, paving the way for the design of new and personalized therapies. Citation Format: Lindsey J. Maccoux, Emma M. Weinberger, Scott M. Berry, Elaine T. Alarid, David J. Beebe. Investigating gene expression alterations in the tumor microenvironment based on receptor status in breast cancer using a microfluidic multiculture platform. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A20.

Inflammatory arthritis increases mouse osteoclast precursors with myeloid suppressor function

Increased osteoclastic bone resorption leads to periarticular erosions and systemic osteoporosis in RA patients. Although a great deal is known about how osteoclasts differentiate from precursors and resorb bone, the identity of an osteoclast precursor (OCP) population in vivo and its regulatory role in RA remains elusive. Here, we report the identification of a CD11b(-/lo)Ly6C(hi) BM population with OCP activity in vitro and in vivo. These cells, which can be distinguished from previously characterized precursors in the myeloid lineage, display features of both M1 and M2 monocytes and expand in inflammatory arthritis models. Surprisingly, in one mouse model of RA (adoptive transfer of SKG arthritis), cotransfer of OCP with SKG CD4+ T cells diminished inflammatory arthritis. Similar to monocytic myeloid-derived suppressor cells (M-MDSCs), OCPs suppressed CD4+ and CD8+ T cell proliferation in vitro through the production of NO. This study identifies a BM myeloid precursor population with osteoclastic and T cell-suppressive activity that is expanded in inflammatory arthritis. Therapeutic strategies that prevent the development of OCPs into mature bone-resorbing cells could simultaneously prevent bone resorption and generate an antiinflammatory milieu in the RA joint.

PP064. M1 Monocyte subpopulation is associated with pro-inflammatory cytokineproduction in pregnant women with preeclampsia

Monocytes from peripheral blood of pregnant women with preeclampsia are endogenously activated and secrete high levels of free radicals and inflammatory cytokines. This study aimed at evaluating whether the inflammatory state of monocytes observed in preeclampsia is associated with the polarization of monocyte to M1 profile in peripheral blood, correlating the expression of surface receptors CD64, TLR2, TLR4, and CD163 and CD206 with cytokine production. We studied 90 pregnant women, 30 normotensive and 60 with preeclampsia, matched for gestational age. Peripheral blood monocytes obtained from normotensive pregnant or preeclamptic pregnant women were cultured for 18h, and the expression of surface receptors on M1 inflammatory monocyte subpopulation (TLR2, TLR4 and CD64) and M2 suppressor monocyte subpopulation (CD163 and CD206) were evaluated by flow cytometry, using specific monoclonal antibodies, labeled with fluorochromes. The values were expressed as ??the mean fluorescence intensity. Moreover, the production of proinflammatory cytokines associated with M1 profile (TNF-α, IL-12p70 and IL-23) and the anti-inflammatory cytokine associated with M2 profile (IL-10) were evaluated in the monocyte supernatant of culture by enzyme immunoassay. Results were analyzed using nonparametric tests with significance level set at 5%. The expression of CD4 and TLR4 on monocyte surface, from women with preeclampsia was significantly higher, while the expression of CD163 and CD206 was significantly decreased compared with normotensive pregnant women, suggesting the predominance of monocyte M1 profile. Endogenous production of TNF-α, IL-12p70 and IL-23 by monocytes was increased, while synthesis of IL-10 was lower in women with preeclampsia compared with normotensive pregnant women. Positive correlations between TLR4 and CD64 (r=0.5849), TLR4 and TNF-α (r=0.5126) or TLR4 and IL-23 (r=0.8095), as well as between CD64 and TNF-α (r = 0.7133) or CD64 and IL-23 (r = 0.6051) were observed in the preeclamptic group. The results confirm the activated state of monocytes from women with preeclampsia by increased production of proinflammatory cytokines and the expression of receptors characteristic of the M1 subpopulation. This study provides evidence that monocytes from women with preeclampsia are classically activated and the systemic inflammatory environment may differentiate and polarize these cells to the M1 profile. CNPq, FAPESP 2009/11924-3 and 2010/20207-0.

M2b monocytes predominantly appear in patients with advanced hepatocellular carcinoma (HCC patients) (162.29)

Abstract In response to tumor environments, resident Mϕ differentiate into M1Mϕ or M2Mϕ. Recently, three different subsets of M2Mϕ (M2aMϕ, M2bMϕ and M2cMϕ) have been reported. Therefore, in this study, the properties and subsets of monocytes isolated from early and advanced HCC patients were characterized. CD14(+) monocytes were isolated from peripheral blood of HCC patients by Ficoll-Hypque sedimentation and magnetic beads. HCC patients, who were confirmed as carriers of hepatic cancer cells, were enrolled in this study. Monocytes (1 x 10(6) cells/ml) were cultured for 48 hrs and culture fluids harvested were assayed for cytokines by ELISA. Tumoricidal activity of monocytes against hepatic cancer cells was assessed using a standard chromium release assay. In the results, monocytes from advanced HCC patients were shown to be M2b monocytes, because they produced IL-10 (188 ± 21 pg/ml) and CCL1 (24 ± 0.6 ng/ml), and did not produce IL-12, CCL17 and CXCL13. Monocytes from early HCC patients were characterized as M1 monocytes, and healthy donor monocytes were determined as resident monocytes. Monocytes from early HCC patients killed syngeneic hepatic cancer cells (73 ± 7%), while monocytes from advanced HCC patients did not (10 ± 3%). These results indicate that advanced HCC patients are carriers of M2b monocytes and early HCC patients are M1 monocyte carriers. M2b monocytes detected in advanced HCC patients may play a role on the decreased host’s antitumor resistances.

A predominance of PMN-II and M2b monocytes in alcoholics (117.9)

Abstract Alcoholics are well-known to be immunocompromised hosts, and are susceptible to various infections. To determine the properties of effector cells in host antibacterial innate immunities, in the present study the functions and subsets of neutrophils and monocytes in peripheral blood of alcoholics were characterized. It has been reported that IL-12(+) neutrophils (PMN-I) and IL-12(+)IL-10(-) monocytes (M1 monocytes) are major effector cells in antibacterial innate immunities, while CCL2(+) neutrophils (PMN-II) and IL-12(-)IL-10(+) monocytes (M2 monocytes) are inhibitory on the generation of antibacterial effector cells. Neutrophils and CD14(+) monocytes, isolated from 10 alcoholics by Ficoll-Hypaque/dextran sedimentations and magnetic beads, were adjusted to 1 x 106 cells/ml and stimulated with or without 0.0075% SAC for 18 hrs (neutrophils) or 48 hrs (monocytes). Culture fluids harvested were assayed for cytokines by ELISA. In the presence of SAC, CCL2 was produced by neutrophils from alcoholics, while IL-12 was not detected in culture fluids of these neutrophils. Healthy donor neutrophils stimulated with SAC produced IL-12 (but not IL-10). Also, IL-10 and CCL1 were produced by monocytes from alcoholics, while healthy donor monocytes produced IL-12 under the stimulation. CCL17 and CXCL13 were not produced by monocytes derived from alcoholics. These results indicate that alcoholics are carriers of PMN-II and M2b monocytes.

The generation of M2 monocytes in cultures of human peripheral blood mononuclear cells exposed to 137Cs γ-rays (117.2)

Abstract Thermally injured patients who are carriers of M2MΦ are greatly susceptible to various opportunistic infections. Since the similarly increased susceptibility to the infection is seen in patients with irradiated injuries, in this study we investigated the effect of 137Cs γ-irradiation on the M2MΦ generation in cultures of human peripheral blood mononuclear cells. Peripheral blood mononuclear cells (PBMC), isolated from healthy volunteers by Ficoll - Hypaque density gradient centrifugation, were exposed to 2 - 8 Gy of 137Cs γ-rays. CD14 positive monocytes were isolated from the irradiated PBMC using magnetic beads, and stimulated with Staphylococcus aureus Cowan strain (0.0075% SAC) for 24 hours. Culture fluids were harvested and analyzed for cytokines by ELISA. In the results, IL-10 was detected and IL-12 was not detected in the culture fluids of monocytes from 137Cs γ-irradiated PBMC, while IL-12 was detected and IL-10 was not detected in the culture fluids of healthy donor monocytes. Also, CCL1 was detected and CXCL13/CCL17 were not detected in the culture fluids of monocytes isolated from 137Cs γ-irradiated PBMC, while all of these chemokines were not produced by healthy donor monocytes. These results suggest that patients with irradiated injuries may be carriers of M2b monocytes.

Surface HLA-DR Expression in Monocyte Subpopulations During Adverse Events After Hematopoietic Stem Cell Transplantation

Abstract 2161 Background:Human leukocyte antigen DR (HLA-DR) surface expression in CD14++ monocytes reflects the activation state of these cells. Decreased HLA-DR expression levels have been described as an indicator of immunosuppression. According to CD14 and CD16 expression levels, monocyte subpopulations have been classified into “classical” CD14++/CD16- (M1), “non-classical” CD14+/CD16++ (M3), “intermediate” CD14++/CD16+ (M2) and CD14-/CD16++ (M4) monocytes (Figure 1). This study analyzes HLA-DR expression in these monocyte subpopulations after hematopoietic stem cell transplantation (HSCT) and in particular during transplant-related adverse events. Patient and Methods:30 pediatric patients (< 15 years) (n = 23) and young adults (> 15 years) (n = 7) (7 autologous, 12 allogeneic and 11 allogeneic haploidentical transplanted) with hemato-oncological malignancies and immunodeficiency disorders were consecutively included after informed written consent. Median age was 9.5 years (range 0.5–38 years). Flowcytometric assessment of HLA-DR expression was started the day before conditioning therapy and continued up to 379 days after HSCT. Normal values of HLA-DR expression in monocyte subpopulations were established from a control group of healthy children and young adults (n = 20). Surface marker expression of CD14, CD16, CD56, and HLA-DR was determined by four-color flowcytometry. Results:Median data acquisiton period was 228 days (range of 43–379 days). By analysis of covariance (ANCOVA), HLA-DR expression in healthy controls varied in dependency of age. In young adults (> 15 years), HLA-DR expression was significantly (P < 0.05) lower in M2, M3 and M4 but not in M1 monocyte subpopulations (P = 0.084) compared to children and adolescents (< 15 years). After HSCT, a significant decrease of HLA-DR expression in M1 and M4 monocytes was observed during sepsis or bacteremia (P < 0.01). The onset of graft-versus-host-disease (GvHD)°III-°IV was associated with a significant increase of HLA-DR expression in M1 monocytes (P < 0.05). HLA-DR expression in monocyte subpopulations was not altered before or during veno-occlusive disease (VOD), a relapse of underlying disease or before death. Conclusion:The distinct HLA-DR expression pattern in monocyte subpopulations during sepsis and GvHD may facilitate identification of patients at risk after HSCT. Moreover, HLA-DR expression in monocyte subpopulations may be used to monitor treatment response in these entities. [Display omitted] CD14++/CD16- (M1), CD14++/CD16+ (M2), CD14+/CD16++ (M3) and CD14-/CD16++(M4) Disclosures:No relevant conflicts of interest to declare.

Microparticles from apoptotic platelets promote resident macrophage differentiation

Platelets shed microparticles not only upon activation, but also upon ageing by an apoptosis-like process (apoptosis-induced platelet microparticles, PMap). While the activation-induced microparticles have widely been studied, not much is known about the (patho)physiological consequences of PMap formation. Flow cytometry and scanning electron microscopy demonstrated that PMap display activated integrins and interact to form microparticle aggregates. PMap were chemotactic for monocytic cells, bound to these cells, an furthermore stimulated cell adhesion and spreading on a fibronectin surface. After prolonged incubation, PMap promoted cell differentiation, but inhibited proliferation. Monocyte membrane receptor analysis revealed increased expression levels of CD11b (integrin αMβ2), CD14 and CD31 (platelet endothelial cell adhesion molecule-1), and the chemokine receptors CCR5 and CXCR4, but not of CCR2. This indicated that PMap polarized the cells into resident M2 monocytes. Cells treated with PMap actively consumed oxidized low-density lipoprotein (oxLDL), and released matrix metalloproteinases and hydrogen peroxide. Further confirmation for the differentiation towards resident professional phagocytes came from the finding that PMap stimulated the expression of the (ox)LDL receptors, CD36 and CD68, and the production of proinflammatory and immunomodulating cytokines by monocytes. In conclusion, interaction of PMap with monocytic cells has an immunomodulating potential. The apoptotic microparticles polarize the cells into a resident M2 subset, and induce differentiation to resident professional phagocytes.

Propranolol as a modulator of M2b monocytes in severely burned patients

A role of immunosuppressive M2 monocytes (IL-12(-)IL-10(+)) on the increased susceptibility of severely burned patients to various opportunistic pathogens has been described. Among M2 monocyte subpopulations, M2b monocytes (IL-17(-)CCL1(+)CXCL13(-)) are predominantly present in the peripheral blood of severely burned patients. In the present study, the rise and fall of M2b monocytes were examined in severely burned patients treated with propranolol. Catecholamine is known as an inducer of M2 monocytes, and propranolol is a competitive blocker of catecholamine binding to β-adrenergic receptors. Twenty-two children with 30% or more TBSA burn were enrolled in the study. Propranolol at a dose of 4 mg/kg/day was administered to these patients by feeding-tube or mouth. Burn patient monocytes exhibited weak bactericidal activity. IL-12 was produced by propranolol-treated patient monocytes after stimulation with Staphylococcus aureus antigen, and the production of IL-10, CCL1, CCL17, or CXCL13 by these monocytes was not demonstrated. These results indicate that a predominance of M2b monocytes in severely burned patients is intervened by the propranolol treatment. The increased susceptibility, to be associated with the appearance of M2b monocytes, of severely burned patients to opportunistic pathogens might be controlled by propranolol.

M2b Monocytes Predominated in Peripheral Blood of Severely Burned Patients

Severely burned patients were shown to be carriers of M2 monocytes, and all of the monocytes isolated from peripheral blood of severely burned patients (19 of 19 patients) were demonstrated as M2b monocytes (IL-12(-)IL-10(+)CCL1(+) monocytes). Low levels of M2a (IL-12(-)IL-10(+)CCL17(+) monocytes) and M2c monocytes (IL-12(-)IL-10(+)CXCL13(+) monocytes) were demonstrated in peripheral blood of severely burned patients (M2a, 2 of 19 patients; M2c, 5 of 19 patients). M2b, M2a, and M2c monocytes were not detected in peripheral blood of healthy donors. However, M2b monocytes appeared when healthy donor monocytes were cultured in media supplemented with burn patient serum (15%). CCL2 was detected in sera of all burn patients, and M2b monocytes were not generated from healthy donor monocytes cultured with media containing 15% burn patient sera that were previously treated with anti-CCL2 mAb. In addition, M2b monocytes were generated from healthy donor monocytes in cultures supplemented with rCCL2. These results indicate that M2b monocytes are predominant in peripheral blood of severely burned patients who are carriers of CCL2 that functions to stimulate monocyte conversion from resident monocytes to M2b monocytes.

Altered M1/M2 activation patterns of monocytes in severe relapsing experimental rat model of multiple sclerosis. Amelioration of clinical status by M2 activated monocyte administration

Objectives:We investigated proinflammatory M1 and immunomodulatory M2 activation profiles of circulating monocytes in relapsing experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, and tested whether altered M1/M2 equilibrium promotes CNS inflammation. Results:Approaches of MRI macrophage tracking with USPIO nanoparticles and expression patterns of M1/M2 macrophages and microglia in brain and M1/M2 monocytes in blood samples at various disease stages revealed that M1/M2 equilibrium in blood and CNS favors mild EAE, while imbalance towards M1 promotes relapsing EAE. We consequently investigated whether M2 activated monocyte restoration in peripheral blood could cure acute clinical EAE disease. Administration of ex vivo activated M2 monocytes both suppressed ongoing severe EAE and increased immunomodulatory expression pattern in lesions, confirming their role in the induction of recovery. Conclusion:We conclude that imbalance of monocyte activation profiles and impaired M2 expression, are key factors in development of relapses. Our study opens new perspectives for therapeutic applications in MS.

Vagus Nerve Stimulation Produces an Anti-Inflammatory Monocyte Phenotype in Blood. (138.25)

Abstract Cytokine production and immune cell migration are under the control of the nervous system. Electrical stimulation of the vagus nerve attenuates cytokine production in endotoxemia, and down-modulates neutrophil migration in animal models of inflammation. The effect of vagus nerve stimulation (VNS) on PBMC phenotypes has not been characterized. We performed VNS or sham surgery in mice (n=5 group) and 24 hours later collected blood for analysis by flow cytometry. The total number of CD11b+ expressing cells was not affected, but CD11b+/Ly-6C+ monocytes significantly decreased in frequency (0.5% vs. 3.2%; p=0.005). VNS decreased dendritic cell numbers defined as CD11b+, CD11c+ (0.4% vs. 1.8%; p=0.042), or CD11c+, F4/80+ (1.6% vs. 2.7%; p=0.025). VNS did not affect the total number of circulating T cells (CD3+). VNS did not affect CD11b+ or Ly-6Clow, which are known to be VEGF-producing and anti-inflammatory. The present results indicate that VNS reduced the proportion of circulating pro-inflammatory monocytes and dendritic cells, and decreased the expression of monocytic lineage cells, in particular those expressed by M1 monocytes which are known to secrete TNF, Il-6, IL-12 (and reduced amounts of IL-10). Together, this indicates that direct activation of a neural circuit can down-modulate inflammatory cell phenotypes in blood, producing an anti-inflammatory cellular milieu.

Human Embryonic Stem Cell-Derived and Fetal Macrophages Exhibit Mainly Features of Alternative (M2) Polarization.

Abstract 4598Monocyte heterogeneity has long been recognized and 2 functional subsets of human monocytes that exert specific roles in homeostasis and inflammation in vivo, M1 and M2 monocytes, has been described. The different monocyte subsets seem to reflect developmental stages with distinct physiological roles but few is known whether the macrophage diversity arises in early ontogeny.Human embryonic stem cells (hESC) provide an unique model for in vitro studies of the early ontogeny of the hematopoietic system. Human embryonic monocytes were obtained from embryoid bodies cultured for 3 weeks in the presence of BMP4, VEGF and a mixture of hematopoietic cytokines. The sorted CD14+CD45+ cells were further cultured for 7 -10 days in the presence of GM- and M-CSF. Embryonic stem cells derived monocytes and macrophages were identified by several criteria including morphology, cytochemical staining, ultrastructural and functional features. In contrast to early mouse hematopoiesis that bypass the series of monoblaste/monocyte precursors human embryonic monocyte lineage developped by process similar to definitive monopoiesis. Two distinct subpopulations of monocytes, first CD14+CD16- then CD14+CD16+ could be successively identified. Transcriptional and functional assays suggest that CD14+CD16- population is not an embryonic counterpart of CD14+CD16- “inflammatory” adult monocytes but a precursor for CD14+CD16+ macrophages and dendritic cells. Moreover, both tested populations possess phenotypical markers of M2 polarization and were positive for a expression of scavenger receptors CD163+ and CD36+ of CCR5 and negative for CD62L. Additional transcriptional studies and chemokine pattern confirmed their IL10 high IL12low phenotype, clearly indicating their M2 polarization state. M2 polarization was confirmed in fetal macrophages derived from CD34+ cells issued from livers of first-trimester fetuses. Involvment of embryonic and foetal monocytes in homeostasis are under investigation.Collectively, our data provide insight into alternative macrophage polarization during early embryogenesis and adds further data to the growing body of evidence that establishment of macrophage heterogeneity is related to early ontogeny. Disclosures:No relevant conflicts of interest to declare.