Regulation of chemokine and chemokine receptor expression by PPARγ in adipocytes and macrophages

Background

PPARγ plays a key role in adipocyte biology, and Rosiglitazone (Rosi), a thiazolidinedione (TZD)/PPARγ agonist, is a potent insulin-sensitizing agent. Recent evidences demonstrate that adipose tissue inflammation links obesity with insulin resistance and that the insulin-sensitizing effects of TZDs result, in part, from their anti-inflammatory properties. However the underlying mechanisms are unclear.

Methodology and Principal Findings

In this study, we establish a link between free fatty acids (FFAs) and PPARγ in the context of obesity-associated inflammation. We show that treatment of adipocytes with FFAs, in particular Arachidonic Acid (ARA), downregulates PPARγ protein and mRNA levels. Furthermore, we demonstrate that the downregulation of PPARγ by ARA requires the activation the of Endoplamsic Reticulum (ER) stress by the TLR4 pathway. Knockdown of adipocyte PPARγ resulted in upregulation of MCP1 gene expression and secretion, leading to enhanced macrophage chemotaxis. Rosi inhibited these effects. In a high fat feeding mouse model, we show that Rosi treatment decreases recruitment of proinflammatory macrophages to epididymal fat. This correlates with decreased chemokine and decreased chemokine receptor expression in adipocytes and macrophages, respectively.

Conclusions and Significance

In summary, we describe a novel link between FAs, the TLR4/ER stress pathway and PPARγ, and adipocyte-driven recruitment of macrophages. We thus both describe an additional potential mechanism for the anti-inflammatory and insulin-sensitizing actions of TZDs and an additional detrimental property associated with the activation of the TLR4 pathway by FA.     

Modulation of cisPlatin cytotoxicity by interleukin-1α and resident tumor macrophages

The modulation of cisPlatin cytotoxicity by interleukin-1 (IL-1α) was studied in cultures of SCC-7 tumor cells with and without tumor macrophages to examine potential mechanisms for the synergistic antitumor activity of cisPlatin and IL-1α in SCC-7 solid tumors. Neither IL-1α nor tumor macrophages affected the survival of clonogenic tumor cells and IL-1α had no direct effect on tumor cell growthin vitro. Macrophages had no direct effect on cisPlatin sensitivity (IC₉₀=6.0 μM), but, the addition of IL-1α (500–2000U/ml) to co-cultures of cisPlatin pretreated tumor cells and resident tumor macrophages increased cell killing (IC₉₀=3.1 μM). Similar responses were seen in primary cultures treated with cisPlatin before IL-1α. The modulation of cisPlatin cytotoxicity by IL-1α exhibited a biphasic dose response that paralleled the IL-1α dose dependent release of H₂O₂by resident tumor macrophages. Further, IL-1α modification of cisPlatin cytotoxicity was prompt and inhibited by catalase. CisPlatin and exogenous H₂O₂ (50 μM) produced more than additive SCC-7 clonogenic cell kill and hydroxyl radicals played an important role in the response. Interleukin-1 modulation of cisPlatin cytotoxicity was schedule dependent. IL-1α treatment for 24 hrs, before cisPlatin, produced drug resistance (IC₉₀=11.1 μM). Our study shows that IL-1α can stimulate tumor macrophages to release pro-oxidants that modify cellular chemosensitivity in a schedule and dose dependent fashion. Our findings may also provide a mechanistic explanation for the synergistic antitumor activity of cisPlatin and IL-1αin vivo.     

Binding of β-endorphin and its fragments to the nonopiod receptor of murine peritoneal macrophages

The tritium-labeled selective agonist of the nonopioid β-endorphin receptor the decapeptide immunorphin ([³H]SLTCLVKGFY) with a specific activity of 24 Ci/mmol was prepared. It was shown that [³H]immunorphin binds with a high affinity to the non-opioid β-endorphin receptor of mouse peritoneal macrophages (K _d 2.4 ± 0.1 nM). The specific binding of [³H]immunorphin to macrophages was inhibited by unlabeled β-endorphin (K _i of the [³H]immunorphin-receptor complex 2.9 ± 0.2 nM) and was not inhibited by unlabeled naloxone, α-endorphin, γ-endorphin, and [Met⁵]enkephalin (K _i > 10 μM). Thirty fragments of β-endorphin were synthesized, and their ability to inhibit the specific binding of [³H]immunorphin to macrophages was studied. It was found that the shortest peptide having practically the same inhibitory activity as β-endorphin is its fragment 12–19 (K _i 3.1 ± 0.3 nM).     

Inhibition of proliferation of retrovirus-immortalized macrophages by LPS and IFN-γ: Possible autocrine down-regulation of cell growth by induction of IL1 and TNF

The GG₂EE macrophage tumor cell line was previously established by immortalization of C3H/HeJ mouse bone marrow cells with the J2 retrovirus which contains the v-myc and v-raf oncogenes. Studies on the control of GGZEE cell proliferationin vitro have recently been performed. We observed that the combination of 5–25 U/ml recombinant mouse interferon- (rmIFN-) plus 0.03 – 0.3 µg/ml lipopolysaccharide (LPS) markedly inhibited the proliferation of GG₂EE cells (by >95%)in vitro, while either agent alone inhibited only by <40% and 0–10%, respectively. Subsequent studies established that biologically active ILI-like (2–4 U/ml) and TNF-like (50–100 U/ml) activities were released into the supernatants of LPS-treated GG₂EE cells. The combination of IFN- + LPS induced more (6–8 U/ml) ILI release. These results suggested that the inhibition of proliferation of GG₂EE cells by IFN- + LPS could have been mediated in part by cytokines produced by the cells themselves. rhIL1 at a concentration of 10 U/ml inhibited GG₂EE proliferation by 25–30%, while rmIFN- (25 U/ml) + rhIL1 (10 U/ml) inhibited proliferation by 98%. Thus, 10 U/ml rhIL1 could completely replace LPS in the LPS + rmIFN- combination. Further, the combination of low doses of rhIL1 (0.1 to 1 U/ml) plus rmTNF (250 U/ml), which together inhibited proliferation by <20% synergized with doses of 5 to 25 U/ml rmIFN- to inhibit proliferation of GG₂EE cells by 98–99%. These results suggest that cytokines produced by the cells themselves can synergize with rmIFN- to inhibit the oncogene-driven proliferation of GG₂EE cells.     

Suppression of TNF-α and IL-1 signaling identifies a mechanism of homeostatic regulation of macrophages by IL-27

IL-27 is a pleiotropic cytokine with both activating and inhibitory functions on innate and acquired immunity. IL-27 is expressed at sites of inflammation in cytokine-driven autoimmune/inflammatory diseases, such as rheumatoid arthritis, psoriasis, inflammatory bowel disease, and sarcoidosis. However, its role in modulating disease pathogenesis is still unknown. In this study, we found that IL-27 production is induced by TNF-α in human macrophages (MΦ) and investigated the effects of IL-27 on the responses of primary human MΦ to the endogenous inflammatory cytokines TNF-α and IL-1. In striking contrast to IL-27-mediated augmentation of TLR-induced cytokine production, we found that IL-27 suppressed MΦ responses to TNF-α and IL-1β, thus identifying an anti-inflammatory function of IL-27. IL-27 blocked the proximal steps of TNF-α signaling by downregulating cell-surface expression of the signaling receptors p55 and p75. The mechanism of inhibition of IL-1 signaling was downregulation of the ligand-binding IL-1RI concomitant with increased expression of the receptor antagonist IL-1Ra and the decoy receptor IL-1RII. These findings provide a mechanism for suppressive effects of IL-27 on innate immune cells and suggest that IL-27 regulates inflammation by limiting activation of MΦ by inflammatory cytokines while preserving initial steps in host defense by augmenting responses to microbial products.     

Generation of retroviruses for the overexpression of cytosolic and mitochondrial glutathione reductase in macrophages in vivo

Retroviral gene transfer and bone marrow transplantation has been used by many investigators to study the role of macrophage proteins in different mouse models of human disease. While this approach is faster and less expensive than generating transgenic mice with macrophage-specific promoters and applicable to a wider array of mouse models, it has been hampered by two major drawbacks: labor-intensive cloning procedures involved in generating retroviral vectors for each gene of interest and low viral titers. Here we describe the construction of a MSCV-based retroviral vector that can serve as an acceptor vector for commercially available Cre-lox-compatible donor vectors. Using this new retroviral vector in combination with a FACS approach to enhance viral titers, we generated high-titer retroviruses carrying either EGFP-tagged cytosolic or EGFP-tagged mitochondria-targeted glutathione reductase. We show that the introduction of these constructs via retroviral gene transfer and bone marrow transplantation into atherosclerosis-prone LDL receptor-null mice results in the long-term increase in macrophage glutathione reductase activity.     

Tumor necrosis factor–related apoptosis-inducing ligand induces the expression of proinflammatory cytokines in macrophages and re-educates tumor-associated macrophages to an antitumor phenotype

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is a promising candidate for cancer therapy, because it can induce apoptosis in various tumor cells but not in most normal cells. Although it is well known that TRAIL and its receptors are expressed in many types of normal cells, including immune cells, their immunological effects and regulatory mechanisms are still obscure. In the present study, we demonstrated that TRAIL affected the activity of NF-κB (nuclear factor-κB) and the expression of its downstream proinflammatory cytokines IL-1β (interleukin-1β), IL-6, and tumor necrosis factor α in macrophages. TRAIL also induced microRNA-146a (miR-146a) expression in an NF-κB–dependent manner. As a result, miR-146a was involved as a negative-feedback regulator in the down-regulation of proinflammatory cytokine expression. In addition, the suppression of histone deacetylase (HDAC) activities by trichostatin A improved miR-146a expression due to the up-regulation of the DNA-binding activity of NF-κB at the miR-146a promoter in TRAIL-induced macrophages, suggesting that histone acetylation was involved in the suppression of miR-146a expression. Further investigation revealed that the HDAC subtype HDAC1 directly regulated the expression of miR-146a in TRAIL-stimulated macrophages. Finally, the TRAIL-sensitive human non small cell lung carcinoma cell line NCI-H460 was used to elucidate the physiological significance of TRAIL with respect to tumor-associated macrophages (TAMs). We demonstrated that TRAIL re-educated TAMs to an M1-like phenotype and induced cytotoxic effects in the tumor cells. These data provide new evidence for TRAIL in the immune regulation of macrophages and may shed light on TRAIL-based antitumor therapy in human patients.     

Maintenance of the activation of peritoneal macrophages in patients with ovarian cancer by sizofiran and recombinant interferon-γ

Four patients with ovarian cancer received 20 mg of sizofiran, a -1,3-glucan (molecular weight: 450,000), intramuscularly one day before and 4, 7, 11, 14, 18 and 21 days after second look laparotomy and recombinant interferon- (rIFN-) intraperitoneally on the day of second look laparotomy and 4, 7, 11, 14, 18 and 21 days thereafter.The peritoneal cavity was washed with physiological saline and peritoneal macrophages (Mø) were isolated. The number of Mø increased 30-1600 times during the treatment period. The concentrations of interleukin-1, interferon-, tumor necrosis factor and prostaglandin E₂ were also found increased in the supernatant fluid of Mø cultured for 24 hours with 10 µg/ml of lipopolysaccharide. The present study demonstrated that the activation of peritoneal Mø could be maintained and its number was increased by repeated dosing of sizofiran and rIFN- in combination every three or four days in patients with ovarian cancer. Peritoneal Mø thus activated may exert an antitumor effect on ovarian cancer.     

Phase I trial of adoptive immunotherapy of cancer patients using monocyte-derived macrophages activated with interferon γ and lipopolysaccharide

 Cells of the monocyte/macrophage lineage have shown antitumor activity in vitro and in murine models after activation with interferon (IFN) γ. In vitro data suggest an additional effect on macrophage antitumor activity when IFNγ is combined with endotoxin (lipopolysaccharides; LPS). In this study we treated nine cancer patients with a total of 62 MAK infusion cycles with autologous macrophages given intravenously (i.v.) after in vitro activation with IFNγ and LPS. Low-grade fever (WHO I/II) was the commonest side-effect. Chills, nausea, and headache were noted when the number of transfused macrophages exceeded 2×10⁸. One WHO IV toxicity occurred, consisting of hypotension after transfer of 3×10⁸ cells, defining this dose as the maximum cell number tolerated. After pretreatment with ibuprofen, however, the maximum cell number could be increased without reaching dose-limiting toxicity. The highest number of cells reinfused was 15×10⁸. Circulating interleukin(IL)-6 increased in a dose-dependent manner as did IL-1 receptor antagonist (IL-1RA) and IL-8. Tumor response consisted of one case of stable disease (12 weeks) in a patient with formerly progressing colorectal cancer and progressive diseases in eight patients. This study indicates that reinfusion of autologous LPS-activated macrophages upon pretreatment with ibuprofen is feasible and tolerated without major side-effects. Received: 22 May 1997 / Accepted: 2 October 1997     

Activation of peritoneal macrophages in patients with gynecological malignancies by sizofiran and recombinant interferon-γ

We investigated the influence of the combined use of sizofiran, a-1,3-glucan and a recombinant interferon- (rIFN-) upon biological activities of peritoneal macrophages (M). The number of peritoneal M and the production of cytokines (interleukin-1, interferon- and tumor necrosis factor) was increased by the combined treatment. Fully activated peritoneal M based on the increased number of elongated pseudopods were observed by electromicroscope. Sizofiran seems to assure a sufficient supply of M to kill tumor cells in the peritoneal cavity and co-administered rIFN- seems to directly stimulate the accumulated M in addition to its direct cytotoxicity against tumor cells. This combination therapy may be a step to the prevention of the recurrence of gynecological malignancies including ovarian cancer, after a negative second-look laparotomy.Abbreviations rIFN- recombinant interferon- - IL-1 interleukin-1 - TNF tumor necrosis factor - SLL second look laparotomy     

Inhibition of Rac and Rac-linked functions by 8-oxo-2′-deoxyguanosine in murine macrophages

Rac is a protein involved in the various functions of macrophages (Mφ), including the production of reactive oxygen species (ROS), phagocytosis, chemotaxis and the secretion of cytokines (such as γ-INF). This study tested the effects of nucleosides containing 8-oxoguanine(8-hydroxyguanine) such as 8-oxo-2′-guanosine (8-oxoG) or 8-oxo-2′-deoxyguanosine (8-oxodG), on Rac and the above-listed Rac-associated functions of Mφ using mouse peritoneal Mφ (MpMφ). It is reported that 8-oxodG was able to effectively inhibit Rac and the Rac-associated functions of MpMφ. Compared to 8-oxodG, 8-oxoG showed negligible effects. Furthermore, normal nucleosides such as deoxyguanosine (dG), guanosine (G) and adenosine (A) did not exert any effects. These results suggest that 8-oxodG could be used as a potential tool to modulate the functions of Mφ that are intimately related to various pathological processes.     

Overexpression of TGF-ß1 in macrophages reduces and stabilizes atherosclerotic plaques in ApoE-deficient mice

Although macrophages represent the hallmark of both human and murine atherosclerotic lesions and have been shown to express TGF-ß1 (transforming growth factor β1) and its receptors, it has so far not been experimentally addressed whether the pleiotropic cytokine TGF-ß1 may influence atherogenesis by a macrophage specific mechanism. We developed transgenic mice with macrophage specific TGF-ß1 overexpression, crossed the transgenics to the atherosclerotic ApoE (apolipoprotein E) knock-out strain and quantitatively analyzed both atherosclerotic lesion development and composition of the resulting double mutants. Compared with control ApoE^−/− mice, animals with macrophage specific TGF-ß1 overexpression developed significantly less atherosclerosis after 24 weeks on the WTD (Western type diet) as indicated by aortic plaque area en face (p<0.05). Reduced atherosclerotic lesion development was associated with significantly less macrophages (p<0.05 after both 8 and 24 weeks on the WTD), significantly more smooth muscle cells (SMCs; p<0.01 after 24 weeks on the WTD), significantly more collagen (p<0.01 and p<0.05 after 16 and 24 weeks on the WTD, respectively) without significant differences of inner aortic arch intima thickness or the number of total macrophages in the mice pointing to a plaque stabilizing effect of macrophage-specific TGF-ß1 overexpression. Our data shows that macrophage specific TGF-ß1 overexpression reduces and stabilizes atherosclerotic plaques in ApoE-deficient mice.     

Suppression of LPS-induced inflammatory and NF-κB responses by anomalin in RAW 264.7 macrophages

The treatment of inflammatory diseases today is largely based on interrupting the synthesis or action of the mediators that drive the host's response to injury. It is on the basis of this concept that most of the anti-inflammatory drugs have been developed. In our continuous search for novel anti-inflammatory agents from traditional medicinal plants, Saposhnikovia divaricata has been a focus of our investigations. Anomalin, a pyranocoumarin constituent of S. divaricata, exhibits potent anti-inflammatory activity. To clarify the cellular signaling mechanisms underlying the anti-inflammatory action of anomalin, we investigated the effect of anomalin on the production of inflammatory molecules in LPS-stimulated murine macrophages. The anomalin dose-dependently inhibited inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA and protein expression in LPS-stimulated RAW 264.7 macrophage. Molecular analysis using quantitative real time polymerase chain reaction (qRT-PCR) revealed that several pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), were reduced by anomalin, and this reduction correlated with the down-regulation of the NF-κB signaling pathway. In addition, anomalin suppressed the LPS-induced phosphorylation and degradation of IκBα. To further study the mechanisms underlying its anti-inflammatory activity, an electrophoretic mobility shift assay (EMSA) using a (32) P-labeled NF-κB probe was conducted. LPS-induced NF-κB DNA binding was drastically abolished by anomalin. The present data suggest that anomalin is a major anti-inflammatory agent and may be a potential therapeutic candidate for the treatment of inflammatory disorders.     

Spleen and liver pigmented macrophages of Rana esculenta L. A new melanogenic system?

The present study reports the results of a morpho-functional analysis of spleen pigmented cells from Rana esculenta L. and comparison with liver melanin-synthesizing cells, belonging to the macrophage cell lineage. Cytological and cytochemical analyses show that parenchymal pigmented cells of the spleen, like those of the liver, are positive to peroxidase and lipase reactions and have phagocytic properties. The observation of premelanosomes in various stages of differentiation, together with the demonstration of dopa oxidase activity in the melanosome proteins, indicate that spleen pigmented macrophages have endogenous melanogenic ability as do liver pigmented macrophages. Attempts to demonstrate tyrosinehydroxylase activity in melanosome protein extracts from frog spleen and liver, using the same protocol as for mammalian tyrosinases, gave negative results. As regards the dopa oxidase activity revealed, some of its properties differ from the typical behaviour observed for tyrosinases from different sources. Peroxidase activity is shown in spleen and liver melanosome proteins with p-phenylenediamine-pyrocatechol (PPD-PC), and not with typical peroxidase substrates. Suitable inhibition tests revealed that dopa oxidase and peroxidase activities might be supported by two different proteins. Liver melanosome extracts display a very strong laccase (dimethoxyphenoloxidase) activity but spleen extracts do not. Differences observed in the enzymatic properties of the spleen and liver melanosomes suggest that pigmented macrophages may undergo tissue-specific differentiation. These preliminary data show that the melanin pathway of pigmented macrophages is different from that of melanocytes and may pave the way to identification of a new melanogenic pathway in vertebrates.     

Lipid synthesis in macrophages during inflammation in vivo: Effect of agonists of peroxisome proliferator activated receptors α and γ and of retinoid X receptors

The effects of peroxisome proliferator activated receptors α and γ (PPAR-α and PPAR-γ) and retinoid X receptor (RXR) agonists upon synthesis and accumulation of lipids in murine C57B1 macrophages during inflammation induced by injection of zymosan and Escherichia coli lipopolysaccharide (LPS) have been studied. It is significant that intraperitoneal injection of zymosan (50 mg/kg) or LPS (0.1 mg/kg) in mice led to a dramatic increase of [¹⁴C]oleate incorporation into cholesteryl esters and triglycerides and [¹⁴C]acetate incorporation into cholesterol and fatty acids in peritoneal macrophages. Lipid synthesis reached its maximum rate 18–24 h after injection and was decreased 5–7 days later to control level after LPS injection or was still heightened after zymosan injection. In macrophages obtained in acute phase of inflammation (24 h), degradation of ¹²⁵I-labeled native low density lipoprotein (NLDL) was 4-fold increased and degradation of ¹²⁵I-labeled acetylated LDL (AcLDL) was 2–3-fold decreased. Addition of NLDL (50 μg/ml) or AcLDL (25 μg/ml) into the incubation medium of activated macrophages induced 9–14-and 1.25-fold increase of cholesteryl ester synthesis, respectively, compared with control. Addition of NLDL and AcLDL into the incubation medium completely inhibited cholesterol synthesis in control macrophages but had only slightly effect on cholesterol synthesis in activated macrophages. Injection of RXR, PPAR-α, or PPAR-γ agonists—9-cis-retinoic acid (5 mg/kg), bezafibrate (10 mg/kg), or rosiglitazone (10 mg/kg), respectively—30 min before zymosan or LPS injection led to significant decrease of lipid synthesis. Ten hour preincubation of activated in vivo macrophages with the abovementioned agonists (5 μM) decreased cholesteryl ester synthesis induced by NLDL and AcLDL addition into the cell cultivation medium. The data suggest that RXR, PPAR-α, or PPAR-γ agonists inhibited lipid synthesis and induction of cholesteryl ester synthesis in inflammatory macrophages caused by capture of native or modified LDL. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 3, pp. 364–374.     

Regulation of phagocytic process of macrophages by noradrenaline and its end metabolite 4-hydroxy-3-metoxyphenyl-glycol. Role of α- and β- adrenoreceptors

The regulatory capacity of noradrenaline and its end metabolite 4-hydroxy-3-metoxyphenylglycol (HMPG) on the complete phagocytic process of macrophages were investigated. Either noradrenaline or HMPG did not modify adherence. However, 10^–12 M of noradrenaline stimulated the chemotaxis of macrophages, mainly mediated by -adrenergic receptors. In contrast, 10^–12 M of HMPG induced an opposed effect on this stage of the phagocytic process. To stimulate phagocytosis, it is necessary to employ a higher concentration (10^–5 M) of noradrenaline and this effect was blocked with either 10^–6 M propranolol or 10^–6 M phentolamine, and maintained by HMPG. Noradrenaline and HMPG did not modify the microbicide capacity of macrophages (measured by O₂ ^– production after phagocytosis). In conclusion, noradrenaline modulates the phagocytic process of macrophages, and this modulation is completed by HMPG, maintaining the phagocytic functions at physiologically optimal levels. Modulation of chemotaxis is mainly mediated by a-receptors and phagocytosis needs both - and -receptor-stimulation.     

Visualization of actin polymerization in invasive structures of macrophages and carcinoma cells using photoconvertible β-actin-Dendra2 fusion proteins

Actin polymerization controls a range of cellular processes, from intracellular trafficking to cell motility and invasion. Generation and elongation of free barbed ends defines the regions of actively polymerizing actin in cells and, consequently, is of importance in the understanding of the mechanisms through which actin dynamics are regulated. Herein we present a method that does not involve cell permeabilization and provides direct visualization of growing barbed ends using photoswitchable β-actin-Dendra2 constructs expressed in murine macrophage and rat mammary adenocarcinoma cell lines. The method exploits the ability of photoconverted (red) G-actin species to become incorporated into pre-existing (green) actin filaments, visualized in two distinct wavelengths using TIRF microscopy. In growing actin filaments, photoconverted (red) monomers are added to the barbed end while only green monomers are recycled from the pointed end. We demonstrate that incorporation of actin into intact podosomes of macrophages occurs constitutively and is amenable to inhibition by cytochalasin D indicating barbed end incorporation. Additionally, actin polymerization does not occur in quiescent invadopodial precursors of carcinoma cells suggesting that the filaments are capped and following epidermal growth factor stimulation actin incorporation occurs in a single but extended peak. Finally, we show that Dendra2 fused to either the N- or the C-terminus of β-actin profoundly affects its localization and incorporation in distinct F-actin structures in carcinoma cells, thus influencing the ability of monomers to be photoconverted. These data support the use of photoswitchable actin-Dendra2 constructs as powerful tools in the visualization of free barbed ends in living cells.     

Elevated COX2 expression and PGE2 production by downregulation of RXRα in senescent macrophages

Increased systemic level of inflammatory cytokines leads to numerous age-related diseases. In senescent macrophages, elevated prostaglandin E2 (PGE2) production contributes to the suppression of T cell function with aging, which increases the susceptibility to infections. However, the regulation of these inflammatory cytokines and PGE2 with aging still remains unclear. We have verified that cyclooxygenase (COX)-2 expression and PGE2 production are higher in LPS-stimulated macrophages from old mice than that from young mice. Downregulation of RXRα, a nuclear receptor that can suppress NF-κB activity, mediates the elevation of COX2 expression and PGE2 production in senescent macrophages. We also have found less induction of ABCA1 and ABCG1 by RXRα agonist in senescent macrophages, which partially accounts for high risk of atherosclerosis in aged population. Systemic treatment with RXRα antagonist HX531 in young mice increases COX2, TNF-α, and IL-6 expression in splenocytes. Our study not only has outlined a mechanism of elevated NF-κB activity and PGE2 production in senescent macrophages, but also provides RXRα as a potential therapeutic target for treating the age-related diseases.     

Rapid recruitment and activation of macrophages by anti-Gal/α-Gal liposome interaction accelerates wound healing

Macrophages are pivotal in promoting wound healing. We hypothesized that topical application of liposomes with glycolipids that carry Galα1-3Galβ1-4GlcNAc-R epitopes (α-gal liposomes) on wounds may accelerate the healing process by rapid recruitment and activation of macrophages in wounds. Immune complexes of the natural anti-Gal Ab (constituting ～1% of Ig in humans) bound to its ligand, the α-gal epitope on α-gal liposomes would induce local activation of complement and generation of complement chemotactic factors that rapidly recruit macrophages. Subsequent binding of the Fc portion of anti-Gal coating α-gal liposomes to FcγRs on recruited macrophages may activate macrophage genes encoding cytokines that mediate wound healing. We documented the efficacy of this treatment in α1,3galactosyltrasferase knockout mice. In contrast to wild-type mice, these knockout mice lack α-gal epitopes and can produce the anti-Gal Ab. The healing time of excisional skin wounds treated with α-gal liposomes in these mice is twice as fast as that of control wounds. Moreover, scar formation in α-gal liposome-treated wounds is much lower than in physiologic healing. Additional sonication of α-gal liposomes resulted in their conversion into submicroscopic α-gal nanoparticles. These α-gal nanoparticles diffused more efficiently in wounds and further increased the efficacy of the treatment, resulting in 95-100% regeneration of the epidermis in wounds within 6 d. The study suggests that α-gal liposome and α-gal nanoparticle treatment may enhance wound healing in the clinic because of the presence of high complement activity and high anti-Gal Ab titers in humans.