Lipocalin-2 Receptor Research Articles

Lipocalin-2 expression identifies an intestinal regulatory neutrophil population during acute graft-versus-host disease.

Acute graft-versus-host disease (aGVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation (allo-HCT), for which therapeutic options are limited. Strategies to promote intestinal tissue tolerance during aGVHD may improve patient outcomes. Using single-cell RNA sequencing, we identified a lipocalin-2 (LCN2)-expressing neutrophil population in mice with intestinal aGVHD. Transfer of LCN2-overexpressing neutrophils or treatment with recombinant LCN2 reduced aGVHD severity, whereas the lack of epithelial or hematopoietic LCN2 enhanced aGVHD severity and caused microbiome alterations. Mechanistically, LCN2 induced insulin-like growth factor 1 receptor (IGF-1R) signaling in macrophages through the LCN2 receptor SLC22A17, which increased interleukin-10 (IL-10) production and reduced major histocompatibility complex class II (MHCII) expression. Transfer of LCN2-pretreated macrophages reduced aGVHD severity but did not reduce graft-versus-leukemia effects. Furthermore, LCN2 expression correlated with IL-10 expression in intestinal biopsies in multiple cohorts of patients with aGVHD, and LCN2 induced IGF-1R signaling in human macrophages. Collectively, we identified a LCN2-expressing intestinal neutrophil population that reduced aGVHD severity by decreasing MHCII expression and increasing IL-10 production in macrophages. This work provides the foundation for administration of LCN2 as a therapeutic approach for aGVHD.

Lipocalin-2-mediated astrocyte pyroptosis promotes neuroinflammatory injury via NLRP3 inflammasome activation in cerebral ischemia/reperfusion injury

BackgroundNeuroinflammation is a vital pathophysiological process during ischemic stroke. Activated astrocytes play a major role in inflammation. Lipocalin-2 (LCN2), secreted by activated astrocytes, promotes neuroinflammation. Pyroptosis is a pro-inflammatory form of programmed cell death that has emerged as a new area of research in stroke. Nevertheless, the potential role of LCN2 in astrocyte pyroptosis remains unclear.MethodsAn ischemic stroke model was established by middle cerebral artery occlusion (MCAO) in vivo. In this study, in vitro, oxygen–glucose deprivation and reoxygenation (O/R) were applied to cultured astrocytes. 24p3R (the LCN2 receptor) was inhibited by astrocyte-specific adeno-associated virus (AAV-GFAP-24p3Ri). MCC950 and Nigericin sodium salt (Nig) were used to inhibit or promote the activation of NLRP3 inflammasome pharmacologically, respectively. Histological and biochemical analyses were performed to assess astrocyte and neuron death. Additionally, the neurological deficits of mice were evaluated.ResultsLCN2 expression was significantly induced in astrocytes 24 h after stroke onset in the mouse MCAO model. Lcn2 knockout (Lcn2−/−) mice exhibited reduced infarct volume and improved neurological and cognitive functions after MCAO. LCN2 and its receptor 24p3R were colocalized in astrocytes. Mechanistically, suppression of 24p3R by AAV-GFAP-24p3Ri alleviated pyroptosis-related pore formation and the secretion of pro-inflammatory cytokines via LCN2, which was then reversed by Nig-induced NLRP3 inflammasome activation. Astrocyte pyroptosis was exacerbated in Lcn2−/− mice by intracerebroventricular administration of recombinant LCN2 (rLCN2), while this aggravation was restricted by blocking 24p3R or inhibiting NLRP3 inflammasome activation with MCC950.ConclusionLCN2/24p3R mediates astrocyte pyroptosis via NLRP3 inflammasome activation following cerebral ischemia/reperfusion injury.

Targeting Oncolytic Adenoviruses to Cancer Cells Using a Designed Ankyrin Repeat Protein Lipocalin-2 Fusion Protein.

Oncolytic viruses are a promising technology to attack cancer cells and to recruit immune cells to the tumor site. Since the Lipocalin-2 receptor (LCN2R) is expressed on most cancer cells, we used its ligand LCN2 to target oncolytic adenoviruses (Ads) to cancer cells. Therefore, we fused a Designed Ankyrin Repeat Protein (DARPin) adapter binding the knob of Ad type 5 (knob5) to LCN2 to retarget the virus toward LCN2R with the aim of analyzing the basic characteristics of this novel targeting approach. The adapter was tested in vitro with Chinese Hamster Ovary (CHO) cells stably expressing the LCN2R and on 20 cancer cell lines (CCLs) using an Ad5 vector encoding luciferase and green fluorescent protein. Luciferase assays with the LCN2 adapter (LA) showed 10-fold higher infection compared with blocking adapter (BA) in CHO cells expressing LCN2R and in cells not expressing the LCN2R. Most CCLs showed an increased viral uptake of LA-bound virus compared with BA-bound virus and for five CCLs viral uptake was comparable to unmodified Ad5. Flow cytometry and hexon immunostainings also revealed increased uptake of LA-bound Ads compared with BA-bound Ads in most tested CCLs. Virus spread was studied in 3D cell culture models and nine CCLs showed increased and earlier fluorescence signals for LA-bound virus compared with BA-bound virus. Mechanistically, we show that the LA increases viral uptake only in the absence of its ligand Enterobactin (Ent) and independently of iron. Altogether, we characterized a novel DARPin-based system resulting in enhanced uptake demonstrating potential for future oncolytic virotherapy.

Lipocalin-2 activates hepatic stellate cells and promotes nonalcoholic steatohepatitis in high-fat diet-fed Ob/Ob mice.

In obesity and type 2 diabetes mellitus, leptin promotes insulin resistance and contributes to the progression of NASH via activation of hepatic stellate cells (HSCs). However, the pathogenic mechanisms that trigger HSC activation in leptin-deficient obesity are still unknown. This study aimed to determine how HSC-targeting lipocalin-2 (LCN2) mediates the transition from simple steatosis to NASH. Male wild-type (WT) and ob/ob mice were fed a high-fat diet (HFD) for 20 weeks to establish an animal model of NASH with fibrosis. Ob/ob mice were subject to caloric restriction or recombinant leptin treatment. Double knockout (DKO) mice lacking both leptin and lcn2 were also fed an HFD for 20 weeks. In addition, HFD-fed ob/ob mice were treated with gadolinium trichloride to deplete Kupffer cells. The LX-2 human HSCs and primary HSCs from ob/ob mice were used to investigate the effects of LCN2 on HSC activation. Serum and hepatic LCN2 expression levels were prominently increased in HFD-fed ob/ob mice compared with normal diet-fed ob/ob mice or HFD-fed WT mice, and these changes were closely linked to liver fibrosis and increased hepatic α-SMA/matrix metalloproteinase 9 (MMP9)/signal transducer and activator of transcription 3 (STAT3) protein levels. HFD-fed DKO mice showed a marked reduction of α-SMA protein compared with HFD-fed ob/ob mice. In particular, the colocalization of LCN2 and α-SMA was increased in HSCs from HFD-fed ob/ob mice. In primary HSCs from ob/ob mice, exogenous LCN2 treatment induced HSC activation and MMP9 secretion. By contrast, LCN2 receptor 24p3R deficiency or a STAT3 inhibitor reduced the activation and migration of primary HSCs. LCN2 acts as a key mediator of HSC activation in leptin-deficient obesity via α-SMA/MMP9/STAT3 signaling, thereby exacerbating NASH.

Lipocalin‐2 Stimulates Proliferation and a Profibrotic Cholangiocyte Phenotype during Cholestasis

Lipocalin‐2 (LCN2), also known as neutrophil gelatinase‐associated lipocalin (NGAL), is a secreted glycoprotein that has been shown to regulate cell proliferation, innate immunity, metabolism, and tumor metastasis in several cell types. LCN2 expression is elevated in hepatic tissues in experimental liver injury models and patients with alcoholic hepatitis. LCN2 has been shown to stimulate proliferation and metastasis of cholangiocarcinoma. However, the role of cholangiocyte LCN2 and LCN2 receptors (NGALR/24p3‐R) during cholestasis and primary sclerosing cholangitis (PSC) has not been evaluated. Therefore, we aimed to assess the role of LCN2 and its receptor, 24p3‐R, in acute and chronic cholestatic models.MethodsLCN2 and 24p3‐R were evaluated in liver sections, total liver, and isolated cholangiocytes from wild‐type (WT; C57) normal and BDL (7 days) mice and WT (FVB/NJ) and Mdr2‐/‐ mice (model of primary sclerosing cholangitis, PSC) by immunohistochemistry and qPCR, respectively. LCN2 levels were evaluated by ELISA in cholangiocyte supernatants (6 hrs) collected from animal models and serum from BDL and Mdr2‐/‐ and WT controls and late‐stage PSC patients and healthy human controls. Serum bile acid (BA) levels were measured using a commercial BA assay kit in BDL, Mdr2‐/‐, and control mice. In vitro, both human (H69) and mouse pooled (MPC) cholangiocytes were treated with either 50 ng/ml LCN2 or 100 mM taurocholic acid (TCA) for 24 hrs after which the expression of proliferation, fibrosis, LCN2, and 24p3‐R genes was determined via qPCR.ResultsThere were significant increases in LCN2 and 24p3‐R immunostaining in BDL mice compared to controls. By qPCR significant increases were observed for both LCN2 and 24p3‐R in total liver and isolated cholangiocytes from BDL and Mdr2‐/‐ mice compared to WT. Increased levels of LCN2 were found in serum from PSC patients and serum and supernatants from isolated cholangiocytes from BDL and Mdr2‐/‐ mice compared to control. In vitro, MPC cells treated with LCN2 had increases in fibrotic marker Col1a1, proliferation marker PCNA, and inflammatory markers IL‐1b and IL‐6. Also, significant increases were seen in fibrotic markers (aSMA, FN‐1, and TGF‐b1) and LCN2 receptor 24p3‐R in H69 cells treated with LCN2 compared to untreated cells. As expected, increased serum BA levels were observed in both BDL and Mdr2‐/‐ mice compared to controls. H69 cells treated with TCA also had significant increases in FN‐1, TGF‐b1, and the LCN2 receptor 24p3‐R, indicating that BA may regulate 24p3‐R expression.ConclusionLCN2 and 24p3‐R expression is elevated in cholestatic models and stimulates a profibrotic cholangiocyte phenotype. These findings suggest that LCN2 may play a crucial role in the pathogenesis of cholestatic liver injury.

Silencing of lipocalin-2 and its receptor improved cardiomyocytes viability via decreasing iron uptake, mitochondrial fission, mitophagy and apoptosis under iron overload condition

Abstract Background Iron overload cardiomyopathy is a common cause of death in iron overload patients. L-type calcium channels (LTCC) and T-type calcium channels (TTCC) have been shown to play important roles for iron uptake into the heart under iron overload condition. Recently, cardiomyocytes which exposed to lipocalin-2 (LCN-2) have been shown to increase apoptosis due to excessive intracellular iron accumulation. However, the mechanistic roles of LCN-2 and LCN-2 receptor (LCN-2R) as iron transporters in cardiomyocytes under iron overload condition have never been investigated. Purpose We hypothesized that the LCN-2 and LCN-2R are alternate iron uptake pathways into cardiomyocytes under iron overload condition. Methods H9c2 cardiomyocytes were treated with either LCN-2 siRNA or LCN-2R siRNA for 72 hr or LTCC blocker (verapamil), TTCC blocker (TTA-P2), or iron chelator deferiprone (DFP) for 1 hr. After treatment, cells were exposed to ferric ammonium citrate (FAC, Fe3+) or FAC + 1mM ascorbic acid (Fe2+) at 200 μM for 48 hr. Intracellular iron level, cell viability, mitochondrial dynamics, mitophagy and apoptosis were determined. Results Both Fe2+ and Fe3+ treated groups showed significantly increased intracellular iron uptake, decreased cell viability, increased mitochondrial fission, mitophagy and apoptotic protein expression in cardiomyocytes. Under Fe2+ overload condition, treatments with LTCC blocker, TTCC blocker, and DFP could significantly decrease intracellular iron accumulation and increase cell viability via decreasing mitochondrial fission, mitophagy and cleaved caspase-3 (Figure), whereas both LCN-2 and LCN-2R siRNA treatment had no beneficial effects on these parameters. Under Fe3+ overload condition, treatment with LCN-2 siRNA, LCN-2R siRNA, and DFP showed beneficial effects on those parameters, whereas neither LTCC nor TTCC blocker provided these benefits (Figure 1). Conclusion Silencing of LCN-2 and LCN-2R increased cardiomyocyte viability via decreasing iron uptake, mitochondrial fission, mitophagy and apoptosis under Fe3+ iron overload condition. Meanwhile, treatment with calcium channel blockers improved cardiomyocytes viability via decreasing iron uptake, mitochondrial fission, mitophagy and apoptosis under Fe2+ iron overload condition. All of these findings suggested that LTCC and TTCC played important roles for Fe2+ uptake, whereas LCN-2 and LCN-2R were essential for Fe3+ uptake into the cardiomyocytes under iron overload conditions. Figure 1. Cell viability and apoptosis Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Thailand Research Fund and NSTDA Research Chair Grant (NC)

Lipocalin 2 induces neuroinflammation and blood-brain barrier dysfunction through liver-brain axis in murine model of nonalcoholic steatohepatitis

BackgroundRecent clinical and basic research implicated a strong correlation between NAFLD/NASH phenotypes with ectopic manifestations including neuroinflammation and neurodegeneration, but the mediators and critical pathways involved are not well understood. Lipocalin 2 (Lcn2) is one of the important mediators exclusively produced in the liver and circulation during NASH pathology.MethodsUsing murine model of NASH, we studied the role of Lcn2 as a potent mediator of neuroinflammation and neurodegeneration in NASH pathology via the liver-brain axis.ResultsResults showed that high circulatory Lcn2 activated 24p3R (Lipocalin2 receptor) in the brain and induced the release of high mobility group box 1 (HMGB1) preferably from brain cells. Released HMGB1 acted as a preferential ligand to toll-like receptor 4 (TLR4) and induced oxidative stress by activation of NOX-2 signaling involving activated p65 protein of the NF-κB complex. Further, the HMGB1-derived downstream signaling cascade activated NLRP3 inflammasome and release of proinflammatory cytokines IL-6 and IL-1β from brain cells. In addition, to advance our present understanding, in vitro studies were performed in primary brain endothelial cells where results showed high circulatory Lcn2 influenced HMGB1 secretion. Mechanistically, we also showed that elevated Lcn2 level in underlying NASH might be a likely cause for induction of blood-brain barrier dysfunction since the adipokine decreased the expression of tight junction protein Claudin 5 and caused subsequent elevation of pro-inflammatory cytokines IL-6 and IL-1β.ConclusionIn conclusion, the NASH-induced brain pathology might be because of increased Lcn2-induced release of HMGB1 and accompanying neuroinflammation.

Lipocalin 2 Induces Neuroinflammation through Liver‐Brain Axis in Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is chronic liver disorder characterized by accumulation of excess fat in liver due to consumption of high fat/lipid diets. A multiple hit paradigm that include lipotoxicity, environmental and genetic causes for prolonged period often leads to nonalcoholic steatohepatitis (NASH) with scarring and inflammation in liver that consequently causes irreversible hepatic damage. Both clinical and basic research implicated a strong correlation between NAFLD/NASH phenotypes with several ectopic manifestations like type 2 diabetes, cardiovascular disease, chronic kidney disease and more recently neuroinflammation and neurodegeneration. Notably, mechanisms of neuroinflammation in underlying NAFLD are poorly understood. In this report we identified adipokine Lipocalin 2 (Lcn2) induced neuroinflammation by acting through liver‐brain axis in diet induced murine NAFLD/NASH model. We found that Lcn2 is overexpressed in liver and in portal circulation that activated 24p3R (Lipocalin2 receptor) in brain and induced the release of high mobility group box 1 (HMGB1) from brain cells. Released HMGB1 acted as a preferential ligand to Toll like receptor 4 (TLR 4) and induced oxidative stress by activation of NOX‐2 signaling. NOX2 downstream signaling activated NF‐kB by phosphorylation of p65 protein. Activation of p65 and NOX‐2 led to NLRP‐3 inflammasome activation which subsequently released proinflammatory cytokines IL‐6 and IL‐1β from brain cells. Furthermore, we show that elevated Lcn2 level from neurons in underlying NAFLD caused a non disruptive blood brain barrier dysfunction by over production of TNFα, IL‐1β and MMP 9 without alteration of tight junction proteins Claudin 5 and Occludin. Based on the above, the above findings identify Lcn2 as a potent molecule inducing neuroinflammation in underlying NAFLD.Support or Funding InformationNIH#P01ES028942‐01 to Dr. Saurabh Chatterjee

Mechanisms of Iron Clearance from the Joint in FVIII-Deficient Mice after Induced Hemarthrosis

Introduction Hemarthrosis in hemophilia causes toxic iron accumulation in the joint, which contributes to synovitis. This study aimed to explore mechanisms and timing of iron clearance from the joint space in mouse models of induced hemarthrosis. Methods Hemarthrosis was induced by sub-patellar puncture in FVIII-deficient mice and in hypocoagulable BALB/c (HypoBALB/c) mice treated with 10 µg/ml warfarin for 7 days and 0.25 mg/kg anti-FVIII antibody 2 hours before knee puncture. Warfarin was reversed on day 2 post-injury with 100 IU/Kg 4-factor prothrombinase complex concentrate and the hematocrit determined in all mice as a measure of joint bleeding. Joint tissue was harvested at baseline and 2 or 4 weeks post-injury for analysis by histology. Ferric iron (Fe3+) was detected by Prussian Blue staining either during or after joint decalcification. Macrophages and macrophage-like synoviocytes were detected in joint tissue by immunohistochemistry with an anti-CD68 antibody. Synovial tissue was harvested from FVIII-deficient mice on day 3 and 2 weeks post-injury for gene expression studies by RNA sequencing (single-end; 75 bp) on an Illumina NextSeq500 platform. The limma-voom method (R BioConductor) was used for differential expression analyses. Results Knee injury caused substantial and comparable hemarthrosis in FVIII-deficient and HypoBALB/c mice (mean day 2 hematocrit: 27 % and 28 %, respectively). Post-decalcification Prussian Blue staining detected ferric iron accumulation in FVIII-deficient mice at week 4 only (5.3-fold increase compared to baseline, p=0.003). No ferric iron was detected in HypoBALB/c mice despite similar bleed volumes. In FVIII-deficient mice, Prussian Blue staining during decalcification was more sensitive and preserved detection of extracellular ferric iron, revealing a significant increase in ferric iron at 2 weeks post-injury relative to baseline (38-fold, p=0.005), which persisted at 4 weeks (23-fold, p=0.03). These findings coincided with increased CD68 staining at 2 weeks (36-fold, p=0.0002) and 4 weeks (8-fold, p=0.1). CD68-positive cells were dispersed throughout synovium at 2 weeks but appeared more clustered at 4 weeks and co-localized with iron staining, suggesting migration and iron uptake between 2 and 4 weeks post-injury. In HypoBALB/c mice, CD68 staining increased at 2 weeks (11-fold, p=0.008) but to a lesser extent than in FVIII-deficient mice, and was comparable to baseline at 4 weeks. Together, this suggests an altered mechanism of iron clearance in hemophilia. RNA sequencing revealed differential expression of 11/57 genes relating to iron transport in synovium on day 3, persisting somewhat at 2 weeks. Upregulated genes on day 3 included heme-oxygenase-1 (heme-degrading enzyme; 31-fold, p=3x10-6), lipocalin-2 (iron-binding protein; 10-fold, p=0.001) and solute carrier family (slc) 11 member 1 (macrophage iron transporter; 3-fold, p=0.0004). Down-regulated genes on day 3 included ceruloplasmin (efflux of cellular iron; 17-fold, p=5x10-5) and its homolog hephaestin (5-fold, p=0.002), CD163 (macrophage scavenging receptor for hemoglobin; 5-fold, p=0.03) and slc22 member 17 (lipocalin-2 receptor; 2-fold, p=0.03). Gene expression changes revealed key players involved in scavenging, degradation and transport of iron in synovium after hemarthrosis, and may expose mechanisms of impaired iron clearance in hemophilia pending further studies. Conclusions Iron handling after hemarthrosis, including uptake and transport in synovium and/or delivery to plasma transferrin, may be impaired in hemophilia and contribute to the evolution of hemophilic arthropathy. Unbiased RNA sequencing created several hypotheses that can be tested to further to elucidate mechanisms and timing of aberrant iron handling. Disclosures von Drygalski: Hematherix Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Founder; University of California San Diego: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; UniQure, Bayer, Bioverativ/Sanofi, Pfizer, Novo Nordisk, Biomarin, Shire, CSL Behring: Consultancy.

Expression of uterine lipocalin 2 and its receptor during early- to mid-pregnancy period in mares.

From previous cDNA subtraction studies analyzing gene expression in equine endometrium, high lipocalin 2 (LCN2) mRNA expression was found in the gravid endometrium. In the uterus, LCN2 may transport hydrophobic molecules and siderophores with iron, or may form a complex with another protein, however, the expression of uterine LCN2 beyond day 20 of equine pregnancy and its receptor has not been characterized. To study the expression and potential roles of uterine LCN2 from pre-implantation to mid-gestation period, stage-specific endometrial samples were obtained from day 13 (day 0 = ovulation) cyclic and days 13, 19, 25, and 60 to 131 pregnant mares. Expression of LCN2 mRNA increased in day 19 gravid endometrium and was abundant from day 60 onward. The expression of LCN2 mRNA was localized to the glandular epithelium. LCN2 protein was detected in day 25 gravid endometrium and luminal fluid, and the protein was localized to the glandular epithelium and luminal cavity, whereas LCN2 receptor expression was found in luminal and glandular epithelium and trophectoderm throughout the experimental period. The presence of matrix metalloproteinase-9 (MMP9) was also examined because MMP9 is known to form a complex with LCN2. Although MMP9 and LCN2 were both found in luminal fluid from day 25 pregnant uterus, the complex of these proteins was not detected. Localization of the receptor in the trophectoderm suggests that endometrial LCN2 could play a role in carrying small substances from the mother to fetus in the equine species.

Cell autonomous and noncell‐autonomous role of NF‐κB p50 in astrocyte‐mediated fate specification of adult neural progenitor cells

In previous work, we demonstrated that NF-κB p50 acts as crucial regulator of adult hippocampal neural progenitor cells (ahNPC). Indeed, NF-κB p50 knockout (KO) mice are characterized by remarkably reduced hippocampal neurogenesis. As a follow up to that work, herein we show that when cultured in vitro, ahNPC from wild type (WT) and p50KO mice are not significantly different in their neurogenic potential. This observation prompted us to investigate cell-autonomous and noncell-autonomous consequences of p50 absence on neuronal fate specification of ahNPC. In particular, we focused our attention on astrocytes, known to provide soluble proneurogenic signals, and investigated the influence of WT and p50KO astrocyte conditioned media (ACM) on WT and p50KO ahNPC differentiation. Interestingly, while WT ACM promoted both neuronal and astroglial differentiations, p50KO ACM only supported astroglial differentiation of WT ahNPC. By using a LC-MS/MS approach, we identified some proteins, which are significantly upregulated in p50KO compared with WT astrocytes. Among them, lipocalin-2 (LCN-2) was recognized as a novel astroglial-derived signal regulating neuronal fate specification of ahNPC. Interestingly, LCN-2 proneurogenic effect was greatly reduced in p50KO NPC, where LCN-2 receptor gene expression appeared downregulated. In addition to that, we demonstrated p50KO NPC unresponsiveness to both neuronal and astroglial fate specification signals from WT and p50KO ACM, and we identified a reduced expression of α2δ1, a thrombospondin-1 receptor, as another phenotypic change occurring in ahNPC in the absence of p50. Altogether, our data suggest that dysregulated NPC-astrocyte communication may contribute to a reduced hippocampal neurogenesis in p50KO mice in vivo. GLIA 2016 GLIA 2017;65:169-181.

The level of nitric oxide regulates lipocalin-2 expression under inflammatory condition in RINm5F beta-cells

We previously reported that proinflammatory cytokines (interleukin-1β and interferon-γ) induced the expression of lipocalin-2 (LCN-2) together with inducible nitric oxide synthase (iNOS) in RINm5F beta-cells. Therefore, we examined the effect of nitric oxide (NO) on LCN-2 expression in cytokines-treated RINm5F beta-cells. Additionally, we observed the effect of LCN-2 on cell viability. First, we found the existence of LCN-2 receptor and the internalization of exogenous recombinant LCN-2 peptide in RINm5F and INS-1 beta-cells. Next, the effects of NO on LCN-2 expression were evaluated. Aminoguanidine, an iNOS inhibitor and iNOS gene silencing significantly inhibited cytokines-induced LCN-2 expression while sodium nitroprusside (SNP), an NO donor potentiated it. Luciferase reporter assay showed that transcription factor NF-κB was not involved in LCN-2 expression. Both LCN-2 mRNA and protein stability assays were conducted. SNP did not affect LCN-2 mRNA stability, however, it significantly reduced LCN-2 protein degradation. The LCN-2 protein degradation was significantly attenuated by MG132, a proteasome inhibitor. Finally, the effect of LCN-2 on cell viability was evaluated. LCN-2 peptide treatment and LCN-2 overexpression significantly reduced cell viability. FACS analysis showed that LCN-2 induced the apoptosis of the cells. Collectively, NO level affects LCN-2 expression via regulation of LCN-2 protein stability under inflammatory condition and LCN-2 may reduce beta-cell viability by promoting apoptosis.

Role of lipocalin 2 in intraventricular haemoglobin-induced brain injury

ObjectiveOur recent studies have shown that blood components, including haemoglobin and iron, contribute to hydrocephalus development and brain injury after intraventricular haemorrhage (IVH). The current study investigated the role of...

Lipocalin 2 and Blood-Brain Barrier Disruption in White Matter after Experimental Subarachnoid Hemorrhage.

We reported previously that subarachnoid hemorrhage (SAH) causes acute white matter injury in mice. In this study, we investigated lipocalin 2 (LCN2) mediated blood-brain barrier (BBB) disruption in white matter, which may lead to subsequent injury. SAH was induced by endovascular perforation in wild-type (WT) and LCN2-knockout (LCN2(-/-)) mice. Sham mice underwent the same procedure without perforation. Mice underwent magnetic resonance imaging (MRI) 24 h after SAH to confirm the development of T2-hyperintensity in white matter. Western blotting and immunohistochemistry were performed to elucidate the mechanisms of LCN2-mediated white matter injury and BBB disruption. It was confirmed that LCN2 expression was significantly increased in white matter of WT mice after SAH by Western blotting (versus sham; p < 0.05). Immunohistochemistry showed that LCN2 receptor 24p3R was expressed in oligodendrocytes, astrocytes, endothelial cells, and pericytes in the white matter. In WT mice with SAH, albumin leakage along the white matter was prominently observed and was consistent with T2-hyperintensity on MRI. As with our previous report, LCN2(-/-) mice scarcely developed T2-hyperintensity on MRI or albumin leakage in white matter. Our results suggest that BBB leakage occurs in white matter after SAH and that LCN2 contributes to SAH-induced BBB disruption.

Neutophil Gelatinase –Associated Lipocalin (LCN2) Affects Primary Melofibrosis Hematopoietic Progenitor Cells As Well As Microenvironmental Cells

Primary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm (MPN) characterized by preferential proliferation of malignant hematopoietic progenitor cells which leads to excessive proliferation of marrow microenvironmental cells which are not involved by the malignant process. These events result in a clinical disorder characterized by anemia, a leukoerythroblastic blood picture, constitutive mobilization of CD34+ cells, extramedullary hematopoiesis, dacrocytosis, marrow megakaryocytic hyperplasia, progressive splenomegaly and reticulin and collagen marrow fibrosis. Several cytokines elaborated by PMF hematopoietic cells including TGF-b, vascular endothelial growth factor and tumor necrosis factor a have been implicated as playing a role in creation of the MF clinical phenotype. Neutrophil gelatinase associated Lipocalin-2 (LCN2) has been reported to have two distinct roles in the pathobiology of chronic myeloid leukemia (CML), suppressing residual normal HPC development and promoting CML proliferation (Devireddy LR et al, 2005, Cell). We, therefore hypothesized that LCN2 might also play a role in the development of the phenotypic features of PMF. Plasma LCN2 levels were measured by ELISA in 77 patients with PMF and were shown to be elevated as compared to 16 normal plasmas (P<0.001) Media conditioned by PMF MNC contained higher levels of LCN2 than media conditioned by normal MNC (p=0.03). The LCN2 receptor level was flow cytometrically analyzed and a significantly greater percentage of normal MNC and CD34+ cells than PMF MNC and CD34+ cells expressed the LCN2 receptor. The effect of increasing concentrations of recombinant LCN2 on CFU-GM and BFU-E derived colony by normal BM CD34+ cells as well as the PMF CD34+ cells was evaluated. The addition of LCN2 inhibited CFU-GM and BFU-E derived colony formation by normal CD34+ cells at a dose of 100 ng/ml (p=0.048 and p=0.043, respectively), while a similar dose of LCN2 did not influence the number of colonies cloned from PMF CD34+ cells. Previously our laboratory has reported that the expression of the chemokine receptor CXCR4 was decreased in PMF CD34+ cells which we hypothesized contributed to abnormal trafficking of CD34+ cells. LCN2 has been reported to affect CXCR4 expression by marrow CD34+ cells (Costa D, et al. 2010, Cytokine). CXCR4 expression by PMF CD34+ cells was lower than that of normal BM CD34+ cells. Incubation with LCN2, however, further reduced the expression of CXCR4 of PMF CD34+ cells by 10 to 50 % (p=0.012). By contrast, LCN2 increased CXCR4+ expression by normal CD34+ cells. We next evaluated the effects of LCN2 on the BM microenvironment. Normal BM mononuclear cells were plated in dishes exposed to the vary doses of LCN2 (10, 50, 100, 200 ng/ml) and the formation of adherent cell layers was monitored. Low doses of LCN2 (10 and 50 ng/ml) promoted the formation and proliferation of adherent cell layers composed of fibroblast-like cells after 1-3 weeks of culture. The fibroblast-like cells expressed vimentin and von Willebrand factor, indicating that they resembled mesenchymal stem cells, fibroblast and endothelial cells. We then examined the formation of adherent cell layers by normal BM MNC co-cultured with PMF, PV or normal MNCs separated by a 0.4 um trans-well for three weeks. The proliferation of confluent fibroblast-like cells was observed solely in BM MNC co-cultured with PMF MNC. These data indicate that LCN2 is generated in increased amounts by PMF mononuclear cells and likely plays a role in PMF biology by promoting malignant hematopoiesis but suppressing normal hemaopoiesis, suppressing CXCR4 expression by PMF CD34+ cells and promoting marrow fibroblast proliferation. Disclosures:No relevant conflicts of interest to declare.

Role of Lipocalin-2-Chemokine Axis in the Development of Neuropathic Pain following Peripheral Nerve Injury

Lipocalin 2 (LCN2), which is also known as 24p3 and neutrophil gelatinase-associated lipocalin (NGAL), binds small, hydrophobic ligands and interacts with cell surface receptor 24p3R to regulate diverse cellular processes. In the present study, we examined the role of LCN2 in the pathogenesis of neuropathic pain using a mouse model of spared nerve injury (SNI). Lcn2 mRNA levels were significantly increased in the dorsal horn of the spinal cord after SNI, and LCN2 protein was mainly localized in neurons of the dorsal and ventral horns. LCN2 receptor 24p3R was expressed in spinal neurons and microglia after SNI. Lcn2-deficient mice exhibited significantly less mechanical pain hypersensitivity during the early phase after SNI, and an intrathecal injection of recombinant LCN2 protein elicited mechanical pain hypersensitivity in naive animals. Lcn2 deficiency, however, did not affect acute nociceptive pain. Lcn2-deficient mice showed significantly less microglial activation and proalgesic chemokine (CCL2 and CXCL1) production in the spinal cord after SNI than wild-type mice, and recombinant LCN2 protein induced the expression of these chemokines in cultured neurons. Furthermore, the expression of LCN2 and its receptor was detected in neutrophils and macrophages in the sciatic nerve following SNI, suggesting the potential role of peripheral LCN2 in neuropathic pain. Taken together, our results indicate that LCN2 plays a critical role in the development of pain hypersensitivity following peripheral nerve injury and suggest that LCN2 mediates neuropathic pain by inducing chemokine expression and subsequent microglial activation.

Abstract 5326: CCL2 is critical for immunoresistance in association with cancer EMT

Abstract Purpose: Epitherial-to-mesenchymal transition (EMT) is a key step toward cancer metastasis, which is still a critical issue in cancer therapy. We previously reported that Snail-induced EMT accelerates cancer metastasis through multiple immunosuppressions using murine and human melanoma cells. However, the molecular mechanism is not completely understood. In this study, we confirmed the Snail-induced immunosuppressions using various human cancer cell lines, and further elucidated the mechanisms by identifying the specific mediators. Results: We first established human pancreatic cancer cell line Panc-1 cells showing typical EMT features such as reduced adhesion and enhanced invasion after transduction with snail gene, which is a transcription factor governing EMT. When human PBMCs were cocultured with the Snail transfectants, CD11c+ cell differentiation was significantly inhibited, and immunosuppressive CD4+FOXP3+ Treg cells were increased as similarly observed using Snail+ melanoma, suggesting the general features of the Snail-induced immunosuppressive mechanisms. We next conducted GeneChip analysis to identify other possible mechanisms by comparing gene profiles between the Snail transfectant and the parental cells. CCL2 was significantly upregulated in various Snail+ tumor cells after transduction with snail or treatment with TGFβ, a potent EMT inducer. CCL2 not only enhanced tumor invasion but also caused immunosuppressions through induction of Lipocalin 2 (LCN2), which was reported to induce apoptosis in various cells. CCL2 also increased LCN2 receptor Megalin and immunosuppressive molecules IDO and PD-L1 in DCs; in turn, LCN2 increased CCR2 in DCs. Utilizing the cooperative mechanism induced by CCL2 and LCN2, Snail+ cancer cells severely hampered APC function through induction of apoptosis and PD-L1/IDO expressions followed by Treg generation and then CTL attenuation. CCL2 blockade utilizing the specific siRNA or antibody significantly inhibited both tumor invasion and those immunosuppressions in vitro. Intratumoral injection with these anti-CCL2 reagents significantly inhibited tumor growth and metastasis by enhancing anti-tumor response systemically in vivo. Conclusion: These results demonstrate that CCL2 plays a vital role in the Snail-induced immunosuppressions. CCL2 blockade in tumor microenvironment may be an attractive strategy to overcome metastasis and immunoresistance of Snail+ cancers by restoring immunocompetence in cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5326.