Activation Of Bone Marrow-derived Macrophages Research Articles

Ceramide Imbalance and Impaired TLR4-Mediated Autophagy in BMDM of an ORMDL3-Overexpressing Mouse Model.

Increased orosomucoid-like 3 (ORMDL3) expression levels, due to single nucleotide polymorphisms (SNPs), have been associated with several inflammatory diseases, including asthma and inflammatory bowel diseases. ORMDL proteins inhibit serine palmitoyltransferase (SPT), the first rate-limiting enzyme in de novo sphingolipid synthesis and alter cellular calcium homeostasis. Both processes are essential for immune response. The present study addresses ORMDL3 protein involvement in macrophage physiology using an overexpressing knock-in mouse model. Ceramide content was notably different in the bone-marrow-derived macrophages (BMDM) from the transgenic mouse model compared with the wild type (WT) macrophages. Our data revealed an alteration of de novo production of sphinganine upon BMDM activation in the transgenic mouse. Gene-expression analysis showed that alteration in ORMDL3 expression levels did not affect activation or macrophage polarization. Nevertheless, we studied phagocytosis and autophagy—crucial processes that are dependent on lipid membrane composition. Phagocytosis in transgenic macrophages was not affected by ORMDL3 overexpression, but we did find a reduction in toll-like receptor 4 (TLR-4)-mediated autophagy. Both genetic and functional studies have pointed to autophagy as an essential pathway involved in inflammation. We believe that our work provides new insights into the functional link between ORMDL3 expression and inflammatory diseases.

Aryl hydrocarbon receptor activation by benzo(a)pyrene inhibits proliferation of myeloid precursor cells and alters the differentiation state as well as the functional phenotype of murine bone marrow-derived macrophages

Intensive research during the past decade has highlighted the impact of the regulatory function of the aryl hydrocarbon receptor (AhR) in immunity. In this study, we focused on the influence of AhR activation on the differentiation of murine bone marrow-derived myeloid precursor cells into mature macrophages. Our results show that the activation of AhR by subtoxic doses of the AhR ligand benzo(a)pyrene (BaP) impaired the proliferation of bone marrow cells (BMCs) whereas the proportion of resulting adherent cells was not affected. Flow cytometric analysis revealed that the number of mature bone marrow-derived macrophages (BMMs) was significantly decreased by AhR activation. However, expression of the murine macrophage marker F4/80, the major histocompatibility complex class II (MHC-II) and the Fcγ receptor I (FcγRI/CD64) were upregulated on BaP-exposed BMMs in an AhR-dependent manner. Analysis of cytokine secretion after BMM activation with heat-killed (hk) salmonellae showed that BaP exposure resulted in suppressed secretion of interleukin (IL)-1β, IL-6 and the chemokine CXC motif ligand 1 (CXCL1). In contrast, the release of tumor necrosis factor (TNF)-α and IL-10 was increased following BaP exposure. In addition, the production of antimicrobial nitric oxide (NO) was increased AhR-dependently. Bacterial stimulation of BaP exposed BMMs also induced the expression of MHC-II and CD64, while the expression of F4/80 was dramatically decreased. In summary, this study demonstrates for the first time that sustained exposure over 6 days of bone marrow-derived myeloid precursors to subtoxic doses of BaP critically interferes with differentiation and activation of BMMs. We could convincingly show that AhR-induced gene regulation is crucial for homeostasis of pro- and anti-inflammatory cytokines during macrophage activation.

Abstract 3796: Substrate specificity of human kallikrein-related peptidase 2 (KLK2) towards beta chain of interleukin-10 receptor

Abstract The kallikrein-related peptidases (KLKs) comprise a family of 15 homologous secreted trypsin- or chymotrypsin-like serine proteases. KLK2 and KLK3 have the most organ restricted expression profile of all KLKs; specifically, they are abundantly expressed in the luminal epithelium of the prostate. The expression of KLK2 gene is regulated by the androgens and androgen receptor (AR) signaling and like human PSA, serum KLK2 can also function as a biomarker for prostate cancer (PCa). The present work provides biochemical data related to the hydrolytic activities of KLK2 on libraries of fluorescence resonance energy transfer (FRET) peptides in the presence of sodium citrate, a kosmotropic salt that is present in normal prostate tissue and seminal fluid in high concentration. The peptide SYR↓IF (cleaved at↓) was the highly hydrolyzed and best substrate so far reported for KLK2. The sequence YRIF is present in the N-terminal segment (59YRIF62) in the β chain of interleukin-10 receptor. In order to verify the effect of KLK2 in the IL10RB surface expression in the bone marrow-derived macrophages (BMDM) cells, they were incubated with KLK2 at 37ºC for 2 hours. BMDM treated with KLK2 showed a significant reduction in the IL10RB surface expression compared with control group, indicating that KLK2 acts directly on this receptor, decreasing its expression. The effect of this reduction was evaluated trough BMDM activation assay. After incubation of BMDM cells with KLK2, they were washed and incubated with or without LPS, IFN-γ and recombinant IL10. When incubated with recombinant IL10, control macrophage showed a significant reduction of NO, TNF-α and IL-12p40 levels indicating the IL10RB functionality and according to IL10 immunosuppressive effect. However, KLK2 pretreated macrophage maintained NO, TNF-α and IL-12p40 levels, even in the presence of recombinant IL10, leading a proinflammatory status in BMDM culture, while KLK3 doesn't show the same effect. Taken together, the data indicates that KLK2 decreases the surface expression of IL10RB on macrophage cells and contributes to a proinflammatory environment. Citation Format: Luiz J. Neto, Maria A. Juliano, Juliana R. Oliveira. Substrate specificity of human kallikrein-related peptidase 2 (KLK2) towards beta chain of interleukin-10 receptor [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 3796.

Statins regulates inflammatory macrophage phenotype through the activation of AhR

Abstract Statins are 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase competitive inhibitors, that lower blood cholesterol, but also exert potent anti-inflammatory effects. The xenobiotic receptor, aryl hydrocarbon receptor (AhR), plays crucial role in the control of inflammatory responses. Evidences suggest that selected statins promote the transcription of the main AhR target gene, cytochrome P450 family 1 (Cyp1a1). Macrophages are essential components of innate and adaptive immunity, characterized by AhR expression and activity. Based on these evidences, we investigated potential AhR-mediated immune regulatory properties of statins in Bone Marrow Derived Macrophages (BMDM). We found that BMDM activation with the pro-inflammatory cytokine TNFa induced the inflammatory marker, inducible nitric oxide synthase (iNOS), which was completely prevented in BMDMs treated with TNFa in combination with specific statins, namely, atorvastatin and rosuvastatin. We confirmed high AhR expression in BMDM that was increased upon treatment with TNFa. We investigated the ability of different statins to activate AhR using a luciferase reporter assay cell lines and in BMDCs. We found that selected statins, such as atorvastatin and rosuvastatin, activated AhR in a dose dependent manner, an activation prevented in cells expressing selected AhR mutants. Notably, AhR deficient BMDMs, expressed undetectable levels of the main statin target protein HMG-CoA, which was instead highly expressed in wild type BMDMs. These data demonstrate that natural or synthetic statins can play regulatory roles in macrophages. Moreover, our results suggest a potential involvement of AhR in statin-mediated anti-inflammatory effects in macrophages.

Glia Maturation Factor-Gamma Modulates M2 Activation of Macrophage Via Suppression of Mitochondria Function

Macrophages are innate immune cells of dynamic phenotype as they can acquire distinct phenotypes and biological functions depending on the microenvironment and the metabolic state. In response to various signals, macrophages can undergo M1 activation (stimulated by TLR ligand and interferon-gamma) or M2 activation (stimulated by IL-4/IL-13, immunocomplexes, TGFbeta, and IL-10). M1 and M2 macrophages have distinct metabolic phenotypes that differ from those of resting macrophages. M1 macrophages rely on aerobic glycolysis, while M2 macrophages use fatty acid oxidation to fuel mitochondrial oxidative phosphorylation. IL-4/IL13 induced M2 macrophages polarization has an enhanced mitochondrial oxygen-consumption rate, whereas M1 has a significantly decreased oxygen-consumption rate. This indicates that macrophage phenotypes can be regulated by the different aspects of cellular metabolism. Thus, the identification of molecules and mechanisms associated with the phenotypic switch of them provides a molecular basis for macrophage-centered therapeutic strategies. Glia maturation factor gamma (GMFG), a novel regulator of the actin-related protein-2/3 (Arp2/3) complex, is predominantly expressed in inflammatory cells. We have previously found that GMFG-knockdown macrophages enhanced IL-4/IL-13-stimulated M2 markers and promoted iron metabolism change to iron recycling states, but its underlying molecular mechanism remains unclear. In this study, to explore the possible role of GMFG on mitochondrial function, we measured mitochondrial respiration by determining the oxygen consumption rate (OCR) and glycolytic flux in GMFG-knockdown M0 (unactivated), M1 and M2 activated bone marrow-derived macrophages using an extracellular flux analyzer. We observed that decreased basal oxygen consumption (ORC), diminished maximal respiratory capacity (MRC) and reduced cellular ATP levels in GMFG-knockdown M0 and M2 macrophages compared with control siRNA transfected macrophages. Whereas, there were no detectable changes in the rate of extracellular acidification (ECAR) in GMFG-knockdown cells. Importantly, oxygen consumption in GMFG-knockdown cells were reduced to the same level of TGFbeta-stimulated cells, but not to the level of M1 macrophages, reflecting the loss of mitochondria fitness similar to TGFbeta-stimulated M2 macrophages. Additionally, we observed a moderately increased mitochondrial ROS levels in GMFG-knockdown cells which is similar to increased levels in M2 macrophages, but significantly lower than the increased level in M1 macrophages. We did not observe decreased mitochondrial membrane potential, but found moderately increased mitochondria mass in the GMFG-knockdown cells, which may be due to the accumulation of dysfunctional mitochondria. Moreover, we found that suppressed OCR in GMFG-knockdown cells were accompanied by suppressed mitochondrial biogenesis as shown by decreased protein expression levels of mitochondrial complex I, ISCU, MnSOD2 and CuZnSOD1 by immunoblotting analysis. We also found that GMFG expression was downregulated by treatment with hydrogen peroxide at a concentration of 0.25 mM, which accompanied with increased protein levels of TfR1 and IRP1 in macrophages. Increased mROS, TfR1 and decreased MnSOD2, CuZnSOD1 in GMFG-knockdown cells can be attenuated by treatment of 5 mM NAC and 0.01 mM MitoTEMPO for 1 hour. Furthermore, we found that the remarkable decreased protein level of Dynamin-related protein 1(Drp1), which promoted mitochondria fission, in GMFG-knockdown cells. Consistent with this, confocal microscope analysis exhibited predominantly punctate mitochondria in GMFG-knockdown cells compared with the elongated tubules mitochondria in control cells. Finally, immunoblotting analysis showed that GMFG-knockdown macrophages showed an increase of phosphorylated-CREB, which might be contributed to enhanced M2 markers in GMFG-knockdown cells. Together, these results suggest that downregulation of GMFG skewing macrophages toward a M2 phenotype through suppression of mitochondrial function, which associated with impaired mitochondria fission/fusion balance. Our results indicate that GMFG plays an important role in maintaining mitochondrial function and dynamic, which is necessary for macrophage reprogramming. DisclosuresNo relevant conflicts of interest to declare.

Tumor necrosis factor alpha derived from classically activated "M1" macrophages reduces interstitial cell of Cajal numbers.

Delayed gastric emptying in diabetic mice and humans is associated with changes in macrophage phenotype and loss of interstitial cells of Cajal (ICC) in the gastric muscle layers. In diabetic mice, classically activated M1 macrophages are associated with delayed gastric emptying, whereas alternatively activated M2 macrophages are associated with normal gastric emptying. This study aimed to determine if secreted factors from M1 macrophages could injure mouse ICC in primary culture. Cultures of gastric ICC were treated with conditioned medium (CM) from activated bone marrow-derived macrophages (BMDMs) and the effect of CM was quantified by counting ICC per high-powered field. Bone marrow-derived macrophages were activated to a M1 or M2 phenotype confirmed by qRT-PCR. Conditioned medium from M1 macrophages reduced ICC numbers by 41.1%, whereas M2-CM had no effect as compared to unconditioned, control media. Immunoblot analysis of 40 chemokines/cytokines found 12 that were significantly increased in M1-CM, including tumor necrosis factor alpha (TNF-α). ELISA detected 0.697±0.03ngmL-1 TNF-α in M1-CM. Recombinant mouse TNF-α reduced Kit expression and ICC numbers in a concentration-dependent manner (EC50=0.817ngmL-1 ). Blocking M1-CM TNF-α with a neutralizing antibody preserved ICC numbers. The caspase inhibitor Z-VAD.fmk partly preserved ICC numbers (cells/field; 6.63±1.04, 9.82±1.80 w/Z-VAD.fmk, n=6, P<.05). This work demonstrates that TNF-α secreted from M1 macrophages can result in Kit loss and directly injure ICC in vitro partly through caspase-dependent apoptosis and may play an important role in ICC depletion in diabetic gastroparesis.

Polyanhydride Nanoparticle Interactions with Host Serum Proteins and Their Effects on Bone Marrow Derived Macrophage Activation.

An in-depth understanding of the interactions of vaccine delivery vehicles with antigen presenting cells is important for tailoring optimal adjuvant properties. Polymeric nanoparticles have been widely studied as adjuvants and delivery vehicles; however, there is little information regarding the effect of serum protein adsorption onto biomaterials and the effect of this adsorption upon interactions with antigen presenting cells. The current studies analyzed effects of polyanhydride chemistry on serum adsorption to nanoparticles with respect to their uptake by and activation of bone marrow-derived macrophages. Differential effects of serum adsorption based on nanoparticle chemistry were shown to enhance (for 1,6-bis(p-carboxyphenoxy)hexane and sebacic anhydride-based) or reduce (for 1,6-bis(p-carboxyphenoxy)hexane and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane-based) nanoparticle uptake. The observed complex interdependence between nanoparticle chemistry and serum protein adsorption on macrophage activation provided insights that will facilitate the rational design of single-dose nanovaccines developed to induce robust immune responses.

Mycobacterium fortuitum induces A20 expression that impairs macrophage inflammatory responses.

Mycobacterium fortuitum is a rapidly growing mycobacterium that has been regarded as an etiological agent of a variety of human infections. However, little is known about the host inflammatory responses and the molecular mechanisms by which MF-induced inflammation is regulated in macrophages. In this study, we report that MF infection leads to the induction of an anti-inflammatory molecule, A20 (also known as TNFAIP3), which is essential for the regulation of MF-induced inflammatory responses in murine bone marrow-derived macrophages (BMDMs). MF triggered the expression of tumor necrosis factor-α and interleukin-6 in BMDMs through signaling of the Toll-like receptor 2 (TLR2)-myeloid differentiation primary response gene 88. Additionally, MF rapidly induced the expression of A20, which inhibited proinflammatory cytokine expression and nuclear factor (NF)-κB reporter gene activities in BMDMs. Notably, MF-induced activation of NF-κB signaling was required for A20 expression and proinflammatory responses in BMDMs. Furthermore, the rough morphotype of the MF clinical strain induced a higher level of proinflammatory signaling activation, but less A20 induction in BMDMs, compared to the smooth morphotype. Taken together, these results suggest that MF-induced activation of host proinflammatory responses is negatively regulated through TLR2-dependent A20 expression.

Proinflammatory signal suppresses proliferation and shifts macrophage metabolism from Myc-dependent to HIF1α-dependent

As a phenotypically plastic cellular population, macrophages change their physiology in response to environmental signals. Emerging evidence suggests that macrophages are capable of tightly coordinating their metabolic programs to adjust their immunological and bioenergetic functional properties, as needed. Upon mitogenic stimulation, quiescent macrophages enter the cell cycle, increasing their bioenergetic and biosynthetic activity to meet the demands of cell growth. Proinflammatory stimulation, however, suppresses cell proliferation, while maintaining a heightened metabolic activity imposed by the production of bactericidal factors. Here, we report that the mitogenic stimulus, colony-stimulating factor 1 (CSF-1), engages a myelocytomatosis viral oncogen (Myc)-dependent transcriptional program that is responsible for cell cycle entry and the up-regulation of glucose and glutamine catabolism in bone marrow-derived macrophages (BMDMs). However, the proinflammatory stimulus, lipopolysaccharide (LPS), suppresses Myc expression and cell proliferation and engages a hypoxia-inducible factor alpha (HIF1α)-dependent transcriptional program that is responsible for heightened glycolysis. The acute deletion of Myc or HIF1α selectively impaired the CSF-1- or LPS-driven metabolic activities in BMDM, respectively. Finally, inhibition of glycolysis by 2-deoxyglucose (2-DG) or genetic deletion of HIF1α suppressed LPS-induced inflammation in vivo. Our studies indicate that a switch from a Myc-dependent to a HIF1α-dependent transcriptional program may regulate the robust bioenergetic support for an inflammatory response, while sparing Myc-dependent proliferation.

IL-4Rα-dependent alternative activation of macrophages is not decisive for Mycobacterium tuberculosis pathology and bacterial burden in mice.

Classical activation of macrophages (caMph or M1) is crucial for host protection against Mycobacterium tuberculosis (Mtb) infection. Evidence suggests that IL-4/IL-13 alternatively activated macrophages (aaMph or M2) are exploited by Mtb to divert microbicidal functions of caMph. To define the functions of M2 macrophages during tuberculosis (TB), we infected mice deficient for IL-4 receptor α on macrophages (LysMcreIL-4Rα-/lox) with Mtb. We show that absence of IL-4Rα on macrophages does not play a major role during infection with Mtb H37Rv, or the clinical Beijing strain HN878. This was demonstrated by similar mortality, bacterial burden, histopathology and T cell proliferation between infected wild-type (WT) and LysMcreIL-4Rα-/lox mice. Interestingly, we observed no differences in the lung expression of inducible nitric oxide synthase (iNOS) and Arginase 1 (Arg1), well-established markers for M1/M2 macrophages among the Mtb-infected groups. Kinetic expression studies of IL-4/IL-13 activated bone marrow-derived macrophages (BMDM) infected with HN878, followed by gene set enrichment analysis, revealed that the MyD88 and IL-6, IL-10, G-CSF pathways are significantly enriched, but not the IL-4Rα driven pathway. Together, these results suggest that IL-4Rα-macrophages do not play a central role in TB disease progression.

Mouse hepatocyte overexpression of NF-κB-inducing kinase (NIK) triggers fatal macrophage-dependent liver injury and fibrosis.

Damaged, necrotic, or apoptotic hepatocytes release damage-associated molecular patterns that initiate sterile inflammation, and liver inflammation drives liver injury and fibrosis. Here we identified hepatic nuclear factor kappa B (NF-κB)-inducing kinase (NIK), a Ser/Thr kinase, as a novel trigger of fatal liver inflammation. NIK is activated by a broad spectrum of stimuli. It was up-regulated in injured livers in both mice and humans. In primary mouse hepatocytes, NIK overexpression stimulated, independently of cell injury and death, release of numerous chemokines and cytokines that activated bone marrow-derived macrophages (BMDMs). BMDMs in turn secreted proapoptotic molecules that stimulated hepatocyte apoptosis. Hepatocyte-specific expression of the NIK transgene triggered massive liver inflammation, oxidative stress, hepatocyte apoptosis, and liver fibrosis, leading to weight loss, hypoglycemia, and death. Depletion of Kupffer cells/macrophages reversed NIK-induced liver destruction and death. the hepatocyte NIK-liver immune cell axis promotes liver inflammation, injury, and fibrosis, thus driving liver disease progression.

T-cell immunoglobulin and mucin domain 4 (TIM-4) signaling in innate immune-mediated liver ischemia-reperfusion injury.

Hepatic ischemia-reperfusion injury (IRI), an innate immunity-driven inflammation response, occurs in multiple clinical settings including liver resection, transplantation, trauma, and shock. T-cell immunoglobulin and mucin (TIM)-4, the only TIM protein not expressed on T cells, is found on macrophages and dendritic cells. The regulatory function of macrophage TIM-4 in the engulfment of apoptotic/necrotic bodies in innate immunity-mediated disease states remains unknown. This study focuses on the putative role of TIM-4 signaling in a model of liver warm ischemia (90 minutes) and reperfusion. The ischemia insult triggered TIM-4 expression by stressed hepatocellular phosphatidylserine (PS) presentation, peaking at 6 hours of reperfusion, and coinciding with the maximal hepatocellular damage. TIM-4-deficient or wild-type WT mice treated with antagonistic TIM-4 monoclonal antibody (mAb) were resistant against liver IRI, evidenced by diminished serum alanine aminotransferase (sALT) levels and well-preserved hepatic architecture. Liver hepatoprotection rendered by TIM-4 deficiency was accompanied by diminished macrophage infiltration/chemoattraction, phagocytosis, and activation of Toll-like receptor (TLR)2/4/9-dependent signaling. Correlating with in vivo kinetics, the peak of TIM-4 induction in lipopolysaccharide (LPS)-activated bone marrow derived-macrophages (BMM) was detected in 6-hour cultures. To mimic liver IRI, we employed hydrogen peroxide-necrotic hepatocytes, which readily present PS. Indeed, necrotic hepatocytes were efficiently captured/engulfed by WT (TIM-4+) but not by TIM-4-deficient BMM. Finally, in a newly established model of liver IRI, adoptive transfer of WT but not TIM-4-deficient BMM readily recreated local inflammation response/hepatocellular damage in the CD11b-DTR mouse system. These findings document the importance of macrophage-specific TIM-4 activation in the mechanism of hepatic IRI. Macrophage TIM-4 may represent a therapeutic target to minimize innate inflammatory responses in IR-stressed organs.

Ninjurin1 Deficiency Attenuates Susceptibility of Experimental Autoimmune Encephalomyelitis in Mice

Ninjurin1 is a homotypic adhesion molecule that contributes to leukocyte trafficking in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. However, in vivo gene deficiency animal studies have not yet been done. Here, we constructed Ninjurin1 knock-out (KO) mice and investigated the role of Ninjurin1 on leukocyte trafficking under inflammation conditions such as EAE and endotoxin-induced uveitis. Ninjurin1 KO mice attenuated EAE susceptibility by reducing leukocyte recruitment into the injury regions of the spinal cord and showed less adhesion of leukocytes on inflamed retinal vessels in endotoxin-induced uveitis mice. Moreover, the administration of a custom-made antibody (Ab26-37) targeting the Ninjurin1 binding domain ameliorated the EAE symptoms, showing the contribution of its adhesion activity to leukocyte trafficking. In addition, we addressed the transendothelial migration (TEM) activity of bone marrow-derived macrophages and Raw264.7 cells according to the expression level of Ninjurin1. TEM activity was decreased in Ninjurin1 KO bone marrow-derived macrophages and siNinj1 Raw264.7 cells. Consistent with this, GFP-tagged mNinj1-overexpressing Raw264.7 cells increased their TEM activity. Taken together, we have clarified the contribution of Ninjurin1 to leukocyte trafficking in vivo and delineated its direct functions to TEM, emphasizing Ninjurin1 as a beneficial therapeutic target against inflammatory diseases such as multiple sclerosis.

Differential Immune Responses to Segniliparus rotundus and Segniliparus rugosus Infection and Analysis of Their Comparative Virulence Profiles

Two closely related bacterial species, Segniliparus rotundus and Segniliparus rugosus, have emerged as important human pathogens, but little is known about the immune responses they elicit or their comparative pathophysiologies. To determine the virulence and immune responses of the two species, we compared their abilities to grow in phagocytic and non-phagocytic cells. Both species maintained non-replicating states within A549 epithelial cells. S. rugosus persisted longer and multiplied more rapidly inside murine bone marrow-derived macrophages (BMDMs), induced more pro-inflammatory cytokines, and induced higher levels of macrophage necrosis. Activation of BMDMs by both species was mediated by toll-like receptor 2 (TLR2), followed by mitogen-activated protein kinases (MAPK) and nuclear factor κB (NF-κB) signaling pathways, indicating a critical role for TLR2 in Segniliparus-induced macrophage activation. S. rugosus triggered faster and stronger activation of MAPK signaling and IκB degradation, indicating that S. rugosus induces more pro-inflammatory cytokines than S. rotundus. Multifocal granulomatous inflammations in the liver and lung were observed in mice infected with S. rugosus, but S. rotundus was rapidly cleared from all organs tested within 15 days post-infection. Furthermore, S. rugosus induced faster infiltration of innate immune cells such as neutrophils and macrophages to the lung than S. rotundus. Our results suggest that S. rugosus is more virulent and induces a stronger immune response than S. rotundus.

RANKL Enhances Macrophage Paracrine Pro-Calcific Activity in High Phosphate-Treated Smooth Muscle Cells: Dependence on IL-6 and TNF-α

Background: Vascular calcification is highly correlated with cardiovascular disease (CVD) morbidity and mortality, and it is associated with inflammation. Receptor activator of NF-ĸB ligand (RANKL) inhibition in vivo has been shown to reduce vascular calcification in a mouse model of atherosclerosis. Therefore, we tested the hypothesis that RANKL regulates smooth muscle cell (SMC) calcification by modulating macrophage production of pro-calcific cytokines. Methods: We used a bone marrow-derived macrophage (BMDM)/SMC co-culture system and examined the effects of RANKL on BMDM activation and SMC matrix calcification. Results: Treatment with RANKL alone did not stimulate SMC calcification induced by elevated phosphate. BMDMs differentiated with macrophage colony-stimulating factor and placed in co-culture with SMCs increased phosphate-induced SMC calcification. RANKL added to the BMDM/SMC co-cultures further enhanced SMC calcification. Treatment of BMDMs with RANKL resulted in increased expression of IL-6 and TNF-α. Thus, increased expression of these pro-calcific cytokines in macrophages may mediate RANKL-induced SMC calcification in a paracrine fashion. Addition of neutralizing IL-6 and TNF-α antibodies together with RANKL treatment significantly reduced the RANKL induction of SMC calcification. Conclusion: RANKL activation of pro-inflammatory and pro-calcific pathways in macrophages may contribute to vascular calcification and inflammation.

Multiple sclerosis or multiple possibilities

Statins are 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase competitive inhibitors, that lower blood cholesterol, but also exert potent anti-inflammatory effects. The xenobiotic receptor, aryl hydrocarbon receptor (AhR), plays crucial role in the control of inflammatory responses. Evidences suggest that selected statins promote the transcription of the main AhR target gene, cytochrome P450 family 1 (Cyp1a1). Macrophages are essential components of innate and adaptive immunity, characterized by AhR expression and activity. Based on these evidences, we investigated potential AhR-mediated immune regulatory properties of statins in Bone Marrow Derived Macrophages (BMDM). We found that BMDM activation with the pro-inflammatory cytokine TNFa induced the inflammatory marker, inducible nitric oxide synthase (iNOS), which was completely prevented in BMDMs treated with TNFa in combination with specific statins, namely, atorvastatin and rosuvastatin. We confirmed high AhR expression in BMDM that was increased upon treatment with TNFa. We investigated the ability of different statins to activate AhR using a luciferase reporter assay cell lines and in BMDCs. We found that selected statins, such as atorvastatin and rosuvastatin, activated AhR in a dose dependent manner, an activation prevented in cells expressing selected AhR mutants. Notably, AhR deficient BMDMs, expressed undetectable levels of the main statin target protein HMG-CoA, which was instead highly expressed in wild type BMDMs. These data demonstrate that natural or synthetic statins can play regulatory roles in macrophages. Moreover, our results suggest a potential involvement of AhR in statin-mediated anti-inflammatory effects in macrophages.

A Small Molecule Deubiquitinase Inhibitor Increases Localization of Inducible Nitric Oxide Synthase to the Macrophage Phagosome and Enhances Bacterial Killing

Macrophages are key mediators of antimicrobial defense and innate immunity. Innate intracellular defense mechanisms can be rapidly regulated at the posttranslational level by the coordinated addition and removal of ubiquitin by ubiquitin ligases and deubiquitinases (DUBs). While ubiquitin ligases have been extensively studied, the contribution of DUBs to macrophage innate immune function is incompletely defined. We therefore employed a small molecule DUB inhibitor, WP1130, to probe the role of DUBs in the macrophage response to bacterial infection. Treatment of activated bone marrow-derived macrophages (BMM) with WP1130 significantly augmented killing of the intracellular bacterial pathogen Listeria monocytogenes. WP1130 also induced killing of phagosome-restricted bacteria, implicating a bactericidal mechanism associated with the phagosome, such as the inducible nitric oxide synthase (iNOS). WP1130 had a minimal antimicrobial effect in macrophages lacking iNOS, indicating that iNOS is an effector mechanism for WP1130-mediated bacterial killing. Although overall iNOS levels were not notably different, we found that WP1130 significantly increased colocalization of iNOS with the Listeria-containing phagosome during infection. Taken together, our data indicate that the deubiquitinase inhibitor WP1130 increases bacterial killing in macrophages by enhancing iNOS localization to the phagosome and suggest a potential role for ubiquitin regulation in iNOS trafficking.

PS2-053. Interleukin 27 ameliorates atherosclerosis by inhibiting bone marrow-derived macrophage activation in arterial walls in mice

PS2-053. Interleukin 27 ameliorates atherosclerosis by inhibiting bone marrow-derived macrophage activation in arterial walls in mice

Suppression of Chronic Damage in Renal Allografts by Liver X Receptor (LXR) Activation: Relevant Contribution of Macrophage LXRα

Liver X receptors (LXR)-α,β regulate intracellular cholesterol homeostasis and inhibit inflammatory gene expression. We studied the effects of the LXRα,β-agonist GW3965 on acute and chronic organ damage in the F344-LEW rat kidney transplantation model. In addition, to gain LXR isoform and cell-specific insights BALB/c kidneys were transplanted into mice with macrophage overexpression of LXRα (mLXRα-tg) and evaluated 7 and 42 days after transplantation. After 56 days GW3965 improved significantly function and morphology of rat kidney allografts by substantial reduction of mononuclear cell infiltrate and fibrosis; in vitro GW3965 reduced inflammatory activity of bone marrow-derived macrophages (BMDMs) and alloreactivity of T cells. Kidneys transplanted into mLXRα-tg mice were also protected from development of chronic allograft dysfunction. Similarly to GW3965-activated BMDMs, mLXRα-tg macrophages secreted significantly less monocyte chemoattractant protein 1 and macrophage inflammatory protein 1β. Interestingly, 7 days after transplantation, when the total number of intragraft macrophages did not differ, evidently more arginase 1- and mannose receptor C type 1-positive cells were found in LXR rat and mice kidney allografts; in vitro both LXR activation by GW3965 and mLXRα overexpression accentuated the induction of alternative activation of BMDMs by IL-4/IL-13, suggesting an additional mechanism by LXRs to prevent graft damage. The results highlight the relevance of macrophage LXRα in allograft rejection and prevention of fibrosis.

Abstract 465: Alternatively activated bone marrow-derived macrophages enhance tumor growth and metastasis in a 4T1 mammary cancer model

Abstract Tumor microenvironment consists of a number of components including malignant cells, stromal cells, endothelial cells, infiltrating immune cells, and extracellular matrix. Because inflammation plays an important role in cancer development and progression, tumor-associated macrophages (TAMs) can be one of key regulators of tumor microenvironment. Recent evidence has shown that TAM is associated with poor prognosis in a variety of cancers, including breast, lung, and pancreatic cancer. TAMs account for up to 50% of the total tumor mass, resemble M2-macrophages, and exhibit a number of pro-tumoral functions, including the expression of growth factors and matrix proteases, the promotion of angiogenesis, and the suppression of adaptive immunity. In the present study, we examined the effect of M2 macrophages on the development of and metastasis of mammary cancer using a 4T1-orthotopic tumor model. Monocytes were isolated from mouse bone marrow and treated with IL-4 to induce differentiation into an M2 phenotype. In order to determine whether or not M2 microphages stimulate mammary cancer growth and progression, 4T1 mammary carcinoma cells were injected into the inguinal mammary fat pad of syngeneic BALB/C mice, either alone or coupled with bone marrow-derived alternatively-activated macrophages (BMDAM). Co-injection of 4T1 cells with BMDAM increased solid tumor volumes (at 2 weeks after inoculation) and lung metastasis (at 3 weeks after inoculation). We prepared 4T1-luc cells that stably express firefly luciferase which allows for direct examination of tumor growth in animals by estimating the luciferase activity of 4T1 cancer cells via an in vivo imaging system. Luminescence was quantified, and the results demonstrated that the accumulation of 4T1-luc cells was significantly more pronounced when 4T1 cells were co-injected with BMDAM. Immunohistochemical staining of tumor tissues revealed that the proportion of Ki67 (proliferation index)-positive cells was increased in tumor tissues when 4T1 cells were co-injected with BMDAM. We also observed that co-injection of BMDAM with 4T1 cells increased the expression of hypoxia-inducible factor-1α and CD31 (a glycoprotein expressed on endothelial cells and in platelets)-positive cells. These results indicate that TAM increases the proliferation of mammary tumor cells, which may lead to the stimulation of lung metastasis in the 4T1 mammary carcinoma model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 465. doi:10.1158/1538-7445.AM2011-465