Thioglycollate-elicited Macrophages Research Articles

Role of nitric oxide and signaling pathways modulating the stimulatory effect of snake venom secretory PLA2S on non-opsonized zymosan phagocytosis by macrophages

The phagocytic activity of macrophages activated with MT-II, a Lys-49 PLA2 homolog, and MT-III, an Asp-49 PLA2, from Bothrops asper snake venom, was investigated in this study using a pharmacological approach. Stimulating thioglycollate-elicited macrophages with both venom components enhanced their ability to phagocytose non-opsonized zymosan particles. MT-II and MT–III–induced phagocytosis was drastically inhibited by pretreating cells with L-NAME, aminoguanidine or L-NIL, cNOS or iNOS inhibitors, or with ODQ (sGC inhibitor) or Rp-cGMPS (PKG inhibitor). These results indicate that the NO/sGC/GMP/PKG pathway plays an essential role in the β-glucan-mediated phagocytosis induced in macrophages by these venom-secretory PLA2s.

Light-emitting diode (LED) photobiomodulation exerts anti-inflammatory action in murine thioglycolate-elicited macrophages stimulated by Bothrops jararacussu venom and by isolated PLA2s

Although the treatment currently recommended for snakebite accidents is serum therapy using antivenom, a need for adjunctive therapy associated with serum therapy for treating the local effects caused by snakebites is an effort of the WHO to reduce local signals and symptoms. Photobiomodulation with laser or LED therapy is one of the primary examples of adjuvant therapy to serum therapy to lessen these local effects caused by snakebite envenoming. For this purpose, the project aims to study the action of photobiomodulation with LED therapy in isolated thioglycolate-elicited macrophages stimulated with Bothrops jararacussu venom (BjV) and isolated bothropstoxins BthTX-I and BthTX-II focusing on cell dead mechanism such as necrosis and apoptosis, mitochondrial membrane potential, and cytokines [Interleukin (IL)-1β, IL-10, IL-6], and [tumor necrosis factor (TNF)-α] and lipid mediator [prostaglandin (PG)E2] liberation. Briefly, thioglycollate-elicited macrophages were harvested from Swiss male mice incubated with BjV or BthTXs irradiated or not with LED, and the following parameters were analyzed: necrosis and apoptosis, mitochondrial membrane potential, cytokines, and lipid mediator liberation. Herein, results showed that LED therapy was able to decrease necrosis cell death, caspase-3 activity, and TNF-α liberation. In addition, LED therapy induces mitochondrial membrane potential and modulates gene expression of lipid mediators. In conclusion, the data of this study support the use of phototherapy as an adjuvant therapeutical approach in combination with serum therapy to mitigate the local effects resulting from snakebite envenoming.

Methyl and Isopropyl N-Methylanthranilates Affect Primary Macrophage Function - An Insight into the Possible Immunomodulatory Mode of Action.

To complement the knowledge on the anti-inflammatory activity of methyl and isopropyl N-methylanthranilates, two natural products with panacea-like properties, we investigated their effects on thioglycolate-elicited macrophages by evaluating macrophage ability to metabolize MTT, macrophage membrane function, and macrophage myeloperoxidase and phagocytic activities. Moreover, two additional aspects of the inflammatory response of these compounds, their inhibitory activity on xanthine oxidase and catalase, were studied. It was found that these two compounds regulate elicited macrophage functions, most probably by interfering with the function of cell membranes and changing the reducing cellular capacity or enzyme activity of macrophages. Nonetheless, no significant inhibitory action either towards xanthine oxidase or catalase was found, suggesting that the inhibition of these enzymes is not involved in the anti-inflammatory mode of action of these two esters.

Loss of Mir146b with aging contributes to inflammation and mitochondrial dysfunction in thioglycollate-elicited peritoneal macrophages.

Macrophages undergo programmatic changes with age, leading to altered cytokine polarization and immune dysfunction, shifting these critical immune cells from protective sentinels to disease promoters. The molecular mechanisms underlying macrophage inflammaging are poorly understood. Using an unbiased RNA sequencing (RNA-seq) approach, we identified Mir146b as a microRNA whose expression progressively and unidirectionally declined with age in thioglycollate-elicited murine macrophages. Mir146b deficiency led to altered macrophage cytokine expression and reduced mitochondrial metabolic activity, two hallmarks of cellular aging. Single-cell RNA-seq identified patterns of altered inflammation and interferon gamma signaling in Mir146b-deficient macrophages. Identification of Mir146b as a potential regulator of macrophage aging provides novel insights into immune dysfunction associated with aging.

Adhesion to E-selectin primes macrophages for activation through AKT and mTOR.

The endothelial adhesion protein E-selectin/CD62E is not required for leukocyte homing, unlike closely related family member P-selectin/CD62P. As transmigration through the endothelium is one of the first steps in generating a local immune response, we hypothesized that E-selectin may play additional roles in the early stages of immune activation. We found contact with E-selectin, but not P-selectin or vascular cell adhesion molecule 1 (CD106), induced phosphorylation of protein kinase B (AKT) and nuclear factor-κB in mouse bone marrow-derived macrophages (BMDMs) in vitro. This occurred within 15 min of E-selectin contact and was dependent on phosphatidylinositol-3 kinase activity. Binding to E-selectin activated downstream proteins including mammalian target of rapamycin, p70 ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E-binding protein 1. Functionally, adhesion to E-selectin induced upregulation of CD86 expression and CCL2 secretion. We next asked whether contact with E-selectin impacts further BMDM stimulation. We found enhanced secretion of both interleukin (IL)-10 and CCL2, but not tumor necrosis factor or IL-6 in response to lipopolysaccharide (LPS) stimulation after adhesion to E-selectin. Importantly, adhesion to E-selectin did not polarize BMDMs to one type of response but enhanced both arginase activity and nitric oxide production following IL-4 or LPS stimulation, respectively. In cultured human monocytes, adhesion to E-selectin similarly induced phosphorylation of AKT. Finally, when E-selectin was blocked in vivo in mice, thioglycollate-elicited macrophages showed reduced CD86 expression, validating our in vitro studies. Our results imply functions for E-selectin beyond homing and suggest that E-selectin plays an early role in priming and amplifying innate immune responses.

Bothrops erythromelas venom and its action on isolated murine macrophages

Snakebite is a universally neglected public health problem. It victimizes approximately 2.5 million people annually and kills around 125 thousand. In Brazil, the Bothrops genus is responsible for 87% of the envenoming. The species Bothrops erythromelas is endemic in the northeast region. Its venom induces local haemorrhage, coagulopathy, oedema, and necrosis and can lead to permanent disability or death. The in vitro effects of Bothrops erythromelas venom (BeV) on thioglycollate-elicited macrophages were investigated in this study. At non-cytotoxic concentrations, BeV did not interfere with the adhesion and detachment of thioglycollate-elicited macrophages. However, BeV induced lipid body formation and the activation of respiratory burst and TNF-α, but not IL-1β and IL-6. The study aimed to extend the knowledge on the mechanism of action of BeV and its contribution toward a better characterisation of macrophage functionality under the action of Bothrops venom.

IRF3 signaling in stromal cells contributes to sepsis pathogenesis

Abstract Interferon regulatory factor 3 is a transcription factor that induces type 1 interferons and related inflammatory genes following activation of pattern recognition receptors. Our prior report demonstrates that IRF3-KO mice are substantially protected from sepsis in a lethal mouse model of cecal ligation and puncture (CLP). Here, we demonstrate that IRF3 remains relevant in the context of a CLP model incorporating clinical treatments (lactated ringer’s solution and antibiotics). 92.3% of IRF3-KO mice survived sepsis in this model, compared to 61.5% of wild-type mice. Septic IRF3-KO mice also had an improved disease score and lower levels of serum cytokines (IL-6 and IL-12), relative to wild-type counterparts. To examine if IRF3 exerts its effect within leukocytes, we generated bone marrow chimeras. We found that chimeras with IRF3-KO leukocytes did not exhibit a significant survival advantage over chimeras with wild-type leukocytes. However, chimeras with IRF3-KO stroma exhibited substantially improved survival relative to chimeras with wild-type stroma, paired with lower disease score and serum cytokines (IL-6 and IL-12). In vitro experiments with thioglycollate-elicited macrophages from IRF3-KO and wild-type mice demonstrate that IRF3 does not directly reduce the amount of IL-6 produced in response to cecal bacteria. However, wild-type monocytes that are adoptively transferred into septic hosts produce less IL-6 when the host is IRF3-KO vs wild-type. These experiments reveal the surprising finding that IRF3 exerts its major effect on sepsis pathogenesis through its presence in stromal cells, with indirect effects on leukocytes.

Regular exercise improves inflammatory responses by resident or recruited macrophages against bacterial pathogens

Regular, moderate-intensity exercise is beneficial to host defenses against infections. Such exercise regimes improve the inflammatory and bactericidal potencies of macrophages, cells that are crucial in activating both innate and adaptive immune responses. The M1 type (classical) macrophage activation after recognition of the pathogen-associated molecular patterns, such as lipopolysaccharide (LPS), induces pro-inflammatory responses in infected foci; these responses are necessary to effectively clear the pathogens. We reported previously that a 3-week-long treadmill exercise program enhanced LPS-induced interleukin (IL)‑12 mRNA expression and IL-12 secretion by downregulating β 2 -adrenergic receptor expression in the murine peritoneal resident macrophages, suggesting that regular exercise promotes the Toll-like receptor signaling and expression of its target genes in resident macrophages. We also reported that an 8-week-long, voluntary wheel-running exercise program potentiated LPS-induced IL-1β and IL-18 secretion without affecting the intracellular pro-IL-1β or pro-IL-18 mRNA or protein levels in the thioglycollate-elicited peritoneal murine macrophages. This program increased the intracellular caspase-1 protein levels. These results suggest that regular exercise promotes pro-IL-1β and pro-IL-18 maturation by increasing the levels of the inflammasome-activated caspase-1 in the recruited macrophages. Here, we review our collective studies and compare the mechanistic differences by which regular exercise improves pro-inflammatory responses to bacterial pathogens by resident or recruited macrophages.

Distinct Transcriptional Profile Activated by SCYE1 Through JAK‐mediated STAT3 in Partially Activated Macrophages

Small inducible cytokine subfamily E, member 1, SCYE1 (also known as EMAP II) is a secreted factor that is locally expressed near sites of injury, labeled as a ‘find‐me’ signal, and recruits macrophages and neutrophils. Our previous studies identified that macrophages sustained protein expression of SCYE1. The transcriptional events that occur within the recruited macrophages in response to SCYE1 are unknown. The objective was to identify the transcription factor activated in partially activated thioglycollate macrophages upon exposure to SCYE1.Partially activated murine thioglycollate‐elicited macrophages (TEPMs) were exposed to SCYE1. Following 2 hours of exposure to either vehicle or SCYE1, fractionated nuclear proteins from TEPMs were used for a luminescence based transcription factor activation screen. The acute SCYE1‐mediated transcription factor activation was assessed by immunoblotting and gene expression by quantitative PCR. Secreted cytokines were measured in supernatants of TEPMs exposed to either vehicle or SCYE1 while pre‐treated with +/− JAK1/2 inhibitor, Ruxolitinib.In this study, we identify STAT3 (Signal Transducer and Activator of Transcription 3) as the most activated transcription factor mediated by SCYE1 in a model of partially activated, recruited thioglycollate‐elicited peritoneal macrophages. The acute and dose‐dependent phosphorylation of STAT3 (Y705) occurs between 5 and 15 minutes in response to SCYE1. Its activation is SCYE1‐specific and could be prevented by inhibition of Janus Associated Kinase (JAK) 1/2.We profiled not only anti‐ and pro‐inflammatory genes but also that of STAT3 target genes upon exposure to SCYE1. STAT3 target genes Arg1 and Socs3, commonly activated in anti‐inflammatory macrophages, are maximal by 1 to 2 hours of exposure to SCYE1. Yet Il10 expression decreased by 1 hour suggesting that any persisting STAT3 activation was not due to Il10. There was also rapid increase of chemokine STAT3 target genes Ccl5 along with other genes activated that are typically found in pro‐inflammatory macrophages.Unlike activated transcription, neither secreted IL‐6 nor IL‐10 was elevated by SCYE1. Consistent with a JAK‐STAT signature, both IL‐1a and G‐CSF secretion by SCYE1 was elevated and could be prevented by JAK inhibition.Our results identify a rapid SCYE1‐mediated STAT3 activation that coincides with both pro‐ and anti‐inflammatory gene expression in partially activated macrophages.Support or Funding InformationFunding: This publication was made possible in part by 5R01HL114977 (MAS) from NIH and the Lilly Endowment, Inc. Physician Scientist Initiative (MAS).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Of Mice and Men: Proteasome's Role in LPS-Induced Inflammation and Tolerance.

The molecular basis responsible for tolerance following inflammatory response to lipopolysaccharide (LPS) is not well understood. We hypothesized that inflammation/tolerance in monocytes/ macrophages is dependent on the proteases of proteasome. To test our hypothesis, first, we examined the expression of different proteasome subunits in different human and mouse monocytes/macrophages. Secondly, we investigated the effect of proteasome subunits/ proteases on LPS-induced expression of tumor necrosis factor-α (TNF-α) and nitric oxide (NO) during inflammation and tolerance using mouse RAW 264.7 macrophages, THP1 cells, and cluster of differentiation 14 positive (CD14) human monocytes. We found that RAW 264.7 cells (XYZ), mouse peritoneal resident, thioglycollate-elicited macrophages, primed RAW 264.7 (XYZ, LMP), and human monocytes (LMP) expressed different types of proteasome subunits/activities. Cells containing predominantly either LMP subunits (such as THP-1 and human monocytes), or only X, Y, Z subunits (RAW 264.7 cells not primed) could only induce TNF-α, but not NO, while cells containing all five to six subunits (XYZ, LMP) of the proteasome could induce both mediators in response to LPS. Distinct states of inflammation/tolerance in LPS treated cells, strongly correlated with an upregulation or downregulation of proteasome's subunits (proteases), respectively. Moreover, interferon-γ treatment of tolerant cells caused robust induction of proteasome's subunit expression in mouse macrophages and human monocytes, and cells regained their ability to respond to LPS. These studies are vital for understanding function of proteasome's subunits during inflammation/tolerance in mouse and human cells, and for design of therapeutic strategies for all diseases based on inflammation.

Flippases and Scramblases at Plasma Membranes that Regulate Phosphatidylserine Exposure

One of the hallmarks of apoptosis is the caspase-dependent exposure of phosphatidylserine (PtdSer) on cell surface, which is recognized by macrophages for engulfment of dead cells (1). How PtdSer is exposed to the cell surface had been elusive for a long time. We recently identified two membrane proteins (TMEM16F and Xkr8) that are involved in scrambling of phospholipids in plasma membrane (2, 3). TMEM16F carries 8 transmembrane regions, and requires Ca2+ to mediate phospholipid scrambling. It plays a role in the PtdSer-exposure in activated platelets for blood clotting, and patients of Scott Syndrome who suffer bleeding disorder carry a mutation in TMEM16F gene. Xkr8 is a protein carrying 6 transmembrane regions. Caspase 3 and 7 cleave off the C-terminal tail of Xkr8, and the cleaved Xkr8 promotes the PtdSer-exposure. In addition to the activation of scramblase, the flippase that translocates PtdSer from outer to inner leaflets was thought to be inactivated during apoptosis. In fact, we recently found that a pair of molecules, ATP11C of a P4-type ATPase and its chaperon CDC50A work as a flippase at plasma membrane (4, 5). ATP11C carries three caspase recognition sites in the middle of the molecule, and is cleaved during apoptosis. When ATP11C gene is mutated, the cells lose most of the flippase activity, but the asymmetrical distribution of PtdSer was still maintained at plasma membrane. Whereas, the cells lacking CDC50A completely lost the flippase activity and constitutively exposed PtdSer. The PtdSer-exposing living CDC50A-null cells were engulfed by thioglycollate-elicited macrophages, indicating that PtdSer exposed on the cell surface is necessary and sufficient to be recognized by macrophages for engulfment. Several molecules such as MFG-E8, Tim-4, Gas6, and Protein S specifically bind to PtdSer with high affinity, and promote the engulfment of PtdSer-exposing cells. However, how they work for the engulfment of apoptotic cells in certain macrophages has not been clear. We recently found that that resident peritoneal macrophages require both Tim4 and Protein S for engulfment, and Tim4, PtdSer-receptor, was involved in tethering of apoptotic cells, while Protein S promoted the engulfment of apoptotic cells by binding to MerTK, a tyrosine kinase receptor (6, 7). Here, I discuss how PdtSer is exposed during apoptotic cell death, and how dead cells are engulfed by macrophages. 1. Nagata S, Hanayama R, Kawane K. Autoimmunity and the clearance of dead cells. Cell. 2010;140:619-630. 2. Suzuki J, Umeda M, Sims PJ, Nagata S. Calcium-dependent phospholipid scrambling by TMEM16F. Nature. 2010;468:834-838. 3. Suzuki J, Denning DP, Imanishi E, Horvitz HR, Nagata S. Xk-related protein 8 and CED-8 promote phosphatidylserine exposure in apoptotic cells. Science. 2013;341:403-406. 4. Segawa K, Suzuki J, Nagata S. Flippases and scramblases in the plasma membrane. Cell Cycle. 2014;13:2990-2991. 5. Segawa K, Kurata S, Yanagihashi Y, Brummelkamp T, Matsuda F, Nagata S. Caspase-mediated cleavage of phospholipid flippase for apoptotic phosphatidylserine exposure. Science. 2014;344:1164-1168. 6. Nishi C, Toda S, Segawa K, Nagata S. Tim4- and MerTK-mediated engulfment of apoptotic cells by mouse resident peritoneal macrophages. Mol Cell Biol. 2014;34:1512-1520. 7. Toda S, Segawa K, Nagata S. MerTK-mediated engulfment of pyrenocytes by central macrophages in erythroblastic islands. Blood. 2014;123:3963-3971. DisclosuresNo relevant conflicts of interest to declare.

Aging affects the responsiveness of rat peritoneal macrophages to GM-CSF and IL-4.

Macrophages undergo significant functional alterations during aging. The aim of the present study was to investigate changes of rat macrophage functions and response to M1/M2 polarization signals with age. Therefore, resident and thioglycollate-elicited peritoneal macrophages from young (3-month-old) and aged (18-19-month-old) rats were tested for phagocytic capacity and ability to secrete inflammatory mediators following in vitro stimulation with LPS and GM-CSF, and IL-4, prototypic stimulators for classically (M1) and alternatively activated (M2) macrophages, respectively. Aging increased the frequency of monocyte-derived (CCR7+ CD68+) and the most mature (CD163+ CD68+) macrophages within resident and thioglycollate-elicited peritoneal macrophages, respectively. The ability to phagocyte zymosan of none of these two cell subsets was affected by either LPS and GM-CSF or IL-4. The upregulated production of IL-1β, IL-6 and IL-10 and downregulated that of TGF-β was observed in response to LPS in resident and thioglycollate-elicited macrophages from rats of both ages. GM-CSF elevated production of IL-1β and IL-6 in resident macrophages from aged rats and in thioglycollate-elicited macrophages from young rats. Unexpectedly, IL-4 augmented production of proinflammatory mediators, IL-1β and IL-6, in resident macrophages from aged rats. In both resident and thioglycollate-elicited macrophages aging decreased NO/urea ratio, whereas LPS but not GM-SCF, shifted this ratio toward NO in the macrophages from animals of both ages. Conversely, IL-4 reduced NO/urea ratio in resident and thioglycollate-elicited macrophages from young rats only. In conclusion, our study showed that aging diminished GM-CSF-triggered polarization of elicited macrophages and caused paradoxical IL-4-driven polarization of resident macrophages toward proinflammatory M1 phenotype. This age-related deregulation of macrophage inflammatory mediator secretion and phagocytosis in response to M1/M2 activators may lead to the deficient control of infectious and/or inflammatory diseases in advanced age.

Abstract 4495: Small molecule approach for targeting tumor-associated macrophages via a functional FRβ

Abstract Tumor-associated macrophages (TAMs) are considered an attractive target for oncotherapy because of their important role in tumor microenvironment. While TAMs mostly arise from inflammatory blood monocytes, they become quickly programmed to the pro-tumor phenotype. In response to tumor-derived factors, these M2-biased TAMs are essentially tumor helper cells, as both cancer cells and TAMs promote each another through a paracrine signaling loop involving epidermal growth factor and colony stimulating factor-1. This important cellular interaction is critical at all stages of tumor progression including angiogenesis, invasion, growth and metastasis. In recent years, TAMs (CD68+, CD163+) have been found to express a functional folate receptor-β (FRβ) that displays high-affinity ligand binding and subsequent ligand-initiated endocytosis. FRβ-expressing TAMs are present in various cancer subtypes, the perivascular region, and the tumor invasive front. The abundance of FRβ+ TAMs has been linked to poor patient survival in pancreatic ductal adenocarcinoma. In the present study, we first characterized the phenotype of FRβ+ TAMs ex-vivo from both syngeneic and xenographic tumor models (breast, lung, melanoma, and pancreatic cancers). Using thioglycollate-elicited macrophages as positive controls, we estimated FRβ levels on TAMs with monoclonal antibodies and functional folate ligands. Using a ligand conjugation approach, we focused on targeting FRβ+ TAMs in tumors arising from cancer cells that do not express functional FR themselves. Preliminary proof-of-concept was established in the highly aggressive FR-negative 4T1 mammary tumor with a folate-targeted DNA alkylating agent. In addition, immunohistochemical analysis showed high FRβ expression in human anaplastic thyroid cancer, a TAM-rich disease with very poor prognosis. Collectively, our data suggest that targeting a TAM-rich cancer via FRβ may be a viable therapeutic approach with potential applications in FR-negative tumors. Citation Format: Leroy W. Wheeler, Yingjuan Lu, Vicky Cross, Alex Lloyd, Nikki Parker, Longwu Qi, Kevin Wang, Ian Wang, Spencer Hahn, Jeremy Vaughn, Iontcho P. Vlahov, Philip S. Low, Christopher P. Leamon. Small molecule approach for targeting tumor-associated macrophages via a functional FRβ. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4495. doi:10.1158/1538-7445.AM2015-4495

Erratum: Analysis of infl ammatory and lipid metabolic networks across RAW264.7 and thioglycolate-elicited macrophages (J. Lipid Res. (2013) 54 (2525-2542)

Author(s): Maurya, Mano R; Gupta, Shakti; Li, Xiang; Fahy, Eoin; Dinasarapu, Ashok R; Sud, Manish; Brown, H Alex; Glass, Christopher K; Murphy, Robert C; Russell, David W; Dennis, Edward A; Subramaniam, Shankar

25-Hydroxycholesterol Activates the Integrated Stress Response to Reprogram Transcription and Translation in Macrophages

25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.

An Altered Macrophage Migration In Adipose Tissue Of Integrin Mac-1 (CD11b/CD18) Deficient Mice

It has recently been recognized that obesity, a critical factor contributing to the number of abnormalities constituting the metabolic syndrome, is associated with adipose tissue inflammation which is attributable in large part to the increased numbers of infiltrating macrophages. Integrins and, in particular, the major myeloid-specific integrin αMβ2 (Mac-1) plays an essential role in leukocyte migration. We have found that during the period of 12-36 weeks, Mac-1-/- mice gained more weight on normal chow diet than control mice, which was due to the increase of white fat mass (∼1.7- and 3.3-fold for males and females, respectively) and not a result of increased food intake. To gain insight into the mechanisms that regulate fat storage in Mac-1-/- mice, we determined the number of macrophages present in omental fat band (OFB) which, similar to other fat depots, underwent the mass increase. Unexpectedly, the number of macrophages per mg of OFB was less in Mac-1-/- mice than that in wild-type mice at all ages examined with the largest difference observed at 6 weeks (∼2.9- and 2.5-fold for males and females, respectively), implicating Mac-1 in the control of macrophage influx and/or retention within OFB. To examine the role of Mac-1 in migration, resident and thioglycollate-elicited inflammatory macrophages were isolated from the peritoneum, labeled with PKH-26 fluorescent dye and their migration to and from OFB were determined by measuring tissue fluorescence. For the “walk-in” experiments, OFB was made “empty” by incubating the tissue for 24 h in a cell culture medium to release OFB-associated macrophages and lymphocytes. “Walk-out” experiments were performed by first filling OFB with labeled macrophages and then tracking their emigration from the tissue. The analyses of the kinetics of macrophage migration showed that both resident and inflammatory Mac-1-/- macrophages arrive to and exit from OFB faster than their wild-type counterparts. For example, the number of Mac-1-/- resident and inflammatory macrophages that accumulated in OFB after 4 h was 11±2.8 and 2.4±0.38 -fold higher than that of wild-type cells. Likewise, the rate of emigration from OFB for both resident and inflammatory was higher for Mac-/- macrophages than that of wild-type cells (4.4±0.55 and 2.8±0.15 after 6 h for inflammatory and resident macrophages, respectively). The distinction in migration was not due to the differences in cell adhesive properties as wild-type and Mac-/- macrophages adhered to the same extent to plastic dishes. To examine macrophage migration in vivo, PKH-26-labeled wild-type and Mac-/- macrophages were injected into the peritoneum of wild-type mice and their migration to and emigration from OFB was tracked by determining the fluorescence intensity of OFB excised after selected time points. Similar to their behavior in vitro, both resident and inflammatory Mac-/- macrophages migrated faster to and emigrated from OFB than wild-type cells. In addition, after the arrival to OFB, wild-type macrophages organized in large clusters, a pattern distinct from Mac-/- cells which were scattered on the surface and within the tissue. These results suggest that the reduced number of macrophages observed in OFB of Mac-/- mice may be due to their enhanced mobility and reduced capacity to establish adhesive bonds within the OFB tissue resulting in the reduced retention and increased turnover. Since less macrophages is present in fat in the overweight Mac-/- mice than in normal wild-type mice, the data also indicate that the increase in body fat can be dissociated from the increase in macrophage numbers in fat. Disclosures:No relevant conflicts of interest to declare.

Analysis of inflammatory and lipid metabolic networks across RAW264.7 and thioglycolate-elicited macrophages

Studies of macrophage biology have been significantly advanced by the availability of cell lines such as RAW264.7 cells. However, it is unclear how these cell lines differ from primary macrophages such as thioglycolate-elicited peritoneal macrophages (TGEMs). We used the inflammatory stimulus Kdo2-lipid A (KLA) to stimulate RAW264.7 and TGEM cells. Temporal changes of lipid and gene expression levels were concomitantly measured and a systems-level analysis was performed on the fold-change data. Here we present a comprehensive comparison between the two cell types. Upon KLA treatment, both RAW264.7 and TGEM cells show a strong inflammatory response. TGEM (primary) cells show a more rapid and intense inflammatory response relative to RAW264.7 cells. DNA levels (fold-change relative to control) are reduced in RAW264.7 cells, correlating with greater downregulation of cell cycle genes. The transcriptional response suggests that the cholesterol de novo synthesis increases considerably in RAW264.7 cells, but 25-hydroxycholesterol increases considerably in TGEM cells. Overall, while RAW264.7 cells behave similarly to TGEM cells in some ways and can be used as a good model for inflammation- and immune function-related kinetic studies, they behave differently than TGEM cells in other aspects of lipid metabolism and phenotypes used as models for various disorders such as atherosclerosis.

Peritoneal mast cell degranulation differently affected thioglycollate-induced macrophage phenotype and activity in Dark Agouti and Albino Oxford rats

AimsMacrophages are heterogeneous population of inflammatory cells and, in response to the microenvironment, become differentially activated. The objective of the study was to explore macrophage effector functions during different inflammatory conditions in two rat strains. Main methodsWe have investigated the effects of in vivo treatment with mast cell-degranulating compound 48/80 and/or thioglycollate on peritoneal macrophage phagocytosis and capacity to secrete hydrogen peroxide (H2O2), tumor necrosis factor-α (TNF-α) and nitric oxide (NO) in Dark Agouti (DA) and Albino Oxford (AO) rat strains. Besides, fresh peritoneal cells were examined for the expression of ED1, ED2 and CD86 molecules. Key findingsIn thioglycollate-elicited macrophages, increased proportion of ED1+ cells was accompanied with elevated phagocytosis of zymosan (DA strain), whereas increased expression level of CD86 molecule on ED2+ macrophages matched elevated secretory capacity for H2O2, TNF-α and NO (AO rats). Although mast cell degranulation induced by compound 48/80 increased the percentages of ED2+ macrophages in both rat strains, the proportion of ED2+ cells expressing CD86 molecule was decreased and increased in DA and AO rats, respectively. Furthermore, in DA strain compound 48/80 diminished macrophage secretion of NO, but stimulated all macrophage functions tested in AO strain. If applied concomitantly, the compound 48/80 additively increased macrophage activity induced by thioglycollate in AO rats. SignificanceMacrophages from DA and AO rat strains show different susceptibility to mediators released from mast cells, suggesting that strain-dependant predisposition(s) toward particular activation pattern is decisive for the macrophage efficacy in response to inflammatory agents.

Connexin43 Is Dispensable for Phagocytosis

Macrophages that lack connexin43 (Cx43), a gap junction protein, have been reported to exhibit dramatic deficiencies in phagocytosis. In this study, we revisit these findings using well-characterized macrophage populations. Cx43 knockout (Cx43(-/-)) mice die soon after birth, making the harvest of macrophages from adult Cx43(-/-) mice problematic. To overcome this obstacle, we used several strategies: mice heterozygous for the deletion of Cx43 were crossed to produce Cx43(+/+) (wild type [WT]) and Cx43(-/-) fetuses. Cells isolated from 12- to 14-d fetal livers were used to reconstitute irradiated recipient animals. After reconstitution, thioglycollate-elicited macrophages were collected by peritoneal lavage and bone marrow was harvested. Bone marrow cells and, alternatively, fetal liver cells were cultured in media containing M-CSF for 7-10 d, resulting in populations of cells that were >95% macrophages based on flow cytometry. Phagocytic uptake was detected using flow cytometric and microscopic techniques. Quantification of phagocytic uptake of IgG-opsonized sheep erythrocytes, zymosan particles, and Listeria monocytogenes failed to show any significant difference between WT and Cx43(-/-) macrophages. Furthermore, the use of particles labeled with pH-sensitive dyes showed equivalent acidification of phagosomes in both WT and Cx43(-/-) macrophages. Our findings suggest that modulation of Cx43 levels in cultured macrophages does not have a significant impact on phagocytosis.

Abstract 105: Timely Removal of Lipid Oxidation Products is Mediated by Macrophages.

Reciprocity of inflammation and oxidative stress is now significantly considered as an important mechanism underlying numerous diseases and cancers. As a mechanistic link connecting them, we have shown that one of the end products of lipid oxidation, 2-(ω-carboxyethyl)pyrrole (CEP) is generated during inflammation and wound healing and recognized by Toll-like receptor 2 (TLR2) on endothelial cells. It leads to angiogenesis, which is independent of vascular endothelial growth factor. Importantly, the levels of CEP are reduced back to normal when the wound is healed, but remain constitutively high in pathological conditions, including aging and cancers. Thus, for most tissue repair processes, it appears critical not only to generate CEP, but also to provide its timely removal. Accordingly, our hypothesis is that in order to avoid tissue damage by inflammation-induced oxidative products, CEP needs to be cleared from injured tissues when its role is complete. We have sought to determine a cellular and molecular mechanism of CEP clearance from injured tissues. At first, through CEP binding and internalization assay, comparing with other cell types, we verified that macrophage is the responsible cell type of CEP clearance. Specifically, resident, F4/80 hi , and M2 macrophages are much more efficient for CEP clearance rather than thioglycolate-elicited, F4/80 lo , and M1 macrophages, respectively. Secondly, by using several knockout mouse lines, we identified that both TLR2 and CD36 are largely involved in CEP clearance. To examine the in vivo relevance of CEP clearance besides skin injury model, we used ApoE deficient mice as an atherosclerotic model because atherosclerosis is known to be intimately associated with inflammation and oxidative stress. Using a CEP-specific antibody, we have been screening the CEP appearance and disappearance in early-to-late stages of atherosclerosis. Recently, we examined that CEP is strongly stained only in and adjacent to the atherosclerotic lesion of ApoE deficient mice fed with western diet for 20 weeks, but barely stained in 30 weeks. Moreover, in the plaque, macrophage-enriched region exhibits weak signals of CEP staining. Therefore, we suggest that timely removal of CEP is mediated by macrophages ex vivo and in vivo .